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Scientific Journal Published by the College of Dentistry University of Baghdad

Vol. 24 No. 3 2012 ISSN ISSN 1680-0087

A quarterly peer reviewed published scientific journal of the College of Dentistry, University of Baghdad.

Editor in chief: Prof. Dr. Nabil Abdulfatah Hatoor, M.Sc Vice editor in chief: Prof. Dr. Hussain Faisal Al-Huwaizi M.Sc., PhD
National Members Prof. Dr. Adel Farhan MSc Prof. Dr. Zainab Al-Dahan MSc Prof. Dr.Wasan Hamdi M.Sc, PhD Prof. Dr. Lekaa Mahmood M.Sc Prof. Dr. Fakhri Abid Ali M.Sc. Prof. Dr. Nidhal Hussain MSc Assist. Prof. Dr. Sahar Shaker MSc Assist. Prof. Dr. Ghassan Abdulhameed MSc Assist. Prof. Dr. Saif Seham MSc Assist. Prof. Dr. Abbas Fadhil PhD Assist. Lecturer Dr. Ammar Salim MSc Board of editorial consultants: Prof. Dr. Khulood Al-Safi Prof. Dr. Ausama Al-Mulla Prof. Dr. Athraa Mostafa Prof. Dr. Maan Rasheed Secretarial committee: 1- Lecturer Dr. Mohammad Nahidh MSc 2- Lecturer Dr. Yassir AbdulKadum MSc 3- Assist. Lecturer Dr. Ahmed Fadhil MSc 4- Assist. Lecturer Dr. Ayad M. Al-Obaidi MSc For consultation, please contact: Website: www.codental.uobaghdad.edu.iq E-mail: baghdad_dentistry@yahoo.com Telephone: (+9641)4169375 Fax: (+9641)4140738 Prof. Dr. Khalid Hamdan Assist. Prof. Lamiaa Hamid Assist. Prof. Intesar Jameel Assist. Prof. Adel Al-Khaeeat International Members Prof. J. L. Gutmann D.D.S., Ph.D.(USA) Prof. Dr. M. Goldberg PhD (France)

Contents
i ii v
Editor and Editorial Board Contents Instructions for the Authors Restorative Dentistry

1 8 13 18 25 29 36 42 47

The shaping effects of three rotary Nickel-Titanium systems in simulated curved canals Hamid Kassim, Abdul Karim J. Al-Azzawi Effect of glass fiber reinforcement surface treatment on the soft liner retention and longevity. Inas Abdul-Sattar, Nabeel Abdul-Fattah The effect of autoclave processing on some properties of heat cured denture base material. Salwan S. Abdulwahhab, Widad A.H. Alnakkash Tooth movement in maxillary complete dentures fabricated with fluid resin polymer using different investment materials. Sanaa R. Abd Al-Aaloosi, Nabeel Abdul Fatah An evaluation of water absorption of Giomer in comparison to other resin-based restorative materials Shatha Abdul Kareem, Rasha H. Jehad The effect of plasma treatment on the bonding of soft denture liners to heat cured acrylic resin denture base material and on some surface properties of acrylic resin polymer. Shaymaa H. Masood, Salah A. Mohamed. Influence of SOLO disinfectant on some properties of different denture lining materials. Shorouq M. Abass, Reem A. Nassif, Bayan S. Khalaf Evaluation of shear bond strength of silicon-based soft liner to the acrylic resin denture base using different polymerization technique with different storage periods in distilled water. Thikra M. Hachim The influence of different pH of saliva and thermal cycling on the adaptation of different denture base materials. Yasir A. Hussein, Shatha S. Al-Ameer

Oral Diagnosis

54

Differential infiltration of CD4, CD8 and macrophage in oral squamous cell carcinoma (Immnoistochemical study). Ahmed A. Ali, Riyadh O. Alkaisi

ii

59 63 67 71 78

Detection of acid fast bacilli in the saliva of patients having pulmonary tuberculosis. GassanYassen, Jamal Noori, Nadia Sabri Yas Trace elements and oxidative stress markers in saliva of subjects with amalgam fillings. Huda Sh. Ahmed, Taghreed F. Zaidan. Ali Yakub A cytopathological study of the effect of smoking on the oral epithelial cells in relation to oral health status by the micronucleus assay. Saeed H. Saeed, Wasen H. Younis Evaluation of the effect of platelet-rich plasma on intrabony defect repair in glucocorticoids-induced osteoporosis in rabbits (Histological and biochemical study). Sudad H. M. Al-Shehabi, Nada M. Hassan Al-Ghaban A specific focus imaging for dental implant planning (CT-Based). Zainab H. Al-Ghurabi, Shafaa H. A-Nuaemi

Oral and Maxillofacial Surgery and Periodontology

82 87 93

Effects of low-energy laser in the treatment of myofascial pain dysfunction syndrome of the temporomandibular joint. Ali H. Abbas. Assessment of salivary elements (Zinc, Copper and Magnesium) among groups of patients with rheumatoid arthritis and chronic periodontitis and its correlation to periodontal health status. Alyamama Mahmood, Maha Shukri. Inorganic ions level in saliva of patients with chronic periodontitis and healthy subjects. Wasan A. Abid Aun.

Orthodontics, Pedodontic, and Preventive Dentistry

98 106 113 116 122 129

The efficiency and stability of maxillary expansion with Quadhelix; a longitudinal study. Anfal AbdulMajeed Al-Ani Effect of casein phosphopeptide-amorphous calcium phosphate on surface roughness of a siloranebased and methacrylate-based composite resin (In vitro comparative study). Baydaa Hussein Effect of repeated pregnancies on periodontal health status. Baydaa A. Yas

Effect of thymus vulgaris extract on streptococci and mutans streptococci, in comparison to chlorhexidine gluconate (in vivo study). Eman A. Al-Timimi, Mohammed AL-Casey. Pain intensity and control with fixed orthodontic appliance therapy (A clinical comparative study on Iraqi sample). Hayder Fadhil Saloom

Salivary vitamins and total proteins, in relation to caries-experience and gingival health, according to nutritional status of a group of five-year old children. Nada J. Radhi

iii

137 140

Orthodontic bracket failure rate; A comparative clinical study between light cured and chemically cured (no mix) bonding systems. Natheer A. Rasheed Correlation between caries related bacteria in plaque and saliva in different age group children. Zainab J. Jafar, Yasameen A.A. Al-Bayati, Ghada I. Taha

Basic Sciences

145 149 154 158

Quantitative analysis of IgG antinuclear antibody in chronic periodontitis patients. Batool H. AlGhurabei, Hind Wael Al-Alousi, Ahmed A. Al-Hassan Comparison between severe haemophilic A and healthy children in Streptococcus mutans, oral Lactobacilli and Candida albicans counts. Zainab A. AlDhaher, Maha F. Almelan, Luma M. Alhadi

Effect of Nigella Sativa L. extracts against Streptococcus mutans and Streptococcus mitis in Vitro. Najah A. Mohammed

Evaluation of Entamoeba gingivalis and Trichomonas tenax in patients with periodontitis and gingivitis and its correlation with some risk factors. Sumaiah Ibrahim, Rasha Abbas

iv

Instruction for the Authors


The Journal of the College of Dentistry accepts manuscripts that address all topics related to dentistry. Manuscripts should be prepared in the following manner: Typescript. Type the manuscript on A4 white paper, with page setup of 2.5 cm margins. Type the manuscript with English language font Times New Roman and the sizes are as follows: 1) Font size 18 and Bold for the title of the manuscript. 2) Font size 14, Bold and capital letters for the headings as ABSTARCT, INTRODUCTION, MATERIALS AND METHODS, RESULTS and REFERENCES. 3) Font size 12 Bold and italic for the names and addresses of the authors ex. Ahmed G. Husam 4) Font size 11 for the legends of the tables and figures. 5) Font size 10.5 for the text in the manuscript. 6) Font size 10 for the text inside the tables. 7) Font size 9 for the references at the end of the manuscript. Use single spacing throughout the manuscript and numbering of the pages should be in the lower right hand corner. Title of the manuscript: The title should be written with a capital letter for the first word as (Effect of the retention and stability.etc). Abstract and key words. The abstract should contain no more than 250 words. The abstract should be divided to the following categories: Background: (It contains a brief explanation about the problem for which the research was done as well as the aim of the study), Materials and methods:, Results:, and Conclusion:. Below the abstract, write 3-5 key words that refer as close as possible to the article. The abstract should be written by the font Century Gothic size 8. Text. The body of the manuscript should be divided into sections preceded by the appropriate major headings (INTRODUCTION, MATERIALS AND METHODS, RESULTS and REFERENCES) which are written in bold and capital. Minor headings should be typed in bold and subheadings should be not bold but underlined. References. References are placed in the text using the Vancouver system (Numbering system). Number references consecutively in the order in which they are first mentioned in the text. Identify references in the text, tables, and figures by Arabic numerals, and place them in parentheses within the sentence as superscription ex. (2). Use the style of the examples given below in listing the references at the end of the manuscript: Book 1. Hickey JC, Zarb GA, Bolender CL. Bouchers prosthodontic treatment for edentulous patients. 9th ed. St. Louis: CV Mosby; 1985. p.312-23. Journal article 4. Jones ER, Smith IM, Doe JQ. Occlusion. J Prosthet Dent 1985; 53:120-9. Tables. All tables must have a title placed above the table. Identify tables with Arabic numbers (e.g. Table 1). The tables should be done with a width of no more than 8 cm. Figures and illustrations. All figures must have a title placed below the figure. Identify figures with Arabic numbers (e.g. Figure 1). The figures should be done with a width of no more than 8 cm. The article should not exceed 7 pages. The author should submit three copies of the article (one original and two copies) and a (CD) containing the article.

J Bagh College Dentistry

Vol. 24(3), 2012

The shaping effects

The shaping effects of three rotary Nickel-Titanium systems in simulated curved canals
Hamid Kassim, B.D.S. (1) Abdul Karim J. Al-Azzawi, B.D.S., M.Sc. (2)

ABSTRACT
Background: The purpose of this study is to compare three rotary endodontic nickel-titanium systems (ProFile, GT and ProTaper) with stainless steel hand K-flexofile in simulated curved canals at different levels, this includes: Incidence of canal aberrations (apical zipping associated with elbow, ledge and perforation), Changes of working length, Preparation time for each system and Breakage and permanent deformation of instruments. Materials and method: Eighty simulated curved canals made of clear polyester resin were used to assess instrumentation. The acrylic blocks were divided into four groups, 20 simulated canals for each group were enlarged from #10 to # 25. In the first three groups all NiTi rotary instruments were set into a permanent rotation with a 16:1 reduction hand piece powered by a torque-limited electric motor set at 300 rpm. All the instruments were used in a crown down manner using a gentle in-and-out (pecking) motion. In the fourth group the simulated canals were instrumented with stainless steel K-flexo-files by using balanced force technique. Each simulated canal was filled with a drawing ink using to increase the color contrast for photographic documentation. Photographs of the unprepared canals were taken by the aid of stereomicroscope and digital camera at magnification of 40 times. When instrumentation of the canals was completed, the canals were injected again with the drawing ink and the image procedure is repeated. Preand postoperative digital photographs of the resin blocks were accomplished using Adobe Photoshop CS2 software program. At this stage the amount of resin removed i.e. the difference between the canal configuration before and after instrumentation was determined for both the inner and the outer side of the curvature at five reference points. Assessments were made under the stereomicroscope according to the presence of different types of canal aberrations (apical zip associated with elbow, ledge and perforation). The changes of working length were determined by subtracting the length of master apical file from the original length (16mm). The time taken to prepare each canal was recorded in minutes with the aid of a stop timer. Throughout the study a record was kept of the numbers and sizes of instruments that fractured or became permanently deformed during use. R e s u l t s : there were significant differences among the groups, even though more zips and ledge were created with K-flexofile followed by ProTaper. NiTi instruments caused a significantly greater loss of working length than K-flexofile; while there was no significant difference among NiTi groups. The shortest mean of preparation time was recorded when ProTaper were used; while the longest time was documented for GT. No permanent deformation occurred with NiTi groups. None of the stainless steel K-flexofile was separated. C o n c l u s i o n : K-flexofiles created significantly more aberration than NiTi systems. ProTaper caused more morphological changes compared with ProFile and GT systems. Small mean changes in working length occurring with rotary NiTi instruments . The preparation time of ProTaper was faster than with other groups. NiTi instruments may be more susceptible to separation than stainless-steel instruments. Most failures of the hand stainless-steel files were deformations rather than fractures. Fracture rate was approximately 30% for ProTaper, 16.6% for GT and 8.3% for Profiles. Keywords: Canal aberrations, preparation time, working length change, instrument fracture and permanent deformation, ProTaper, GT, ProFile. (J Bagh Coll Dentistry 2012;24(3):1-7).

INTRODUCTION
The aim of root canal preparation is to clean and shape the root canal while maintaining its special relationship within the root. The desired result is a uniformly tapering canal that results from cutting the canal circumferentially as evenly as possible, maintaining the original canal outline with a definite apical seat which facilitates a hermetic seal at the obturation stage. This is especially difficult in curved canals, where procedural errors such as transportation, zipping, elbow formation, ledging, perforation, stripping perforation, and instrument fracture can occur (1,2).
(1) (2) M.Sc. Student, department of conservative dentistry college of dentistry, university of Baghdad. Professor , department of conservative dentistry, college of dentistry, university of Baghdad.

Various techniques have been used to avoid or minimize these errors, though none has been universally accepted as the answer to the maintenance of root canal curvature. Similarly, modification to instrument tip design, and flute alteration have not provided a solution to the management of the apical section of the curved root canal (1) . However, in particular when used in severely curved canals, traditional stainless steel instruments often fail to achieve the tapered root canal shapes needed for adequate cleaning and filling (3). In 1988, nickel titanium was first introduced in endodontics to overcome the limitations of stainless steel ISO instruments and make the preparation of curved canals much easier because of its superior elasticity and shape memory effect. During the last 10 years, scientific evidence has clearly shown that rotary Ni-Ti
1

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The shaping effects

instruments used in a crown-down fashion produce consistent canal shape, less debris extrusion and stay well centered inside the root canal (4). It is expected from modern engine-driven rotary root canal instruments made of nickel-titanium (Ni-Ti) to allow adequate and acceptable preparation of even severely curved root canals. Procedural errors were reported to be rather infrequent when root canals of extracted teeth were shaped with rotary Ni-Ti instruments (2) The purpose of this study is to compare three rotary endodontic nickel-titanium systems (ProFile, GT and ProTaper) with stainless steel hand K-flexofile in simulated curved canals at different levels, this includes: a) Incidence of canal aberrations (apical zipping associated with elbow, ledge and perforation). b) Changes of working length. c) Preparation time for each system. d) Breakage and permanent deformation of instruments.

each sequence. Prior to use, each instrument was coated with glycerin to act as a lubricant and copious irrigation with tap water was performed repeatedly before and after the use of each instrument using disposable syringes and 27 gauge tips.

Rotary NiTi instruments


In the first three groups all NiTi rotary instruments were set into a permanent rotation with a 16:1 reduction hand piece powered by a torque- limited electric motor set at 300 rpm. And the torque at 1.2 Ncm. All the instruments were used in a crown down manner using a gentle inand-out (pecking) motion until resistance was felt and changed for the next instrument.

Manual technique
In the fourth group the simulated canals were instrumented with stainless steel K-flexofiles by using balanced force technique described by Roan et al in 1985 (6). Which continue until an apical stop of size 25 was achieved. Then stepping backs the preparation with # 30, # 35 and # 40files and used also in balanced force motion.

MATERIALS AND METHODS


Simulated curved canals made of clear polyester resin were used to assess instrumentation. The diameter and the taper of all simulated canals were equivalent to an ISO standard size 10 root canal instrument. The canals were 16 mm long, the straight part being 11 mm and the curved part 5 mm with angle of 40 (5) (Fig.1).

Assessment of canal preparation Postoperative canal shape


Prior to their preparation, each simulated canal was filled with a drawing ink using a 27 gauge needle to increase the color contrast for photographic documentation.' In order to achieve a standardized position of the resin blocks against the lens of the microscope, a holder was constructed from stone for this purpose with a hole in the center in which the resin blocks could be placed and repositioned in exactly the same position. The central hole was covered with a transparent paper on which the five chosen levels were drawn and the artificial canal could be measured easily. Photographs of the unprepared canals were taken by the aid of stereomicroscope and digital camera at magnification of 40 times. One image on screen corresponded to 2 mm of the real canal length. Therefore eight images were needed to assemble the entire canal. Both X and Y coordinates on the microscope's nonius scale were recorded for each image, allowing repositioning and reproduction of the pictures at any given moment (i.e. pre- and postoperative). The images were standardized by securing the camera at a fixed distance from a microscope lens. After that the simulated canals were cleaned using tap water with irrigating syringe. When instrumentation of the canals was completed, the canals were
2

Figure 1: Angle and radius of canal curvature

Preparation of artificial canals


Eighty acrylic blocks were divided into four groups, 20 simulated canals for each group were enlarged from #10 to # 25. The first penetration in the simulated canal was performed with #10 K-file hand instrument to the full working length (16 mm). Patency of the resin blocks was checked with the same size after
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The shaping effects

injected again with the drawing ink and the image procedure is repeated. Pre- and postoperative digital photographs of the resin blocks were stored in a Pentium 4 computer and measurements were accomplished using Adobe Photoshop CS2 software program. At this stage, the amount of resin removed, i.e. the difference between the canal configuration before and after instrumentation was determined for both the inner and the outer side of the curvature at five reference points, using a method described by Calberson et al (7). All measurements were made at right angles to the surface of the canal (Fig.-2). Point 1 (O): the canal orifice. Point 2 (HO): the point half-way from the beginning of the curve to the orifice. Point 3 (BC): the point where the canal deviates from the long axis of its coronal portion and is called the beginning of the curvature. Point 4 (AC): the point where the long axes of the coronal and the apical portions of the canal intersect and are called the apex of the curve. Point 5 (EP): the end point of preparation.

The time was logged from the beginning of the preparation procedure and included total active instrumentation, instruments changes within the sequence and irrigation. Throughout the study a record was kept of the numbers and sizes of instruments that fractured or became permanently deformed during use by examining the instruments after each use under magnifying lens (10X).

RESULTS
Canal aberrations The results concerning the assessment of canal aberrations are summarized in Table-1.

Table 1: Incidence of canal aberrations by instruments types.


System Zip/elbow Ledge Perforation 4 4 0 PF 6 2 0 GT 8 8 0 PT 10 8 0 KO 0 Chi-square 2.627 2.11 0.042 0.049 P-value S S

With respect to the different types of aberration evaluated; there were significant differences among the groups, even though more zips and ledge were created with Kflexofile followed by ProTaper. ProFile created the lowest percentage of elbow; while GT produced the least number of ledges. No perforations were observed during preparation.

Figure 2: The five levels of measurement


Furthermore, basing on the superimposition of pre-and postoperative images, assessments were made under the stereomicroscope according to the presence of different types of canal aberrations (apical zip associated with elbow, ledge and perforation). These different types of canal aberrations were defined according to the detailed descriptions published by Thompson and Dummer (8). When the preparation was completed, a Kfile size 10 was inserted into each canal to the apical stop to prove that working length had been reached. The changes of working length w.ere determined by subtracting the length of master apical file from the original length (16mm). The time taken to prepare each canal was recorded in minutes with the aid of a stop timer. Restorative Dentistry

Figure 3: Composite image of the simulated resin canal with apical zip and elbow

Changes of working length


All canals remained patent following instrumentation; thus, none of the canals became blocked with resin shavings. None of the canals showed overextension of preparation, whereas a loss of working distance was found in
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The shaping effects

several canals. The mean changes of working length that occurred as a result of the preparation of the canals are given in Table -2. The difference among the four groups was statistically significant as in Table -3.

Table -6: ANOVA table of time


System F-test P-value 0.022 PF 3.39 P<0.05 GT S PT KO The statistical analysis of the data by ANOVA test showed significant difference among the four groups (Table -6). By Student t-test (Table-6) a significant difference was found between ProTaper and other groups. The ProFile was significantly quicker than GT; while there was no significant difference with K-fiexofile. Finally there was no significant difference between GT and K-flexofile.

Table 2: Mean changes of working length (mm) and standard deviation with the four different instruments.
System PF GT PT KO Mean 0.275 0.275 0.250 0.100 SD 0.255 0.256 0.255 0.205

Table-3: ANOVA table of change of working length.


System PF GT PT KO F-test 2.38 P-value 0.049 P<0.05 S

Table 7: t-test of time between groups


System PF&GT PF&PT PF&KO GT&PT GT&KO PT&KO t-test 2.52 2.14 1.20 3.89 0.03 2.08 P-value 0.017 0.04 0.24 0.004 0.98 0.048 S S NS S NS S

NiTi instruments caused a significantly greater loss of working length than K-flexofile; while there was no significant difference among NiTi groups (Table-4).

Table 4: t-test of change of working length (mm) among groups


System PF&GT PF&PT PF&KO GT&PT GT&KO PT&KO t-test 0.31 0.00 2.39 0.31 2.04 2.39 P-value 0.760 1.000 0.022 0.760 0.049 0.022 NS NS S NS S S

Instrument fracture and permanent deformation


The number of fractured and permanently deformed instruments that occurred during the study is listed in Table -8.

Table 8: Number of fractured and permanently deformed instruments


System PF GT PT KO Chi-square P-value Fracture 2 4 6 0 3.628 0.029 S Deformation 0 0 0 4 14.03 0.007 S

Preparation time
The mean time taken to prepare the canals with the different instruments is shown in Table-5. The shortest mean of preparation time was recorded when ProTaper were used (7.304 min.); while the longest time was documented for GT (8.309 min.).

Table- 5: Mean and standard deviation of preparation time (min.)


System PF GT PT KO Mean 7.744 8.309 7.304 8.295 SD 0.508 0.866 0.766 1.983

Throughout the preparation no permanent deformation occurred with NiTi groups. In term of fracture all happened at the tip region of the files. Six instruments of ProTaper were fractured (two Fl in the fourth use and four F2, two during the fourth manipulation and two throughout the fifth use).Two pieces of ProFile were broken (both were taper.04 size 25 in the fifth use) and four files of GT were separated (one taper .04 size 20 during the preparation of the fifth canal and three instruments taper .04 size 25 in the fourth handling).
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Vol. 24(3), 2012

The shaping effects

In the manual technique none of the stainless steel K-flexofile was separated on the other hand four files were permanently deformed (two pieces size 20 and two pieces size 25 during the fourth and fifth use respectively).

DISCUSSION
Canal aberration From Table-1 it is appear that the preparation with ProFile resulted in a significantly fewer zipping /elbow (n=4) than other tested instruments followed by GT (n=6), ProTaper (n=8) and K-flexofiles (n=10) respectively; While the incidence of ledges was as follow: GT (n=2), ProFile (n=4), ProTaper (n=8) and Kflexofiles (n=8). K-flexofiles created significantly more aberration than NiTi systems. Similar results have been established by most of reports concerned shaping ability of endodontic instruments (3) (9) (10). This highly incidence may be attributed to the inherited rigidity of these instruments. On the other hand ProTaper caused more morphological changes compared with ProFile and GT systems. This result is consistent with investigations of Schafer and Vlassis , 2004 a, b (11) ; Sonntag et al, 2007 2). This could be related to two reasons :(a) ProTaper is less flexible than other NiTi instruments due to progressive tapering of this system, (b) convex triangular cross-section design of ProTaper with no radial lands unlike U-shape cross-section of ProFile and GT with three radial lands which plane the canal walls rather than engaged and screwing into them and to cut of dentin evenly along the canal wall (13). The number of morphological changes in the present study was higher when compared with similar studies on NiTi instruments ( Hulsmann et al, 2001 (14); Schafer and Vlassis , 2004 a, b (1I)). This may be due to the sever curvature degree of artificial canals were used in this study (40) compared with other reports (28-35). Changes of working length In the present investigation the only statistically significant differences were found between rotary groups and the manual technique, whereas no significant differences were found among NiTi systems (Table-2). This finding is in agreement with observations of other studies whose observed only small mean changes in working length occurring with rotary NiTi instruments ( Kum et al, 2000 05) ; (8) Thompson and Dummer , 2000 ; Schafer Restorative Dentistry

and Florek , 2003a 10); Schafer and Vlassis , 2004a,b (11); Paque'et al, 2005 16); Guelzow et a1,2005 (3)). A number of reasons have been discussed to explain possible reasons for alteration of the working distance. Thompson and Dummer in 2000 (8) reported that these changes may probably be due to a minor canal straightening during canal enlargement. Other factors may be explain the significant difference of working length between NiTi groups and K-flexofiles, these related to the fact that, NiTi instruments caused early flaring of the coronal portion of the canal due to employment of crown-down technique and greater tapering of these instruments ending with great changes of working distance compared with balance force technique and ISO tapering of Kflexofiles. Therefore it is advisable to measure the working length after preparing the coronal part of the canal to avoid alteration of the working distance. Preparation time Although a brushing action had to be used with ProTaper before further advancing the files, the preparation time of this system was substantially faster than with other groups (Table-5). This outcome corroborates the results of previous studies (3) (16) This may be due to the convex triangular cross-sectional design of ProTaper resulting in a very aggressive cutting edges and a positive rake angle which is known to require less energy to cut dentine than blades with a neutral or negative rake angle as present in ProFile and GT. In other word the cutting ability of the active instruments is superior when compared with passive instruments with radial lands (16) .Furthermore, the finding that ProTaper took less working time was largely to the fact that the fewer number of instruments used (PT: 5; PR 6; GT: 6 KO: 9). The assumption that instrumentation times with rotary NiTi files were substantially faster than with stainless-steel hand files could not confirmed in the present study which demonstrated that root canal preparation was less time consuming when using K-flexofiles compared with GT, although the number of instruments was larger with K-flexofiles than GT .This outcome disagree with the findings of the most similar reports ( Schafer and Florek , 2003a (10); Guelzow et al, 2005 (3)). This longer preparation time for GT could be attributed to the operator's influence as well as to the cutting efficacy of the files. Additionally most of the previous investigations employed
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reaming motion with hand files unlike the present research that exercised balanced force technique which is recommended for the preparation of curved canals as it resulted in better configuration and less time consuming (6). Fracture and permanent deformation Several earlier studies indicating that NiTi instruments may be more susceptible to separation than stainless-steel instruments. This is because of their flexibility, there is a loss of tactile sensation as compared with the more rigid stainless-steel files, also when used NiTi instruments in a mechanical rotary motion to prepare narrow curved canals, the tips can bind and not move in the narrow portion of the canal, whilst the shaft and the coronal part of the file are still active according to the direction of the force exerted by the operator. Under these circumstances unpredictable separation might occur. This is in contrast to what has been observed with hand instruments which tend to unwind before a fracture occurs. So, most failures of the hand stainless-steel files were deformations rather than fractures (17). The given data of Table-8 shown that a total of 4 out of 36 instruments of Kflexofiles permanently deformed and this may reflect their rather inflexible nature a factor which may well explain their relatively high percentage of failure (11.12 %). Related to the total number of instruments used a fracture rate was approximately 30% for ProTaper (6 out of 20 files), 16.6% for GT (4 out of 24 files) and 8.3% for Profiles (2 out of 24 files). Possible explanations for the results noted with the ProTaper might be related to: (a) excessive taper of this system .The taper for finishing files used to prepare the apical portion of the canals in the ProTaper were .07and ,08 respectively; while the taper of the ProFile and GT was .04. According to a study by Haikel et at in 1999 (18) , taper was found to be a significant factor in determining fracture probability for files, (b) convex triangular cross-section of ProTaper which results in a more massive core, this may reduce the flexibility of the instruments when compared to U-shaped cross-sections of ProFile and GT systems. The degree of influence of the individual geometric characteristics of the instruments, however, remains speculative (12). The highly percentage of fracture of F2 files of ProTaper is in agreement with other study which has been shown that, F2 possess lower resistance to fracture because of cyclic fatigue
Restorative Dentistry 6

than the other instruments in the ProTaper series. It is known that instruments with low taper have lower resistance to torsional stress, whereas those with greater size and taper are more subjected to fracture because of cyclic fatigue (5) (19) . It is worth emphasizing that all fractured files were occurred when used for the fourth or fifth canal no fractures occurred when instruments were used to enlarge three canals only. This observation is in accordance with previous reports ( Thompson and Dummer , 2000 (8); Schafer and Florek , 2003 a (10) Schafer and Vlassis , 2004 a,b (12) ; Paque " et al, 2005 (16)). With regard to the incidence of file breakage, the amount of fracture in this study was higher than that reported by others ( B r y a n t and Dummer , 1999 (20); Yancy et al, 2001 (21)) and was most likely due to differences in the methodology. This study used curvatures much larger than that of the other studies, thus higher breakage would be expected due to increased stresses. In addition the above mentioned investigations used human extracted teeth; while plastic blocks were used in the present study .The simulated canals may have been a main cause of incidence of failure. -Obviously the instruments tend to attach to the plastic and pull into the canal, which leads to a higher failure rate than is seen when extracted teeth are used. The generated heat may sometimes soften the resin material so that cutting blades may bind and consequently break (22) During this study no NiTi file was deformed .This is in contrary with most of similar investigation which resulted in a high deformation percentage of NiTi instruments ( Eha b et al, 2005 (23) ; Ug ur et al, 2007 (24)). This is may be due to the difference in the procedures that has been depended. In the research the magnifying lens (10X) has been used to examine the files after each use. Clearly this power of enlargement was insufficient to detect the deformation of NiTi files which is usually observe under scanning electron microscope.

REFERENCES
1. Lam T, Lews D, Atkins D, Macfarlance R, Clarkson R, Whitehead M, Blockhurst P, Moule A. Changes in root canal morphology in simulated curved canals over-instrumented with a variety of stainless steel and Nickel-Titamiun files. Austral Dent J 1999, 44: (1): 12-19. 2. Bartha et al, 2006. 3. Guelzow A, Stamm 0, Marius P, Kielbassa A. Comparative study of six rotary nickel-titanium

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systems and hand instrumentation for root canal preparation. International Endodontic Journal 2005, 38 (10), 743-752. Webber & Machtou, 2001. Pruett J, Clement D, Games D. Cyclic fatigue testing of nickel- titanium endodontic instruments. J of Endod 1997; 23, 77-85. Roan J, Sabala C, Duncanson M. The balanced force concept for instrumentation of curved canals. J of Endod 1985; 11 (5), 203-11. Calberson F, Deroose C, Hommez G, Raes H, DeMoor R. Shaping ability of GT rotary files in simulated resin root canals. Intern Endod J 2002; 35: 607-14. Thompson S, Dummer P. Shaping ability of HER0642_rotary_nickle- titanium instruments in simulated root canals: Part 2. Intern Endod J 2000; 33:(3) 255-61. Schafer E, Lohmann D .Efficiency of rotary nickel titanium FlexMaster instruments compared with stainless steel hand KFlexofile Part 1. Shaping ability in simulated curved canals. Intern Endod J 2002; 35: 505-13. Schafer E, Florek H. Efficiency of rotary nickel-titanium K3 instruments compared with stainless steel hand K-flexofile. Part l.Shaping ability in simulated curved canals. Intern Endod J 2003; 36 (3), 199-207. Schafer E, Vlassis M .Comparative investigation of two rotary nickels titanium instruments: ProTaper versus RaCe. Part 1.Shaping ability in simulated curved canals. Intern Endod J 2004; 37(4) 229-38. Sonntag D, Mareike 0, Kathrin K, Vitus S. Root canal preparation with the NiTi systems K3. Mtwo and ProTaper. Aust Endod J 2007. Tepel J, Schafer E. Endodontic hand instruments: cutting efficiency, instrumentation of curved canals, bending and torsional properties. Endod Dent Traumatol 1997;13,201-210 Hulsmann M, Schade M, Schafers F. A comparative study of root canal preparation with

HERO 642 and Quantec SC rotary NiTi instruments. Intern Endod J 2001; 34: 538-46. 15. Kum K, Spangberg L, Cha B, Il-Young J, Seung J, Chan Young L. Shaping ability of three ProFile rotary instrumentation techniques in simulated resin root canals. J of Endod 2000; 26:719-23. 16. Pa que F, Musc h U, Hul sma nn M. Compa r i son of r o o t c a na l preparation using RaCe and ProTaper rotary NiTi instruments. Intern Endod J 2005; 38: 8-16. 17. Kavanagh D, Lumley P. An in vitro evaluation of canal preparation using Profile .04 and .06 taper instruments. Endod Dent Traumatol 1998; 14: 16-20. 18. Haikel Y, Serfaty R, Bateman G, Senger B, Allemann C. Dynamic a n d c y c l i c f a t i g u e o f engine-driven rotary nickel titanium endodontic instruments. J of Endod 1999; 25: 43440. 19. Fife D, Gambarini G, Britto L. Cyclic fatigue testing of ProTaper NiTi rotary instruments after clinical use. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endod 2004;97 (2) 251-6. 20. Bryant S, Dummer P. Shaping Ability of .04 and .06 Taper ProFile Rotary Nickel-Titanium Instruments in Simulated Root Canals. Intern Endod J 1999; 32:155-64. 21. Yancy A, Tygesen H, Steiman R, Ciavarro C. Comparison of distortion and separation utilizing Profile and Pow-R Nickel-Titanium rotary files. J of Endod 2001; 27:762-4. 22. Thompson S, Dummer P. Shaping ability of Profile .04 taper Series 29 rotary nickel-titanium instruments in simulated root canals. Part 1. Intern Endod J 1997; 30 (1) 1-7. 23. Ehab E, Azza H, Abeer M. Defects in Hero-Shaper Rotary Nickel Titanium Instruments before and after use. An SEM study. Ainshams Dent J 2005; 8: 2: June. 24. Ugur I, Cumhur A, Ozgur U, Ozgur T, Tayfun A. Evaluation of the surface characteristics of used and new ProTaper instruments: An atomic force microscopy study. J of Endod 2007; 33 (11).

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Vol. 24(3), 2012

Effect of glass fibe r

Effect of glass fiber reinforcement surface treatment on the soft liner retention and longevity
Inas Abdul-Sattar BDS, MSc (1) Nabeel Abdul-Fattah BDS, MSc (2)

ABSTRACT
Background: Denture liners have been used in dentistry for many years. Soft denture liner is one of the denture liners which used to enhance the fit of poor fitting dentures and prevent trauma to sensitive mucosa. One of the disadvantages of the soft denture liner is the frequent debonding from the denture during clinical use thus reducing the longevity of such prosthesis. Glass fibers integrated at or on the fitting surface of the denture used to improve the bonding of the silicone soft liner to the acrylic surface. Materials and methods: the shear bond strength calculated to evaluate the effect of the glass fiber surface treatment on the bonding between the acrylic surface and the silicon soft liner. 120 samples were prepared and divided into 3 major groups: Group I for the conventional heat cure acrylic, Group II for the pour acrylic and Group III for the light cure acrylic. Each one of these major groups divided into 2 subgroups, the first one for the Mollosil silicon soft liner and the second one is for the Molloplast-B silicon soft liner. Each one of these sub groups consist of 2 types of the acrylic surface: smooth (control) and glass fiber net surface treatment. Results: this study revealed that some types of the surface treatment exhibited a highly significant improvement in the bonding between the acrylic surface and the soft liner. Conclusion: glass fiber surface treatment could improve the bonding between the acrylic surface and the soft liner. Key words: silicon soft liner, glass fiber, shear bond strength, surface treatment. (J Bagh Coll Dentistry 2012;24(3):8-12).

INTRODUCTION
Denture liners have been used in dentistry for many years. They are classified into hard reline material, permanent tissue liners and tissue conditioners. (1) They are used to enhance the fit of poor fitting dentures and prevent trauma to sensitive mucosa by forming a cushioned layer between the denture base and the oral mucosa.(2) Soft liners suffer from low tear strength, porosity, water absorption and frequent de bonding from denture during clinical use, thus reducing the longevity of such prosthesis.(3) Silicon soft liners have been used as resilient liners for denture bases since the early 1960s. their resilience at mouth temperature is not derived from the use of plasticizer but from an intrinsic of this type of polymer therefore they retain resilience throughout their working life, also Silicon type may be chemically or heat activated. Bond strength between denture bases and resilient materials has been evaluated by several tests such as tensile,(4) shear(5) and peel tests.(6) Shear tests are suitable for examining bond strength of liner to acrylics, as masticatory forces in the oral cavity are similar to tear and shear forces rather than tensile forces.(7)

Glass fibers are used to increase the impact strength of heat cured acrylic denture bases especially when the minimum required acrylic thickness is unobtainable. The present study is not concerned with fiber reinforcement of the acrylic. Rather, the focus is on the potential for the fiber, integrated at or on the fitting surface of the acrylic, to improve bonding of the liner to the acrylic surface.

MATERIALS AND METHODS


A total of 120 samples were prepared. These 120samples divided into 3 major groups: I, II and III (100 sample for each major group) . Group I for the heat cure acrylic, group II for the pour acrylic and group III for the light cure acrylic. Each one of the 3 major groups divided into 2 subgroups ( 20 specimen for each subgroup) : group I divided into subgroup A(heat cure acrylic relined with Mollosil) & subgroup B( heat cure acrylic samples relined with Moloplast B), group II divided into subgroup C(pour acrylic samples relined with Mollosil)& subgroup D(pour acrylic samples relined with Moloplast B) and group III divided into subgroup E(light cure samples relined with Mollosil)&subgroup F(light cure samples relined with Moloplast B). Each one of these subgroups consist of 2 types of acrylic interface (10 specimen for each type): Smooth surface (control group), surface without any treatment. Treatment with glass fiber net. Each specimen consist of 2 acrylic blocks with dimensions of (3 inch 1 inch 3/16 inch
8

(1) (2)

M.Sc. Student, department of prosthodontics, college of dentistry, university of Al-Mustansirya. Professor, department of prosthodontics, college of dentistry, university of Baghdad.

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Effect of glass fibe r

length, width, depth respectively) with stopper of depth about 3mm.(8) One block of the acrylic put over the other block leaving a space between them of dimensions ( 1 inch 1 inch 3mm length, width and depth respectively). The thickness of the handle of the acrylic specimen is 5 mm, this thickness is important for the clamping of the specimen by the Instron machine. Preparation of the heat cure acrylic specimens The heat cure acrylic specimens prepared as that for the complete denture preparation. Heat cure acrylic interface preparation (3) Smooth surface The acrylic surface was smoothed by pressing the acrylic while it is in the dough stage against the mould and after polymerization it was left without polishing or any treatment. (4) Glass fiber net addition In this group before curing of the acrylic a multidirectional glass fiber net was cut that fit the acrylic interface (of 0.1g weight) then this net impregnated in the thin mix of the acrylic of weight of 0.02gm and the net was put in the mould in the area of the acrylic interface.

cleaned by the ultrasonic cleaner and stored in distilled water for 48h. 2. Addition of glass fiber net A multidirectional glass fiber net that fit the acrylic interface (0.1g in weight) was impregnated in the thin mixture of the pour acrylic (0.2g powder/0.2 g liquid) and applied to the putty 1:1 mould in the area that will form the acrylic interface. After that the pour acrylic is poured in the mould and the steps of the sample preparation completed as usual.

Figure 2: glass mould used for the light cure acrylic specimens preparation
Preparation of the light cure acrylic specimens A glass mould constructed for the purpose of the study was used for the preparation of the specimens. The separating medium was applied to the mould and the sheet of the light cure acrylic was knead and applied to the glass mould and then put it in the light cure acrylic polymerization unit for 10min. to each one of the 2 sides of the glass mould. Light cure acrylic interface preparation 1. Smooth surface Acrylic surface smoothed by pressing the acrylic dough against the glass mould then after complete polymerization the acrylic surface left without any treatment. 2. Glass fiber net application A multidirectional glass fiber net cut to the size that fit the acrylic surface which will face the soft liner (0.1g in weight)was added to the glass mould in the area of the acrylic interface. Then the light cure acrylic dough added to the glass mould and polymerized as usual. Soft Relining Material Application Mollosil application (silicon-based cold curing chair side soft liner) The acrylic surface was coated with the adhesive material. Let the adhesive dry for 1min. A homogeneously equal lengths of mollosil base material and catalyst were mixed for 30 seconds. The mixed material was applied with spatula onto the acrylic surface. Then each pair of the acrylic specimen joined together and the excess of the material was removed by using sharp knife

Figure 1: acrylic sample with glass fiber net surface treatment


Pour acrylic specimens preparation The same metal pattern which was used for the construction of the heat cure acrylic specimens was used for the construction of the pour acrylic specimens. 2 wax sprues were added to the metal pattern for the purpose of pouring the acrylic. After finishing with pouring wait for 3min. before placing the model in the pressure vessel (Ivomet). Polymerize at 55oC for 30 min. and pressure to 2.5 bars. After polymerization take out the model and remove the putty 1:1 , remove the sprues and start to trim and finish the specimen. Pour acrylic interface preparation 1. Smooth surface The acrylic interface was left without any treatment. It takes the shape and smoothness from the mould of the putty material which was prepared from the metal pattern. The samples

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Effect of glass fibe r

and the specimen was put under weight of 2.5Kg (9) and left for 30min for bench cure. After complete curing the specimens were stored into distilled water containers in an incubators at 37oC for 24 hours. Molloplast-B soft liner application This material is supplied as a one paste system. The primo adhesive (chemical bonding of Molloplast B) was brushed uniformly onto the acrylic surface and let the primo adhesive air dry for approximately 60min prior to applying Molloplast B. take Molloplast B with a clean spatula from the jar and apply it evenly on the acrylic surface. Then join the pair of acrylic specimen and put it in the flask. The Molloplast B polymerized by placing the flask in cold water and heat up slowly up to 100oC for approximately 2hours. After complete polymerization the flask cooled down slowly. After opening the excess material was cut with a sharp knife.

0.618N/mm2. The results of the t-test show that there is a significant increase in the bonding between the heat cure acrylic and the Mollosil soft liner with the glass fiber net addition. The samples of the smooth surface of the pour acrylic showed a mean value of the shear bond strength equal to 0.483 and for the glass fiber net group equal to 0.714. The results of the t-test show that there is a highly significant increase in the bonding between the pour acrylic and the Mollosil soft liner with the glass fiber net addition. The samples of the smooth surface of the light cure acrylic showed a mean value of the shear bond strength equal to 0.294 while the glass fiber net group showed a mean value of the shear bond strength equal to 0.284 and the results of the t-test show a non significant difference from the control.

Figure 3: Two acrylic samples joined together with the soft liner between them
Shear bond testing procedure Each specimen was tested after 24hours after processing (10) using universal Instron testing machine with a suitable grips for the test specimens. The specimens subjected to 1 KN load at a cross head speed 10mm/min until failure occurred. The maximum load required for the failure was recorded in order to calculate the value of shear bond strength for each test specimen Bond strength(N/mm2 )= Maximum load Cross sectional area =F A

Figure 4: Bar chart for the mean distribution of the shear bond strength of three types of the acrylic relined with the Mollosil soft liner.
Shear bond test of the three types of acrylic relined with Molloplast-B soft liner The samples of the control group of the conventional heat cure acrylic showed a mean value of the shear bond strength equal to 1.233N/mm2 while the mean value of shear bond strength for the glass fiber net group equal to 1.199N/mm2. The results of the t-test show that there is a non significant difference in the bonding between the heat cure acrylic and the Molloplast-B soft liner with the glass fiber net addition.

RESULTS
Shear bond test of the three types of acrylic relined with Mollosil soft liner The samples of the control group of the conventional heat cure acrylic showed a mean value of the shear bond strength equal to 0.508N/mm2 while the mean value of shear bond strength for the glass fiber net group equal to
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Effect of glass fibe r

Figure 5: Bar chart for the mean distribution of the shear bond strength of three types of the acrylic relined with the Molloplast-B soft liner. Table 1: Modes of the bonding failure of different types of the acrylic with the Mollosil soft liner
Acrylic type Surface treatment Failure type (in numbers) C/M/A 7/3/0 8/2/0 8/2/0 9/1/0 0/1/9 0/9/1

The samples of the smooth surface of the pour acrylic showed a mean value of the shear bond strength equal to 1.581N/mm2 and for the glass fiber net group equal to 1.516N/mm2. The results of t-test show that there is a non significant difference in the bonding between the pour acrylic and the Molloplast-B soft liner with the glass fiber net addition. The samples of the smooth surface of the light cure acrylic showed a mean value of the shear bond strength equal to 1.021N/mm2 and for the glass fiber net group equal to 0.832N/mm2. The results of the t-test show that there is a highly significant reduction in the bonding between the pour acrylic and the Molloplast-B soft liner with the glass fiber net addition.

DISCUSSION
The bonding of silicon elastomers depend on the strength of the silicon elastomers and the adhesive primers used. Besides, the strength of the bond also depends on the treatment modalities of the acrylic resin surface.(11) In the conventional heat cure acrylic relined with the Mollosil soft liner, the Glass fiber net group show significant difference, the increase in the bond strength can be attributed to changes in the topography of the acrylic interface due to the presence of the glass fiber. The adhesive used penetrated the resin matrix that impregnates the net shaped glass fibers leading to enhanced chemical adhesion with the liner this in agree with Hatamleh M.M.(2010) finding that the sticktech net fiber-reinforced surface exhibited a stronger bond to the soft liner. In the pour acrylic relined with Mollosil soft liner the adhesive used prior to the application of the Mollosil penetrate the resin matrix which impregnate the glass fiber net which was used in the surface treatment so that increase the bonding between the pour acrylic surface and the soft liner this in agree with Hatamleh M.M. (2010) finding that the sticktech net fiber-reinforced surface exhibited a stronger bond to the soft liner. In the light cure acrylic relined with the Mollosil soft liner, the glass fiber net affect the surface of the light cure acrylic and likely act as impurities and loose particles on the light cure acrylic surface which reduce the bonding between the two materials. Also it reduce the surface area of the bonding site. In the conventional heat cure acrylic relined with the Molloplast-B soft liner, the Glass fiber net surface treatment show a non significant difference from the control because the same chemical bonding occur. Which is attributed to
11

Smooth Conventional heat surface cure acrylic G.F. net Smooth surface Pour acrylic G.F. net Smooth Light cure acrylic surface G.F. net
C=cohesive failure M = mixed failure A = adhesive failure G.F.= glass fiber

Table 2: Modes of bonding failure of different types of the acrylic with the Molloplast-B soft liner
Acrylic type Conventional heat cure acrylic Pour acrylic Surface treatment Smooth surface G.F. net Smooth surface G.F. net Smooth surface G.F. net Failure type (in numbers) C/M/A 10/0/0 10/0/0 3/7/0 10/0/0 9/1/0 10/0/0

Light cure acrylic

C=cohesive failure M = mixed failure A = adhesive failure G.F.= glass fiber

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the acryloxyalkylsilicone which as well as improving the cross linking of the silicon soft liner , it intended to adhere to PMMA, besides; the adhesive contained a -Methacryloxy propyl trimethoxysialie which improve the adhesion and cross linking to the underlying PMMA this is according to Wright (1984). In the pour acrylic relined with MolloplastB soft liner, The glass fiber net surface treatment affect the bonding in non significant difference from the control because the same adhesion and cross linking of the soft liner adhesive to the underlying pour acrylic surface occur. In the light cure acrylic relined with the Molloplast-B soft liner, the glass fiber net surface treatment reduce the bonding because it reduce the bonding area because these glass fibers weren't covered by light cure acrylic matrix because the light cure acrylic supplied as sheet so the glass fibers couldn't be covered by the matrix and applied directly to the surface so it reduce the surface area of the bonding site. Most of specimens of the conventional heat cure acrylic and pour acrylic bonded to the Mollosil soft liner and the conventional heat cure acrylic, pour acrylic and light cure acrylic bonded to the Molloplast-B show cohesive failure. It means that the bond strength between the acrylic and the soft liner were stronger than the strength of the soft liner itself. While in the case of light cure acrylic relined with Mollosil the mode of failure is adhesive because of low adhesion between the two materials due to the low compatibility between them.

9.

10.

11.

12.

13.

liners: part II-Effect of aging. J Prosthet Dent 1995;74:299-304. Sarac YS, Basoglu T, Ceylan GK, Sarac D, Yapici O. Effect of denture base surface pretreatment on microleakage of a silicon-based resilient liner. J Prosthet Dent 2004; 92(3):283-287. El-Hadary A, Drummond JL. Comparative study of water sorption, solubility and tensile bond strength of two soft lining materials. J Prosthet Dent 2000;83:356-361. Xiao-na L, Yi-min Z. Effect of surface treatment on the bonding of silicon elastomer to acrylic resin. Journal of US-China Medical Science 2008;5:54-58. Hatamleh MM, Maryan CJ, Silikas N , Watts DC. Effect of net fiber reinforcement surface treatment on soft denture liner retention and longevity. J Prosthodont 2010;19:258-262. Wright PS. Composition and properties of soft lining materials for acrylic dentures. J Dent 1981;9:210-223.

REFERENCES
1. 2. Braden M, Wright PS, Parker S. Soft lining materials- a review. Eur J Prosthodont Restor Dent 1995;3:163-174. McCabe JF, Carrick TE, Kamohara H. Adhesive bond strength and compliance for denture soft lining materials. Biomaterials 2002;23:1347-1352. Elias CN, Henriques FQ. Effect of thermocycling on the tensile and shear bond strengths of three soft liners to denture base resins. J Appl Oral Sc 2007;15:18-23. Mutluay MM, Ruyter IE. Evaluation of bond strength of soft relining materials to denture base polymers. Dent Mater 2007;23:1373-1381. Hatamleh MM, Watts DC. Fiber reinforcement enhances bonding of soft lining to acrylic dental and maxillofacial prostheses. Eur J Prosthodont Restor Dent 2008;16:116121. Taft RM, Cameron SM, Knudson RC, et al. The effect of primers and surface characteristic on the adhesion-in-peel force of silicon elastomers bonded to resin materials. J Prosthet Dent 1996;76:515-518. Al-Athel MS, Jagger RG. Effect of test method on the bond strength of a resilient denture lining material. J Prosthet Dent 1996;76(5):535-540. Wagner WC, Kawano F, Dootz ER, Koran III A. Dynamic viscoelastic properties of processed soft denture

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The effect of autoclave

The effect of autoclave processing on some properties of heat cured denture base material
Salwan S. Abdulwahhab, B.D.S. (1) (2) Widad A.H. Alnakkash, B.D.S., D.D.H., M.Sc.

ABSTRACT
Background: Although most of the physical and mechanical properties of denture base resin polymerized by the conventional heat polymerization have been studied, the effect of autoclave processing in these properties has not been fully determined. The aim of the present study is to investigate the effect of two different cycles of autoclave processing on the transverse strength, impact strength, surface hardness and the porosity of acrylic denture base material. Materials and methods: Vertex was the heat- cured acrylic denture base material included in the study. A total of 120 specimens were prepared, the specimens were grouped into: Control groups (Group A) in which acrylic resins processed by conventional water- bath processing technique (74C for 1.5 hours then boil for 30 minutes) and experimental groups in which acrylic resins processed by autoclave at 121C,210KPa.The experimental groups were divided into Group B(Fast) for15min. , and Group C (Slow) for 30min... To study the effect of the autoclave processing (Tuttnauer 2540EA), four tests were conducted transverse strength (Instron universal testing machine), impact strength (charpy tester), surface hardness (shore D), and porosity test. The results were analyzed to ANOVA and LSD test. Results: There were no significant differences between the results of the processing techniques regarding transverse, impact, and hardness tests. While, there were a highly significant difference in porosity test results. Conclusions: The autoclave processing technique might also be a good alternative to the conventional water bath processing technique. Regarding to autoclave processing technique, the slow (long) curing cycle provide better denture bases material including the tested physical and mechanical properties as compared with the fast (short) curing cycle. Keywords: Autoclave, Transverse, Impact, Hardness, Porosity. (J Bagh Coll Dentistry 2012;24(3):13-17).

INTRODUCTION
Poly (methyl methacrylate) (PMMA) is the most commonly used material in construction of denture base since 1936. (1) Attempts to improve the mechanical properties of poly (methyl methacrylate) have taken the researcher through many avenues. (2) Over the years, curing procedures have been modified with a view to improve the physical and mechanical properties of resin materials. Different polymerization methods have been used: heat, light, chemical and microwave energy. (3) The water bath processing technique has been the most conventionally used polymerization technique. In spite of the advantages provided by this technique like the ease, simplicity and costeffectiveness, a major disadvantage has been the long processing time required. (4) Indian researchers extensively investigated the pressure cooker polymerization technique, Conventional acrylic resin material can be used for this technique and requires less than 1h for polymerization and utilizes conventional equipment.
(1) (2) M.Sc. Student. Department of Prosthodontics. College of Dentistry, University of Baghdad. Professor. Department of Prosthodontics, College of Dentistry, University of Baghdad, Baghdad, Iraq.

Previous studies of pressure cooker polymerization have shown comparable physical and mechanical properties to the water bath technique. (5) There is no previous Iraqi study deal with the investigation of the effect of varied autoclave processing conditions on the final properties of acrylic resins. Therefore, the aim of this study was to investigate the effect of different time durations of autoclave processing on some physical and mechanical properties of acrylic denture base material.

MATERIALS AND METHODS


A total of 120 specimens were prepared, the specimens were grouped into: Control groups (Group A) in which acrylic resins processed by conventional water- bath processing technique and experimental groups in which acrylic resins processed by autoclave. The experimental groups were divided into Group B (Fast), and Group C (Slow). 40 specimens were divided for each group. Three different metal patterns were prepared, a bar shaped specimen with dimensions of (65mm X 10mm X 2.5mm) length, width, thickness respectively (6) for transverse strength test and surface hardness test; then, a bar shaped specimen
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with dimensions of (80mm X 10mm X 4mm) length, width, thickness respectively (7) for impact strength test; finally, a disk shaped specimen with dimensions of (30mmX 3mm) diameter & thickness respectively (8,9) for Porosity test. By the use of dental stone (Bluejey, Extra hard type IV, Italy) as investment material, these metal patterns were invested in their corresponding flasks (Broden, Sweden) After metal patterns removal, a fine brush was used to spread the separating medium (Isodent, Spofa Dental, Czechoslovakian, Europe) on to the exposed surfaces of a warm, clean stone moulds. Powder and liquid of heat cured acrylic resin (Vertex, Vertex-Dental by J.v.Oldenbamevetin Zeist, The Netherlands) were proportioned and mixed according to the manufacturers instructions. The specimens were packed at the dough stage according to the conventional dough molding method. The flasks were trial closed and then final closed under pressure using hydraulic press (Germany); then, Flasks were clamped with clamps (HANUA, Engineering corp. U.S.A) and be ready for processing. Curing cycle for conventional water-bath processing technique: For control specimens, curing was carried out by placing the clamped flask in a thermostatically controlled water-bath (memmert, Germany) and processed by short curing cycle (74C for 1.5 hours then boil for 30 minutes.).(6) Curing cycle for autoclave processing technique: For experimental specimens, Curing was carried out by placing the clamped flask in a fully automatic autoclave (Tuttnauer 2540EA, Tuttnauer USA Co., NY, USA) and processed by the preprogrammed cycles (Fast 121C/210KPa, 15 min. & Slow 121C/210KPa, 30 min.). Before placing the clamped flask inside the autoclave, the autoclave must be leveled and filled. Then, the clamped flask placed in the tray and pushed inside the chamber, then closed and secured the door. Fast curing cycle In this cycle, held and selected the program (Fast 121C).The stages of operation of autoclaves include air removal, steam admission and sterilization cycle (includes heating up, holding/exposure, and cooling stages) (10,11). The autoclave operated and started heating the water, then the temperature and pressure were raised till its reached (121C &210 KPa) respectively. When the temperature reached (121 C), temperature and pressure held automatically at (121C &210KPa) respectively for 15 min., then
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automatically exhausted the steam and the programmed cycle was finished. Unsecured and opened the door, then removed the clamped flask. Slow curing cycle In this cycle, held and selected the program (Fast 121C). The autoclave operated and started heating the water, then the temperature and pressure were raised till its reached (121C &210KPa) respectively. When the temperature reached (121 C), temperature and pressure held automatically at (121C &210KPa) respectively for 30 min., then automatically exhausted the steam and the programmed cycle was finished. Unsecured and opened the door, then removed the clamped flask. All the specimens were carefully finished and polished according to (6). After that, all the specimens were stored in distilled water at 37C by the use of an incubator (Gallen bamp, England) for 48 hours. (6) Transverse strength test: The total number of specimens for Transverse strength was 30: Ten acrylic control specimens and twenty experimental specimens. The test was achieved by using instron testing machine (instron corporation, 1122, canton mass), each specimen was positioned on bending fixture, consisting of 2parallel supports (50) mm apart, the full scale load was 50kg, and the load was applied with cross head speed of 1mm/min by rod placed centrally between the supports making deflection until fracture occurred. The transverse bend strength was calculated in N/mm using the following formula: Transverse strength= 3Pl/2bd P: is the peak load in Newton. l: is the span length in millimeter. b: is the sample width in millimeter. d: is the sample thickness in millimeter.(12) Impact strength test: The total number of specimens for Transverse strength was 30: Ten acrylic control specimens and twenty experimental specimens. Impact strength test was conducted with charpy type impact testing instrument (IZOD CHARPY, testing machines, Inc.) The specimen was supported horizontally at its ends and struck by a free swinging pendulum which released from a fixed height in the middle. A pendulum of 2 joules testing capacity was used. The scale reading gave the impact energy absorbed to fracture the specimen in joules when struck by a sudden blow. The charpy impact strength of unnotched specimen was calculated in KJ/mm as given by the following equation: Impact strength= E/b.d 10 E: is the impact absorbed energy in joules.

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b: is the width in millimeters of the test specimens. d: is the thickness in millimeters of the test specimens.(12) Surface hardness test The total number of specimens for Transverse strength was 30: Ten acrylic control specimens and twenty experimental specimens. Surface hardness was determined using (shore D) durometer hardness tester (TH210, TIME group Inc. company, Italy.) according to (6) which is suitable for acrylic resin material. The instrument consists of blunt-pointed indenter 0.8mm in diameter that tapers to a cylinder 1.6mm. The indenter is attached to a digital scale that is graduated from 0 to 100 units. The usual method is to press down firmly and quickly on the indenter and record the maximum reading as the shore D hardness measurements were taken directly from the digital scale reading. Five readings with 1 cm apart between each two indentation along the specimen (the same selected area of each specimen), and an average of five readings was calculated. Porosity test: The total number of specimens for Transverse strength was 30: Ten acrylic control specimens and twenty experimental specimens. The specimens were immersed in a solution of permanent black ink for 30 min., then washed for 10 seconds and dried with absorbent paper. A surface area of 1 cm was limited in the center of each specimen and observed under 40X in light microscope (OLYMPUS, Japan). The number of pores per area were determined and calculated for each specimen. (8, 9) The results of this study were analyzed by the following statistical methods: Descriptive statistic which includes (Mean, Standard deviation (S.D)). Inferential statistics: ANOVA (one-way analysis of variance test) was used for assessing the differences between more than two groups; LSD (Least Significant Difference test) was used for examining the differences between each group. P-value more than 0.05 statistically considered as a non-significant (P>0.05) while P-value less than 0.05 accepted as a significant (P<0.05) and Pvalue less than 0.01 accepted as a highly significant (P<0.01).

Vertex by using F-test in porosity test showed in (Table 2).

Table 1: Mean and standard deviation values of examined tests results.


Transver Impact se strength Surface Porosity strength test hardnes test test (KJ/mm s test (N/mm) ) Mea Mea S. Mea S. Mea S. S.D. n n D. n D. n D. 3.3 86.4 2.2 11.6 4.7 Contr 142.4 23.3 8.87 2 6 1 4 8 0 6 ol 148.8 21.6 1.3 85.6 3.8 2.3 Verte 8.90 4.60 Fast 2 6 6 7 9 6 x 154.1 23.4 1.5 83.8 1.6 1.5 9.40 2.10 Slow 2 8 8 7 5 2

Table 2: F-test by ANOVA table of examined test results. Between groups of Vertex Impact Transvers strength Surface Porosit e strength hardnes test y test test (KJ/mm s test (N/mm) ) 0.65 0.16 2.25 23.74 F-test 0.52 0.85 0.12 0.00 P-value Significan NS NS NS HS t
P>0.05 non-significant (NS). P<0.01 highly significant (HS).

Table 3: LSD test between groups of Vertex in porosity test


Vertex groups P-value Significant HS Control & Fast 0.00 HS Control & Slow 0.00 0.09 NS Fast & Slow
P>0.05 non-significant (NS). P<0.01 highly significant (HS

The LSD test between groups of Vertex in porosity test showed a highly significant difference between control and fast groups and control and slow groups; while, a non-significant difference between fast and slow groups showed. As shown in (Table 3).

RESULTS
Mean and standard deviation (S.D.) values of examined tests results were shown in (Table 1). A non-significant difference between groups of Vertex by using F-test in transverse strength, impact strength, and surface hardness tests; while, a highly significant difference between groups of
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DISCUSSION
An autoclave is a pressurized device designed to heat aqueous solutions above their boiling point to achieve sterilization. It was invented by Charles Chamberland in 1879. Under ordinary circumstances (at standard pressure), liquid water cannot be heated above 100C in an open vessel.

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Further heating results in boiling, but does not raise the temperature of the liquid water. However, when water is heated in a sealed vessel such as an autoclave, it is possible to heat liquid water to a higher temperature. As the container is heated, the pressure rises due to the constant volume of the container. The boiling point of water is then raised because the amount of energy needed to form steam against the higher pressure is increased. (13).Tuttnauer 2540EA autoclave is an example of a gravity displacement. The autoclave is a pressure cooker. A pressure cooker is a container with an airtight lid that traps steam from boiling water. The steam increases the pressure inside the cooker, which raises the water's boiling point. The higher temperature kills bacteria much faster than at lower temperatures. The autoclave is no different than the simple pressure cooker used in home cooking which less costs as compared to the autoclave (14). Transverse strength test: The transverse (flexural) strength test is especially useful in comparing denture base materials in which a stress of this type is applied to the denture during mastication. The transverse (flexural) strength is a combination of compressive, tensile, and shear strengths, all of which directly reflect the stiffness and resistance of a material to fracture.(2)The results in Vertex showed that there was a non-significant difference in transverse strength between autoclave and water-bath methods, but there were a slight increase in slow curing and fast curing values than the control values as a result of high heat treating could be attributed to increase in cross linking. Cross-linkage provides a sufficient number of bridges between linear macromolecules to form a three-dimensional network that decreases water sorption, decreases solubility, and increases the strength and rigidity of the resin. (12) Durkan et al in 2008 studied the effect of autoclave polymerization on the transverse strength of denture base polymers, Under 3atm (303KPa) pressure, the specimens were subjected to one of the processing cycles: autoclave-cured for 60C/ 30 min. followed by130C/10 min., or autoclave-cured for 60C/ 30 min. followed by130C/20 min., the results revealed that polymerization in an autoclave led to a statistically significant increase in transverse strength for the two materials evaluated when compared to the control. However, there were no statistically significant differences in transverse strength between the two time durations of autoclave polymerization system, and there were no statically significant differences between the
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two acrylic resin control groups. Curing cycles that used by Durkan et al in 2008 were different from this study. Impact strength test: Impact strength defined as the energy required fracturing a material under an impact force (12), impact strength is an important property for acrylic denture base materials which have tendency to fracture if accidentally dropped on to a hard surface. (15), the results in Vertex showed that there was a non-significant difference in impact strength between autoclave and water-bath methods, but there were a slight increase in slow curing and fast curing values than the control values due to the pressure. The pressure played an important role in speeding up the initial polymerization and elevating the boiling temperature of the monomer and thus might reduce the residual monomer content (5) the most important determinant of resin strength is the degree of polymerization exhibited by the material. As the degree of the polymerization increase, the strength of the resin also increases; In this regard, the polymerization cycle employed with a heat activated resin is extremely important.
(12)

Surface hardness test Hardness may be broadly defined as the resistance to permanent surface indentation or penetration. (16) In this study, shore (D) hardness tester was used which is suitable for measuring the hardness of acrylic resin, Shore durometer type (D) hardness tester eliminate problem with elastic recovery owing to its use of a method that measures the depth of the loaded indentation under loading condition directly by screen which show the number of it (17). In Vertex, The results showed that there was a non-significant difference in surface hardness between autoclave and water-bath methods. The results of Vertex in this study coincide with the results obtained by Ming et al in1996 when the resin was polymerized in automatic pressure cooker with different pressure and time duration than this study, the pressure cooker was filled with water and inflated with 6KgF/cm (580KPa) air pressure and heated to 120C (248 F) for 10 min., the results revealed no statistically significant differences between the pressure cooker and conventional polymerization methods. Porosity test: Porosity in acrylic resin is a complex phenomenon of multifactorial origin. It appears to depend partly on the material/ polymerization method combination and the flasking technique used. (18). A number of methods have been used to study porosity, including microscopic observation

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of a cut specimen (19, 20), a photographic method, and mercury porosimetry, which is generally regarded as the best method available for the routine determination of pore size. (18).There are two major causes of porosity: polymerization shrinkage-associated contraction porosity, and volatilization of the monomer, termed gaseous porosity.(21) The results in Vertex showed that there was a highly significant difference in porosity between autoclave and water-bath methods. The lower value of porosity in slow curing cycle and fast cycle than the highest value in control curing cycle could be attributed to the role of the pressure accelerating the polymerization, the higher pressure is instantly transmitted to the resin dough and prevents the monomer from boiling (22) The results of porosity in Vertex were coincide with the results of Undurwade and Sidhye in 1989 demonstrated a reduction in porosity in a denture base resin polymerized in the domestic pressure cooker with steam pressure rise to 1520mm Hg (197KPa) and held for 30min.While, Ming et al in1996 cured 10 acrylic resin in an automatically controlled pressure cooker at 0.58MPa (580KPa) of 120C for 10min. Ming et al in1996 showed no porosity in any specimens.

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REFERENCES
1. 2. Sideridou ID. Polymeric Materials in Dentistry. New York, Nova Science Publishers, Inc; 2011. p.7-8. Jagger DC, Jagger RG, Allen SM, Harrison A. An investigation into the transverse and impact strength of high strength denture base acrylic resins. J of Oral Rehabilitation, Blackwell Science Ltd 2002; 29; 2637. Azzari MJ, Cortizo MS, Alessandrini JL. Effect of the curing conditions on the properties of an acrylic denture base resin microwave polymerized. J Dent; 2003. 31: 463-8. Banerjee R, Banerjee S, Prabhudesai PS, Bhide SV. Influence of the processing technique on the flexural fatigue strength of denture base resins: An in vitro investigation. Indian Dent Assoc; 2010. 21: 391-5. Undurwade JH, Sidhaye AB. Curing acrylic resin in a domestic pressure cooker: A study of residual monomer content. Quintessence Int 1989; 20(2): 123-9. American Dental Association Specification No.12 Guide to dental materials and devices.10th Ed., Chicago; 1999. p. 32. ISO 179-1. International organization for standardization. Plastics. Determination of charpy impact properties-Part 1: Non-instrumented impact test; 2000. Rodrigues Garcia, RCM. & Del Bel Cury AA. Accuracy and porosity of denture bases submitted to two polymerization cycles. Indian J Dent Res 1996; Vol.7, 122126, ISSN 0970-9290 Al-Fahdawi IH. The effect of the poly vinyl pyrrolidone (PVP) addition on some properties of heat- cured acrylic resin denture base material. PhD 21. 22.

3.

thesis, College of dentistry, University of Baghdad, 2009. Judelson HS. Operation of the autoclaves. An excellent overview of autoclave operation posted by Dr. Howard Judelson at University of California at Riverside; 2004. Oyawale FA and Olaoye AE. Design and Construction of an Autoclave. Pacific J Science and Technology 2007; 8(2):224-30. Anusavice KJ. Phillipss sciences of dental materials. 11th Ed. Saunders Co. Philadelphia; 2007. p.162-9. Durkan R, Ozel MB, Bagis B, and Usanmaz A. In vitro, Comparison of autoclave polymerization on the transverse strength of denture base resins. Dental Materials J 2008; 27(4): 640-2. Monson MH. The pressure cooker as a steam sterilizer. Trop Doct 1988; 18(4):159-60. McCabe JF, Walls AWG. Applied dental material. 9th Ed. Blackwell Publishing Ltd; 2008. p. 110-112. Powers J M, Sakaguchi R L. Craigs Restorative dental materials. 13th Ed. Philadelphia, PA Mosby Co; 2012. p.84-5. Unalan F , Dikbas I. Effects of mica and glass on surface hardness of acrylic tooth material. Dent. Material J 2007; 26(4): 545-8. Yannikakis S, Zissis A, Polyzois G, Andreopoulos Evaluation of porosity in microwave-processed acrylic resin using a photographic method. J Prosthet Dent 2002; 87: 613-9. Reitz PV, Sanders JL, Levin B. The curing of denture acrylic resins by microwave energy. Physical properties. Quintessence Int. 1985; 16: 547-51. Wolfaardt JF, Cleaton-Jones P, Fatti P. The occurrence of porosity in a heat-cured poly (methyl methacrylate) denture base resin. J Prosthet Dent 1986; 55:393-400. Noort RV. Introduction to dental materials. 3rd Ed. Elsevier limited; 2008. p.217. Ming XC, Changxi S, Weizhou H. Rapid processing procedure for heat polymerization of poly (methyl methacrylate) in a pressure cooker with automatic controls. J Prosthet Dent 1996; 76: 445-7.

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Tooth movement in maxillary complete dentures fabricated with fluid resin polymer using different investment materials
Sanaa R. Abd Al-Aaloosi BDS, HDD, MSc (1) Nabeel Abdul Fatah BDS, MSc (2)

ABSTRACT
Background: This study investigates the effect of different investments on tooth movement of new fluid resin and compares the results with that of heat-cured resin. Besides, comparison between the right and left changes in vertical distances of maxillary complete dentures that had been made. Materials and methods: A maxillary complete denture with acrylic teeth was waxed to full contour on the master cast and replicated to make 40 wax dentures. Metallic reference points were placed for linear and vertical measurements. Ten dentures were allocated for each of 4 groups; group I was processed using conventional heatcured resin (RegularTM), group II was processed with cold-cured fluid resin (CastavariaTM) in a reversible hydrocolloid mold, group III and IV processed with the same fluid resin in addition silicone duplicating mold, and in a dental stone type III mold, respectively. Measurements had been made at the wax stage immediately before flasking, 24 hours after deflasking, and after decasting, finishing and storage in water at 37 C for 1 week, the measurement had been made with a micrometer microscope accurate to 0.005 mm. Results: the results showed a significant tooth displacement in the different investments and in different types of acrylic resin used in this study. The right and left vertical measurements did not differ significantly from each other. Conclusions: Maxillary dentures processed from fluid resin in silicone molds, produce the least linear dimensional changes comparable to those constructed from heat-cured resins. The decrease in vertical dimension of occlusion still exists as a disadvantage of the new fluid resin systems. Key words: dimensional stability, fluid or pour acrylic resin, investments. (J Bagh Coll Dentistry 2012;24(3):18-24).

INTRODUCTION
Maintaining achieved occlusal scheme from the time of the try-in appointment until delivery has always been a difficult goal in complete denture prosthodontics. Inaccuracies in the occlusal harmony of completed prostheses may be the result of technical or clinical judgment errors made by the dentist; technical errors made in the dental laboratory; and deficiencies of the techniques and materials used in the construction of the denture. All of the errors and inaccuracies should be corrected before the patient is permitted to wear the prostheses (1). Such corrections can create a time-consuming occlusal adjustment sequence that often results in the disfigurement of the anatomy of the artificial teeth (2). In spite of the development of various denture base materials, acrylic resin remains the principle choice. Many processing methods had been developed for the purpose of minimizing polymerization shrinkage, but some warpage after processing is inevitable (3). To overcome these undesirable processing effects,various flaskingand polymerizationtechniques and materials had been studied (4).
(1) (2) MSc Student, Department of Prosthetic Dentistry, College of Dentistry, University of Baghdad. Professor, Department of Prosthetic Dentistry, College of Dentistry, University of Baghdad.

Several techniques are utilized in resin polymerization method, such as heat polymerization method, pouring method, injection pressing method, and microwave activated polymerization method. Improvements have been made in each method to strive towards a system with high accuracy and reliability (5, 6). The pouring method of denture base resin was developed in the 1960s using agar hydrocolloids as investment material (7) and has been one of the most popular polymerization techniques because of three merits. It is simple to use, less time consuming, and it offers better adaptation accuracy than the heat polymerization method (8, 9,10) . Different types of investments had been used with pour acrylic resin. These include: agar hydrocolloids, alginate hydrocolloids and a quick-setting modified gypsum investing material(11). The use of gypsum products like type III dental stone as an investment material for pour acrylic resin has been used (12), but its effect on the dimensional stability of the resin was not studied yet. Besides, the introduction of new polyvinyl siloxane duplicating material which gained popularities in many dental laboratories because of its excellent properties, but again its effect on the accuracy of the pour or fluid resin has not being yet investigated. However, it is important to investigate the

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investment material that can yield minimal tooth displacement. The present study was designed to evaluate the effect of using agar hydrocolloids, duplicating silicone, and type III dental stone, as investments for processing pour acrylic resin, on the dimensional stability of the resin and compare the results with that of the heat-cured acrylic.

MATERIAL AND METHODS


The materials used were (Heat-cured and coldcured pour type denture base materials, Castagel duplicating reversible hydrocolloids, Castasil 21 duplicating vinyl polysiloxane (PVS) addition silicone, type III dental stone, base plate wax, stainless steel saw needle, Unimetric post screw, pressure vessel, thermostatically controlled water path, hydraulic press, autoduplicator, laboratory engine and hand piece, micrometer microscope, vernier caliper, metal flasks and clamps, castaflask clear, modified flask made from teflon and plastic cover, semiadjustable articulator, plastic mold for upper arch, base of surveyor attached to plastic base, rubber bowel, spatulas, bakers, and incubator). Sample Grouping: G I (Control group): 10 maxillary complete dentures were processed with heat cured acrylic denture base material for linear measurements and it was further subdivided into right and left sample (5 samples for each side) for vertical measurements. G II: 10 maxillary complete dentures were processed with pour type acrylic denture base material in reversible hydrocolloid and it was further subdivided into right and left samples (5 samples for each side). GIII: 10 maxillary complete dentures were processed with pour acrylic base material invested in duplicating silicone and it was further subdivided into right and left samples (5 samples for each side). GIV: 10 maxillary complete dentures were processed in pouring type acrylic denture base invested in type III dental stone and it was again subdivided into right and left samples (5 samples for each side). Pilot study: Selecting an appropriate and reliable reference points were very critical step in this study, as the whole dimensional measurements accuracy depends on this point. Two different types of references point were needed. The first one was embedded or placed on the occlusal surface of the teeth for linear measurements and a section of stainless steel needle was selected. A second references point was needed to be placed in the
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flange area of the denture-the Unimetric post screw was selected- and the facial surface of the teeth for vertical measurements. Specimen preparation:This includedpreparing the initial denture with the following steps: cast preparation, construction of record base and bite rim, mounting on the articulator, arrangement of artificial teeth with monoplane concept of occlusion, and finally insertion of 4 screws above the canines and first molars on both sides. The initial denture was completely waxed and fastooned. The sample was removed from the articulator and two wax sprues had been prepared and attached to the posterior area of the buccal flanges, distal to the second molar.The simulation denture was duplicated using a pourable silicone duplicating material (Elite double 22 fast, Zhermack technical, Italy) supported by plastic container to provide rigidly for the duplicating material 13. After complete setting of the silicone, the container was discarded, the sample was removed, and the silicone mold was ready to be used figure 1.

Figure1: Silicone mold for denture duplication.


Reference points preparations: Six cavities were prepared in the occlusal surface of the teeth for linear measurements figure 2. Another two cavities were prepared in the buccal surface of the canine and first molar teeth above the screws for vertical measurements figure 3. These cavities were prepared with round bur in a slow speed contra-angle hand piece and then modified with fissure bur with 1.18mm diameter. A stainless steel saw needle with a diameter of 1.00mm was sectioned using carborundum disc and placed in the prepared cavities with cyanoacrylate adhesives. Formation and preparation of the mold: The maxillary dentures were smoothed after removing of the wax sprue, measurements were completed and the samples were flasked conventially using a standard metallic flask and type III dental stone to form the gypsum mold for

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expansion, table 1. One way ANOVA test was used to measure the difference in mean between the studied groups, as well as the experimental groups.Paired t test was used to analyze the difference between the right and left vertical distances.

Figure 2: Linear distances to be measured.

Figure 3: Vertical distances to be measured.


G I samples. For GII and GIII samples, the molds were prepared from reversible hydrocolloids and duplicating silicone PVS, respectively, and according to the manufacturer instructions. For G IV samples, a technique described by Kolitz et al in 1973 was used to create the molds 14. Processing acrylic denture base materials were accomplished according to the manufacturers instructions for both heat-cured and cold-cured pour type acrylic resins. Then the samples were deflasked carefully without decasting and stand on the bench for 24 hours for the second measurements, then they were decasted, finished and stored in water at 37 C for one week to be prepared for the third and last measurements 15.

Figure 4: Bar chart showing mean of change distribution of studied groups in RPM-LPM distance.

Figure 5: Bar chart showing mean of change distribution of studied groups in RM-LM distance.

RESULTS
The measurements at the wax stage are used as the baseline reading. Changes in the measured distances are calculated for all dimensions in each sample and between the three pairs of stages: comparison of the mean measurement at the deflasking stage versus wax stage (D-W), comparison of the mean measurement at the processing stage versus wax stage (P-W), and comparison of the mean measurement at the processing stage versus deflasking stage (P-D). In each case, the difference taken was between the measurement of the later stage and the measurement at the earlier stage, so that a negative change score reflects a diminution of the measured dimension, representing linear shrinkage, and a positive change score represents

Figure 6:Bar chart showing mean of change distribution of studied groups in RM-RI distance.

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Table 1: Descriptive statistics for changes between stages in denture base distances in mm for the eight distances and the four groups.
Distance RPMLPM (N=10) RM-LM (N=10) RM-RI (N=10) LM-LI (N=10) RC-R (N=5) RM-R (N=5) LC-L (N=5) LM-L (N=5) GI
-0.125 (0.025) -0.126 (0.020) -0.103 (0.020) -0.120 (0.025) -0.102 (0.107) -0.070 (0.060) 0.010 (0.021) 0.028 (0.096)

D-W stage G II G III


-0.301 (0.065) -0.083 (0.079)

G IV
0.085* (0.071)

GI

P-W stage G II G III

G IV

GI

P-D stage G II G III

G IV

-0.099 -0.326 -0.110 0.034* (0.039) (0.072) (0.080) (0.086)

-0.027 0.025 0.027 0.051* (0.027) (0.018) (0.018) (0.044) 0.296 (0.040) -0.078 (0.060) -0.157 (0.091) -0.079 (0.046) 0.050 (0.115) -0.011 (0.037) -0.033 (0.263) 0.355* (0.146) -0.092 (0.049) -0.110* (0.064) 0.073 (0.203) 0.0004 (0.059) -0.031* (0.006) 0.003 (0.294)

-0.340 -0.183 0.138* -0.221 -0.557 -0.480 -0.218* 0.094 0.217 (0.138) (0.139) (0.068) (0.029) (0.117) (0.147) (0.154) (0.042) (0.104) -0.177 -0.140 -0.013* -0.029 -0.061 -0.062 0.079* -0.074 -0.116 (0.067) (0.100) (0.074) (0.190) (0.098) (0.094) (0.079) (0.179) (0.120) -0.361 -0.207 -0.036* -0.088 -0.264 -0.051 0.074* -0.032 -0.097 (0.122) (0.081) (0.027) (0.060) (0.144) (0.139) (0.052) (0.049) (0.064) -0.469 -0.363 -0.195* -0.051 -0.571 -0.284 -0.268* -0.051 0.102 (0.236) (0.105) (0.042) (0.053) (0.317) (0.085) (0.221) (0.109) (0.097) -0.499 -0.403 -0.220* -0.046 -0.611 -0.454 -0.220* -0.025 0.112 (0.148) (0.168) (0.033) (0.133) (0.241) (0.090) (0.090) (0.097) (0.160) -0.289 -0.140 -0.104* 0.051 -0.311 -0.130 -0.073* -0.040 0.022 (0.087) (0.105) (0.043) (0.016) (0.088) (0.081) (0.042) (0.026) (0.035) -0.399 -0.267 -0.201* 0.194 -0.435 -0.234 -0.204* -0.166 0.036 (0.095) (0.285) (0.036) (0.020) (0.220) (0.157) (0.285) (0.096) (0.184) The values represent means (standard deviation values are given in parentheses). * Represents ANOVA test between studied groups significant at the 0.05 levels. Represents ANOVA test between experimental groups significant at the 0.05 levels.

Figure 7: Bar chart showing mean of change distribution of studied groups in LM-LI distance.

Figure 9:Bar chart showing mean of change distribution of studied groups in RM-R distance.

Figure 8: Bar chart showing mean of change distribution of studied groups in RC-R distance.

Figure 10: Bar chart showing mean of change distribution of studied groups in LC-L distance.

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Figure 11: Bar chart showing mean of change distribution of studied groups in LM-L distance.

Figure 12: Bar chart showing mean of change distribution of studied groups that compare between the RC-R and LC-L distances in the P-W stage.

Figure 13: Bar chart showing mean of change distribution of studied groups that compare between the RM-R and LM-L distances in the P-W stage.

DISCUSSION
Dimensional changes in a complete maxillary denture may be the result of a variety of factors such as polymerization shrinkage, thermal shrinkage, swelling due to water absorption, relaxation of stresses, etc. Therefore, the overall shape of the denture is not likely to change according to the referred parameters, but may
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distort in unpredictable forms 16. The dimensional characteristics of processed denture bases are affected by many factors, these are: type of acrylic; type of investing medium selected; method of resin introduction; and temperature used to activate polymerization process 17. Changes in the mediolateral directions of the complete denture samples in D-W stage, shrinkage occurs in these distances in all groups except G IV which show expansion, figure 4 and 5. The effect of investment on polymerization of fluid resin is obvious. G II show more shrinkage due to evaporation and syneresis of reversible hydrocolloid sol that cause farther shrinkage in the mold. G III show less shrinkage of the resin as the PVS show better dimensional stability than reversible hydrocolloids 18. The shrinkage in G III occurred because of polymerization contraction of PVS during setting which is mainly due to cross-linking and rearrangement of bonds within and between the polymer chains 19. G IV show expansion and this is attributed to two reasons. First, the investment material used here is type III dental stone, which has a setting expansion of 0.15 - 0.25% 19. Once the investment has been set, the mold become slightly larger than the original dimensions. Second, the method of investing used in this study was without clamping and this will allow for more expansion of the mold. Tooth movement is restricted when the two halves of the flask are clamped together after completion of the investment. Apparently the effect of setting expansion of the gypsum is reduced by confining it within the flask 20. Thus, the amount of expansion of the gypsum mold exceeds the amount of polymerization shrinkage of fluid resin and causing expansion in these distances. The effect of clamping is obvious in this study in G I samples, where the same investment was used but here the flasks were clamped together. In the P-D stage, the effect of stress relaxation due to thermal contraction and polymerization contraction is obvious in figure 4 and 5 with more shrinkage in the posterior region than the premolar region, because the posterior region was flatter and less restrictive than the anterior region which allowed more strain to be released producing shrinkage and deformation 21. The RPM-LPM distance in G I show expansion, which mainly due to water sorption. In the RMLM distance the amount of water sorption does not overcome the shrinkage due to relaxation of stresses, resulting in reduction of this distance. This result was in agreement with 22. For the experimental groups, both the premolar and

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molar distances show shrinkage. This is attributed to the fact they are constructed from fluid resin which show both strain due stress relaxation and shrinkage when stored in water due to leaching out of residual monomer23. The differences within the experimental group are so small that they are not statistically significant. Converse situation exists in the molar region which is attributed to the geometry of the sample. The type of investment and its effect on the amount of stress relaxation is obvious in this study. The harder the investment the more difficult the deflasking procedure which inherits an additional stress within the resin that subsequently released after decasting and water storage. This explain the least amount of shrinkage in G II samples followed by G III which was harder to be deflasked compared to G II and finally G IV samples with gypsum investment which showed more shrinkage as the deflasking procedure was the most difficult in the experimental groups. Further studies are required to evaluate the effect of deflasking procedure on stress relaxation of fluid resin. The situation in anteroposterior directions are the same as in the mediolateral directions except for G IV which show shrinkage instead of expansion but with the least amount compared to G II, G III and even G I, figure 6 and 7. This could be attributed to the geometry of the samples with more thickness of acrylic resin in these directions. The thickness of the resin is more in these areas compared to the cross arch distances, where the thickness of the palatal portion was maintained to be 2 mm. It has been stated that dimensional shrinkage occurs toward the area of greatest bulk. However, the shrinkage relates more to the intrinsic qualities of the resin24.This thickness produces an amount of shrinkage that exceeds the expansion of type III dental stone that has been explained previously. In P-D stage, there is no general trend as seen in mediolateral direction, where all groups in both sides show expansion due to water sorption and due to the thickness of these distances; this thickness made these areas stiffer to prevent release of some stresses 25,26; with different rates which might be the result of different rates of stress relaxation because of the different investments used. In general, these differences are so small that are statistically not significant. Effect of different investments on the final dimensions of complete denture samples constructed from fluid resin: For the linear measurements, all groups show shrinkage except G IV, which show expansion in all distances except molar-molar
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region where shrinkage takes place. The setting expansion of type III dental stone accompanied with clamping effect and water sorption are responsible for this expansion and as discussed in details previously. In molar region, G IV shows slight more shrinkage then G I, but this difference is statistically not significant. Hardy 27 had pointed out to such conclusion with the exception that shrinkage was more in the heatcured resin than the fluid resin, may be due to different gypsum investment used ( the modified type) and different curing conditions for the fluid resin. The maximum amount of contraction for the linear measurements occurs in G II samples.For the vertical distances and as shown in figure 8,9,10 and 11, the whole experimental groups show more shrinkage than the control one. A similar result was obtained in the previous studies 28,29.G III show comparable dimensions with the control group except in the molar region which is mainly due to the nature of fluid resin. The rigid investment in G IV samples produces the least amount of shrinkage in the vertical distances compared to other resilient investments used in this study. Several workers had pointed out to such conclusion 11,27G III show comparable results with G IV in the vertical distances. Comparison between the right and left vertical distances: The result of this study shownonly significant difference between the right and left side exist in the final dimension of the vertical distance in the control group, and in P-D stage in the molar region of the same group, figures 12 and 13. This behavior could be attributed to the position of the denture within the flask24, or due to water sorption by acrylic resin during processing 30. G III in the molar region also shows a significant difference between the two sides, this could be attributed to the erratic release of internal stress after water storage due to stress induced during deflasking procedures.

REFERENCES
1. Sykora O, Sutow EJ. Comparison of dimensional stability of two waxes and two acrylic resin processing techniques in the production of complete dentures. J Oral rehabil 1990; 17: 219-27. 2. Nogueira SS, Ogle RE, and Davis EL. Comparison of accuracy between compression-and injection- molded complete dentures. J Prosthet Dent 1999; 82: 291300. 3. Kobayashi N, Komiayama O, Kimoto S, and Kawara M. Reduction of shrinkage on heat-activated acrylic denture base resin obtaining gradual cooling after processing. J Oral Rehabil 2004; 31: 710-16.

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4. Shibayama R, Filho HG, Mazaro TVQ, Vedavatto E, and Assun ao WG. Effect of flasking and polymerization technique on tooth movement in complete denture processing. J Prosthodont 2009; 18: 259-64. 5. Teraoka F and Takahashi J. Controlled polymerization system for fabricating precise dentures. J Prosthet Dent 2000; 83: 514-20. 6. Laughlin GA, Eick JD, Glaros AG, Young L, and Moore DJ. A comparison of partial adaptation in acrylic resin denture bases using conventional and anchored polymerization technique. J Prosthodont 2001; 10, 204-211. 7. Shepard WL. Denture bases processed from a fluid resin. J Prosthet Dent 1968; 19: 561-72. 8. Goodkind RJ, Schutte RC. Dimensional accuracy of pour acrylic resin and conventional processing of cold-curing acrylic resin bases. J Prosthet Dent 1970; 24: 662-68. 9. Takamata T, Sectos JC, Phillips RW, and Boone ME. Adaptation of acrylic resin dentures as influenced by the activation mode of polymerization. JADA 1989; 119: 271-75. 10. Lee CJ, Bok SB, Bae JY, and Lee HH. Comparative adaptation accuracy of acrylic denture bases evaluated by two different methods. Dent Mater J 2010; 29: 411-17. 11. Winkler S and Puengphob R. Gypsum investing medium for pour resins. J Dent Res 1974; 53: 769. 12. Ono T, Kita S, and Nokubi T. dimensional accuracy of acrylic resin maxillary denture base polymerized by a new injection pressing method. Dent Mater J 2004; 23: 348-52. 13. Keenan PLT, Radford DR, and Clark RKF. Dimensional change in complete denture fabricated by injection molding and microwave processing. J Prosthet Dent 2003; 89: 37-44. 14. Koblitz FF, Smith RA, and Wolfe HE. Fluid denture resin processing in a rigid mold. J Prosthet Dent 1973; 30: 339-46. 15. American dental association Specification No. 12 for denture base polymers. Council on Dental Material and Devices, 4th edition, 1976. 16. de Gee AJ, ten Harkel, and Davidson CL. Measuring procedure for the determination of the threedimensional shape of dentures. J Prosthet Dent 1979; 62: 149-53. 17. Anusavice KJ. Phillips science of dental materials. 11th edition. St.Louis: W.B. Saunders; 2008. P. 722. 18. Bahannan S, Abd El-Hamid A, and Abd Al-Halim M. Accuracy and reproducibility of reversible hydrocolloids versus elastomers duplicating materials. The Saudi Dent J 1995; 7: 7-11. 19. Craig RG and Powers TM. Restorative dental materials. 11th edition. St.Louis: Mosby; 2002.p.36465,403. 20. Grant AA. Effect of the investment procedure on tooth movement. J Prosthet Dent 1962; 12: 1053-8. 21. Consani RLX, Domitti SS, and ConsaniS. Effect of a new tension system, used in acrylic resin flasking, on the dimensional stability of denture bases. J Prosthet Dent 2002; 88: 285-9. 22. Consani RLX, Mesquita MF, Consani S, Sobrinho LC, and Sausa-Neto MD. Effect of water storage on tooth displacement in maxillary complete dentures. Braz Dent J 2006; 17: 53-7.

23. Winkler S, Ortman HR, Morris HF, and Plezia RA. Processing changes in complete dentures constructed from pour resins. JADA 1971; 82: 349-53. 24. Wolfaardt J, Cleaton-Jones P, and Fatti P. The influence of processing variables on dimensional changes of heat-cured poly (methyl methacrylate). J Prosthet Dent 1986; 55: 518-25. 25. Woelfel JB, Paffenbarger GC, and Sweeney WT. Dimensional changes occurring in denture during processing. JADA 1960; 61: 413-30. 26. Chen JC, LacefieldWR,and Castleberry DJ. Effect of denture thickness and curing cycle on the dimensional stability of acrylic resin denture bases. Dent Mater 1988; 4: 20-24. 27. Hardy F. Comparison of fluid resin and compression molding methods in processing dimensional changes. J Prosthet Dent 1978; 39: 375-77. 28. Grant AA, Atkinson HF. Comparison between dimensional accuracy of denture produced with pourtype resin and with heat-processed materials. J Prosthet Dent 1971; 26: 296-301. 29. Antonopoulos AN. Dimensional and occlusal changes in fluid resin dentures. J Prosthet Dent 1978; 39(6), 605-15. 30. Ristic B, Carr L. Water sorption by denture acrylic resin and subsequent changes in vertical dimension. J Prosthet Dent 1987; 58: 689-93.

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An evaluation of water

An evaluation of water absorption of Giomer in comparison to other resin-based restorative materials


Shatha Abdul Kareem B.D.S., M.Sc. (1) Rasha H. Jehad B.D.S., M.Sc. (1)

ABSTRACT
Background: Polymeric composites have been widely used as dental restorative materials. A fundamental knowledge and understanding of the behavior of these materials in the oral cavity is essential to improve their properties and performance. The goal of this study was to measure water sorption of four composite resins containing different filler and resin matrix contents. Materials and method: Resin composite specimens giomer (Beautifil II) Filtek P90, Filtek Z350 XT, and Tetric NCeram were prepared in a cylindrical mould of 3mm thickness and 6mm diameter (n=10) and light cured . All specimens placed in silica-gel desiccators at 37 C for seven days, a constant weight was obtained. All samples were immersed in deionized distilled water at 37 C and weighed at suitable time interval once a week for 30 days. Water sorption was calculated based on ISO 4049. Data were subjected to student t- test. Results: Silorane and Giomer composites showed the lowest values of water sorption, while Z350 and Tetric N-Ceram displayed the highest values at a period of 4 weeks. Conclusion: Each resin- matrix composite varied in water sorption which may affect clinical service. The attained water sorption values are mainly influenced by the generic type of material and variations occurring between materials of the same type may result from differences in resin matrix compositions. Keywords: Water sorption, Giomer, dental resin, silorane. (J Bagh Coll Dentistry 2012;24(3):25-28).

INTRODUCTION
Restorative dental composites are becomingly more popular and extensively used in dentistry due to their esthetic and good in physical and mechanical properties. However, in a wet oral environment the composites may absorb water or other liquids such as saliva, food components or beverages which can have an appreciable influence on the degradation of dental composite. Excessive of liquids uptake may produce deleterious effects on the structure and function of the resin , as these can reduce the mechanical and physical properties that lead to a shortened service life of dental restoration. ISO 4049 is a standard method which is commonly used by researchers to determine water sorption and solubility of restorative dental composites (1) In an aqueous oral environment, polymer composites absorb water and release unreacted monomers. The release of unpolymerized monomers from polymer composites may stimulate the growth of bacteria around the restoration and promote allergic reactions in some patients. Also the water ingress into dental composites in the oral cavity can, over time, lead to deterioration of the physical/mechanical properties due to hydrolytic breakdown of the bond between the silane-filler particles, filler matrix debonding or even hydrolytic degradation of the fillers.

However, some water ingress may have a positive side effect, such as the expansion of the composite compensating for polymerization shrinkage leading to improved marginal sealing. Thus the solvent uptake by dental composite is generally a very important property which must be investigated (2) Giomers, a new class of materials in which the glass is pre-reacted with polyacid then blended with resin to form a composite-type structure. The pre-reacted zone may affect only the surface of the glass or may consume almost the whole of the glass particles and this difference creates a further sub-division of the products.Giomer bear the advantages of both composite resin and glass ionomers, they have excellent esthetics, good polishability, and biocompatibility and also render glass ionomer properties, including fluoride release and fluoride recharge potential. Proper seal against bacterial microleakage and minimal mechanical and chemical irritation of the pulp are other advantages of Giomers (3) This study emphasized in determination of water storage on water sorption of 4 different types of composite resin materials.

MATERIALS AND METHOD


Forty disk shape specimens of Giomer (Beautifil II, Shofu), FiltekTM P90 (3M ESPE,USA), FiltekTM Z350 XT (3M ESPE,USA), and Tetric N-Ceram (Ivoclar Vivadent, Liechtenstein) of (6mm internal diameter and 3 mm height) shade A3 were fabricated using a
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(1) Lecturer, Department of Conservative Dentistry, College of Dentistry, University of Baghdad.

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specially designed cylindrical mold (6mm diameter and 3mm depth). A transparent matrix strip covered with a glass slide of 1mm in thickness was applied at the top of the surface of each composite sample with a constant pressure to extrude excess material, to flatten the surface and to reduce voids at the surface.All specimens were light cured for 40 seconds using LED light curing device with a light intensity of 500mW/cm2 according to manufacturer instructions. Specimens cured under the glass slide had a mirror smooth surface that didnt require further finishing and polishing (4).The specimen was carefully removed from the mould and flash cut away using a sharp blade (1) All samples were transferred to a desiccator with silica gel maintained at 37 C, where they were kept for seven days until weight loss change stabilized. Constant weight was obtained with an accuracy of 0.0001g using calibrated electronic microbalance (Sortorious_Germany) (5) The specimens were then individually placed in a sealed plastic container containing about 10 ml of deionized distilled water in incubator at 37 C. The media used were changed twice a week to avoid microbial activity. The weight measurements were taken from the second day (48 hours) after incubation and continue as one measurement every week for 30 days. Prior to weighing, the specimens were removed, blotted dry to remove excess liquid, weighed and returned to the liquid bath (6) The percentage weight changes were calculated using the following formula: Weight change = WA WB\ WB X 100 % Where WA is the weight of the sample after immersion and WB is the original weight of the sample before immersion.

two materials after 2 weeks, 3 weeks, and 4 weeks immersion in water p> 0.05 table (3) Student t-test also showed that there was no significant difference in water sorption between Giomer and Z350 after 1 week, 2 weeks , and 3 weeks immersion in water p>0.05, while there was a significant difference between the two materials after 4 weeks immersion in water p<0.05 table (4).

DISCUSSION
There are several factors that influence water sorption, for instance the hydrophilicity of the polymer matrix, crosslinking density, fillers, porosity and solvents (7). Water molecules induce the degradation of composites via two mechanisms. Firstly, water molecules diffuse into the polymer network and occupy at the free volume between polymer chains and microvoids, causing plasticization and swelling of polymer matrix and also initiate the chains scission causing monomer elution. The water molecules also tend to degrade the siloxane bonds (bond between silanol groups of the silica surface and the silane coupling agent) via a hydrolysis reaction, causing filler debonding(1). These occurances lead to the degradation or softening of resin composites which may diminish some physical and mechanical properties such as hardness, strength and modulus of elasticity. Thus the dimensional change of a polymer composite in a solvent is complex and difficult to predict and depends on the chemical structure of the polymer matrix. Several data for water sorption for composite materials have been published, but it is difficult to correlate them as the results are often for different time periods and are expressed in different units. Moreover comparisons are difficult to make due to differences in reported specimen size, since different sizes of specimen will take different periods of time for water to completely infiltrate throughout the polymer matrix. The smaller the specimens, the shorter the period for equilibration with water, and the materials which absorbed more water also took longer to stabilize (8). Results of the present study reported that Silorane was found to have statistically the lowest water sorption among the test materials. Silorane is a new monomer with the combination of hydrophobic siloxane and low shrinkage ringopening oxirane. Its photo-activated cationic polymerization is relatively intensive than with radical polymerization to oxygen. Not only is the polymerization shrinkage reduced, but also this effect increases the degree of conversion. Thus, the water sorption of Silorane will be expected to be low (9).
26

RESULTS
The mean, standard deviation and standard error of water sorption for all groups are illustrated in table (1). All disk specimens exhibited percentage mass changes as a function of time when immersed in de-ionized water. Statistical analysis of data using t-test between Giomer and Silorane groups showed that there was a significant difference in water sorption at 1 week, 2 weeks and 3 weeks of immersion in water p<0.05, while there was no significant difference at 4 weeks immersion in water p>0.05 table (2) There was a significant difference in water sorption between Giomer and Tetric N-Ceram after one week immersion in water p<0.05, while there was no significant difference between the

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Giomer showed statistically lower water sorption than Tetric N-Ceram and Z350, this may be due to the incorporation of higher amount of fillers into polymer matrix of Giomer, this can reduce the available free volume for water uptake that lowers the rate of water sorption. It is generally understood that highly filled composites are more resistant to degradative reactions, based on more limited spaces and pathways available for water molecules to diffuse within the polymer structure (7). The main difference in microstructure between Giomer and the other tested composite materials is the presence of pre-reacted glass polyacid zones which become part of the filler in the Giomer structure (10). Results of this study also revealed that Z350 showed higher water sorption than giomer , this may be attributed to the porous nature of zirconia and silica nanocluster, in addition, nonagglomerated nanosilica in Z350 could also provide a large surface area to volume ratio, which allows the fluids accumulated around the filler-polymer interface and leads to an increase in the water sorption . It could be stressed that the total mass of water absorbed was not only diffused through the polymer matrix but also largely diffused into the filler-matrix interface or microvoids of the composite(6). Z350 also contains comparatively small nanoclusters, producing a greater surface area to volume ratio and hence a larger area of hydrophilic silane available for water sorption. Consequently, the physicchemical properties of the intermediate phase will become more critical since a higher degree of silanisation will be required for resin-based composite with a high volume percentage of nano particles (11). Z350 contains hydrophilic monomers which have the following order in hydrophilicity: TEGDMA > BisGMA >UDMA. Mohsen et al., suggested a mechanism of water sorption in UDMA polymer and composite. At an early stage, strongly bound water will form intramolecular hydrogen bonds with the UDMA polymer. Progressively, loosely bound water will interact with intermolecular hydrogen bonds of adjacent polymer chains, inducing chain slippage and polymer plasticization (12). Tetric N-Ceram showed statistically higher water sorption than Giomer but the difference is statistically not significant, because the monomers in these materials are hydrophilic in nature due to the presence of polar groups in their structure which tends to be attracted by water molecules to form hydrogen bonding. Nevertheless, the degree of hydrophilicity of monomers varies, depending on the type of functional groups obtained in
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monomer structure. The hydroxyl group present in BisGMA would form a strong hydrogen bonding with water molecules (13). The high viscosity of Bis-GMA polymer requires the addition of diluents monomers, such as TEGDMA. Such diluents monomers, coupled with the presence of hydroxyl groups in the BisGMA molecules, result in an increase in water sorption because TEGDMA polymer have high network flexibility and heterogenicity creating spaces which accommodate a larger amount of water (14). As conclusion, within the limitation of this study, it has been shown that resin-composite specimens investigated were found to undergo progressive water sorption over a period of 4 weeks. A Silorane composite, however, showed statistically the greatest stability in an aqueous environment followed by Giomer restorative material. By contrast, Z350 and Tetric N-Ceram was the least stable material, due to the incorporation of hydrophilic monomers.

REFERENCES
1. Curtis AR, Shortall AC, Marquis PM, Palin WM, Water uptake and strength characteristics of a nanofilled resin-based composite. J Dent 2008; 36(3):186-93. 2. Karabela MM, Sideridou ID. Effect of the structure of silane coupling agent on sorption characteristics of solvents by dental resin-nanocomposites. Dent Mater 2008; 24:1631-9. 3. Sunico MC, Shinkai K, Katoh Y. Two-year clinical performance of occlusal and cervical giomer restorations. Oper Dent 2005; 30: 282-9. 4. Whitehead SA, Shearer AC, Watts DC, Wilson NHF. Surface texture changes of a composite brushed with tooth whitening dentifrice. Dent Mater 1996;12:315-8. 5. Costella AM, trochmann JL, Oliveira WS.Water sorption and diffusion coefficient through an experiemental dental resin. J Mater Sci: Mater Med 2010; 21:67-72. 6. Rahim TNAT, Mohamad D, Akil HM, Ab Rahman I. Water sorption characteristics of restorative dental composites immersed in acidic drinks. Dent Mater 2012; doi: 10.1016/ J Dental 2012.03.011. 7. Ferracane JL. Hygroscopic and hydrolytic effects in dental polymer networks. Dent Mater 2006; 22(3):211-22. 8. Toledano M, Osorio R, Osorio E, Fuentes V, Prati C, Garcia-Godoy F. Sorption and solubility of resinbased restorative dental materials. J Dent 2003; 31: 43-50. 9. Wei Y, Silikas N, Zhang Z, Watts DC. Diffusion and concurrent solubility of self- adhering and new resinmatrix composites during water sorption/ desorption cycles. Dent Mater 2011(27):197-205. 10. Mc Cabe JF, and Rusby S. Water absorption, dimensional change and radial pressure in resin matrix dental restorative materials. Biomaterial 2004; 25: 4001-7.

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11. Wilson KS, Zhang K, Atonucci JM. Systematic variation of interfacial phase reactivity in dental nanocomposites. Biomaterials 2005; 26:5095-103 12. Mohsen NM, Graig RG, Filisko FE. The effects of moisture on the dielectric relaxation of urethane dimethacrylate polymer and composites. J Oral Rehabil 2001; 28: 376-92.

13. Moraes RR, Sinhoreti MAC, Correr-Sobrinhol, Ogliari FA, Piva E, Petzhold CL. Preparation and evaluation of dental resin luting agents with increasing content of bisphenol-A ethoxylated dimethacrylate. J Biomater Appl 2010; 24(5): 453-73. 14. Martin N, Jedynakiewiez MM, Fisher AC. Hygroscopic expansion and solubility of composite restoratives. Dent Mater 2003; 19: 77-86.

Table 1: Descriptive statistics of water sorption values for all groups


Material Silorane Giomer Tetric N- Ceram Z 350 Mean 0.001583 0.0041 0.01169 0.00658 1 week SD 0.000953 0.003351 0.006954 0.001994 SE 0.000302 0.001061 0.002201 0.000631 Mean 0.00608 0.00865 0.0114 0.01224 Water absorption at specified time (g/cm3) 2 weeks 3 weeks SD SE Mean SD 0.001625 0.000514 0.00709 0.001383 0.002321 0.000734 0.00909 0.002315 0.003967 0.001255 0.01192 0.004128 0.003058 0.000968 0.01417 0.004094 SE 0.000438 0.000733 0.001306 0.001296 Mean 0.00816 0.01125 0.01268 0.0174 4 weeks SD 0.001583 0.003446 0.004281 0.005852 SE 0.000501 0.001091 0.001355 0.001852

Table 2: Student t-test between Giomer and Silorane groups at specified times
Material Giomer & Silorane Time 1 week 2 weeks 3 weeks 4 weeks t-test 4.542 3.114 3.888 1.129 p-value 0.001 0.012 0.004 0.288 Sig S S S NS

P<0.05 Significant P>0.05 Non significant

Table 3: Student t-test between Giomer and Tetric N-Ceram groups at specified times
Material Giomer & Tetric N-Ceram Time 1 week 2 weeks 3 weeks 4 weeks t-test 2.088 1.286 0.893 0.411 p-value 0.049 0.230 0.395 0.690 Sig S NS NS NS

Table 4: Student t-test between Giomer and Z350 groups at specified times
Material Giomer & Z350 Time 1 week 2 weeks 3 weeks 4 weeks t-test 2.088 1.286 0.893 0.411 p-value 0.049 0.230 0.395 0.690 Sig NS NS NS S

Figure 1: Bar chart showing means of water sorption values for Silorane, Giomer, Tetric NCeram, and Z350 materials after 1, 2, 3, and 4 weeks immersion in water

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The effect of plasma

The effect of plasma treatment on the bonding of soft denture liners to heat cured acrylic resin denture base material and on some surface properties of acrylic resin polymer
Shaymaa H. Masood B.D.S. (1) Salah A. Mohamed B.D.S., M.Sc. (2)

ABSTRACT
Background: Acrylic resin polymers used in dentistry, are usually with problems in bonding, especially failure of the bond with soft denture lining materials. The aim of this study is to investigate the effect of plasma treatment on tensile bond strength, wettability and on physical surface changes for acrylic resin polymer. Materials and methods: Heat cured acrylic resin specimens with dimensions 81030 mm were prepared for tensile bond strength test, in which each two acrylic specimens were joined by a 3-mm thick soft liner (Vertex Soft, Molloplast-B). Another heat curd acrylic resin specimens were prepared with dimensions 2830 mm for wettability test and physical surface analysis. For each test done in this study, the specimens were grouped as control, oxygen plasma treated and argon plasma treated acrylic specimens. Results: Plasma treatment increased the tensile bond strength for both Vertex and Molloplast-B soft lining materials, also induced a decrease in water contact angle values (i.e., increase in wettability) for oxygen and argon plasma treated groups compared with control group, with highly significant difference (P <0.01) among groups. AFM images showed a collection of new distinct nanograins and numerous grooves (pitlike-structures) after oxygen and argon plasma treatment with argon plasma treatment showed more new nanograins, deepest grooves and highest protuberances which increased the surface-roughness (i.e. nano-roughness) when compared with control and oxygen plasma treated groups. Conclusion: Plasma treatment was an effective method for increasing tensile bond strength, wettebility, and induced physical topographical surface changes that increased the surface roughness(mainly after argon plasma treatment) for plasma treated heat cured acrylic resin specimens. Key words: plasma treatment, tensile bond strength, wettability, AFM analysis. (J Bagh Coll Dentistry 2012;24(3):2935).

INTRODUCTION
Polymer surfaces usually present problems in bonding and finishing due to their low hydrophilicity (1). Soft polymers (Denture Liners) have been used for almost 40 years as lining materials for dentures in the short-term prosthodontic management of denturesupporting mucosa. In general, the most common drawbacks with regard to the use of soft liners are poor adhesion to the denture base and the loss of softness over time (2). Many studies have revealed improved adhesion of polymers by plasma treatment (3). Plasma is a partially or wholly ionized gas with a roughly equal number of positively and negatively charged particles (4). With plasma treatment, the surfaces of polymers can be improved in terms of hydrophilicity by forming oxygen-containing functional groups, such as C=O and OH, (5). These effects result in acid-base interactions and covalent linkages.
(1) (2) MSc student. Department of Prosthodontic, College of Dentistry, University of Baghdad. Assistant professor. Department of Prosthodontic, College of Dentistry, University of Baghdad.

Where some of these effects overlap, bonding enhancement is thus successfully achieved through plasma treatment (6).

MATERIALS AND METHODS


Heat cured acrylic resin (PMMA) specimens were prepared for each test done in this study. These specimens were prepared from SR Triplex hot, heat-cured acrylic denture base material (Ivoclar Vivadent, Germany).For each test done in this study, the specimens were grouped as control plasma untreated, oxygen plasma treated and argon plasma treated acrylic specimens. Plasma treatment In this study a plasma apparatus with parameters: 800 V, 75 mA, power 60 W, with 2 minutes exposure time, with the plasma source was kept 4cm above the test specimens, were used for all tests of this study with the application of two types of plasma treatments (oxygen and argon plasma treatments).

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Figure 1: Plasma apparatus.


Tensile bond strength test: Two hundred and forty rectangular heat cured acrylic resin specimens with dimensions 8mm10mm30mm as width, height, and length respectively, were prepared, in which each (2) specimens were joined by a 3-mm thick soft liner disk (7), to finally reproduce (120) specimens which were grouped as: 40 control specimens without plasma treatment, 40 oxygen plasma treated specimens, and 40 argon plasma treated specimens. each 40 specimens for each group was subdivided into 20 specimens with application of Molloplast-B soft liner and 20 specimens with application of Vertex Soft liner. For tensile bond strength test the following procedures were done: 1. Preparation of acrylic resin blocks: A mold was made by investing a metal piece of dimensions 810 mm cross-sectional area and 30 mm length, in hard but flexible silicone rubber (Addition silicone duplicating material). The obtained mold was then used to fabricate rectangular wax blocks, which in turn were used to produce the rectangular acrylic resin blocks. Proportioning and mixing of acrylic was done according to the manufacturers instructions with ratio 2.25 gm (powder): 1 ml (liquid). The packing process was performed while the acrylic was in the dough stage. According to the manufacturers instructions, polymerization was carried out by placing the clamped flasks in cold water, heat them up to 100 C, and let boil for 45 minutes. All the other acrylic blocks were prepared as the previously mentioned method. After that, the bonding surfaces of all the acrylic blocks were smoothened using 240-grit aluminum oxide paper. All specimens should be cleaned by means of ultrasonic cleanser machine, and dried. Only the 40 rectangular acrylic resin blocks for oxygen plasma treatment group and the 40 rectangular acrylic resin blocks for argon plasma treatment were exposed to plasma treatment. 2. Preparation of tensile bond strength test specimens: A second mold was made by
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investing a metal sample of 810 mm crosssectional area and 63 mm length. The 3 mm represent the spacer length between each two acrylic blocks for packing of soft liner material (Vertex Soft, Molloplast-B), in hard but flexible silicone rubber investment material. The obtained mold was used to fabricate the tensile bond strength test specimens by processing the soft liner against the two rectangular acrylic resin blocks, so each 2 acrylic blocks were placed back into the mold and the soft liner was packed into the space between two blocks, trial packed, and polymerized according to the manufacturers instructions.

Figure 2: Rubber mould for preparation of tensile bond strength test specimens.

Figure 3: Tensile test specimen with Molloplast-B.


Soft Liner Materials polymerization procedure was done according to manufacturers instructions for each material as illustrated in table (1). 3. Preparation of artificial saliva: An electrolyte composition similar to that of human saliva was used in this study (8) which includes: 1. [1 g] Sodium carboxy-methyl-cellulose. 2. [4.3 g] Sorbitol. 3. [0.1 g] Potassium chloride. 4. [0.1 g] Sodium chloride. 5. [0.02 mg] Sodium fluoride. 6. [5 mg] Magnesium chloride. 7. [5 mg] Calcium chloride. 8. [40 mg] Potassium phosphate. 9. [1 mg] Potassium thio-cyanate. 10. [100 ml] Distilled de-ionized water. Before tensile bond strength testing, all tensile bond strength specimens were stored in artificial saliva substitute (9) in an incubator at (37C) for two periods in which half the specimens were stored for 48h and the other were stored for 12 weeks. 4. Tensile bond strength test procedure: The rectangular tensile bond strength test specimens

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were tested using Instron testing machine with a suitable grips for the test specimens. The specimen was subjected to tensile bond with crosshead speed 5 mm/min with maximum load capacity 1000 N. Force at failure was recorded in Newton. The value of tensile bond strength were calculated for each test specimen as the force at the de-bonding divided by a cross-section area of interface according to the following formula:

Figure 5: Atomic Force Microscope for AFM analysis.

Where: F= force at failure (N) A= surface area of the cross section (mm) Wettability test (water contact angle measurement): Three wax specimens were prepared with dimensions 2830 mm, which in turn were used to prepare the (3) heat cured acrylic resin specimens. Immediately after preparation, the control specimen was measured for wettability test. The other two specimens were measured immediately after oxygen and argon plasma treatments. For wettability test, a versatile digital microscope was used. Each specimen was placed onto a glass microscope slide using double-sided tape to ensure a flat viewing surface. The glass slide was then placed onto a stage, where a 10- L deionized water drop was dispersed from the pipette onto the surface. For each specimen, five measurements were taken immediately after plasma treatment. The values were averaged to obtain a final contact angle value.

RESULTS
Tensile bond strength test: The effect of oxygen and argon plasma treatments, on the tensile bond strength was tested and evaluated, as show in tables 2-9. Wettability Test:

Figure 6: Polygon Illustrates the Mean Values of Water Contact Angles for Acrylic Polymer Specimens with Different Types of Surface Treatment.

AFM Analysis:

Figure 4: Dino-Lite Digital Microscope.


Physical or Topography Surface Analysis (Atomic Force Microscopy or AFM Analysis): The surface topography/morphology of the untreated and plasma treated acrylic polymer specimens was analyzed and compared by atomic force microscopy. Also, the specimen dimensions for (AFM) analysis were, 2830 mm, as the same dimensions which were used for wettability test.

Figure 7: AFM Image for Control Acrylic Specimen: 3 Dimensions Image.

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Figure 8: AFM Image for Oxygen Plasma Treated Acrylic Specimen: 3 Dimensions Image.

Figure 9: AFM Image for Argon Plasma Treated Acrylic Specimen: 3 Dimensions Image.

DISCUSSION
The Effect of Plasma Treatment on Tensile Bond Strength: Based on the results obtained in this present study, both the oxygen and argon plasma treatments were increased and improved the tensile bond strength. As a discussion for these findings, for oxygen plasma treatment, these results might be due to the effects of chemical oxidation reactions and/or chemical etching process. During the oxidation reactions, plasma promotes adhesion by inducing further chemical reactions which generated new chemical functional groups on the O2 plasma treated surface such as the hydroxyl group (-OH) which increased the hydrophilicity that enhanced the penetration of Vertex and Molloplast-B soft liners into the irregularities on the acrylic resin surface and contributed latter to the increase in the tensile bond strength that result after the oxygen plasma treatment, while during the chemical etching process, this process results in chemical removal of surface material that increased the effective surface area of the polymer (i.e., surface roughening). This roughening in turn promotes more intimate molecular contact between the plasma exposed polymer surface and the applied soft liner allowing for stronger bonds to occur. For argon plasma treatment, the increase in the tensile bond strength might be due to the effect of physical
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sputtering (i.e. physical removal of surface material) which increased the surface roughness that caused by the bombardment of highly energetic and high molecular weight argon particles that enhanced micromechanical interlocking between the Vertex or the Molloplast soft liners and the acrylic denture base material. These results were in agreement with the findings of study by (10) which showed that, argon and oxygen plasmas have also been shown to participate in surface sputtering in addition to modification, resulting in the physical removal of material from the surface, also in agreement with the findings of study by (11) which showed that, chemical etching process, physical sputtering occurs frequently as well when polymers are exposed to plasma. Also the results of this study were in agreement with the findings of studies by (12); (13); and (14) which revealed that, through the oxygen plasma treatment, oxygen-containing groups of C-O and C=O were effectively introduced onto the polymer surface due to the highly reactive property of oxygen plasma. The presence of oxygen-containing groups then improved the surface hydrophilicity of the plasma treated specimens. The Effect of Plasma Treatment on Wettability: Results of the present study showed that, there was a decrease in water contact angle mean values after the application of oxygen and argon plasma treatments when compared with the control group for the acrylic polymer specimens. This may be due to either surface texture changes or oxidation functionality surface changes. There can be benefits of such interactions, such as the modification of hydrophobic or hydrophilic properties of polymers due to chemical modification and texturing (15). The Effect of Plasma Treatment on Physical Surface Morphology: AFM images for the surfaces of the acrylic (PMMA) specimens (control, oxygen and argon plasma treated specimens) showed a collection of grooves (pitlike-structures), ridges, peaks, crater-like features and surface irregularities. Our results are consistent with the findings of (16). AFM images after argon plasma treatment in this study had produced more new, distinct nanograins with smaller diameters, deepest grooves, highest protuberances and more regularity in granularity normal distribution chart which increased the surface nano-roughness due to physical sputtering as compared with oxygen plasma treated and plasma untreated groups. Differences in the topological features of (PMMA) surfaces

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specimens may be attributed to the differences in their physical and chemical properties. AFM analysis had proved that, plasma treatment produced nano-roughness on the plasma treated surface mainly after the argon plasma treatment, and such roughness was beneficial for enhancing the tensile strength by creating mechanical interlocks between the acrylic resin polymer and the two types of soft liners that were used in this study.

7.

8.

9.

REFERENCES
1. Lai J, Sunderland B, Xue J, Yan S, Zhao W, Folkard M, Michael BD, Wang Y. Study on hydrophilicity of polymer surfaces improved by plasma treatment. Appl Surf Sci 2006; 252: 33753379. Kanie T, Kadokawa A, Arikawa H, Fujii K, Ban S. Effects of adding methacrylate monomers on viscosity and mechanical properties of experimental light-curing soft lining materials based on urethane (meth)acrylate oligomers. Dent Mater J 2008; 27: 856-861. Kusano Y, Mortensen H, Stenum B, Goutianos S, Mitra S, Ghanbari Siahkali A, Kingshott P, Sorensen BF, Bindslev H. Atmospheric pressure plasma treatment of glassy carbon for adhesion improvement. Int J Adhes Adhes 2007; 27: 402408. Chen FF. Introduction to plasma physics and controlled fusion: Vol.1 Plasma Physics, 2nd ed, Springer-Verlag, New York, 1984. p. 1-4. Nishigawa G, Maruo Y, Oka M, Oki K, Minagi S, Okamoto M. Plasma treatment increased shear bond strength between heat cured acrylic resin and selfcuring acrylic resin. J Oral Rehabil 2003; 30: 10811084. Liston EM, Martinu L, Wertheimer MR. Plasma surface modification of polymers for improved adhesion: a critical review. J Adhesion Sci Technol

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1993; 7: 1091-1127. Huaiqin Zhang, Jianglin Fang, Zheng Hu, Junchi Ma, Yi han and Jie Bian. Effect of oxygen plasma treatment on the bonding of a soft liner to an acrylic resin denture material: Dental Materials Journal 2010; 29(4): 398402. Cavalla V, Reis AF, Giannini M, Ambrosan GM.The effect of elapsed time following bleaching on enamel bond strength of resin composites. Operative Dentistry 2001; 26:597-602. Ritchie GM, Fletcher AM, Amin WM. Further mechanical tests on the bond between poly methyl methacrylate teeth and some acrylic denture base polymers. Proc Eur Prosthodont Assoc 1984; 7:107. Grant, J. L.; Dunn, D. S.; McClure, D. J. J. Plasma surface modification techniques. Vac. Sci. Technol. A. 1988; 6: 2213-2220. Inagaki N. Plasma surface modification and plasma polymerization.Basel: Technomic Publishing AG, 1996. Chan CM, Ko TM, Hiraoka H. Polymer surface modification by plasmas and photons. Surf Sci Rep 1996; 24: 1-54. Chen M, Zamora PO, Som P, Pena LA, Osaki S. Cell attachment and biocompatibility of polytetrafluoroethylene (PTFE) treated with glowdischarge plasma of mixed ammonia and oxygen. J Biomater Sci Polymer Ed 2003; 14: 917-935. Lim H, Lee Y, Han S, Kim Y, Song JM, Kim JS. Wettability of poly (styrene-co-acrylate) ionomers improved by oxygenplasma source ion implantation. J Polym Sci Pol Phys 2003; 41: 1791-1797. Banks BA, Rutledge SK, Hunt JD, Drobotij E, Cales MR, Cantrell G, Atomic Oxygen Textured Polymers, paper presented at the Materials Research Society, San Francisco, CA. April, 1995: 17-21. Lombardo M, De Santo MP, Lombardo G, Barberi R, Serrao S. Analysis of intraocular lens surface properties with atomic force microscopy. J Cataract Refract Surg 2006; 32: 1378-1384.

Table 1: Polymerization of Soft liners.


Type of soft liner Vertex Soft Polymerization procedure 1.2 gr (powder): 1 ml (liquid) Polymerization was done by placing the flask in cold water and heat up to 70 C for 90 min, then boil up to 100 C and left at this temperature for 30 min, then cool down flask slowly. Ready-to-use paste Before packing of Molloplast-B, Primo adhesive should be applied by brushing Primo adhesive 1-2 times onto the entire bonding surface of each specimen. Let Primo air-dry for 60 min. prior to applying Molloplast-B. Polymerization was done by placing the flask in cold water and heat up slowly to 100 C. Polymerization in boiling water at 100 C for approximately 2 hours, then cool down flask slowly.

Molloplast-B

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Table 2: Descriptive statistics of tensile bond strength for Vertex Soft.


Type of Soft Liner Type of surface treatment Time of storage Mean SD Vertex-soft Control without plasma treatment 48 h 12 weeks 1.594 0.111 1.394 0.084 (O2) plasma treatment 48h 2.217 0.249 12 weeks 1.876 0.150 Argon plasma treatment 48 h 1.875 0.112 12 weeks 1.799 0.142

Table 3: Descriptive Statistics of Tensile Bond Strength for Molloplast-B.


Type of Soft Liner Type of surface treatment Time of storage Mean SD Molloplast-B Control without plasma treatment 48 h 12 weeks 1.408 0.120 1.228 0.115 (O2) plasma treatment 48h 12 weeks 1.404 0.142 Argon plasma treatment 48 h 12 weeks 1.615 0.230

1.687 0.169

1.775 0.160

Comparisons between groups: Table 4: LSD for Vertex-soft after 48h.


Type of surface treatment Control and Oxygen Control and Argon Oxygen and Argon 48h P-value 0.000 0.000 0.002 Sig HS HS HS

Table 5: LSD for Vertex after 12 Weeks.


Type of surface treatment P-value Control and Oxygen Control and Argon Oxygen and Argon 0.001 0.000 0.251 48h Sig HS HS NS

Table 6: LSD for Molloplast-B after 48h.


Type of surface treatment P-value Control and Oxygen Control and Argon Oxygen and Argon 0.001 0.000 0.251 48h Sig HS HS NS

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Table 7: LSD for Molloplast-B after 12 Weeks.


Type of surface treatment Control and Oxygen Control and Argon Oxygen and Argon P-value 0.008 0.000 0.026 HS S 12 weeks Sig HS

Table 8: Descriptive Statistics for Wettability Test.


Type of surface treatment Control Oxygen plasma treatment Argon plasma treatment water contact angle mean values (degree) 46.161 24.03 31.499 SD 2.201 2.87 2.024

Comparisons between groups: Table 9: LSD between Groups of Acrylic Polymer.


Type of surface treatment Control and Oxygen plasma treatment Control and Argon plasma treatment Oxygen plasma treatment and Argon plasma treatment P-value 0.000 0.000 0.002 Sig. HS HS HS

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Influence of SOLO disinfectant on some properties of different denture lining materials


Shorouq M. Abass, B.D.S., M.Sc. (1) Reem A. Nassif, B.D.S., M.Sc. (1) Bayan S. Khalaf, B.D.S., M.Sc. (1)

ABSTRACT
Background: Denture lining materials are widely used in prosthodontic treatment and management of traumatized oral mucosa. A contaminated prosthesis can provide a source of cross-contamination between patients and dental personnel as well as a cause for denture stomatitis. Therefore, denture disinfection has been recommended as an essential procedure for maintenance of a healthy oral mucosa. This study investigated the effect of SOLO disinfectant solution on some properties of different denture lining materials. Materials and methods: Three different solutions were used in this study; SOLO disinfectant solution, sodium hypochlorite solution, and water on three types of acrylic denture lining materials; hot cure, cold cure, and soft acrylic resin. Twenty seven disk-shaped samples were used to evaluate the color stability and forty five rectangular samples were used for testing the surface micro hardness and surface roughness of the different denture lining materials. Data measurements of the color stability, surface hardness, and surface roughness were analyzed and compared statistically. Results: The color stability for the tested denture lining materials was insignificantly affected (p>0.05) by the immersion in the SOLO disinfectant solution. There was a highly significant difference (p< 0.01) in the surface hardness of the hot cure while it was insignificant (p>0.05) for cold cure denture lining materials when immersed in the SOLO disinfectant solution. For surface roughness there was no significant difference (p>0.05) by immersion in SOLO disinfectant solution for the different denture lining materials. Conclusions: Based on the results of this study SOLO disinfectant solution produced no adverse effect on the color stability, surface hardness, and surface roughness of the hot cure, cold cure, and soft acrylic denture lining materials Keywords: acrylic, immersion, SOLO, color, hardness, roughness. (J Bagh Coll Dentistry 2012;24(3):36-41).

INTRODUCTION
Denture lining materials are widely used as adjuncts in the prosthodontic treatment and management of traumatized oral mucosa. However, the soft-lined dentures have been associated with Candidal growth especially in soft lined mandibular dentures more than unlined maxillary dentures.1 One of the etiological factors involved in denture stomatitis is the lack of denture sanitation. The need to remove denture plaque at regular intervals, especially on the tissue fitting surfaces of dentures, was emphasized to prevent denture stomatitis. In addition, the unpolished surface of the denture was a suitable site for Candida proliferation and C. albicans penetration was greater on the unpolished denture surface.2 Cross contamination of dental personnel may occur during denture repair or adjustment when particles of the internal surface become airborne during grinding.3 Also, dental professionals and patients should be careful of denture-borne microorganisms to cause oral/systemic diseases. Thus, they should take into consideration the appropriate sanitization procedures to reduce the reservoir of microorganisms and to prevent cross contamination.4
(1) Lecturer at Department of Prosthodontics, Collage of Dentistry, Baghdad University.

Denture disinfection has been recommended as an essential procedure for preventing crosscontamination and the maintenance of a healthy oral mucosa. Several studies have investigated the disinfection efficiency of several chemical solutions for denture lining materials. Furukawa et al.5 stated that the immersion technique was more effective than the spray technique, however the Chlorine dioxide did not reach the minimal standard of disinfection for the tested denture liners. Pavarina et al.6 recommended scrubbing the denture with a disinfecting solution combined with immersion for 10 minutes and this was effective in reducing the microbial growth. Barnabe et al.7 also suggested brushing the dentures and they used coconut soap and 0.05% sodium hypochlorite to significantly reduce the clinical signs of denture stomatitis, however the C. albicans counts did not decline. Other examiners were careful to study the effect of the chemical disinfection on the physical and mechanical properties of the different types of denture acrylic resin. Some of these chemical disinfectants caused color shifts and surface damage including increased surface roughness.8-11 Some studies for denture disinfection have been proposed, including immersion in chemical solutions and microwave irradiation. They found that microwave disinfection increased the surface roughness and adversely affected the surface
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texture and produced clinically unacceptable alterations in the adaptation of maxillary acrylic resin denture bases to the stone casts.12-14 Abass et al.15 evaluated the influence of immersion in NaCl solution, immersion in water, and in dry air during microwave disinfection on dimensional stability, water sorption, and water solubility of hot cure, cold cure, and soft acrylic resin. They suggested that immersion in NaCl solution when used for hot cure acrylic resin affected the dimensional stability, while for soft acrylic resin the dimensional stability could be affected when immersed in water during microwave disinfection. Water sorption for cold cure acrylic resin significantly changed when immersed in water and when placed in dry air during microwave disinfection. Also the surface roughness and hardness were evaluated by Ibrahem16 who found that the used of the same methods adversely affected the surface roughness. Our hypothesis was that immersion of the acrylic resin denture lining materials in the SOLO disinfectant solution could adversely affect some of the acrylic resins properties. The study was aimed at assessing the effect of SOLO disinfectant, sodium hypochlorite, and water on three-different types of acrylic denture base materials in association with the surface hardness, surface roughness, and color stability.

Sample preparation was conducted according to the manufacturers recommendations for each type of denture lining material. After finishing and polishing, all the samples were immersed in distal water at 37 C for 50 hours.18 The samples were then divided into nine test groups according to the type of denture lining material and immersion solution used, as shown in table 1. Color stability was evaluated using twenty seven disk-shaped samples with a diameter of 50 mm and thickness of 1 mm in accordance with ADA specification no. 12.19 with the UV-Visible Recording Spectrophotometer (UV-160A, Shimadzu Corp., Kyoto, Japan) was used for evaluation of color stability at a wave length of (400-500 !). Two readings were obtained for each sample; one before the immersion and one after. Surface roughness & surface hardness were assessed with forty-five rectangular samples with dimensions of (20) mm (12) mm (3) mm. The acrylic samples were tested for surface micro hardness test with Vickers hardness test machine (VHN- Kg/mm2) with a load of 10 Kg. Three indentations were made at different points on each sample, and then the mean reading was calculated for each sample. Two readings were evaluated; one before the immersion and one after (figure 1). Acrylic samples were tested for surface roughness, Ra ("m), with the Profilometer device (surface roughness tester). Two measurements were taken for each sample and the average reading was then calculated. Two readings were recorded for each sample; one before the immersion and one after. Statistical analysis included descriptive statistics and paired sample t-test at a significance level of p<.05.

MATERIALS AND METHODS


Three types of denture lining materials were used in this study; hot cure acrylic resin denture base material (SR Triplex Hot, Ivoclar Vivadent, Liechtenstein), soft acrylic resin for relining dentures (Vertex Soft, Vertex-Dental, Netherlands), and cold cure acrylic resin for repair and relining of dentures (MEGA-A, Megadenta Dentalprodukte GmbH, Germany). The properties of these materials were evaluated with the influence of the immersion in water, Sodium hypochlorite solution, & SOLO disinfectant solution on the color stability, surface roughness, and surface hardness of the different denture lining materials. Sodium hypochlorite solution was prepared by using the household bleach of 5.25% hypochlorite solution and diluted with water at a ratio of 1 part of bleach: to 10 parts of water to make 1:10 ratio and the samples were immersed in this solution for 10 minutes according to the ADA recommendation for disinfection.17 Preparation of SOLO Disinfectant solution (SOLO, Ebiox Ltd., Healthcare Enterprise House, UK.) and duration of immersion were according to the manufacturers instructions.
Restorative Dentistry 37

RESULTS
The results of this study revealed that there was no significant change (p > .05) in the color of the test samples for all of the test groups (table 2 & 3). The surface hardness of the hot cure acrylic resin highly significantly increased by immersion in water and the SOLO solution, (p < .01), while it was insignificantly affected by the immersion in the sodium hypochlorite solution, (p > .05). For the cold cure acrylic resin the surface hardness was significantly increased by immersion in water (p < .05) and insignificantly affected by immersion in sodium hypochlorite and SOLO disinfectant solution (p > .05), see table (4&5). The results for the surface hardness

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test for the soft acrylic resin samples were excluded from the statistical analysis because they didnt register any readings due to the elasticity of the material that prevented any permanent deformation. Surface roughness for the samples of all of the test groups was unaffected (p > .05) by immersion in any of the solutions, as shown in table (6 & 7).

Figure 1: Surface Micro-Hardness indention

DISCUSSION
The hypothesis that the immersion of acrylic denture lining in a SOLO disinfectant solution adversely affects the color stability was rejected. It seems that SOLO disinfectant solution did not have any effect on the color of the different denture lining materials. The results of Ma et al.8 were in agreement with this study, but were in disagreement with the finding of Hong et al.11 who found that the influence of denture cleansers on the color stability of denture base acrylic resins varied according to the type of denture cleanser used. This may be related to the fact that the time of immersion in this study was too short to produce any change in the color, while their duration of immersion was 12 hours. Their solutions were used as denture cleansers, while the goal in this study was for disinfection only and according to the manufacturers instructions, for SOLO disinfectant solution, this imposed a short duration of immersion. Also, they used a different device for measurement of color stability and their chemical disinfection solutions were not the same, so this could have had an influence on the difference between the outcomes. The hypothesis that SOLO disinfectant solution could adverse effect the surface hardness was rejected. The surface hardness of hot cure acrylic denture lining material was significantly increased after immersion in SOLO disinfectant solution for 10 minutes, while it was insignificantly affected for the cold cure acrylic resin, although there was some enhancement in surface hardness. This difference in surface
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hardness could be explained by the findings of Mohamed et al.20 who stated that the residual monomer content for hot cure acrylic resin samples was less than that of cold cure and the higher residual monomer content in the cold cure acrylic resin acted as a plasticizer, subsequently reducing the properties of the cold cure acrylic resin.21 The amount of residual monomer that leaked out of the hot cure acrylic ended in a higher surface hardness, but with the cold cure acrylic resin even with the escape of some of the residual monomer into the immersion solution it was not enough to enhance the surface hardness to a highly significant difference. In addition, Machado et al.12 and Braun et al.22 both found that the cold cure reline materials exhibited significantly lower hardness mean values than the hot cure relining materials. Also, Braun et al.22 further stated that the immersion in water caused leaching of residual monomer from denture base materials that the contributed to the higher surface hardness. Asad et al.23 confirmed in their study that disinfection by immersion in chemical solution did not adversely affect the surface hardness of acrylic resin materials. Immersion of the hot and cold cure denture lining materials in the sodium hypochlorite solution resulted in an insignificant increase in the surface hardness. Chau et al.24 observed that sodium hypochlorite penetrated beyond the surface of the acrylic to a depth of 3 mm with ten minutes of immersion in a solution of 1% sodium hypochlorite. In addition, Miranda et al.25 found that mouthwashes containing hydrogen peroxide and/or alcohol reduced the surface hardness of different resins by different immersion solutions. So we can concluded from the previous two studies that sodium hypochlorite could have had an effect on the surface hardness of the acrylic resin by opposing the effects of the reduction of the residual monomer and retarding the increase in surface hardness to an insignificant level. This also confirmed with the finding of Neppelenbroek et al.26 who demonstrated a significant decrease in hardness after immersion in chemical disinfectant solutions, including sodium hypochlorite, regardless of material and disinfectant solution used. The results of their study showed that chemical disinfection with sodium hypochlorite adversely affected the surface hardness of denture base acrylic resin. They assumed that the sodium hypochlorite solution may have penetrated in to the tested materials and resulted in softening of the materials. The hypothesis that the SOLO disinfectant solution could adversely affect the surface

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roughness of the different acrylic denture lining materials was rejected. The surface roughness was not affected for the samples of all the test groups. This was in agreement with the outcome of Ma et al.8 who found that all denture acrylic resins tested could be immersed in some disinfectants for up to 30 minutes without appreciable alteration to surface texture. Also, da Silva et al.9 stated in their study that immersion for 10 minutes produced no significant effect on the surface roughness of acrylic resin. However, this finding was in disagreement with the results of Machado et al.12 who stated that immersion in a chemical disinfectant solution may increased the surface roughness of denture base acrylic resin and the findings of our study differed because of the short duration of immersion of the acrylic samples which may have not been enough to manifest any changes statistically. In conclusion SOLO disinfectant solution could be used as a disinfection solution for hot and cold cure denture lining material as well as for soft acrylic resin lining material, since it had no adverse effect on the color stability, surface hardness, and surface roughness of these materials.

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REFERENCES
1. Makila E, Hopsu-Havu VK. Mycotic growth and soft denture lining materials. Acta Odontol Scand 1977;35(4);197-205. (Abstract) 2. Webb BC, Thomas CJ, Willcox MDP, Harty DWS, Knox KW. Candida associated denture stomatitis. Austral Dent J 1998; 43(3): 160-6. 3. Lin JJ, Cameron SM, Runyan DA, Craft DW. Disinfection of denture base acrylic resin. J Prosthet Dent 1999; 81: 202-6. 4. Glass RT, Conrad RS, Bullard JW, Goodson LB, Mehta N, Lech SJ, Loewy ZG. Evaluation of microbial flora found in previously worn prostheses from the Northeast and Southwest regions of the United States. J Prosthet Dent 2010; 103: 384-9 5. Furukawa KK, Niagro FD, Runyan DA, Cameron SM. Effectiveness of chlorine dioxide in disinfection on two soft denture liners. J Prosthet Dent 1998; 80: 723-29. 6. Pavarina AC, Pizzolitto AC, Machado AL, Vergani CE, Giampaolo ET. An infection control protocol: effectiveness of immersion solutions to reduce the microbial growth on dental prostheses. J Oral Rehab 2003; 30; 5326. 7. Barnabe W, De Mendonc T Neto A, Pimeta FC, Pegoraro LF, Scolaro JM. Efficacy of sodium hypochlorite and coconut soap used as disinfecting agents in the reduction of denture stomatitis, Streptococcus mutans and Candida albicans. J Oral Rehab 2004; 3: 4539. 8. Ma T, Johnson GH, Gordon GE. Effects of chemical disinfectants on the surface characteristics and color of denture resins. J Prosthet Dent 1997; 77: 197-204. 9. da Silva FC, Kimpara ET, Mancini MN, Balducci I, Jorge AO, Koga-Ito CY. Effectiveness of Six

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Different Disinfectants on Removing Five Microbial Species and Effects on the Topographic Characteristics of Acrylic Resin. J Prosthod 2008; 17: 62733. Goiato MC, dos Santos DM, Gennari-Filho H, Zavanelli AC, de Carvalho Dekon SF, Mancuso DN. Influence of Investment, Disinfection, and Storage on the Microhardness of Ocular Resins. J Prosthod 2009; 18: 325. Hong G, Murata H, Li Y, Sadamori S, Hamada T. Influence of denture cleansers on the color stability of three types of denture base acrylic resin. J Prosthet Dent. 2009 ;101: 205-13. Machado AL, Breeding LC, Vergani CE, da Cruz Perez LE. Hardness and surface roughness of reline and denture base acrylic resins after repeated disinfection procedures. J Prosthet Dent 2009;102:115-22. Sartori EA, Schmidt CB, Walber LF, Shinkai RSA. Effect of microwave disinfection on denture base adaptation and resin surface roughness. Braz Dent J 2006; 17(3): 112-7. Pavan S, Arioli Filho JN, Dos Santos PH, Mollo Jr. FA. Effect of Microwave Treatments on Dimensional Accuracy of Maxillary Acrylic Resin Denture Base. Braz Dent J 2005; 16(2): 119-23. Abass SM, Ibrahem RA, Alkafaji AM. Effect of immersion in sodium chloride solution during microwave disinfection on dimensional stability, water sorption, and water solubility of denture base acrylic resin. J Baghdad Col Dent 2010; 22(3): 46-51. Ibrahem RA. The effect of microwave disinfection on surface roughness and hardness of hot, cold acrylic resin and soft liner in different conditions. J Bagh Col Dent 2010; 22(4): 36-40. Council on Dental Therapeutics Council on Prosthetic Services and Dental Laboratory Relations. Guidelines for infection control in the dental office and the commercial dental laboratory. JADA 1985:110; 969-72. International Standards Organization.ISO 1567: 1999. Dentistry- denture base polymers. Cited in Seo RS, Vergani CE, Pavarina AC, Compagnoni MA, Machado AL. Influence of microwave disinfection on the dimensional stability of intact and relined acrylic resin denture bases. J Prosthet Dent 2007; 98:216-23. American Dental Association specification no. 12 for denture base polymers. JADA 1975; 90(2): 451-8. Mohamed SH, Al-Jadi Alb M, Ajaal T. Using of HPLC Analysis fo Evaluation of Residual Monomer Content in Denture Base Material and Their Effect on Mechanical Properties. J Physical Science 2008; 19(2) 12735. Craig RG. Restorative Dental Materials. 10th ed. St. Louis(Missouri): CV Mosby; 1997:521-2. Braun KO, Mello JA, Rached RN, Del Bel Cury AA. Surface texture and some properties of acrylic resins submitted to chemical polishing. J Oral Rehabil 2003;30:91-8. (Abstract) Asad T, Wattkinson AC, Huggett R. The effects of various disinfectant solutions on the surface hardness of an acrylic resin denture base material. Int J Prosthodont 1993; 6: 9-12. (Abstract) Chau VB, Saunders TR, Pimsler M, Elfring DR. Indepth disinfection of acrylic resins. J Prosthet Dent 1995;74:309-13.

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25. Miranda DA, Bertoldo CES, Aguiar FHB, Lima DANL, Lovadino JR. Effects of mouthwashes on Knoop hardness and surface roughness of dental composites after different immersion times. Braz Oral Res 2011; Mar-Apr;25(2):168-73. Test groups HA HB HC CA CB CC SA SB SC acrylic resin Hot cure Hot cure Hot cure Cold cure Cold cure Cold cure Soft acrylic Soft acrylic Soft acrylic

26. Neppelenbroek KH, Pavarina AC, Vergani CE, Giampaolo ET. Hardness of heat-polymerized acrylic resins after disinfection and long-term water immersion. J Prosthet Dent 2005; 93:171-6.

Table 1: Experimental design for the test groups used in this study
Disinfectant solution Immersion in water for 10 min. Immersion in Sodium hypochlorite solution for 10 min. Immersion in SOLO solution for 5min. Immersion in water for 10 min. Immersion in Sodium hypochlorite solution for 10 min. Immersion in SOLO solution for 5min. Immersion in water for 10 min. Immersion in Sodium hypochlorite solution for 10 min. Immersion in SOLO solution for 5min.

Table 2: Mean and Standard Deviation for Color Stability


Test groups HAb HAa HBb HBa HCb HCa CAb CAa CBb CBa CCb CCa SAb SAa SBb SBa SCb SCa Mean Standard Deviation .91033 .086077 .91800 .033451 .065744 .93167 .92833 .109418 .79333 .033081 .063836 .79500 1.11200 .239217 1.09667 .227280 1.16033 .219751 1.05833 .074568 .98100 .045640 .99767 .014468 1.28067 .152762 1.20900 .115013 1.34400 .278253 .137293 1.32233 1.42300 .103131 1.41833 .138500 (b) Before, (a) After Std. Deviation .070401 Std. Error Mean .040646 t -.189 Sig.

Table 3: Paired Sample T-Test of Color Stability


Test groups HAb-HAa Mean Diff. -.007667 .86 8 .91 .003333 .045092 .026034 .128 HBb-HBa 0 .93 .031134 .017975 -.093 HCb-HCa -.001667 5 1.51 .26 .015333 .017502 .010105 CAb-CAa 7 8 1.21 .34 .102000 .145506 .084008 CBb-CBa 4 9 .50 .036088 .020835 -.800 CCb-CCa -.016667 8 3.25 .08 .071667 .038188 .022048 SAb - SAa 0 3 .91 .021667 .316393 .182669 .119 SBb - SBa 6 .84 .035838 .020691 .226 SCb - SCa .004667 3 (b) Before, (a) After, * Significant p < . 05, ** Highly significant p < .01

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Table 4: Mean and Standard Deviation for Surface Hardness


Test groups HAb HAa HBb HBa HCb HCa CAb CAa CBb CBa CCb CCa Standard Deviation 6.03333 .030551 3.98333 .086217 4.32000 .020000 4.17333 .155349 8.04333 .058595 4.47667 .222336 5.46333 .098150 5.05000 .045826 4.03667 .063509 3.65333 .571431 4.33667 .317857 4.16000 .144222 (b) Before, (a) After Mean

Table 5: Paired Sample T-Test of Surface Hardness


Test groups Std. Error t Sig. Mean 2.050000 .105357 .060828 33.702 .001(**) HAb-HAa .146667 .136137 .078599 1.866 .203 HBb-HBa 3.566667 .208167 .120185 29.676 .001(**) HCb-HCa .413333 .136137 .078599 5.259 .034(*) CAb-CAa .383333 .534634 .308671 1.242 .340 CBb-CBa .176667 .461122 .266229 .664 .575 CCb-CCa (b) Before, (a) After, * Significant p < . 05, ** Highly significant p < .01 Mean Diff. Std. Deviation

Table 6: Mean & Standard Deviation of Surface Roughness


Test groups HAb HAa HBb HBa HCb HCa CAb CAa CBb CBa CCb CCa SAb SAa SBb SBa SCb SCa Mean 1. 38333 1.94200 1.21633 2.81100 .85500 .81200 2.27933 1.53567 1.26433 1.16000 1.39067 1.11533 2.64800 2.89567 1.94300 1.67100 2.36067 5.52100 Standard Deviation .382743 1.100768 .835685 1.827104 .124048 .012166 1.003071 .517355 .286280 .81854 .625574 .922042 .328827 1.924886 .649023 .708420 1.508769 .197302

(b) Before, (a) After

Table 7: Paired Sample T-Test for Surface Roughness


Test groups Std. Std Error t Sig. Deviation Mean -,558667 1.252481 .723120 -,773 .521 HAb-HAa -1.594667 1.397909 .807083 -1.976 .187 HBb-HBa .043000 .114053 .065848 .653 .581 HCb-HCa .743667 .493919 .285164 2.608 .121 CAb-CAa .104333 .368058 .212498 .491 .672 CBb-CBa .275333 .728300 .420484 .655 .580 CCb-CCa -,247667 1.673500 .966196 -,256 .822 SAb - SAa .272000 1.205241 .695846 .391 .734 SBb - SBa -3.160333 1.681604 .970875 -3.255 .083 SCb - SCa (b) Before, (a) After, * Significant p < . 05, ** Highly significant p < .01 Mean Diff.

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Evaluation of shear bond

Evaluation of shear bond strength of silicon-based soft liner to the acrylic resin denture base using different polymerization technique with different storage periods in distilled water
Thikra M. Hachim, B.D.S., M.Sc. (1)

ABSTRACT
Background: This study was to investigate the effect of different polymerization techniques of denture base (conventional heat cure acrylic and visible light cure acrylic) and storage duration on shear bond strength of siliconbased soft lining material. Materials and methods: The soft liner investigated was a silicon-based autocured (Ufi Gel P). The soft liners were processed according to manufacturers instruction. The resilient liner specimens for shear bond strength testing were 25 25 3 mm and were processed between two acrylic blocks (size of each block (75x25x5mm). A total of 40 specimens were fabricated, 20 specimens for each type of denture base specimens were stored for 1 day, 1 week, 1 month and 3 months in distilled water at 37 degree oC (n=5). Bond strength was measured by instron testing machine at cross head speed 5mm/min until fracture. ANOVA with multiple comparisons LSD test and student t-test were used to analyze the data (the mean difference is significant at the 0.05) Results: The result indicated that type of denture based affect the bond strength of soft lining material and the shear bond strength of lining material decrease with storage duration (for both groups). Conclusion: Within the limitations of this in Vitro study: Ufi Gel P exhibited as significant higher bond strength to heat cured acrylic denture base when compared to visible light cure acrylic resin. Water storage for 1 month and 3 month result in significant decrease in shear bond strength of Ufi Gel P soft liner to a heat cured and Visible light cured denture base. Key words: Polymerization techniques, denture base materials. (J Bagh Coll Dentistry 2012;24(3):42-46).

INTRODUCTION
Soft lining materials are used on dental prostheses to absorb some of the energy produced by masticatory impact (1). A soft liner would distribute the functional and parafunctional stresses more evenly and thus have a dampening effect due to their elastic behavior, thus acting like a "shock absorber" (2). Therefore these materials are used as long-term denture liner for management of atrophid mucosa or traumatic ulceration, for obturators after maxillofacial surgery and to improve denture retention by engaging undercuts (3). Soft lining materials can be classified as provisional or definitive. Then, depending on their chemical composition-silicon rubber or acrylic resin, they can either be chemically or heat polymerized (4, 5). Despite their advantages the liners posses a few disadvantages also. Among them the chief disadvantages is bond failure occurring between the liner and denture base which will create a potential interface for microleakage which subsequently result in delamination of the liner from the denture thus negating the effective function of liner (6).

A variety of parameters effect the bond between the resilient lining materials and the denture base, including water absorption, surface primer use, and denture base composition. Visible light cure resin technology was developed for use in removable prosthodontic since 1983. In 1984 visible light cure (VLC) become available to profession and marketed under the trade name triad. Visible light cured resin has advantages which are: accuracy of fit, absence of free (methylmethacrylate), color stability, low bacterial adherence, ease manipulation, superior strength, and low exothermic heat (7). The purpose of this study was to evaluate the shear bond strength of silicon-based soft liner to acrylic denture base with different curing method (conventional heat cured acrylic and visible light cured acrylic) and different storage duration in distil water.

MATERIALS AND METHODS


A heat activated poly-methyl methacrylate (PMMA) resin (SR Triplex hot/ivoclar vivadent AG), light cure denture base resin (Pink colored, sheet form, MEGADENTA, Germany) and silicon-based liner (Ufi Gel P/Voco made in Germany) were used. A total of 40 specimens were prepared from two type of resin (20 specimens for each group).

(1) Lecturer, Department of Prosthodontics, College of dentistry, University of Baghdad

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In first group 20 specimens were prepared from 40 blocks of heat cure acrylic resin (two acrylic resin blocks were required to form one specimen) with dimension of (75mm x 25mm x 5mm length, width, depth respectively) with stopper of depth about 3 mm (8). One block of acrylic put over the other block leaving a space between them of dimensions (25mm x 25mm x 3mm length, width, depth respectively) for soft liner material application. The thickness of the handle of the acrylic specimen is (13mm). The dimension of specimens was such that they could be produced in conventional denture flasks and gripped easly in the instron machine (Figure 1).

In second group 20 specimens were prepared from 40 visible light cure acrylic blocks (two visible light cure acrylic blocks were required to from one specimen). A forty silicon rubber molds were prepared as discussed previously. Visible light cure resin were put inside the mold an cured by using visible light cure devise (light cure machine. YETI DENTAL-preci-NT shuttle wavelenath 400nm, supplied by fluorescent lamps qWatt x4, Germany) and curing each 2mm incrementaly for 40 seconds. Two visible light cure resin blocks were required to from one specimen. The mixture of reline material was applied into the space between the two acrylic blocks as discussed previously for heat cure acrylic blocks. Visible light cure specimens were immersion in water at 37oC for 1 day, 1 week, 1 month and 3months. (n=5) for each storage duration. Shear bond strength was determined for 40 specimens using instron testing machine at cross head speed 5mm/min, (Figure 2) recorded in megapascals (MPa).

Figure 1: acrylic specimen


To standardize fabrication of specimens, five equal sized brass dies were prepared and utilized to fabricate a silicon rubber mold (Addition silicon, poly vinylsiloxane/ putty consistency/ Zermack-Italy). Wax blocks of equal size were prepared in mold, invested in dental stone in a denture-processing flask, followed by de-waxing. Heat cure acrylic material was mixed according to manufacturers instructions and packed in dough stage into the mold, flask was compressed and closed using clamps and the excess material was removed. After bench press, the flask was kept for curing at 75oC for 9 hour. After polymerization, acrylic specimens were trimmed to remove all flashes. Polishing was accomplished for the surfaces of specimen except the surface that faces the soft liner material by using bristle brush and pumice with lath polishing machine, then the acrylic specimens were conditioned in distilled water at 37oC for 48 hour according to ADA specification No.12 1999. Acrylic blocks cleaned and dried. A layer of adhesive was applied to the surface which were to retain Ufi Gel P. Allowed to aerate for about 1min. the mixture of reline material was applied into the space between the two acrylic blocks by using spatula, any excess of material was removed by using sharp knife and the specimen was put under weight of 200mg for stability and left until complete setting of reline material was obtained. The twenty specimens were immersion in water at 37oC for 1 day, 1week, 1 month and 3 months, (n=5) for each storage duration.
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Figure 2: Instron testing machine


The rupture peak load was recorded. The shear bond strength I (MPa) was calculated by dividing this load (N) by the adhesive area (9). Group statistics Mean, standard deviation, t-test for Equality of Means between 2 groups, analysis of Variance (ANOVA) test with multiple comparisons LSD test. Debonding site were investigated using an optical microscope at 10 x magnification. Failures that occurred at the soft lining acrylic resin interface were recorded as adhesive, and those that occurred within the soft lining were considered to be cohesive; mixed failure was assigned when both failure mode was observed.

RESULTS
Mean and standard deviation values of shear bond strength (Map) of resilient liner to heat cure

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acrylic and light cure acrylic for different storage duration, 1 day, 1 week, 1 month and 3 months are listed in table (1). The highest mean value was obtained in 1 day water storage of heat cure acrylic denture base group (1.04), while lowest mean value was obtained in 3 months water storage of visible light cure denture base (0.02) table (1) also showed the t-test comparisons of the means, it appear that a highly significant decrease in shear bond strength when soft liner used with visible light cure denture base. In Table (2) showed analysis of variance (ANOVA) test of heat cured acrylic denture base group at different storage duration which showed significant difference between storage duration.

In Table (3) showed analysis of variance (ANOVA) test of visible light cure denture base group at different storage duration which showed significant difference between storage duration. Multiple comparisons LSD test table (4) showed significant difference in shear bond strength between 1 day and 1, 3 months storage duration for both group (heat cured, light cured) of denture base. Lower bond strength in 1 and 3 months storage duration. Table (5) showed the failure mode frequency after debonding. In heat cured group the of failure was cohesive failure in 1 day storage duration and mixed in 1 month and 3 month storage duration, while in light cure group the mode of failure was adhesive failure in all storage duration.

Table 1: Means and standard deviation values of the shear bond strength (MPa) of soft liner material to 2 groups of denture base for 4 storage duration (n=5) ant t-test for Equality of means.
N 1 day 5 1 week 5 1 month 5 3 month 5 Heat cure acrylic denture base Visible light cure denture base Mean S.D. Mean S.D. t df Sig. 1.0402 .3772 17.121 8 .000 .07107 .04946 .3690 24.615 8 .000 1.0438 .05307 .03068 .6796 .2604 13.525 8 .000 .06167 .03164 .2432 17.432 8 .000 .6494 .04744 .02156

Table 2: ANOVA test of shear bond strength of soft liner to heat cure acrylic for different storage duration 1 day, 1 week, 1 month, 3 month.
Between groups Within groups Total Sum of squares df Mean squares F-value Sig. 0.715 3 0.238 0.056 16 0.003 68.469 .000 0.771 19

Table 3: ANOVA test of shear bond strength of soft liner to light cure acrylic for different storage duration 1 day, 1 week, 1 month, 3 month.
Between groups Within groups Total Sum of squares df Mean squares F-value Sig. 0.074 3 0.025 0.019 16 0.001 20.459 .000 0.093 19

Table 4: Multiple comparisons LSD significant difference test for acrylic and light cure denture base group between 4 storage duration.
Heat cure acrylic Light cure (1) time (J) time Mean difference (I-J) Sig. Mean difference (I-J) Sig. -.00360 .924 .00820 .715 1 week .36060* .000 .11680* .000 1 day 1 month .39080* .000 .13400 * .000 3 month .00360 .924 .00820 .715 1 day . 36420* .000 .10860* .000 1 wk 1 month .39440* .000 .12580* .000 3 month -.3606* .000 -.11680* .000 1 day -.36420* .000 -10860* .000 1 M 1 week .03020 .430 .01720 446 3 month -.39080* .000 -.13400* .000 1 day -.39440* .000 -.12580* .000 3M 1 week -.03020 .430 -.01720 .446 1 month
*The mean difference is significant at 0.05 level

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Table 5: Failure mode frequency (%) after debonding


Heat cured acrylic group Light cured acrylic group Adhesive Cohesive Mixed Adhesive Cohesive Mixed 100 100 1 day 80 20 100 1 week 100 100 1 month 100 100 3 month

DISCUSSION
The bonding between (Ufi Gel P) soft liner and two types of denture base (heat cured acrylic resin and visible light cured resin) at different storage duration in distilled water was determined by shear bond test. This study showed that the bond strength of (Ufi Gel P) soft liner to heat cured acrylic denture base was higher than that to visible light cured resin at all storage periods in distilled water as in table (1). The significant differences in bond strength exist between two groups may be attributed to the denture base polymer variables. In a previous study on the adhesion properties of resilient lining material to a visible light cured acrylic resin (VLC), it was shown that adhesive characteristics depended on a variety of factors: chemical properties, physical properties, and mode of polymerization of the resitient lining material and the bonding agent used (10, 11). A study by Takahashi et al showed that a light activated denture base polymer bonded adequately with light activated relin polymer but less well with the other reline polymers tested (12). The findings of the present study agreed with those of Takahashi et al. The processing methods of denture base may also influence the surface properties. The higher bond strength to heat cure acrylic resin base may be attributed to improved adhesive bonding system used with silicon-based soft liner, which usually contain silicon polymer in volatile solvent that is able to penetrate the acrylic resin (13) . The bond strength of Ufi Gel P decreased after storage in distal water for 1 month and 3 month. This find agree with earlier reports by polyzois and Aese et al which stated that water storage reduce the bond strength of resilient liners to heat cured acrylic and visible light cured acrylic resin (11,14) . Water absorbed by soft lining material had effect on its bonding to acrylic resin, because water may directly lead to swelling of reline material and stress builds up at the bond interface which cause reduction bond strength, or indirect lead to change the characteristic properties of material (11).

Graig suggested that storage in water did not affect the bonding of denture liners to acrylic denture base (15). The findings of the present study disagree with Graig. However a direct comparsion of studies can not be made because of different research protocols used. After shear testing of the heat acrylic specimens, a cohesive failure mode was predominantly observed which indicated that shear strength of soft liner was less than bond strength. The failure mode changed to mixed after storage of the specimens in distilled water for 1 month and 3 months which indicated that shear strength of soft liner was nearly equal or equal to its bond strength while in light cure specimens failure mode was adhesive in all storage duration which indicated that shear strength of soft liner was greater than bond strength.

REFERENCES
1. Anusavice KJ. Phillips science of dental material. 11th ed. Sounders: Elsevier; 2003 pp. 750-751. 2. Sara CD, Sarac YS, Basoglu T, Yapici O, Yuzbasioglu E, The elevation of microleakage and bond strength of silicone-based resilient liner following denture base surface pretreatment. 3. Graig RG. Restorative dental material, 7th ed, CV Mosby Co, St. Louis, 1986, pp. 496-598. 4. Wright PS. Composition and properties of soft lining materials for acrylic denture J Dent 1981; 9: 210-223. 5. Qudah S, Harrison A, Huggett R. Soft lining materials in prosthetic dentistry: A review. Int J prothodont 1990; 3: 477-483. 6. Graham BS, Jones DW, Sutow EJ. An in vivo and in vitro study of the loss plasticizer from soft polymer-gel materials. J Dent Res 1991; 70: 870-73. 7. Stipho HD, Talic YF. Repair of denture base resin with visible light polymerized reline material: effect on tensile and shear bond strengths. J Prosthet Dent 2001; 86: 143-8. 8. Yousif AA. The effect of disinfection, tray per and adhesive usage on the tensile and shear bond strength using two different elastomeric impression material (comparative study). A master thesis, prosthetic department. University of Baghdad, 2006. 9. Yaniko tlu N, Denizo lu S. The effect of difference solutions on the bond strength of soft lining material acrylic resin. Dental materials J 2006; 25(1): 39-44. 10. Emmer TJ Jr, Emer TJ Sr, Vaidynathan J, Vaidynatha TK. Bond strength of permanent soft denture liners bonded to denture base. J Prosthet Dent 1995; 74: 595601.

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11. Polyzois GL. Adhesion properties of resilient lining material bonded to light-cured denture resins. J Prosthet Dent 1992: 68: 854-8. 12. Takahashi Y, Chai J. Shear bond strength of denture reline polymers to denture base polymers. Int J prosthodont 2001; 14 (3): 271-275 (Abstract). 13. Wright PS. Characterization of adhesion of soft denture liners to poly(methylmethacrylate). J Dent Res 1982; 61: 1002-5. 14. Mese A, Guzel. KG. Effect of storage duration on the hardness and tensile bond strength of silicon-and acrylic resin-based resilient denture liners to a processed

denture base acrylic resin. J Prosthet Dent 2008; Feb: 99(2): 153-9. 15. Graig RG. Restorative dental Materials. 7th ed, CV Mosby Co, St Louis. 1986, pp. 496-498.

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The influence of different pH of saliva and thermal cycling on the adaptation of different denture base materials
Yasir A. Hussein, B.D.S. (1) Shatha S. Al-Ameer, B.D.S., M.Sc. (2)

ABSTRACT
Background: The difference in the adaptation of denture bases processed from heat cure acrylic, light cure acrylic and nylon materials and the effect of artificial accelerated aging (thermal cycling and pH) on the adaptation was studied. Materials and methods: One hundred eighty stone cast were prepared and assigned into 3 main groups according to the denture base material that processed on them(heat cure, light cure, nylon), each denture base and its cast was sectioned at posterior palatal border and the gap between the denture base and the cast was measured using digital microscope. Half of each group exposed to thermal cycling (2000 cycle) between (5 C-55!C ) 30 second in each bath then the adaptation was evaluated again. All denture bases were stored in artificial saliva of different pH (5.7, 7,8.3) at 37"C for 30 days, then the adaptation was measured again. Results and discussions: The results showed that the adaptation of heat cure acrylic was better than light cure and nylon before and after aging process. The nylon showed the poorest adaptation. Thermal cycling decreases the adaptation of all denture bases. Variation of pH effected the adaptation of heat cure and light cure denture bases and did not affect the adaptation of nylon. Keywords: adaptation, light cured resin, nylon, thermal cycling and the pH of saliva. (J Bagh Coll Dentistry 2012;24(3):47-53).

INTRODUCTION
Adaptation in prosthetic dentistry means the degree of fitness between the prosthesis and supporting structure (1). The adaptation is important because the retention of complete denture depends on the proper fitness (2) and to ensure that the denture should maintain its shape over a period of time (3). There are several methods that are used in the measurement of the adaptation accuracy of upper complete denture like microscopic measurement of the gap space between the denture base and the cast or weighing an elastomeric materials impressed under the denture base (4). Adaptation of the denture is affected by several factors as the previous studies showed which are 1-palatal shape which was found that the better adaptation was on shallow and medium palatal depth than the deeper one (5) 2-liner shrinkage which was found that Linear shrinkage should be less than 2% for the denture to be clinically acceptable and to have minimal effect on adaptation and cuspal integration (6). 3-curing method It was concluded that all types of resin showed processing contraction and the best fitting type were prepared from auto polymerizing and microwave resins while the poorest fitting was for the conventional heat activated resin (7).
(1) (2) M.Sc. Student, Department of Prosthetics Dentistry. Professor, Department of Prosthetics Dentistry, College of Dentistry, University of Baghdad

4-polymerization procedure as the studies showed that the use of tension system and immediate post pressing time improved the adaptation of the denture (8) 5- thickness of the denture which showed statistical significant effect on the adaptation (9) 6-thermal shrinkage as the shrinkage of conventional heat cure resin is mainly thermal and this demonstrate the advantage of low temperature cycle in reducing thermal shrinkage (10) 7-seperating medium produces shrinkage at the thick section and this lead to dimensional changes of the denture (11) 8water sorption which partially compensates the polymerization shrinkage and improve the denture adaptation (12) 9- type of stone as the studies showed the use of type IV dental stone improved fitness of the denture (13) 10-internal stress as poor fitness of the denture may arise from faulty processing as immediate insertion in boiling water results in high internal stress, also rapid cooling tends to increase the stress in the denture which are released later (14) Aging process could also affect the adaptation, as the denture while in use is submitted to rigours clinical conditions as alteration in pH, salivary flow and temperature, therefore in an attempt to simulate this condition, several methods are used like thermal cycling and storage in distilled water and exposure to UV light and water spray (15). PH changes may contribute to dimensional changes. The pH of saliva could be more acidic due to certain types of food like orange juice, sugar like candy, pastries, smoking or disease
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which could cause the saliva to be acidic like Sjgren's Syndrome and chemotherapy (16). Saliva pH could be more alkaline due to food like amaranth or due to disease like problem in digestive functions, including enzyme production and pancreas secretions, and eliminative functions, especially the liver and lymphatic's (16). The purpose of this study was to evaluate the effect of aging process on the adaptation of the denture bases made from the three materials (heat cure acrylic, light cure acrylic, nylon).

MATERIALS AND METHODS


The total number of samples maxillary denture base with their corresponding casts" were 180 samples. They were divided into three main groups according to the type of materials of the denture base (Heat cure acrylic, light cured resin and nylon denture base materials) . Each main group of denture bases was subdivided into two groups (with thermo cycling and without thermo cycling). Each sub group consisted of 30 samples. Each sub group was subdivided into three smaller groups each one contained 10 samples of denture bases stored in artificial saliva of different pH (acid, base and neutral).all the denture base samples were of 2mm thickness Cast preparation Silicon mold of upper edentulous arch and type 3 dental stone were used in preparation of 180 cast following usual conventional procedure. Hot cure denture base preparation 60 denture bases were prepared using biostar machine and biostar plate of 2mm thickness, the biostar plate was trimmed and sealed on the cast with wax in usual manner. Flasking The cast with the corresponding biostar sheet was flasked in the lower half of flask with plaster. The upper half of flask was secured to the lower half and 2nd layer of plaster was poured and allowed to set for 1 h . Then wax was eliminated with the use of boiling water and the biostar plate was removed. One ml of separating medium was applied to the surface of cast and mold and distributed evenly by brush. Packing and curing The powder and liquid was mixed at a ratio of 3:1 by volume and left until it reached the dough stage then placed over the cast and trial closure was made then final closure was made under pressure of 20 bar for 5 min. The flask was immersed in water bath until it reached at 100 oC and kept for 30 min. The flask was removed from the bath and allowed to cool for 3 h then deflasked. The cast with corresponding denture

base was sectioned at 5 mm away from posterior palatal end. Light cure denture base preparation 60 record bases were prepared using the same biostar machine but the procedure was done in two steps, step one produces spacer of 2 mm thickness. The second step produces the biostar plate to be adapted over the light cure material keeping edges of 2nd sheet on the periphery of the cast to act as stopper. Sheet of visible light cure resin was adapted to the cast and the biostar sheet was adapted over them until the stopper touched the border of the cast. The cast with it's corresponding resin sheet and biostar plate was placed in light cure unit and cured for 12 min then the cast was removed and the light cure sheet & the biostar plate was placed again in the curing unit in the reverse direction and cured for another 12 min according to manufacture instruction. The cast with it's denture base was sectioned in the same manner as for heat cure acrylic. Nylon denture base preparation The biostar plates were prepared in the same manner as for heat cure acrylic. The cast with it's corresponding biostar plate was placed in the lower half of the flask. The mixture of water and plaster was poured in the lower half. Sprue (one major 6mm diameter and 3 minor of 4 mm in diameter) were prepared to form passage way for injected nylon, the upper half of the flask was placed and plaster was poured and allowed to set then wax was eliminated and mold was left overnight to get rid of moisture. The nylon was injected inside the flask at 287oC after 12 min of heating the nylon capsule. Then the flask was allowed to cool for 15 min . The flask was opened and the sprues were cut then the cast denture base set was sectioned in the same manner. Thermal cycling Half of the samples in each group (heat cure, light cure, nylon) were thermal cycled between (5oC to 55oC 2oC) for 2000 cycle with an immersion time of 30 seconds at each temperature and a total cycle time of one minute. Artificial saliva preparation The artificial saliva was prepared in three different pH 5.7,7 and 8.3. An electrolyte composition similar to that of human saliva was used in this study which includes(17): Na2HPO4 0.260g/l NaCl 0.700g/l KSCN 0.330g/l KH2PO4 0.200g/l

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NaHCO3 KCl 1.200g/l

1.500g/l

Table 1: The mean distribution for the space(mm) for heat cure acrylic group
points A B C N =60 Mean .231 .088 .238 S. D .089 .049 .063 S. E 0.115 0.063 0.082

Buffer solution was prepared first from KH2PO4 and Na2HPO4. By dissolving each salt in 1 liter of de ionized distilled water . The acidic solution (5.7 0.01) was prepared by placing 500 ml of KH2P04 in graduated flask and Na2 HPO4 solution was added gradually until it reached to the required pH, then the remaining salts of artificial saliva were added and the volume was completed to 1 liter by de ionized distilled water. The basic saliva (8.3 0.01) was prepared by placing 500ml of Na2HPO4 in graduated flask and KH2PO4 solution was gradually added until it reached to the required pH then following previous procedure The neutral saliva solution was prepared as previously and the amount of Na2HPO4 solution was added until it reached pH 7 as it was recorded by the digital pH meter placed in the graduated flask. All the samples were stored in artificial saliva inside the incubator at 37o C for 16 hours and for 8 hours inside the distilled water which simulated the daily usage of denture by the patient. This cycle was repeated for each sample for 30 days Adaptation measurement. The digital micro scope (Dino lite) was used in measuring the space between the cast and the denture base margin at 3 points which are the depth of vestibule A, the crest of the ridge B, and the midline C as in (fig.1). Measurements were made immediately after sectioning and for the half of the samples after thermal cycling and for all the samples after 30 days of storage in artificial saliva of different pH.

Table 2: The mean distribution for the space (mm) for light cure acrylic group
points A B C N =60 Mean .260 .159 .346 S.D .069 .080 .098 S.E .089 .010 .013

Table 3: The mean distribution for the space (mm) for nylon group
points A B C N =60 Mean .982 .707 1.005 S.D .284 .274 .239 S.E .037 .036 .031

Comparing the adaptation of different materials; the light cure material had a larger gap space than the heat cure in (figure 2) this may be due to difference in material composition and technique used for polymerization as no pressure is applied during curing of light cure while the pressure is ordinary used in flasking and curing of heat cure, this result is in agreement with Emre et al (21). The nylon material showed the greatest gap space which is 4 times greater than heat cure at point C and 3 times greater than light cure material at point C as shown in(fig.2) this may be due to the difference in chemical composition between the materials, Nylon shows high mold shrinkage as the result of their crystallinity (22).

Figure 1: The position of three points (A,B,C)

RESULTS AND DISCUSSION


The effect of the material before aging process The result showed that the greatest gap space was at point C for the three materials as in tables (1,2,3). This result is in agreement with Mohamed Tahir (18) ,Abdul Khalik (19) and Abdul Razzaq (20).

Figure 2: The mean space (mm) for the heat cure, light cure and nylon at the three points (A,B,C)
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The effect of thermal cycling on the material Heat cure acrylic. There was significant increase in the mean space in the three points (A,B,C) as shown in (figure 3). This may be due to contraction of denture base due to further polymerization that occur due to exposure to high temperature with reduction in the spaces between the chains of the polymer. This result is in agreement with Ogawa and Hasegawa (23). Light cure resin. Mean gap space was increased significantly at point A,B and non significant effect on point C. This can be explained as point C is in the center of the sample and will receive more light intensities from two sides of the curing chamber more than points A and B as they are located in position that receive less intense light , which might be less effective in curing therefore after thermal cycling there might be more polymerization and more shrinkage at these points A&B . while point C was not affected as it had already been more polymerized before thermal cycling as shown in (figure 3). Nylon. The mean gap space for the three points was increased. This might be due to the reaction between amide and amine terminal groups by heating (24) such reaction could lead to the contraction of denture base and increase in the gap space, or due to exposure of polyamides to heat and oxygen which may cause changes in physical and chemical characteristics due to thermal oxidative degradation (25). There is highly significant difference between heat cure &nylon and light cure &nylon at the three points . There is significant difference at points B and C between heat cure and light cure as shown in (figure 3).

be due to erosion (26) or due to cationic reaction (27) as shown in (figure 4). Base. The increase in the mean gap space of denture base after immersion in basic saliva might be either due to the erosion by basic media or due to hydrolysis of the polymer that will lead to more hydrogen bonding between the chains and more contraction will occur. Point C showed significant difference due to topographic effect (18) as shown in (figure 4). Neutral. The storage of heat cure acrylic denture base for 30 days in neutral saliva did not produce any significant change in the space which means no significant expansion or contraction as shown in (figure 4)

Figure 4: The effect of pH changes on heat cure acrylic groups Light cure resin
Light cure acrylic Acid. There was significant increase in the gap space for the three points after storage 30 days in saliva which might be due to erosion (26) as shown in (figure 5). Base. There was increase in the mean space for the 3 points due to erosion by basic media but it was only significant at point B which is due to strain release of maxillary record base which tends to draw the flange inward, and the resulting premature contact of the denture with the mold in these regions cause the denture to be elevated (28) as shown in (figure 5). Neutral. There was significant increase at point B& C due to strain release and premature contact of the flange (28) or could be due to water solubility of light cure (29) as shown in figure 5.

Figure 3: The effect of thermal cycling on the three materials (heat cure, light cure, nylon) at the three points (A,B,C)
The effect of pH changes Heat cure acrylic. Acid. There was significant increase in the mean gap space for the three point (A,B,C) after storage in saliva for 30 days as shown in graph this might
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Figure 5: The effect of pH changes on light cure acrylic groups


Nylon. In acid, base and neutral saliva there was significant decrease in the mean gap space. This might be due to the polar properties of resin macromolecules through a diffusion process (15) or might be Due to presence of functional groups such as amine, amide and carbonyl in the structure of nylon polymer, therefore water absorption is likely to be higher than PMMA (24) as shown in (fig.6).

Base. Significant decrease in the gap space for the three points (A,B,C)might be due to that thermal cycling might increase the degree of polymerization of heat cure acrylic by which the acrylic might become more resistant to the attack by basic saliva therefore effect of water absorption was more pronounced that antagonize the effect of erosion so the space was decreased because of expansion. Also the effect of acidic saliva was greater than the effect of basic saliva which might support the hypothesis of the negligible effect of basic saliva on heat cure acrylic after thermal cycling as shown in figure 7. Neutral. The decrease in the gap space for the three points after storage in neutral saliva for 30 days might be due to stress relaxation so the better adaptation to the cast and reduction in the space as shown in figure 7.

Figure 7: the effect of aging process (thermal cycling + pH(acid,base,neutral)) on heat cure acrylic groups
Light cure resin Acid. There was non significant effect for the three point after thermal cycling and storage in acidic saliva this might be due to the thermal cycling might increase the degree of polymerization which make the resin more resistant to erosion effect of acidic media. This may explain the non significant difference between after thermal cycling and after immersion in acidic saliva as shown in figure 8). Base. Storage in basic saliva after thermal cycling showed significant decrease in the mean gap space for the three points because thermal cycling increased the degree of polymerization this might cause more resistant to the effect of basic saliva as its effect is weaker than the effect of acidic saliva and so the effect of water absorption was more pronounced which lead to expansion and reduction in the space. As polymerized networks which contain polar groups such as hydroxyl, carbamate (urethane), amide, ester and/or ether linkage are hydrophilic in nature and tends to be

Figure 6: The effect of pH changes on nylon groups


The effect of pH changes and thermal cycling together Heat cure acrylic Acid. The mean distribution showed significant increase in the gap space at point C this may be due to increase the degree of polymerization by the thermal cycling and therefore the erosion effect by acidic media was significant at point C only as this point is the most frequent point for change due to topographic factor(18) as shown in (figure 7) .

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more susceptible to moisture absorption (30,31) as shown in figure 8. Neutral. Significant decrease in the mean gap space for the 3 points after thermal cycling and immersion in neutral saliva might be due to stress relaxation and water absorption as shown in figure 8.

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6. 7.

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Figure 8: The effect of aging process (thermal cycling + pH(acid,base,neutral)) on light cure resin groups Nylon. The nylon material behave the same with different pH after thermal cycling due to water absorption but it was less than that without thermal cycling because of the effect of heat on increasing polymerization and the water absorption became less as shown in figure 9.

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Figure 9: The effect of aging process (thermal cycling + pH(acid, base, neutral)) on nylon groups

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REFERENCES
1. 2. Glossary of prosthodontics term. J Prosthet Dent 2005;94(1),11. Anthony DH, Pyton FA. Dimensional accuracy of various denture base materials. J Prosthet Dent 1962;72(1) ,67-81. Chen J, Lacefield W, Catlberry D. Effect of denture thickness and curing cycle on the dimensional of acrylic resin denture bases. Dent Mat J 1998; 4: 20-4. Lee C,Bok S,Bae J, Hae-hyoung Lee. Comparative 20.

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adaptation accuracy of acrylic denture bases evaluated by two different methods. Dent Mater J 2010; 29(4) 411-17. Nikoukari H. A study of posterior palatal seal with varying palatal forms. J Prosthet Dent1975; 34:60513. Anusavice K J. Philip's science of dental materials" Elsevier , 11th Ed, 2003. Takamata T, Sectos J, Philips R, Boone M. Adaptation of acrylic resin dentures as influenced by activation mode of polymerization. J Am Dent Assoc 1989 ; 199: 271-6. Andrea F, Consani, Marcelo F, Mario A C, Guilherme E. Effect of flask closure method and post-pressing time on the upper denture base adaptation. J Gerodontology 2010; 27: 2249. Yesil D, Yankoglu D. Influence of a Thickness and Processing Method on the Linear Dimensional Change and Water Sorption of Denture Base Resin. J Dent Mater 2004; 23 (1) 8-13. Kawara M, Komiyama D, Kimoto S, Kobayashi K. Distortion behavior of heat activated acrylic denture base resin in conventional and long, low temperature processing methods. J Dent Res 1998 ;77:1446-53. Wolfoardt J, Cleaton-Jones P, Fatti P. The influence of processing variables on dimensional changes of heat cured poly methyl methacrylate. J Prosthet Dent 1986; 55: 518-25. Jagger RG. Dimensional accuracy of thermoformed poly methylmethacrylate. J Prosthet Dent 1996; 76:573- 5. Sykora O, Sutow E. improved fit of maxillary complete dentures processed on high expansion stone cast . J Oral Rehabil 1995; 23(5) 342-5. Craig RG, Powers JM. Restorative dental materials. 11th Ed. St. Louis, The C.V. Mosby Co, Missouri USA, 2002. Garcia LF, Roselino LM, Mundim FM, Consani S. The influence of artificial accelerated aging on dimensional stability of acrylic resin submitted to different storage protocols. J. Prosthodont 2010; 19: 432-37. Minich DM, Bland JS. Acid-alkaline balance: role in chronic disease and detoxification. Alternative Therapies in Health and Medicine 2007; 13: 62-5. Pusz A, Szymiczek M, Michalik K. Ageing process influence on mechanical properties of polyamide glass composites applied dentistry. J Achiv. Mat 2010; 38(1) 49-55. Mohamed Tahir FN. The effect of flasking tension system on the adaptation of acrylic resin denture base in different palatal models and base thickness. A master thesis. Department of prosthodontics, University of Baghdad, 2007. Abdul khalik HK. The effect of different packing methods, silinated glass fiber incorboration and CoCr framework involvement on the fit of acrylic denture base at the posterior palatal region. A master Thesis. Department of Prosthetic Dentistry, College of Dentistry, University of Baghdad, 2009. Abdul Razzaq A. Evaluation and comparison of the effect of repeated microwave irradiation on some mechanical and physical properties of heat cure acrylic resin and valplast denture base materials. A master thesis, Department of prosthodontic, Dentistry College, University of Baghdad, 2011. Emre M, Cilingir A, Gencel B, Tonguc Sulun.

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22.

23.

24. 25. 26.

27. 28.

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Flexural properties of a light-cure and a self-cure denture base materials compared to conventional alternatives. J Adv Prosthodont 2011;3:136-9. Parvizi A, Lindquist T, Schneider R, Boyer D. Comparison of dimensional accuracy of injectionmolded denture base materials to that of conventional pressure pack acrylic resin. J Prosthodont 2004;13: 83-9. Ogawa T, Hasegawa A. Effect of curing environment on mechanical properties and polymerizing behavior of methyl- methacrylate auto polymerizing resin. J Oral Rehabil 2005;32:221-6. Andrew J. Polymer chemistry properties and application. Carl verlag publisher, Munich, 2006. Do C H , Pearce E M, Bulkin B J. Polymer Chemistry. J Polym Sci 1987; 25: 2409-24. Constantinescu IR, Ursache M, Mardarez D. Effect of pH on the surface roughness of heat cured denture base acrylic resins. Rev Med Chir Soc Med Nat Iasi 2007;111(2)477-81. Malcolm P. Polymer chemistry an introduction. Oxford University Press, New York, 1999 Turk MD, Lang BR, Wiclox DE, Meiers JC. Direct measurement of dimensional accuracy with three denture processing techniques. Int J Prosth 1992; 5: 367-72. Sakaguchi R, Powers M. Craig's restorative dental materials. 13 Ed, Mosby, Elsevior,2012 Renald E, Knobloch L, Schricker S, Gregg B. Synthesis and evaluation of modified urethane dimethacrylate resin with reduced water sorption and solubility. Dent Mater J 2009; 25: 302-13. Puffer R, Sebanda J. On the structure and properties of polyamides XXVII. The mechanism of water sorption in polyamide. J Polym Sci 1967; Part C(16),79.

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Differential infiltration of CD4, CD8 and macrophage in oral squamous cell carcinoma (Immnoistochemical study)
Ahmed A. Ali, B.D.S. (1) Riyadh O. Alkaisi, B.D.S., MSc., Ph.D. (2)

ABSTRACT
Background: Oral Squamous cell carcinoma (OSCC) is by far the most common malignant neoplasm of the oral cavity. It is an aggressive and lethal malignancy. The oral squamous cell carcinoma microenvironments contain many immune cells and their secretory products. Cell-mediated immunity and the innate immune system may interact with cancer cells and plays an important role in immune responses against cancer, CD8 cytotoxic T lymphocytes (CTLs) are key effectors cells in antitumor immunity, while CD4 T cell have cardinal role in orchestrating antibody production and the activation of CD8 T cells and macrophages to exhibit antitumor functions. This suggests that immune system-related mechanisms have an effect on the development and spread of malignant diseases in humans Materials and methods: Thirty formalin-fixed, paraffin- embedded blocks of oral squamous cell carcinoma were included in this study. H&E stain was done for each block for reassessment of histological examination. The expression of CD4, CD8 and macrophages were detected by immunehistochemistry. Results: Immunehistochemical mean expression level of CD4, CD8 and macrophage in OSCC was (54.67) %, (51.08) % and (55.93) % respectively. Non significant correlation was obtained among the three studied infiltrates with clinicopathological findings of OSCC and with each other. Conclusion: Immunohistochemical expression of CD4, CD8 and macrophage infiltrates were observed in all studying samples of oral squamous cell carcinoma, however, statistically non significant correlation was found between the mean expression level of these infiltrates with all clinicopathological findings of OSCC and with each other. Increasing expression level of CD4, CD8 and macrophage infiltration in all studied cases of OSCC suggest their important role in oral carcinogenesis, however further studies with larger samples needed to identify their exact correlation with clinicopathological features of tumor. Key words: CD4, CD8, Macrophage, Oral squamous cell carcinoma, Immunehistochemistry. (J Bagh Coll Dentistry 2012;24(3):54-58).

INTRODUCTION
Oral squamous cell carcinoma OSCC is a tumor involving the oral cavity, pharyngeal tube and larynx. It is the most common malignant tumor occurring in the oral cavity and one of the 10th most common causes of death in the world. It arises from dysplastic oral squamous epithelium(1). Many advances in diagnosis and treatment have been made but the survival rate for OSCC (which is less than 5 years) remained stable for the last decade and is rather low (2). The anticancer immune reactions are especially important for localizing the spread of OSCC; reports in the literature have proposed that tumor infiltrating immunocytes within the cancer milieu may play an important role in anticancer immunity (3), that could explain by the role of T cell in anti tumor mechanisms. CD4 T cells may mediate tumor rejection through many mechanisms, including up regulation of Major Histocompatibility MHC molecules expression, inhibition of angiogenesis, induction of tumor dormancy and stimulation of cytotoxic effect on tumor cells by providing help for tumor-reactive CD8 T-cell responses, which are specialized for lytic function. Most of the solid tumors express MHC class I, but not class II molecules; therefore, it is believed that CD8 T cells are the main effectors cells responsible for destroying tumors. So, CD4 T-cell response can augment the accumulation of CD8 T cells within tumor and promote the expansion, trafficking, and differentiation of the tumor-specific CD8 T cells, which enhance antitumor immunity (4). The tumor-reactive CD4 T cells can produce lymphokines that amplify the cytotoxic activity of CD8 T cells and other inflammatory cells (5). On other hand, macrophages are involved in various aspects of host defense mechanisms and pathophysiological conditions, such as chronic inflammatory disease and cancer .The functional competence of macrophages is acquired after the exposure of macrophages to stimuli in the tissue microenvironment .Bacterial cellular components, such as lipopolysaccharide (LPS) and interferongamma (IFN- ) stimuli can activate macrophages to produce large amounts of proinflammatory cytokines, such as IL-12 and tumor necrosis factor-alpha (TNF-!), reactive oxygen intermediates and reactive nitrogen intermediates, which contribute to the antimicrobial and antitumor activities of macrophages (6).
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(1) M.Sc. student, Department of Oral and maxillofacial pathology, College of Dentistry, University of Baghdad (2) Professor, Department of Oral and maxillofacial pathology, College of Dentistry, University of Baghdad.

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The activation of CD4, CD8 T lymphocytes and macrophages will lead to an efficient immune response to destroy tumor cells. That role could be clarified by the analysis of possible compositional significance of CD4 and CD8 T cells and macrophages in the cancer milieu (3).

RESULTS
Immunostaining of CD4,CD8 and macrophage was detected as brown staining on membrane of these immune cells as shown in figure. 1-6. These cells infiltrating the epithelial lining, predominately and observed in tumor stroma and parenchyma in all the grades of tumor specimens and in all specimens, as shown in Tables 1-3.

MATERIALS AND METHODS


A retrospective study was performed on thirtyformalin- fixed paraffin-embedded blocks of OSCC. Twenty cases were collected from the histopathological laboratory in surgical specialties Hospital (SSH) in the period (1998-2010). Ten cases were collected from the archives of Oral Pathology. Laboratory, College of Dentistry, Baghdad University in the period (2006-2010) The diagnosis of each case was confirmed by examining the Hematoxylin and Eosin (H&E) sections by two experienced pathologists. The clinicopathological information regarding age, sex, tumor site, clinical presentation, tumor grading and staging (if present) in addition to any other information was obtained from the case sheets presented with the tumor specimens. The positive tissues control in this study according to antibodies manufacturer was:Tonsil tissue sections used as positive control for CD8 and macrophage.Human skin sections used as positive control for CD4. Positive reading was indicated when the infiltrating CD4 and CD8 Tlymphocyte cell and macrophages display a brown membranous immunostaining,so any number of immunestained cell consider positive ,while negative reading was indicated for absence of immunostaining..All the slides were assessed blindly without prior knowledge of the corresponding clinicopathological parameters, the readings were calibrated by two experienced pathologists, and the average of the two readings was obtained. IHC stained OSCC sections were studied by light microscope under 10 X objectives. The scoring was done through evaluation positively stained cells with each antibody. Then three high-power magnification representative fields with the most abundant distribution of positive cells were selected from each specimen .The positively stained and unstained cells were counted. The data were expressed as the mean percentage of the ratio of the number of positive cells relative to the total number of cells for one microscopic field (400)
(3).

Table 1: Expression of CD4 in OSCC


NO. of cases 30 CD4 Expression% (Mean) 54.67 Standard deviation 15.28

Table 2: Expression of CD8 in OSCC


NO. of cases 30 CD8 Expression% (Mean) 51.08 Standard deviation 12.64

Table 3: Expression of macrophage in OSCC


Macrophage Standard deviation NO. of cases Expression% (Mean) 55.93 13.90 30

The results showed that there were no significant correlation among these infiltrates and clinicopathological findings including tumor grade and stage and among each other. (Table 49).

Table 4: Correlation of CD4 and grade .


Grade NO. CD4 Expression% P- value mean 65.0 52.71 59.0

7 Well Moderate 21 2 Poor

0.320

Table 5: Correlation of CD4 and stage .


Stage NO. I II III IV 8 4 5 13 CD4 Expression % P- value mean 59.62 48.52 59.00 53.52

0.504

The statistical analyses were calculated by SPSS(7) (personal computer). In all multiple comparisons significant p-value was at (p< 0.05).

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Figure 1: Positive control for immunohistochemical staining for CD4 in Human skin (400x)

Figure 4: Positive brown membranous immunohistochemical staining for CD8 in well differentiated OSCC-Tongue (400x). Table 8: Correlation of macrophage and grade.
Grade NO. Macrophage Expression % mean 60.78 53.89 60.00 Pvalue 0.529

7 Well Moderate 21 2 Poor

Figure 2: Positive brown membranous immunohistochemical staining for CD4 in poorly differentiated OSCC-Tongue (400x). Table 6: Correlation of CD8 and grade
CD8 Expression % P-value mean 7 50.57 Well 0.717 50.42 Moderate 21 2 52.50 Poor Grade NO.

Table 9: Correlation of macrophage and stage


Stage NO. I II III IV 8 4 5 13 Macrophage Expression % mean 56.31 56.25 54.00 56.29 Pvalue

0.954

Table 7: Correlation of CD8 and stage .


Stage NO. CD8 Expression% mean P- value 8 54.87 I 4 43.75 II 0.603 43.00 III 5 52.99 IV 13

Figure 5: Positive brown membranous immunohist.ochemical staining for macrophage in moderately differentiated OSCC-Tongue (400x).

Figure 3: Positive brown membranous immunohistochemical staining for CD8 in poorly differentiated OSCC-Tongue (400x) Figure 6: Positive brown membranous immunohistochemical staining for macrophage in poorly differentiated OSCCTongue (400x).
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DISCUSSION
Assessment of CD4, CD8 immunohistochemistry This study showed that the mean expression level of CD4 and CD8 was (54.67%), (51.08%) respectively, statistically there was no significant difference correlation between them and clinicopathological parameters. That result disagree with Meneses and colleagues in 1998(8) which demonstrated, in oral squamous cell carcinoma samples, a significant correlation among tumor size, area of invasion, angiogenesis, and peritumoral inflammatory infiltrate. The renewed interest in TIL studies in the current decade ,Costa L. and colleagues in 2005(9) in a retrospective clinical study with 38 samples of oral squamous cells carcinoma, the TNM classification (tumor staging determined by the size of the primary cancer and the presence of metastases) correlates with peritumoral lymphocytic infiltration. Badoual and colleagues in 2006(10) reported that decreased CD4 T cell, not CD8 T cell, infiltration was significantly correlated with late tumor stage. On other hand,present findings come in accordance with previous study by Kazumasa and colleagues in 2011(3),who found non significant statistical difference relationship between infiltrated CD4,CD8 and the pathological grade of OSCC. The reasons for such discrepancy were difficult to explain, because patterns of tumor-related leukocyte infiltration vary between primary tumors and metastatic lymph nodes in head and neck cancers, that lead to suppression of local cellular immunity which may be a strategy by which tumors may evade host immunity (11).Also, because the infiltration of T cells in the tumor tissue is not necessarily functionally active,previous studies have shown that the tumor-infiltrating T cells are functionally inactivated in certain types of tumors (12).On other hand, because tumor cells expressed high levels of tumor-specific proteins may fail to be recognized and destroyed by cytotoxic T lymphocytes ,as such kind of escape from surveillance of local immune system, this can lead to the progression and metastasis of tumor cells. Other exceplanation is that tumor cells may obtained the ability to evade immune surveillance by other strategies, including a lack of adequate T-cell co stimulation (13) , down regulation of cell-surface MHC class I expression (14), secretion of immunosuppressive factors, such as transforming growth factor-b (15) (16), or interleukin-10 and various antiinflammatory cytokines and growth factors, such as IL-6, IL-13, VEGF, and M-CSF, which may
Oral Diagnosis 57

modulate infiltrating T- lymphocytes (17). Similar to the effects on M2 macrophages, tumor cellderived cytokines and mediators, such as IL-10, TGF-", have immunosuppressive effects on the infiltrating lymphocytes. Therefore, these lines of evidence suggest that the tumor microenvironment in OSCC may create an immunosuppressive microenvironment and that could be reason for present findings . Also OSCC represent a heterogeneous group of tumors characterized by squamous differentiation, arising at diverse anatomical sites with varied clinical presentation and different associated lymphatic drainage(18). Assessment of macrophage immunohistochemistry This study showed that the mean expression level of macrophages was (55.93%). Statically non significant correlation was found with clinical parameters. This result disagree with, Li C. and colleagues in 2002(19) who identified that tumoral accumulation of macrophages is associated with stage of invasion. Sica and colleagues in 2006(20) reported predominance of the macrophage population in the peritumoral infiltrate in OSCC. On other hand, present finding was in the same line with Kazumasa and colleagues in 2011(3), who found non significant statistical difference between infiltrated macrophages and the pathological grade and stage of OSCC. The reasons for these results were difficult to explain, because macrophage cells include M1, M2 and undifferentiated monocytes/macrophages, it is likely that these mixed cell populations are functionally heterogeneous regarding the development and progression of OSCC. Accumulation of macrophages M1,M2 in response to tumor cell- derived signals, either because of tumor selection and evolution or as part of anti-tumor responses of the host, is diverted to pro-tumorigenic responses by stimulate tumor growth and invasion through enhanced cell proliferation mediated through the production of TNFa, IL-6, and other cytokines(3) Assessment of correlation among immunohistochemical expression of the three tumor infiltrates: Concerning the correlations among the three tumor infiltrates, the present study showed statically non significant correlations, which is in same line with Kazumasa and colleagues in 2011(3). This finding may be attributed to small sample size and methods of sample collection; however larger studies with larger number of tumor samples needed to clarify this correlation.

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REFERENCES
1. Delvarian Z, Pakfetrat A, Mohtaham N, Shirazian SH. Oral squamous cell carcinoma with an usual clinical manifestation: a case report. Cases J 2009; 2:6608. McMahon S, Chen AY. Head and neck cancer. Cancer Metastasis Rev 2003; 22(1):2124. Kazumasa M, Miki H, Jun S, Yoshihiro O. Infiltration of M2 Tumer Associated Macrophages in Oral Squamous Cell Carcinoma Correlates With Tumer Malignancy. Cancer J 2011; 3: 3726-39. Bos R, Sherman L A. CD4+ T-cell help in the tumor milieu is required for recruitment and cytolytic function of CD8+ T lymphocytes. Cancer Research 2010; 70( 21):836877. Marzo L, Kinnear B F, Lake R A. Tumor-specific CD4+ T cells have a major post-licensing role in CTL mediated anti-tumor immunity. J of Immunology 2000; 165( 11) : 604755. Gordon S. Alternative activation of macrophages. Nat. Rev. Immunol 2003; 3: 23-35. SPSS: Statistical package of social science; version16 and17(Win/Mac/Linux,user'sguidespssinc.,Chicago# ,UA,website,http://www.spss.com/. Meneses A, Verastegui E, Barrera JL, Zinser J, de la Garza J, Hadden JW. Histologic findings in patients with head and neck squamous cell carcinoma receiving peri lymphatic natural cytokine mixture (IRX-2) prior to surgery Arch Pathol Lab Med 1998; 447-54. Costa L, Araujo RF Junior, Ramos CC. Correlation between TNM classification and malignancy histological feature of oral squamous cell carcinoma. Rev Bras Otorrinolaringol (Engl. ed.) 2005; 71(2):181-87. Badoual C, Hans S, Rodriguez J, Peyrard S, Klein C, Agueznay Nel H, Mosseri V, Laccourreye O, Bruneval P, Fridman WH, Brasnu DF, Tartour E. Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers. Clin Cancer Res 2006; 12:46572. Balkwill FMA. Inflammation and cancer: back to Virchow? Lancet 2001:53945. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol 2007; 25:267-96. Thomas GR, Chen Z, Oechsli MN, Hendler FJ, Van Waes C .Decreased expression of CD80 is a marker for increased tumorigenicity in a new murine model of oral squamous-cell carcinoma. Int J Cancer 1999; 82: 37784. Imboden M, Murphy KR, Rakhmilevich AL, Neal ZC, Xiang R, Reisfeld RA, Gillies SD, Sondel PM .The level of MHC class I expression on murine adenocarcinoma can change the antitumor effector mechanism of immunocytokine therapy. Cancer Res 2001; 61: 150007. Inge TH, Hoover SK, Susskind BM, Barrett SK, Bear HD .Inhibition Of tumor-specific cytotoxic Tlymphocyte responses by transforming Growth factor beta 1. Cancer Res 1992; 52: 138692. Fiorentino DF, Zlotnik A, Mosmann TR, Howard M, OGarra A. IL-10 inhibits cytokine production by activated macrophages. J Immunol 1991; 147: 3815 22. Kawamura K, Komohara Y, Takaishi K, Katabuchi H, Takeya M. Detection of M2 macrophages and

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colony-stimulating factor 1 expression in serous and mucinous ovarian epithelial tumors. Pathol Int 2009; 59:300-05. 18. Rabinovich GA, Gabrilovich D, Sotomayor EM. Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol 2007; 25: 267-96. 19. Li C, Shintani S, Terakado N, Nakashiro K, Hamakawa H. Infiltration of tumor-associated macrophages in human oral squamous cell carcinoma. Oncol Rep 2002; 9(6): 1219-23. 20. Sica A, Schioppa T, Mantovani A, Allavena P. Tumor-associated macrophages are a distinct M2 polarized population promoting tumor progression: potential targets of anti-cancer therapy. Eur J Cancer 2006; 42: 717-27.

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Detection of acid fast bacilli in the saliva of patients having pulmonary tuberculosis
GassanYassen, B.D.S., M.Sc. (1) Jamal Noori, B.D.S., M.Sc. (2) Nadia Sabri Yas, B.D.S., M.Sc., Ph D. (2)

ABSTRACT
Background: Tuberculosis is a serious disease caused by bacteria called Mycobacterium tuberculosis. The disease is readily detected by demonstration of the bacteria in a clinical specimen. The purpose of this study was to determine the density of acid fast bacilli in the mixed and parotid saliva samples and to compare them with the sputum, in addition, to find out the efficacy of the saliva samples in the diagnosis of pulmonary tuberculosis. Subject and Methods: A sample of 25 patients of both sexes, Age ranged from 17-70 participated in this study, Unstimulated mixed saliva and the parotid saliva was collected for direct .smear of acid fast bacilli by Ziehl-Nelson acid fast stain. Five samples were inoculated on Lowenstein Jensen media and storen brink media to determine the presence of the bacilli in the samples. Results: Concerning the mycobacterium tuberculosis, about 60% of unstimulated mixed saliva revealed positive acid fast bacilli, while all samples of parotid saliva showed negative acid fast bacilli. There was no significant relationship between the duration of signs and symptoms of disease and the detection of mycobacterium tuberculosis in the collected specimens. The density of mycobacterium tuberculosis in the mixed saliva mainly was scanty which mean it was not more than 2-9 bacilli in at least 100 fields. This confirms the fact that the body fluids commonly contain only small number of mycobacterium tuberculosis. The five samples of saliva which were inoculated on Lowenstein Jensen media and stonebrink media showed positive cultures. Conclusion: Mixed saliva was less efficient than sputum by direct smear of sputum. Keywords: Mycobacterium tuberculosis, saliva. (J Bagh Coll Dentistry 2012;24(3):59-62).

INTRODUCTION
Tuberculosis is a serious disease caused by bacteria called Mvcobacterium tuberculosis. The disease usually affects the lungs but other organs may also be affected (1) The variable nature of its manifestation, as well as its ability to involve almost every organ system, either singly or multiply, makes it essential that the possibility of extra pulmonary tuberculosis be included in the differential diagnosis of any infectious process in the body (2,3) The disease is considered a worldwide problem. Almost one-third of the world's population is infected with TB, although a healthy immune system can prevent active disease. (4) The disease is readily detected by skin test, chest x-ray, or by demonstration of M. tuberculosis bacteria in a clinical specimen. There are two distinct stages of TB: 1.TB infected individuals are those who are tuberculin test positive, but do not have thebacteria in their saliva and are without clinical symptoms.1 2. TB diseased persons have M. tuberculosis bacteria in their saliva and are symptomatic for the disease. (5)
(1) Private practice. (2) Assistant professor, department of Oral Diagnosis, College of Dentistry, University of Baghdad

Since the disease is infectious in nature, the clinical signs and symptoms of tuberculosis are common to many other diseases which are: . Loss of weight. . Loss of energy. . Poor appetite. . Fever and wet cough. TB is transmitted through the air from exposure to germs in the saliva of infected person from their lungs. (1,6) There are two kinds of active TB : Primary TB: Occurs soon after a person is first exposed to TB. Reactivation TB: occurs in people who were previously exposed to TB if their immune system is weakened, TB can breakout of the tubercles and cause active disease. Most of the cases of TB in people with HIV disease are due to reactivation of a previous TB infection. (7, 8) TB may be of concern to the dentist from at least three standpoints: First: It is an infectious disease and as such is communicable in its active state. The dentist is in a high-risk population and may contract the disease from a patient or patients may contract the disease from the dentist who might have an active case. Second: On rare occasions tuberculosis lesion may be found in the oral cavity; thus the dentist must be alert to include tuberculosis in the differential diagnosis of oral lesions. Third: The dentist may be the first person to discover that a patient has Tuberculosis. (1)
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The treatment usually consists of combination of drugs. Generally, TB drugs are taken daily for 5 to 12 months. It is important that the exact medication plan should be decided by qualified health care providers. If left untreated, an individual with TB disease can become severely ill and also transmit the disease to others. Untreated, TB disease can be fatal. (9) The purpose of this study was finding out the density of acid fast bacilli in the mixed and parotid saliva samples and to compare them with the sputum, in addition, to determine the efficacy of the saliva samples in the diagnosis of pulmonary tuberculosis.

SUBJECTS AND METHODS


A total of 25 subjects who were diagnosed as having pulmonary tuberculosis participated in this study. Their age ranged between 17-65 years. The subjects were not having systemic disease other than tuberculosis. Those patients were collected from Chest and Respiratory Disease Institute, TB Lab Reference. The patients were having duration of the disease between 2-12 months. The samples of the saliva were collected from the patients under standardized condition (between 10-12 a.m., at least 2hrs after eating and oral hygiene procedure). The mixed saliva samples were taken from the floor of the mouth, while parotid saliva was taken after localization of the orifice of the parotid salivary gland duct. The area was dried with a piece of cotton, and then the gland milked gently with the finger. The milked saliva was collected with blunt instrument and distributed on a glass slide for a direct smear. The slides were then processed and stained with Ziehl Nelson acid fast stain and examined under oil immersion (1000x) for the presence of acid fast bacilli. Culture of mycobacterium wasdone by digestion-decontamination method to confirm the presence of the micro-organism. Statistical analysis was done with the assessment of the values at the P>0.05 levels.

the whole number of the patients, as shown in table 1. In the specimens examined, the presence of 2-9 bacilli in 100 fields was considered scanty positive) which were observed in saliva samples, while in the sputum, samples the microorganisms were scanty to sever; as shown in figure 1. The duration of signs and symptoms of the total TB patients ranged between 1-12 months. In order to, compare the duration of signs and symptoms with the results of direct smear of saliva we divided the duration into four groups.The highest percentage of positive acid fast bacilli found in patients with 1-3 months duration of the disease, while the lowest percentage was found in 7-9 months.There was no significant relationship between the duration of signs and symptoms of the disease and the presence of microorganisms (Table 2).

DISCUSSION
In this part of study about 25 patients having pulmonary tuberculosis were diagnosed by direct smear of sputum for acid fast bacilli. By using mixed saliva from those patients with pulmonary tuberculosis, about 15 (60%) revealed the presence of mycobacterium tuberculosis in the saliva. This confirms the fact of presence of mycobacterium tuberculosis in the saliva of patient having pulmonary tuberculosis. (10) We did not identify mycobacterium tuberculosis in the parotid saliva because the microorganism which was identified in the mixed saliva was not present in the saliva primarily but it results from contact of the oral tissue with infected sputum. Up to our knowledge we did not find any study performed on the saliva as a sample for diagnosis pulmonary tuberculosis to compare our finding with these studies, so we did our comparisonbetween the sensitivity of saliva and the sensitivity of sputum. In our study the sensitivity of direct smear of saliva for acid fast bacilli is equal to 60% of the sensitivity of sputum. The sensitivity of direct smear of sputum for the microorganism ranged from 22-80%. (11, 12) The density of mycobacterium tuberculosis in the mixed saliva mainly is scanty which mean it was not more than bacilli in at least 100 fields (2-9). This confirms the fact that the body fluid commonly contains only small number of mycobacterium tuberculosis. (10) Therefore, the mixed saliva was less efficient than sputum because by direct smear of sputum the quantity of the bacilli observed on the smear could be
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RESULTS
The results of mixed saliva showed microorganisms in 15 patients. However, 10 patients showed negative results (absence of microorganisms) in the samples collected. The microorganisms could not be isolated from the parotid saliva of the total number (25 patients with pulmonary tuberculosis) so the results considered negative. However the microorganisms were isolated from the sputum of
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provided which serve in the demonstration of the severity of disease. To overcome this shortage, we had to concentrate the sample of saliva by using, cytocentrifugation, or sequential layering of several drops of uncenterfuged fluid, on-slide, or polycarbonate membrane (10,13) Cultivation To explain the ability of using saliva as sample for culture, five samples of mixed saliva selected randomly to be inoculated on the Lowenstein Jensen media and stone brink media. All samples revealed positive culture. This means that the mycobacterium tuberculosis which was recovered well by Lowenstein Jensen media and mycobacterium bovis which was recovered by stone brink media were present in the saliva. (14) So we can conclude that the sample of saliva can be inoculated on different media and we can use it if the sputum is unavailable. Therefore, we can summarize the difference between sample of saliva and sputum as shown in Table 3.

Table 3: Comparison between the saliva and sputum


Always available Less efficient than sputum because is like other body fluids commonly contain only small numbers of mycobacteria Can be concentrated to maximize the yield of mycobacterium before inoculation on media and direct smear Can be inoculated to liquid and solid media The sample which revealed positive is always scanty so we cannot graduate the severity of disease Sometimes not available

More efficient than the body fluid

Also can be concentrated to maximize the yield of mycobacterium before inoculation on media and direct smear Can be inoculated to liquid and solid media The sample which revealed positive may be scanty or moderate or severe so we can measure the severity of disease

RFERENCES
1. James W, Little D, Falace A. Pulmonary disease in: Dermal management of Medically Compromise Patients. Mosby Company. 5 th ed., 1997; 251-9. 2. Valdasol P, Perez A, Albarracin A. Tuberculosis arthritis, Report of a case with multiple joint involvement and periarticular involvement and periarticular tuberculosis abscess. J Rheumatol 1990; 17; 399-401. 3. Mathew R, George F. Extrapulmonary tuberculosis experience of a community hospital and review of literature. American J Medicine 1985; 79: 467-77. 4. Mehta JB, Burt A, Harvill L, Mathews K. Epidemiology of extra-pulmonary tuberculosis. Chest 1991; 99:1134-8. 5. Lvfalcolm A, Lynch. Diseases of the respiratory system in;Burket's of oral .Medicine diagnosis and treatment. 4 th ed., JB Lippincot Company, Philadelphia, 1994; 435-48. 6. Lucas SB. Histopathology of tuberculosis in; Clinical tuberculosis, 2nd ed. Chapmann and Hall medical, 1998; 113-27. 7. Hang M, Gong JH, Lyer DV, Jones BE, Modlin RL, Barnes PF. T cell cytokineresponses in personswithtuberculosis and HW infection. J ClinInvestig 1994; 94: 2435-42. 8. Jeanne M, Wallace MD, Andrew L, Deutch MD, James H, Harrell MD, Kenneth M, Moser MD. Bronchoscopy and transbronchial biopsy in evaluation of patient with suspected active tuberculosis. Am J Medicine 1981; 70:1189-94. 9. Sharba JA. Tuberculosis in 1990s: Therapeutic challenge. Chest 1995; 108:585-625. 10. Beverly G, Metchock F, Ritchard JR. Mycobacterium In: Manual of clinical microbiology, 7 thed ASM press Washington DC 1998; 399-437. 11. Lipsky BJ, Gats FC, Tenover JJ.Factors affecting the clinical value of microscopy for acid fast bacilli. Rev Infect Dis 1984; 6:214-22.

Table 1: The presence of mycobacterium tuberculosis in different specimens


Sample Positive Negative Total 15 10 25 Mixed saliva 0 25 25 Parotid saliva 25 0 25 Sputum

Table 2: The duration of the disease and presence of microorganisms in the stimulated mixed saliva
Duration Positive Negative Total 12 5 17 1-3 2 2 4 4-6 0 1 1 7-9 1 2 3 10-12

Figure 1: Scanty Mycobacterium TB in a specimen


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Detection of acid fast

12. Murray PR, Elmore K., The acid fast stain A specific and predictive test for mycobactrium disease. Ann Inter Med 1980; 92:512-3. 13. Saceanu C N, Pfeiffer, Miclean T. Evaluation of sputum smear concentrated by cytocentrifugation for detection of acid fast bacilli, J Clin J Micro 1993; 31:2371-3. 14. Zaher F, Mark J. Methods and medium for culture of tubercle bacilli. Tubercle 1997; 58: 143-5.

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Trace elements and oxidative stress markers in saliva of subjects with amalgam fillings
Huda Sh. Ahmed, B.D.S, M.Sc. (1) Taghreed F. Zaidan, B.D.S, M.Sc. Ph.D. (2) Ali Yakub, M.B.Ch.B, M.Sc. F.I.B.M.S (3)

ABSTRACT
Background: Amalgam is the most frequently used restorative material for dental treatment. It is mainly used in posterior teeth, usually on occlusal surfaces as an economical, long lasting and durable filling material and represent the main source of exposure to mercury and other toxic metals (copper, tin, silver, etc.). This study designed to measure oxidative stress marker malondialdehyde (MDA) and anti-oxidants (uric acid and glutathione) concentrations in saliva of subjects with amalgam fillings. And measure trace elements (copper, zinc) concentrations in saliva of subjects with amalgam fillings. Subjects,materials and methods: Fifty subjects were participated in this study, they were between the age of (20-50) years with amalgam fillings (cases group) and fifty one subjects with no amalgam fillings (control group), they were gender and age matched to that of subjects with amalgam fillings. Informed consent and ethical approval was obtained. Each subject fill a case sheet questionnaire then examined by using sterile dental mirror and sterile dental probe to determine any oral manifestations and to calculate the number of amalgam filled teeth and the number of filled surfaces. Results: The results obtained from this study showed that Oxidative stress marker (MDA) were increased while antioxidants (glutathione, uric acid) were decreased in saliva of subjects with amalgam fillings. Trace elements (copper, zinc) were higher in saliva of subjects with amalgam fillings; salivary copper was significantly higher in subjects with > 10 amalgam filled teeth. Salivary total glutathione was significantly correlated (negative correlation) with the number of filled teeth. Salivary copper was significantly correlated (positive correlation) with the number of filled teeth and filled surfaces. Conclusion: This study revealed that amalgam fillings associated with increase in oxidative stress marker(MDA) and decrease in antioxidants (glutathione, uric acid).Trace elements (copper, zinc) increased in saliva of subjects with amalgam fillings. Keywords: Oxidative stress, malondialdehyde , glutathione. (J Bagh Coll Dentistry 2012;24(3):63-66).

INTRODUCTION
Amalgam is the most frequently used restorative material for dental treatment. Concern has been raised about amalgam fillings because they contain elemental mercury, and very small amounts of mercury vapor are emitted from dental fillings, mercury is toxic because it induce production of free radicals and modifies the redox potential of the cells. Amalgam fillings are known to release significant amounts of mercury in saliva which could represent a continuous source of oxidative damage to oral tissues (1). Dental amalgam fillings interacts in a complex way with the environment in the oral cavity as they are subjected to chemical, biological, mechanical and thermal forces. These forces change the restoration appearance and properties, while metal ions, amalgam debris, non metallic corrosion products and mercury vapor are released into the oral cavity (2).
(1) (2) (3) M.Sc. Student, Department of oral Diagnosis, College of Dentistry, University of Baghdad. Professor, Department of oral Diagnosis, College of Dentistry, University of Baghdad. Consultant, Poisoning consultation center, Gazi Al-Hariry Hospital.

Amalgam corrosion is an oxidation-reduction reaction; the metals in the amalgam produce chemical compounds up on reaction with non metallic elements in the mouth. Amalgam corrosion is important because it is one of the factors that determine how much mercury is released into the mouth from the fillings (3). Oxidative stress represent imbalance between the production and manifestation of reactive oxygen species and a biological systems ability to detoxify the reactive intermediates or to repair the resulting damage. The reactive oxygen species degrade unsaturated fatty acids and forming malondialdehyde. The production of this compound is used as a biomarker to measure the level of oxidative stress (4). Glutathione is a protein composed of three amino acids: cystein, glutamic acid and glycine. Glutathione is important antioxidant counteracting the effects of free radicals produced in the body by oxidation reactions (5). Uric acid is one of the most antioxidants in the body, it is heterocyclic compound of carbon, nitrogen, oxygen and hydrogen. It is created when the body breaks down purine during metabolism (6).

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MATERIALS AND METHODS


Unstimulated whole saliva was collected from each individual, all subjects were healthy look without any signs and symptoms of any systemic diseases and were without gingivitis or periodentitis. Each subject asked to collect 5-10 ml saliva in the tube by spitting. After collection of saliva samples, it centrifuged at 3000 rpm for 15 minutes, the clear supernatant were separated and stored frozen at -20 c until assayed. Measurements of oxidative stress marker malondialdehyde (MDA): Principle: Lipid peroxidation end product ,particularlymalondialdehyde (MDA) react with thiobarbituric acid (TBA) under acidic condition and heating to give a pink color that measured spectrophotometricaly at 532 nm (7). Measurement of salivary total glutathione (GSH) (8): Principle: 5,5 dithiobis 2-nitrobenzoic acid (DNTB) is a disulfide chromogen that is readily reduced by sulfhydryl group of GSH to an intensely yellow compound.The absorbance of the reduced chromogen is measured at 405 nm and is directly proportional to the GSH concentration. Salivary Uric acid measurements Principle: Uric acid is oxidized by uricase to allantoin with the formation of hydrogen peroxide.In the presence of peroxidase (POD), a mixture of dichlorophenol-sulphonate (DCPS)and 4aminoantipyrine(4-AA) is oxidized by hydrogen dye proportional to the concentration of uric acid in the sample (9). Measurements of trace elements (Cu, Zn) in saliva: The elements Cu and Zn under examination were determined using air-acetylene atomic absorption spectrophotometer (AAS). The principle of AAS measurement is as follow: the sample for analysis are dispersed in a beam of energy from a hallow-cathod lamp and atoms in the ground state absorb the incident energy of certain wave length. The absorption causes a decrease in emerging energy and with suitable instrumentation the decrease could be measured and the metal ions concentration was determined (10) .

Statistical analysis using t-test showed that the mean of salivary MDA was significantly higher (p<0.001) in subjects with amalgam fillings than that in subjects without amalgam fillings. The mean of salivary total glutathione, uric acid in subjects with amalgam fillings was lower than that in control subjects with statistically no significant differences. The mean of salivary copper, zinc in subjects with amalgam fillings was higher than that of the control group with statistically high significant differences as shown in figure 1. The results showed that no significant correlation has been found between salivary malondialdehyde concentration and the number of filled teeth while a significant negative linear correlation has been found between salivary total glutathione concentration and the number of filled teeth. No significant correlation has been found between salivary uric acid concentration and the number of filled teeth. High significant positive linear correlation has been found between salivary copper and zinc concentrations and the number of filled teeth. No significant correlation has been found between salivary malondialdehyde , total glutathione, uric acid concentrations and the number of filled surfaces. Highly significant positive linear correlation has been found between salivary copper concentration and the number of filled surfaces while no significant correlation has been found between salivary zinc concentration and the number of filled surfaces. The results showed that in the control subjects a significant negative linear correlation has been found between salivary MDA and salivary total glutathione using the correlation coefficient(r), while no significant correlation was found between salivary MDA and salivary uric acid . In subjects with amalgam fillings no significant correlation has been found between salivary MDA and both salivary total glutathione and salivary uric acid as shown in table 1.

RESULTS
The results showed that the mean of salivary MDA in subjects with amalgam fillings was higher than the mean of salivary MDA in control subjects.

Figure 1: Mean levels of chemicals under study in both study groups.


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Table 1: The correlation between salivary MDA concentration and both (total glutathione and uric acid) concentrations in each study group
Cases (N = 50) Chemicals ( mol/L) Total glutathione Uric Acid r P Control (N = 51) r P

-0.138 0.340 *-0.299 0.033 -0.066 0.651 -0.152 0.286

DISCUSSION
This study show that increase in oxidative stress marker (MDA) in saliva of subjects with amalgam fillings. Klinghardt and Mercola 2008(11); Graham et al., 2009(12) found that the positive correlation of salivary malondialdehyde levels with the number of amalgam fillings and also with the number of filled surfaces could be attributed to that amalgam fillings represent a mixture of metals in an electrolyte(saliva),this result in galvanic current that pump mercury and other metals into the gums and oral mucosa from which it is carried throughout the body by the blood and nerves and the released mercury is toxic because it induce the production of free oxygen radicals and modifies the redox potential of the cells so represent a continuous source of oxidative damage to mouth tissues (13). These results were in agree with the findings of (14) who found that there is a continuous release of mercury from amalgam fillings and there is a statically significant correlation between mercury concentration in saliva of subjects with amalgam fillings and the number of amalgam fillings and the release of mercury from amalgam fillings was increased with the increase in the number of filled surfaces. The results of this study also agree with the results of (15) who found that amalgam fillings bearers show significantly increased oxidative stress in saliva which correlates with the number of amalgam fillings. Lower concentration of salivary total glutathione in subjects with amalgam fillings than that of subjects without amalgam fillings have been found with statically no significant differences this due to that glutathione is the main natural chelator for heavy metals in the body due to its sulfhydryl-containing cysteine, mainly mercury which is bound to glutathione to be capable to leave the body via urine or biliary excretion, thus high levels of glutathione is crucial for mercury metabolism. Glutathione enzyme system act as a detoxifier by elimination of numerous toxins from the body including pollutants, heavy metals like mercury
Oral Diagnosis 65

and lead, carcinogens, etc (16) so its level decreased as a response to continuous release of mercury from amalgam fillings. At the same time glutathione act as antioxidant and its level decreased as a result of oxidative Stress caused by mercury release from amalgam fillings (17). These results agreed with (18) who found that very low mercury concentrations which are frequently seen in tissues of many people with dental amalgam leads to increased oxidative stress and reduction of glutathione concentrations which lead to sub cellular damage. Although no significant differences was found in the mean of salivary uric acid between subjects with amalgam fillings and those without amalgam fillings but still it was higher in those with amalgam fillings.These results may be due to that uric acid is an important salivary antioxidants and it increased to counter act the increase in oxidative stress which is represented by saliva MDA, which was increased with the increase in the number of filled teeth, these result was agreed with Becker, 1993(19) also uric acid act as pro-oxidant according to the result of the study of Proctor, 1972(20). The negative correlation between uric acid and the number of filled teeth may be due to that uric acid act as antioxidant against the oxidative stress that caused by mercury from amalgam fillings so uric acid may depleted to provide defense (21). The result of this study showed that the mean of salivary copper was significantly higher in subjects with amalgam fillings than in saliva of subjects without amalgam fillings.These results may be due to electrochemical corrosion of dental amalgam which result in a conversion of the metallic solid components into dissolved metal ions and non metallic corrosion products.These results were agreed with (22, 23). It has been shown that the mean of salivary zinc was significantly higher in subjects with amalgam fillings than that of subjects without amalgam fillings. The positive correlation between salivary zinc and the number of filled teeth / surfaces may be due to electrochemical corrosion of dental amalgam which leads to release of components of amalgam fillings as metal ions and other corrosion products.Zinc thermodynamically is the most active components of dental amalgam and a fast preferential initial dissolution of zinc from zinc-containing amalgams had been reported (24, 25).

REFERENCES
1. Dezwart LL, Meerman JH, Commandeur JN et al. Biomarkers of free radical damage applications in

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2.

3.

4.

5. 6.

7.

8. 9. 10.

11.

12.

13.

14.

15.

16. 17.

18.

experimental animal and humans Free Radic Biol and Med 1999; 26:202-26. Mctigue D, Brice Nada CR, Sarkar NK. The in vivo corrosion of dispersalloy. J Oral Rehabil 1984; 11:351-359. Dodes, John E. The amalgam controversy: An evidence-based analysis. Journal of the American Dental Association 2001; 132:348-356. Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malonaldehyde as toxic molecule and biological marker of oxidative stress Nutr Metab Cardiovasc Dis 2005; 15 (4): 31628. Guoyao, Wu, etal. Glutathione metabolism and it is implications for health. J Nutrition 2004; March. Heinig M, Johnson RJ. Role of uric acid in hypertension, renal disease, and metabolic syndrome. Cleveland Clinic J Medicine 2006; 73 (12): 105964. Shah SV, Walker PD. Evidence suggesting a role for hydroxyl radical in glycerol induces acute renal failure. AM J Physiol, renal, fluid, electrolyte physiol 1989; 24: 3: 438-43. Burits CA and Ashwood ER. Tietz text book of clinical chemistry. 3 rd edition 1999: pp.790. Tietz NW. Clinical guide to laboratory tests. 3rd Edition.1995. Ward FN, Nakagawa MM, Harms TF and Vansickle GH. Atomic absorption methods of analysis useful in geochemical exploration U.S.Geol surv bull 1969; 45-47. Klinghardt D, Mercola J. Heavy metals and chronic disease and mercury toxicity and systemic elimintation agents. J Nutritional and Environmental Medicine 2008; 11:53-62; and Amalgam Detox, Klinghardt Academy of Neurobiology. Graham N George, Satya P Singh, Jay Hoover and Ingrid J Pickering. The Chemical Forms of Mercury in Aged and Fresh Dental Amalgam Surfaces Chem Res Toxicol 2009; 22/11: 17614. Dezwart LL, Meerman JH ,Commandeur JN et al. Biomarkers of free radical damage applications in experimental animal and humans. Free Radic Biol and Med 1999; 26:202-26. Shakir AT. Measurements of mercury concentrations in saliva of selected sample of children in relation to amalgam fillings. A thesis submitted to the college of Dentistry, Baghdad University, 2004. Pizzichini M, Fonzi M, Sugherini L, Fonzi L, Comporti, M, Gasparoni A, and Pompella, M Release of mercury from dental amalgam and its influence on salivary antioxidant activity. Bull Group Int Rech Sci Stomatol Odontol 2000; 42:94100. Sies H Glutathione and its cellular functions. Free Radic Biol Med 1999; 27:916-21. Chad K, and Darry W The antioxidant role of glutathione and N-Acetyl-Cysteine supplements and exercise induced oxidative stress. Journal of the international society of sports nutrition 2005; 2:3844. Olivieri G, Brack C, Muller Spahn, F, Stahelin HB, Herrmann M, Renard P, and Hock C Mercury induces cell cytotoxicity and oxidative stress and increases beta-amyloid secretion and tau phosphorylation in SHSY5Y neuroblastoma cells. J Neuro chem 2000; 71:231-236.

19. Becker BF. Towards the physiological function of uric acid Free Radical Biology & Medicine 1993; 14 (6): 61531. 20. Proctor P. Electron-transfer factors in psychosis and dys-kinesia Physiological Chemistry and Physics 1972; 4 (4): 34960. 21. Glantzounis G, Tsimoyiannis E, Kappas A, Galaris D. Uric acid and oxidative stress Curr Pharm 2005; (32): 414551. 22. Lin JH, Marshall GW, Marshall SJ Corrosion product formation sequence on Cu-rich amalgams in various solutions. J Biomed Mater Res 1983; 17:914-920. 23. Sutow EJ, Jones DW, Hall GC, Owen CG Crevice corrosion products of dental amalgam. J Dent Res 1991; 70:1082-1087. 24. Brune. Corrosion of amalgams. Scand J Dental Res 1981; 89:506-514. 25. Jensen SJ. Corrosion of zinc containing dental amalgam .Scand J Den Res 1983; 91: 325-328.

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A cytopathological study of the effect of smoking on the oral epithelial cells in relation to oral health status by the micronucleus assay
Saeed H. Saeed B.D.S. (1) Wasen H. Younis B.D.S., M.Sc., Ph.D. (2)

ABSTRACT
Background: Micronucleus is a cytoplasmic fragment of DNA reported as a biomarker of cancer. It is a cytoplasmic chromatin mass formed in the basal cells layer of the epithelium. These fragments can form their own membrane. The aims of the study was to detect the micronuclei expression in the oral epithelial cells in cytopathological smears of the non-smokers and the smokers males, correlate the micronuclei expression in the oral epithelial cells with the oral health status variables, and evaluate the efficacy of the micronuclei assay to detect the subjects at high risk of oral mutations. Materials and methods: This study was conducted on 75 males of (35- 40) years of age divided into 25 heavy smokers, 25 light smokers, and 25 non-smokers. A cytobrush was used to obtain the smears. The oral health status was evaluated by using the plaque, gingival, calculus indices in addition to the amalgam and composite restorations. Results: There was a statistically significant difference in the micronuclei expression among the three groups. There was a strong correlation between the oral health status variables and the micronuclei expression in the non- smokers' group, for the Plaque index with (P-value =0.0005) and for the calculus index (P-value = 0.04). The smokers' group had a strong correlation with the amalgam restorations with (P-value =0.0005). Conclusion: The micronucleus assay detected by Pap stain is a useful biomarker to detect the people at high risk of oral mutations due to the harmful effect of the smoking, the calculus and plaque indices, in addition to the amalgam restorations. Key words: Cytopathology, micronucleus assay, smoking effects. (J Bagh Coll Dentistry 2012;24(3):67-70).

INTRODUCTION
The oral epithelial cells represent the preferred target site for the early genotoxic events induced by different types of agents entering the oral cavity (1). The smoking is a complex mixture of different type substances that are with a genotoxic and a carcinogenic effect on the oral epithelial cells. These substances lead to the DNA damage and the nuclear anomalies formation. One of these a nuclear anomaly is the micronuclei formation (2). Micronucleus is a cytoplasmic fragment of DNA reported as a biomarker of mutagenesis. It is a cytoplasmic chromatin masses that can form its own membrane. The micronuclei are formed in the basal cells layer. The micronucleus assay is used to detect the subjects at high risk of malignant transformations in their oral epithelial cells (3). It has been extensively used to evaluate the extent of chromosomal damage in the human population exposed to the genotoxic agents in various occupational settings, in the environment, or as a consequence of the life style (4).

(1) (2)

M.Sc. Student, Ministry of Heath Professor, Chairman of Oral Diagnosis Department, College of Dentistry, University of Baghdad.

The micronuclei test is gaining an increased attention among researchers and laboratories in the field of environmental mutagenesis, and a number of published studies based on this biomarker are increasing rapidly (5). Oral cytopathology is a simple technique that is non-aggressive, relatively painless, and readily accepted by the patient. It is used to obtain cells from the oral epithelium. It is the art and science of interpretation of the cells obtained from the oral cavity. The oral epithelial cells may be detached naturally or artificially as in scrubbing and the cytobrush sampling (6). In the current study, micronuclei assay was sought to be used for the first time on Iraqi sample to evaluate its validity as a biomarker for the early detection of the oral epithelial cells with mutation in relation to the effect of the smoking as a very popular habit, on the oral epithelial cells from different keratinized and non-keratinized oral sites. The smoking is a complex mixture of different types of substances that have a mutagenic and carcinogenic effect on the oral epithelial cells. The smoking effect on the oral epithelial cells was evaluated in relation to the different oral health status variables. The oral health status variables include the plaque, gingival, calculus indices and the amalgam and the composite restorations.
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MATERIAL AND METHODS


Seventy five males volunteers aged (35-40) year attending to the oral diagnosis department/collage of dentistry/Baghdad University, the maxillofacial clinic in Al Hussein hospital, and specialized center for Dentistry in Karbala. The oral examination includes the determination of the types of restorations in each tooth by the application of the FS fraction of the DMFS index. Oral health status for each patient was assessed by using plaque, gingival and calculus index. Oral smear should be obtained from normal mucosa by using the cytopathological brush. The oral sites include: - buccal mucosa, hard palate, gingiva and floor of mouth. The smears were transferred and spread onto the labeled, clean, dry glass slide. Each slide was labeled with the patient's name ,the site from which the sample obtained and the type of stain .For Pap stain method , the slides were fixed at once by 95% ethanol for 20 minutes, whereas, they were air dried for Giemsa stain method. Each oral site has two slides, the first one was stained by Pap stain and the second by Giemsa stain. Each patient has 8 slides and 1000 oral epithelial cells examined by the light microscope.

stained by Giemsa stain for the micronuclei expression (table4). There was a strong correlation between the oral health status variables and the micronuclei expression in the non- smokers' group, for the Plaque index with (P =0.0005) and for the calculus index (P = 0.04). Regarding the smokers' group, they had a strong correlation with the amalgam restorations with (P =0.0005). According to the multiple linear regression model indicated that the non-smokers' group was with (P =0.007) for the calculus index, while in the smokers' group (P = 0.006) for the amalgam restorations.

Figure 1: An oral epithelial cell with micronucleus stained by Pap stain at X100 oil emersion

RESULTS
All the subjects of the study sample were with a positive expression of the micronuclei in different numbers (table1,2, and 3) (Figures1,2,3, and 4). There was a statistically significant difference in the micronuclei expression between the non-smokers and the light smokers, where (P= 0.031) in the floor of mouth stained by Pap stain. There was a statistically significant difference in the micronuclei expression between the non- smokers and the heavy smokers where (P = 0.0005) in both the floor of mouth and the gingiva, (P = 0.002) in the buccal mucosa, and (P= 0.004) in the palate stained by Pap stain. For the slides stained by Giemsa stain, floor of mouth, gingiva, and the buccal mucosa (P = 0.001), while palate was a non-significant difference (P=0.685). There was a statistically significant difference between the light smokers and the heavy smokers where (P = 0.0005) for all the oral sites stained by Pap stain, and floor of mouth stained by Giemsa stain only showed a highly significant difference (P=0.0005). Both gingiva and palate were with a (P=0.521), while buccal mucosa was (P = 0.59) for the slides
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Figure 2: An oral epithelial cell with micronucleus stained by Giemsa stain at X100 oil emersion

Figure 3: An oral epithelial cell with micronucleus stained by Pap stain at X40

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Figure 4: An oral epithelial cell with micronucleus stained by Giemsa stain at X40

DISCUSSION
The study results revealed that, there was a micronuclei expression in all the smears taken from the examined males, but in different proportions. The mean of the micronuclei expression in the non-smokers' group was (2.36) micronucleus in each 1000 oral epithelial cells. This was consistent with the baseline of the micronuclei expression in the healthy subjects which was (0.5-2.5) micronucleus per 1000 oral epithelial cells according to the results of (7) who studied the micronuclei expression in the oral epithelial cells from patients with cancer, precancerous lesions, and healthy controls. They found an 11 fold increase in the micronuclei expression in the patients with cancerous oral lesions (p-value = 0.001) and (10.38) fold increase in the micronuclei expression in the patients with pre-cancerous oral lesions (p-value =0.002). The current study was also related to the oral health status variables and their effect on the oral epithelial cells in relation to the smoking by the micronuclei expression. In the non- smokers' group, there was a strong relation of the plaque index and the calculus index with the increase in the micronuclei expression in the oral epithelial cells since the dental plaque and calculus represent sites for the oral bacteria which produce the chronic bacterial infection, the chronic infection is usually lead to the chronic inflammatory process that is often associated with the human carcinogenesis and the formation of clastogenic and anuploid genetic damage in the oral epithelial cells (8). So these indices have a prominent effect on the oral epithelial cells by increasing the rate of micronuclei. The micronuclei expression in the non-smokers resulted from the effect of the plaque and calculus indices in addition to the effect of the environmental pollutants and the passive smoking or the spicy and hot food.

In the heavy smokers' group and according to the results statistically analyzed especially on the buccal mucosa, there was a strong effect of the amalgam restorations in relation to the smoking on the micronuclei expression in the oral epithelial cells which could be attributed to that the heavy smoking will inhibit the growth of the oral bacteria and enhance the growth of tar resistant bacteria on the oral epithelial cells that have a carcinogenic effect by increase the micronuclei expression. Additional to the path of the poisonous effect of smoking on the buccal mucosa, the unfavorable effect of the amalgam restorations on the oral epithelial cells due to the direct contact of the amalgam restorations on the oral epithelial cells and the metallic ions released from these restorations. The biological interaction of the restorations with the oral epithelial cells is related to the toxic and allergic reactions and the increase in the bacterial adherence which can lead to the inflammatory effects (9).

REFERENCES
1. Nersesyan A, Kundi M, Atefie K, Herman RS, and Knasmuller S. Effect of staining procedure on the results of micronucleus assay with exfoliated oral mucosa cells J cancer Epidemiology, biomarkers, and Prevention 2006; 10: 1055-108. Tolbert PE, Cari MS, Allen JW. Micronuclei and other nuclear anomalies in the buccal smears: method development. J mutation 2003; 271(1) 6977. Ray MR, Basu CM, Mukherjee KS, Chodhury SR, Lahiri TF. The micronuclei frequencies and nuclear anomalies in the exfoliated buccal cells of fire fighters 2005; J Genetics. 5(1) 45-8. Benites CI, Amondo LL, Vianna AP, Roth MD. Micronucleus test on the gas station attendants. J Genetics and molecular 2006; 5(1) 45-54. Buajeeb W, Kraivaphan P, Amronchit C, Suthamajariya K. Reduction of micronuclei in oral Lichen planus supplemented with Beta carotene. J oral science2008; 50(4) 461-7. Johnes AA, Stewart CM. Oral Cytology: indications, contraindications, and techniques. J Gen Dent 1995; 43(1) 74-7. Harshvardhan SJ, Alka DK, Mohan KP. Micronucleus as a potential biomarker of carcinogenesis. J India 2011; 2(2) 67-76. Bloching M, Reich W, Schubert J, Sandner A. Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors. J oral Oncology 2008; 44: 220-6. Albander JM, Streckfus CF, Adesanya MR, Winn DM. Cigar, pipe, and cigarette smoking as risk factor for periodontal diseases and tooth loss. J Periodontology 2000; 71: 1874-81.

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3.

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Table1: Micronuclei expression in the study sample


The stain Pap stain Groups Non-smokers Light smokers Heavy smokers 52 74 380 Giemsa stain 7 18 122 The total of the micronuclei 59 92 502

Table 2: The mean and the SD of the micronuclei expression in Pap stain:
sites Palate Gingiva Buccal mucosa Floor of mouth mean SD No. mean SD 0.36 0 25 1.16 0.37 0.76 0.23 25 1.44 0.50 2.65 1.20 25 6.76 2.42 No. mean SD NO. mean SD No. Groups Non-smokers 7 0.28 1.00 7 0.28 0 9 Light smokers 11 0.44 1.00 9 0.36 0 18 Heavy smokers 25 2.67 2.76 24 3.16 1.62 24

Table 3: The mean and the SD of the micronuclei expression in Giemsa stain:
Floor of mouth Buccal mucosa Gingiva Palate Sites SD mean No. SD mean No. SD mean No. SD mean No. Groups 0 0.24 6 _ 0 0 _ 0 0 _ 0.04 1 Non-smokers 0 0.65 14 _ 0.04 1 0 0.08 2 _ 0 0 Light smokers 0.17 2.16 25 0.42 1.12 22 0.62 1 17 0.36 0.64 14 Heavy smokers

Table4: The statistical difference of the micronuclei expression according to the oral sites.
Giemsa stain Pap stain Stain P-value df F-test P-value df F-test sites 0.71 NS 1 0.14 0.0005 2 15.98 palate 0.31 NS 1 1.08 0.0005 2 15.30 Gingiva 0.61 NS 1 0.26 0.0005 2 22.25 Buccal mucosa 0.0005 2 18.08 0.0005 2 119.41 Floor of mouth
P 0.005is highly significant, NS means non- significant.

Table 5: The multiple linear regression of the micronuclei expression in the non-smokers' group.
Factors Beta t-test P-value 0.08 0.35 0.73 Pl. I. Gi. I. -0.39 -1.57 0.15 Cal. I. 0.72 3.35 0.007 Amalgam -0.04 -2.14 0.83 Composite 0.21 0.92 0.37

Table 6: the multiple linear regression of the micronuclei expression in the smokers' group.
Factors Beta t-test P-value Pl. I. 0.12 0.50 0.62 Gi. I. 0.11 0.50 0.62 Cal. I. 0.15 0.71 0.49 Amalgam 0.73 3.43 0.006 Composite 0.14 0.58 0.57

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Evaluation of the effect of platelet-rich plasma on intrabony defect repair in glucocorticoids-induced osteoporosis in rabbits (Histological and biochemical study)
Sudad H. M. Al-Shehabi B.D.S., M.Sc.(1) Nada M. Hassan Al-Ghaban B.D.S., M.Sc., PhD (2)

ABSTRACT
Background Osteoporosis has an impact on bone healing process, platelets rich plasma (PRP) ameliorated the deleterious effect of osteoporosis on bone healing process. Autologous (PRP) could be used in many clinical fields of oral and maxillofacial bone, implant reconstructive surgery and periodontology. This study was carried out to evaluate histologically the regeneration capacity of autologous (PRP) on defect in the mandible bone of osteoporotic rabbits. Materials and Methods Forty-eight female rabbits were used in this study, divided into four groups (12 rabbits for each group), each rabbit was received intraboney defect in the mandible. Two groups were save as control groups one of them left for normal healing (group A), while other group were receiving PRP treatment (group B). The remaining two groups were given 10 mg/B.W hydrocortisone i.m daily for 8 weeks to induce OP-like condition which save as experimental groups. One of them left for normal healing (group C), while other were receiving PRP treatment (group D).After 2, 4, and 6 weeks postoperatively (4 rabbits from each groups), blood sample was taken from each animal for serum alkaline phosphatase, calcium, and phosphorous analysis. Then the animals were sacrificed and the decalcified sections of the bone were studied histomorphologically. These histometric analyses including counting of bone cells: osteoblast, osteocyte, and osteoclast. Bone trabecular, separation, width, and number; cortical width, blood vessels number, and bone marrow space and volume assed. Results Histological examinations showed that with the use of autologous platelets rich plasma in an osteoporotic and normal rabbit, an obvious enhancement of new bone formation and neovascularization significantly more than that of groups without (PRP) application. The results of osteoporotic group treated with (PRP) nearly reached the levels of normal group without PRP in all the three periods postoperatively.Biochemical serum analysis revealed an increase in serum alkaline phosphatase and calcium concentrations in osteoporotic animals than control one, while serum phosphorous level increased in control animals than osteoporotic ones. Conclusions This study illustrated that the (PRP) has an osteopromotive activity that accelerated bone-healing process in madibular bone defect in an osteoporotic rabbits as well as in normal rabbits. Key words: Platelet-rich plasma, intraboney defect. (J Bagh Coll Dentistry 2012;24(3):71-77).

INTRODUCTION
Repair of bone tissue is a complex process involving a number of cellular functions and mineralization of the defect followed by an eventual remodeling of the defect site to attain the original structure (1). Systemic disease such as diabetic mellitus and osteoporosis (OP) has been suggested as potential conditions that delay bone healing. OP-like conditions often manifested themselves in the geriatric female population, for which bone fracture has become common(2). OP has received attention in the dental field, as it is characterized by reduction of bone mass, structure, and function. OP is thought to be a result of altered bone remodeling capacity, i.e., bone formation decrease while restorative capacity remains relatively constant (3).

(1)Lecture, Department of Oral Diagnosis, Ministry of Health, Baghdad, Iraq (2)Assistant professor, Department of Oral Diagnosis College of Dentistry, University Of Baghdad

Several studies have shown that bone regenerativeprocedures in normal defect or osteoporotic defect may be enhanced by the addition of specific growth factors.(4) Growth factor is a naturally occurring substance capable of stimulating cellular growth, proliferation and cellular differentiation. Usually it is a protein or a steroid hormone ( 5). Platelet-rich plasma (PRP) is blood plasma that has been enriched with platelets. As a concentrated source of autologous platelets, PRP contains several different growth factors and other cytokines that stimulate healing of bone and soft tissue. Based on this principle PRP are introduced to stimulate a supra-physiologic release of growth factors in an attempt to jump start healing in chronic injuries. All of the known clinical applications of PRP highlight an accelerated tissue cicatrization due to the development of effective neovascularization, accelerated bone healing with fast tissue remodeling, and nearly total absence of infectious events ( 6) .

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MATERIALS AND METHODS


Forty-eight female rabbits were divided into 4 groups (12 rabbits for each), Intra bony defect were made for each rabbits in the diastima near the first premolar in the mandible (fig 2-4). These groups are: Group A: (12 rabbits) control group with Group B: (12 rabbits) control group received platelet rich plasma. Group C: (12 rabbits) experimental group osteoporotic group- with normal healing. \Group D:(12 rabbits) experimental group osteoporotic group- received platelet rich plasma Four rabbits from each group were sacrificed at 2, 4 and 6 weeks intervals according to this table.
Healing period Study group Group A Group B Group C Group D Total 2 week postope ratively 4 weeks postope ratively 4 4 4 4 16 6 weeks postope ratively 4 4 4 4 16 T ot al

The hole was made on the mandible at the diastema between central incisors and, premlar of rabbit, its size approximately 2mm in diameter and (2-3mm) in depth (10). Then the hole filled with small piece of gelita tampon immersed in platelet rich plasma in the rabbits of (group B and groupD) While, the hole left unfilled in the rabbits of (group A and group C) for normal healing.

RESULTS
Histological analysis:

4 4 4 4 16

12 12 12 12 48

Figure 1: View of group A at the end of 2nd week showing woven bone (WB) and blood vessels (BV) in the area of bony defect. (H&E stain X100)

Induction of osteoporotic-like condition: Twenty four rabbits were received (10 mg/kg body weight) of hydrocortisone daily by i.m injection for 8 weeks to induced osteoporotic like condition (7) (8) Preparation of Platelet Rich Plasma (PRP): This processes involve the collection of whole blood that is anticoagulant with citrate dextrose before undergoing two stages of centrifugation designed to separate the PRP aliquot from platelet poor plasma and red blood cell. The platelets poor plasma was separated from platelets rich plasma along with puffy coat (9), the P.R.P. was activated by combination with equal volume of sterile solution of 10% CaCl2 Surgical procedure After anesthetizing the rabbit by general anesthesia, the operative field was `properly `draped by sterilized towels. An incision of (1.52.5 cm) was made along the alveolar crest in the naturally edentulous space between the incisors and premolar teeth in the mandibular arch (lower left diastema). Bone penetration was performed by dental engine of low speed hand piece of (2000rpm), round bur (no.012) cooled by a continuous stream of sterile normal saline, used only to perform the orifice in the bone , then with fissure bur ( No. 010 )the cavity deepened to hold the implanted material
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Figure 2: View of group A at the end of 2nd week showing newly osteoid tissue (OT) formation surrounded by osteoblast(OB),fat cells (FC) and haemopoeitic cells. (Gomori Blue Trichrome stain X100

Figure 3: Newly bone trabecula(BT)lined by osteoblast(OB),osteocyte(OC),osteoclast(OC L), numerous blood vessels(BV) and collagen fiber.(H&E stain X200)

Figure 4: View of group B at the end of 2nd week showing bone trabeculae(BT) an d blood vessels. (Gomori Blue Trichrome stain

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Figure (5) View of group C at the endof2nd week showing collagenous connective tissue and blood vessels (BV). (H&E stain X100)

Figure 10: View of group A at the end of 4th week showing (BT) surrounded by collagenous connective tissue (CCT). (Gomori Blue Trichrome stain X100)

Figure 6: View for group C at the end of 2nd weeks showing woven bone(WB) and bloodvessels (BV). (Gomori Blue Trichrome stains X400

Figure 11: View of group B at 4thweeks duration showing new bone formation (H&E stain X100)

Figure 7: View of group D at the end of 2nd week showing bone trabeculae (BT) lined by osteoblast (OB) , collagenous connective tissue (CT) and numerous blood vessels (BV).(H&E stain X100)

Figure 12: View of group B at the end of 4th week showing new bone formation (Gomori Blue Trichrome stain X200)

Figure 8: View for group D at the end of 2nd weeks showing speculae of bone trabeculae, surrounded by blood vessels (BV) (Gomori Blue Trichrome stain X100)

Figure 13: View of group C at 4thweeks duration showing cartilage tissue surrounded by new bone (H&E stain X100)

Figure 9: View of group A (control without PRP) at the end of 4th week showing that the defect filled with bone trabeculae (BT) (H&E stain X100)
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Figure 14: View of group C at the end of 4th weeks showing new bone formation with irregular arranged osteocytes (OS) (Gomori Blue Trichrome stain X400)

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Figure 15: View of group D at 4thweeks duration showing fibrous connective tissue (FCT) surrounded new bone (H&E stain X100)

Figure 20: View of group B at the end of 6th weeks showing new bone formation (Gomori Blue Trichrome stain X200)

Figure 16: View of group D at the end of 4 weeks showing fibrous connective tissue(FCT) and bone trabeculae (BT) (Gomori Blue Trichrome stain X200)

th

Figure 21: Higher magnification of previous slide showing preosteocyte(POS), osteocyte(OS), and osteoclast(OCL) (H&E stain X200)

Figure 17: View of group A at the end of 6 week showing irregular arrangement of osteocytes (OS).(H&E stain X400)

th

Figure 22: View of group C at the end of 6thweeks showing trabecular bone (BT) formationl (Gomori Blue Trichrome stain X100)

Figure 18: View of group A at the end of 6th weeks showing new bone formation filled the defect area (Gomori Blue Trichrome stain X100)

Figure 23: View of group D at the end of 6thweek showing new bone formation with increase Haversian canals (HC) no (H&E stain X100)

Figure 19: View of group B at the end of 6th week showing mature (lamellated) bone and small size lacunae of osteocytes (OS) (H&E stain X200)
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Figure 24: View of group D at the end of 6th weeks showing mature bone formation (Gomori Blue Trichrome stain X100)

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Histomorphometric analysis for bone microarchitecture: Cortical width: The cortical width values increased significantly in group B (control with PRP) than other three groups in all healing periods. (P 0.01).However, there was significant increase in cort.wid in all groups with time lapse in three healing periods P 0.01(Figure 25).

Osteocyte number: The highest mean values of osteocyte number were seen in group B (control group with PRP) & the least mean values were seen in group C (exp without PRP) (figure 29)

Figure 29: Linear chart showing the mean of the osteocyte number Figure 25: Linear chart showingthe mean of cortical width Trabecular width : The results of trabecular width revealed that at 2nd week duration showed least significant increase in Tb.wid. Also the results showed that group B (control with PRP) had higher mean value in all healing periods. (figure 26).
Blood vessel number: The results denoted that there was significant reduction in the number of B.V. in almost all groups, except in group A, which showed nonsignificant differences in B.V. number. On the other hand the highest blood vessels mean number was seen in group B than other groups in all healing period (Figure 30)

Figure 26: Linear chart showing the mean of the trabecular width Volume star bone marrow space (V*m):The results showed that there were highly significant reduction in V*m with time progression. The highest mean vales of V*m was seen in group C in all healing periods, while the least mean in all healing periods was seen in group B. & Figure 28.

Figure 30: Linear chart showing the mean of the blood vessels number
Alkaline phosphatase level: The highest mean levels of alkaline phosphatase were showed in groups C and D (osteoporotic groups) than group A and B (control groups). In addition, there were highly significant increased in alkaline phosphatase with time in groups A and B. (figure 31)

Figure 28: Linear chart showing the mean of the Volume star bone marrow space (V*m)

Figure 31: Linear chart showing the mean of the alkaline phosphatase level
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Histological and Histochemical analysis: At the end of 2nd weeks postoperatively: Group A (control group without PRP) showed primitive bone formation while group C osteoporotic group without PRP) the defect still filled with collagen fiber and beginning of osteoid matrix formation, this mean that there was delay in bone healing related to GC administration which lead to reduce bone formation and increase bone resorption (11). Histological findings in control bony defect treated with PRP (group B) illustrated formation of bone trabeculae with active osteoblast and active blood vessels, the findings were not observed in control group without PRP (group A) which showed only primitive bone formation, this is in agreement with study done by (12), who found platelets can enhance the plasminogen activation capacity of mesenchymal progenitors which responsible for bone formative cell. At the end of 4th weeks postoperatively: - at this healing period, the histological findings illustrate bone trabeculae formation in all groups but they were thinner, spare and randomly oriented in osteoporotic rabbits (group C) than other three groups. Moreover, fibrous connective tissue may be still filled the cortical bone in some area of boney defect in the osteoporotic group without PRP, this delay was attributed to improper cell function related to GC administration. This will lead to diminished bone formation, lower mineral density in newly formed bone, and delay bone healing(13) Furthermore, the delay of bone formation was very obvious in the defects of experimental group without PRP by the presence of cartilgenous tissue with its chondrocyte in some area, while in defects treated with PRP showed new bone formation with large number of osteocyte This histological finding was in agree with (14), who found that PRP has an osteopromotive activity since it contains a concentrated growth factors that increasing cellular proliferation. At the end of 6th weeks postoperatively : The histological features of control groups (with and without PRP) revealed the maturity of bone formation, in which, the osteocytes arranged in circular manner around Haversian canal. In addition, there were fewer amounts of spaces between cortical bones in the control groups (group A and B) than that in the experimental groups (group C and D). This might be attributed to the effect of hydrocortisone in delay bone formation (13). On the other hand, the application of PRP to the bony defect of group B and D had positive effect on the healing process of these defect. These results could be observed by the presence large
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numbers of haversian canals, which mean that there was increased in blood supply.These findings are indicated that the platelets within PRP release growth factors and proteins like osteonectin , fibronectin , and osteocalcin , all of them influence bone healing in different ways. (15) Histomorphometrical analysis: In general, osteoporotic animals showed reduction in mean value of cortical width, trabecular width, trabecular number and osteocyte number when compared with control animals in three healing period(16). On the other hand, group C (experimental without PRP) showed the less value of previous objects and highest mean value of trabeculr separation and volume star bone marrow space when compared with other groups. Although in this study the results nearly showed no significant differences in the bone architecture analysis between control group without PRP and experimental group with PRP These findings coincide with that of (17) who concluded that PRP in combination with an osteoconductive synthetic alloplastic substitute has an effect on bone regeneration more significantly in ovariectomized osteoporotic rats than in normal rats. Results of histomorphometric analysis for bone artchitecture parameters showed that using of autologous PRP in the normal bone defect or osteoporotic one has benefits for organizing the formative cell (specially osteoblast), formation of neovascularization and more rapid and faster apposition of bone matrix with its mineralization process.The more supplement of blood to healing area in animals treated with PRP, accelerate and potentiate two processes (18). *First process include local hemostasis at sites of vascular injury *Second process include providence of nourishment for undifferentiated cells to be differentiate and provide significant effects for their migration to the healing area and activate its biological role. The result of the present study reported that there are a significant increment in trabecular number, trabecular width, cortical width and osteocyte number in the defect area as period progress and that is true on the fact that osteoid formation progress to bone trabeculae formation and their opposition and maturation and their establishment to ideal thickness needs for time (19) Biochemical serum analysis:Alkaline phosphatase may assist in the diagnoses of OP, including high turnover of osteoporotic bone (20). Results of alkaline phosphatase level increased significantly in group C&D (experimental groups) than group A&B (control groups) in all healing

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period. These were in agreement with (16), which recorded an increase in the level of alkaline phosphatase in overiectomized rat to induce OP, when it compared with their controls. These changes in serum total alkaline phosphatase may indicate suppression of bone formation in osteoporotic rabbits. The result suggest that GC inhibits bone growth mainly by decreasing bone formation (21).

REFERENCES
1. Oda T,Kinshita , Ueda M. Effect of cortical bone perforation on periosteal Destruction. J Maxillofacial Surg 2009; 67 (7): 1478-85. 2. Nieves JW. Osteoporosis: the role of micronutrients. Am J ClinNutr 2005; 81 (5): 1232S9S 3. Wong PK, Christie JJ, Wark JD. "The effects of smoking on bone health" Clin Sci 2007; 113 (5): 23341. 4. Jung RE, Glauser R, Scharer P, Hammerle CH, Sailer HF, Weber FE. Effect of rh-BMP-2 on guided bone regeneration in humans Clin Oral Implants Research 2003; 14:556-68. 5. Hauer AD, Habets KL, van Wanrooij EJ, et al. "Vaccination against TIE2 reduces atherosclerosis". J Oral Medicine. 2008; 204 (2): 36571. 6. Choukroun J, Diss A, Simonpieri A, Girard MO, Shoeffler C. Platelet-rich fibrin (PRF) A second generation platelet concentrate. Part: Histologic evaluations of PRF effects on bone allograft maturation in sinus lift .Journal of Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;101: 299-303. 7. Aschraft MB, Southard KA, Tolly EA. The effect of Corticosteroid induced osteoporosis on orthodontic tooth movement. Am J Orthod Dentofac Orthop 1992; 102, 310-319. 8. Al-Ghaban N. Shatha S, Huda M, Effect of glucocorticoids induced osteoporosis on osseointegration of titanium implants in rabbits. M.Sc. Thesis College of Dentistry Baghdad University. (2008). 9. Shayesteh YS, khirsand A, Dehghan M and Ardestani MA Evaluation of platelet rich plasma in combination with deprotinized bovine bone mineral in rabbit cranium. J Dent 2005; 2: 12734. 10. Ayyamkh., intraosseous implantation of gattapercha,silver point and zinc oxide-eugenol based sealar in the mandible bone of Hamste .Master Thesis,Collage of Dentistry ,University of Baghdad .(1996). 11. Ing-Lorenzini, K. et al. Low-energy femoral fractures associated with the long-term use of bisphosphonates: a case series from a Swiss university hospital Drug Saf 2009; 32: 775-785. 12. Agis H, Kandler B, Fischer Mb, Watzek G, Gruber R. Activated Platelets Increase Fibrinolysis Of Mesenchymal Progenitor Cells. J Orthop 2009;36(5):90-6 13. Khan AA. et al. Bisphosphonate associated osteonecrosis of the jaw. J Rheumatol. 2009; 36: 478-90 14. Marx RE "Platelet-rich plasma: evidence to support its use". J Oral and Maxillofac Surg 2004; 62 (4): 48996. 15. Al-Kurikchy M, Shatha S, Rafah S. Histological study of bone healing using organic bovine bone in combination with platelet rich plasma (An Experimental

Study in Rabbit) M.Sc. Thesis College of Dentistry Baghdad University. (2008). 16. Ozawa S, Ogawa T, Iida K, Sukotjo C, Hasegawa H, Nishimura RD, Nishimura I. Ovariectomy hinders the early stage of bone-implant integration: histomorphometric, biomechanical, and molecular analyses. Bone 2002;30:137-43. 17. Sa!nchez AR, Sheridan PJ, Kupp LI. Is plateletrich plasma the perfect enhancement factor A current review. Int J Oral Maxillofacial Implants 2003;18:93-103 18. Anitua M, Snchez E, Nurden A, Nurden P, Orive G, Anda I. New insights into and novel applications for platelet-rich fibrin therapies. Trends Biotechnol. 2006; 24(5):22734. 19. Samuelson DA. Text book of veterinary histology. Saunders, Elsevier Inc China. 2007; 6: 10729. 20. Eberhardt AW, Yeager-Jones A, Blair HC. Regional trabecular bone matrix degeneration and osteocyte death in femora of glucocorticoid-treated rabbits. Endocrinology 2001; 142(3), 1333-40. 21. Ng PC, Lam CWK, Wong GW, lee CH, Cange PS, Fok HIS. Changes in markers of bone metabolism during dexamethasone treatment for chronic lung diseases in preterm infant. Archives of diseases in Childhood fetal and Neonatal Edition 2002; 86, 49- 54

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A specific focus imaging for dental implant planning (CT-Based)


Zainab H. Al-Ghurabi, B.D.S., M.Sc. (1) Shafaa H. A-Nuaemi, B.D.S., H.D.D., M.Sc. (2)

ABSTRACT
Background: Today the availability of real 3D planning software which furthers more allows a reliable transfer to the surgical field through drilling template placement, imaging modalities available preoperative planning purpose with specific focus in the use of software for planning of oral implant surgery. This study was established to shed light on the role of 3D dental planning CT in assessment the bone Quantity (height and width) and quality for dental implant and its relation to opposing tooth. Materials, subjects and method: Oral implant planning was performed for twenty patients with fifteen suspected oral implant area in AL-Karkh general hospital by incorporating the restorative planning during the preoperative assessment of the implant site by using a radiographic template with a radiopaque indicator (inside the patient mouth while the patient mouth was closed during the diagnosis) in conjunction with a CT-based imaging system. Preoperative and postoperative CT images were compared (planned and actual implant positions), and the accuracy of this type of image-guided therapy was assessed. Result: From the collected data (preoperative CT-image) in this study it was shown that, the ability of 3D dental planning CT to supply the surgeon with a high resolution image of anatomical limitations, bone density and true length and width. There were no major surgical complications, 20 implants were available for comparison so the required accuracy of dental implant planning was superior with CT-based surgical guidance template image there was a statistically significant correlation in the accuracy of any implants placed with the same guide Conclusion: CT-based oral implant planning with surgical guidance, when properly utilized, will help of removing the limitations associated with two-dimensional imaging modalities for planning implants, and will empower clinicians with more diagnostic information, it allowed for a physical transfer of the implant planning to the patients mouth safely and predictably. Key words: Multi-detector computed tomography, computer-aided design, and dental implants, surgical Templates . (J Bagh Coll Dentistry 2012;24(3):78-81).

INTRODUCTION
Implant dentistry has evolved into one of the most predictable treatment alternatives for partially and completely edentulous patients. The initial excitement about successful has allowed clinicians to offer an extended set of treatment alternatives that include single tooth replacement to full mouth reconstruction. (1,2,3) A dental implant is a "root" device, usually made of titanium, used in dentistry to support restorations that resemble a tooth or group of teeth to replace missing teeth. Virtually all dental implants placed today are root-form endosseous implants, i.e., they appear similar to an actual tooth root (and thus possess a root-form) and are placed within the bone (endo- being the Greek prefix for in and osseous referring to bone). The bone of the jaw accepts and osseointegrates with the titanium post. Osseointegration refers to the fusion of the implant surface with the surrounding bone. Dental implants will fuse with bone; however they lack the periodontal ligament, so they will feel slightly different than natural teeth during chewing.
(1) Assistant lecturer, Department of Oral and Maxillofacial Surgery. College of Dentistry, University of Baghdad (2) Specialist Radiology, Department of Oral and Maxillofacial radiology. Al-karkh General hospital

Although osseointegration is a predictable consequence of surgical placement, anatomical limitation as well as restorative demands encourages the surgeon to gain Presidion in planning and surgical positioning of implant (4, 5, 6) . The success of dental implant treatment depends on careful preoperative planning. In order to accurately plan an implant procedure it's Essential to obtain information regarding the Volume, quality and quantity of the bone at a potential implant site. It's also important to determine the relationship of the proposed implant to important anatomical structures such as nerves, vessels, teeth, nasal floor and sinus cavities at the implant site. This information can be obtained with clinical examination and appropriate Radiographs. (7,8) . CT scans have been used for medical imaging since 1973. It was not until 1987 that CT scans became available for dental applications. Ct-based dental imaging for implant planning and surgical guidance carries both clinical and radiographic information for implant positioning as far as trajectory, depth and distribution. When reviewing imaging modalities for preoperative assessment of the dental implant site, many conflicting variables need to be considered. The amount of information provided, its accuracy
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and its applicability need to be weighed against cost, convenience, availability, radiation dose and expertise required to produce and read the output of each modality. Because the higher cost and lesser availability of dental CT, spiral or multi slices CT scanner is still a standard investigation in many implant logic centers. (9, 10, 11) Currently, there are a number of software systems that analyze CT scans to aid in planning the surgery and produce the physical surgical drilling template guides. These templates are computer manufactured in such a way that they perfectly match the planned implant location, trajectory and depth. They stabilize the drilling procedure while the dental practitioner placing the implants performs the procedure by restricting the degrees of freedom of the drill trajectory and depth (12). This study was established to shed light on the role of 3D dental planning CT in assessment the bone Quantity (height and width) and quality for dental implant and its relation to opposing tooth.

operative), were done and the accuracy of this type of image-guided therapy was assessed. Two radiologists was study the image of dental planning image pre operative and post operative and the reading was confirmed by the two. After the examination, the axial slice are transfer to a workstation to perform a multiplanar reconstruction, a curve was draw on the center of the jaw and multiple orthoradial reconstruction are calculated perpendicular to the curve line after that a multiple cross section were obtained. , 20 implants were available for comparison. So after 6 month of the implant insertion another CT diagnosis was done for these implants as a fallow up

RESULTS
Table 1: This table shows the mean difference between CT measurements and dental implant
Mea SD Mean SD Mea SD Mean SD mean SD Mean SD Mean SD Length Ct Implant 12.9 12 0.818 0.866 31 10.125 3.199 3.473 12.6 11.5 0 0 13.2 11,666 5.058 4.1633 11.05 10.75 1.484 1.060 18 15 0 0 17.33 14.9 0 0 Width Ct Implant 5.566 4.5 0.945 0.433 5.275 4.387 1.170 0.725 4.7 4.2 0 0 5.9 4.266 2.227 0.837 4.2 3.775 0.565 0.671 6 4.75 0 0 5 4.5 0 0

SUBJECT, METHOD

MATERIALS

AND

Up 1 Up 2 Up 3 Up 4 Up5 Lower1 Lower 2 Lower 3 Lower 4 Lower 5

Oral implant planning was performed for twenty patients with fifteen suspected oral implant area in AL-Karkh general hospital by incorporating the restorative planning during the preoperative assessment of the implant site. Dental CT examination was performed with 3D multi slice spiral CT scanner Brilliance 64 with as small as possible X- ray field 5.4*2.5cm, 80 kv and 30ma and 2.5 second exposure time. Prior to imaging, patients should be informed about the investigation and fill a case sheet. Every patient have especial dental cast and the site of implant was determined on this cast by the surgeon. A radiographic indicator was adhering to this cast and template adaptive over the cast then insert into thermoplastic device to produce a surgical template with radiographic marker. Now temple inserted inside the patient mouth, the patient mouth was closed during the diagnosis and instructed him not to move or swallow during the scan., the area that wanted to investigate determined with a shell which must be as small as possible (10 cm) by aiding of computer, to protect the adjacent anatomical structure from radiation the investigation was performed in the supine position, after that the template removed from patients mouth and adapted over the cast and sent to the surgeon with a report of dental planning. After six months the patients come back to have a second 3D CT scanning to investigate the position of implant postoperatively, compared (planned pre operative and actual implant positions post
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Table 2: T-test and P-value for length and width


Length t-test P-value 0.871 0.184 NS 0.152 0.175 NS 0.178 1.752 NS 0.199 1.893 NS 0.500 1.00 NS 0.425 1.651 NS 0.114 1.02 NS Width t-test P-value 0.226 1.730 NS 0.300 1.708 NS 0.203 1.936 NS 0.186 1.984 NS 0.508 o.947 NS 0.220 0.681 NS 0.563 1.631 NS

Up1 UP 2 UP 3 UP 4 UP 5 Lower 1 Lower 2 Lower 3 Lower 4 Lower 5

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B Figure 1: A show CT-based pre-implant assessment for proposed implant area Bshow the implant (fixture) according CT assessment (width, length and angulations
(bone density (HU
1400 1200 1000 800 600 400 200 0

restorative, functional and aesthetic point, it allow for physical transfer of the implant planning to the patients mouth safely and predictably According to the information obtained from the CT planning to the surgical field with a surgical template, there were no major surgical complications. Statistic analysis by using T- test, its found that there was a non significant difference between the mean of CT scan measurements for proposed implant area (with guide) and the mean of implant size (fixture) placed with the same guide for all selected teeth in this study, as shown in table 1 this was because the CT give a true length and width, and this confirm with many studies com in this projection Valente et al and Jung et al (13,14). According the hounce field unite (the measurement unit of CT) it could be determining the bone density for each tooth specifically, and according this measurement the type of bone could be determined (D1, D2, D3 or D4), as shown in figure 2and this study confirm with Teo (15) . Regarding the relationship of the implant with opposing teeth, the exact angle is determined with CT but till now the angulations of implant depend on the skill of the surgeon. For this reason this subject still under research and study to obtain right transferring of the angle to the surgeon.

REFERENCES
1. Siessegger M, Schneider BT, Mischkowski RA, Lazar F, Krug B, Klesper B, Zoller JE. Use of an imageguided navigation system in dental implant surgery in anatomically complex operation sites. J Craniomaxillofac Surg 2001; 29(5):276-281. 2. Fortin T, Champleboux G, Bianchi S, Buatois H, Coudert JL. Precision of transfer of preoperative planning for oral implants based on cone-beam ctscan images through a robotic drilling machine. Clin Oral Implants Res 2002; 13(6):651-656. 3. Tardieu PB, Vrielinck L, Escolano E. Computerassisted Implant placement. A case report: treatment of the mandible. Int J Oral Maxillofac Implants2003; 18(4):599-604. 4. Gahlert M, Rhling S, Wieland M, Sprecher CM, Kniha H, Milz S "Osseointegration of zirconia and titanium dentanimplants:a histological and histomorphometrical study in the maxilla of pigs". Clinical Oral Implants Research 2009; 20 (11): 1247 535. 5. Gerds TA, Vogeler M. Endpoints and survival analysis for successful osseointegration of dental implants. Statistical Methods in Medical Research 2005; 14 (6): 57990. 6. Fischer K, Stenberg T, Hedin M, Sennerby L. Fiveyear results from a randomized, controlled trial on early and delayed loading of implants supporting fullarch prosthesis in the edentulous maxilla. Clinical Oral Implants Research 2008; 19 (5): 43341.

(bone density (HU

Figure 2: This chart show the relation between tooth type and bone density.

DISCUSSION
Our study represents a combination between the uses of surgical template with multi slice CT dental analysis in dental implant analysis, this planning was optimize from anatomical,
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7. Kling B, Petersson A, Maly P. Location of mandibular canal: Comparison of macroscopic findings, conventional radiography, and computed tomography. Int J Oral Maxillo fac Implants 1989; 4:327-332. 8. Siessegger M, Schneider BT,Mischkowski RA, Lazar F, Krug B, Klesper B, Zoller JE. Use of an imageguided navigation system in dental implant surgery in anatomically complex operation sites. J Craniomaxillofac Surg 2001; 29(5):276-281. 9. Ekestubbe A. Conventional spiral and low-dose computed mandibular Tomography for dental implant planning. Swed Dent J 1999; 138 (Suppl):182 10. Abrahams JJ, Berger SB. Oral maxillary Sinus fistula (oroantral fistula):Clinical features and findings on Multiplanar CT. Am J Roentgenol 1995; 165:1273 127 11. Fortin T, Champleboux G, Bianchi S, Buatois H, Coudert JL. Precision of transfer of preoperative planning for oral implants based on cone-beam ctscan

12.

13.

14.

15.

images through a robotic drilling machine. Clin Oral Implants Res 2002;13(6):651-656 Quirynen M. Lamoral y. Dekeyser C. CT scan standard reconstruction technique for reliable jaw bone volume determination. Int J Oral Maxillofac Implants 1990; 5:384-389. Valente F. Scgiroli G. Sbrenna A. Accurecy of computer-aided oral implant surgery: a clinical and radiographic study Int J Oral Maxillofac Implants. 2009; 24(2):234-42. Jung RE. Pjetursson bE. Glauser R, Zembic A. Zwahlen M.Lang NP.A systemic review of the 5-year survival and complication rates of implant supported single crown. Clin Oral Implants Res. 2008; 19(2):191-30. Epub 2007 Dec 7. Teo JCM. Correlation of cancellous bone micro architectural parameters from micro CT number and bone mechanical properties. Materials science and Engineering 2007; 27:333-339.

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Effects of low-energy laser in the treatment of myofascial pain dysfunction syndrome of the temporo-mandibular joint
Ali H. Abbas, B.D.S, M.Sc. (1)

ABSTRACT
Background: The aim of this study to evaluate the effectiveness of low-energy 904nm gallium-arsenide laser therapy in myofascial pain dysfunction syndrome of the temporo-mandibular joint. Materials and methods: Twenty six patients included in this study who complain from myofascial pain dysfunction syndrome of the temporo-mandibular joints. The patients were randomly divided into two groups. Group 1 consisted of 13 patients who received non steroidal anti inflammatory drug and muscle relaxant for three weeks, and group 2 consisted of 13 patients who received low-energy laser for three weeks. Results: The study reveals significant early improvement in laser group in reducing pain severity and muscle tenderness from first week up to the end of the observation period. Conclusion: Low-energy laser had an effect at the trigger points and significantly reducing the pain and muscle tenderness. Keywords: Low-energy laser, myofascial pain dysfunction syndrome. (J Bagh Coll Dentistry 2012;24(3):82-86).

INTRODUCTION
Myofascial pain dysfunction syndrome (MPDS) is the most common cause of facial pain. Patients with MPDS suffer from headache, earaches, clicking or popping of the temporomandibular joints (TMJ) during mouth opening, restricted jaw movement and masticatory muscle tenderness (1). It is characterized by the presence of one or more hypersensitive site in the involved muscle referred to as myofascial trigger points. The pressure stimulation of this point causes intense pain in terms of both pain and referred pain. The trigger point is the most sensitive spot in a taut band where the muscle appears as tight and rigid structure upon manual palpation (2). Psychological factors, occlusion imbalance and parafunctional habits are the most important (1) predisposing factors . Although the pathophysiology of MPDS is unknown thus, numerous therapeutic approaches have been used with varying success rate and can be categorized into pharmacological and non pharmacological treatment. Pharmacologic therapies include injection of local anesthesia, prescription of non steroidal anti inflammatory drugs (NSAIDS), antidepressant, benzodiazepine and muscle relaxant. Non pharmacologic therapy include selective grinding of the occlusal surfaces of the teeth, orthodontic treatment to correct the occlusion, occlusal splints to prevent bruxism disorder, biofeedback, physiotherapy and acupuncture (3). The ideal therapy should be fast, cheap and long term effective (2).
(1) Lecturer, Department of oral and maxillofacial surgery, College of Dentistry, University of Baghdad.

Modern dentistry utilizes low-energy laser (LEL) in tissue healing acceleration, pain alleviation and reduce inflammation in the orofacial region (4, 5). LEL was introduced as an alternative non invasive, easy and short term treatment for MPDS, but its effectiveness is still controversial. The effectiveness of LEL is affected by wavelength, treatment duration, dosage and site of application over nerves versus joints. Despite lack of consensus over its ideal use, specific test and protocols for LEL suggest it is effective in relieving short-term pain for arthritis, acute and chronic neck pain, tendinopathy, chronic joint disorders and acceleration of wound healing (1, 4, 5, 6) . It is claimed that the laser provides an analgesic and anti inflammatory effect by increasing pain threshold in sensory nerve endings, by stimulating the electrolyte exchange in the cell protoplasm and thus increasing the metabolism (2). In addition to this, laser irradiation stimulates collagen production, alter DNA synthesis and improve the function of damaged neurologic tissues (2). The use of LEL is therefore suggested in various musculoskeletal dysfunctions in the treatment of soft tissue lesion, arthritis and neurologic disorders (1, 2).

MATERIALS AND METHODS


This study was done in private health center in Baghdad, from March 2011, to June 2012. A total of 26 patients with MPDS of the TMJ (15 women and 11 men), the patients age range from 17-45 years old (mean age 31 years). The patients were randomly divided into two groups. Group 1 consisted of 13 patients who received non steroidal anti inflammatory drugs (NSAIDS);

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(Ibuprofen tablets 200mg tid) and muscle relaxant [Norgesic tablets (Orphenadrine 35mg & Paracetamol 450mg) tid] for three weeks, and group 2 consisted of 13 patients who received low energy laser (LEL) for three weeks. The laser was applied over at least three painful or trigger points to the muscles of mastication (masseter, lateral pterygoid and medial pterygoid) and TMJ, on both sides on needed for 3 minutes over each trigger point three times a week(figures 1,2 &3).

power (in optic fiber) 5 mW, focal spot 5.1mm and energy density 7 J/cm2 (figure 4).

Figure 1: Laser irradiation of lateral pterygoid muscle

Figure 2: Laser irradiation of masseter muscle

Figure 4: Laser unit All patients were examined clinically and the pain severity, joint sounds (Clicking), muscle tenderness, and degree of mouth opening were recorded at six stages in the following order; before treatment, one week after treatment, two weeks, three weeks, four weeks and two months follow-up. The effectiveness of treatment was evaluated by Verbal Pain Scale (VPS) and pressure pain threshold. In VPS measurement to register the pain severity (mild, moderate or severe) according to the patients symptoms. The pressure pain threshold was evaluated by increasing the compression gradually on the trigger points by dentists finger. Table 1 demonstrates the demographic data. Using SPSS15 Windows XP for statistical analysis. Chi-square test was used for comparing the clinical variables of pain severity, muscle tenderness and TMJ clicking for both groups before and after treatment. If P < 0.05 was considered statistically significant (S), while if p > 0.05 was considered as non significant (NS) and t-test was used for comparing the clinical variables for degree of mouth opening before and after treatment.

RESULTS
Group 1: The study reveals significant improvement in reducing pain severity from 4th week up to the end of the observation period (table 2). Significant improvement in muscle tenderness from 3rd week, and significant improvement for the TMJ and mouth opening started from the 4th week up to the end of the observation period (table 3&4). Group 2: Significant improvement in reducing pain severity started from the 3rd week up to the end of the observation period (table 2), while there is a significant improvement in muscle tenderness

Figure 3: Laser irradiation of TMJ


The type of laser which is used in this study was low-energy infra red gallium-arsenide laser, wave length 904nm, pulsating beam, average
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from the 1st week and highly significant from the 4th week up to the end of the observation period, and significant improvement in the degree of mouth opening from 3rd week and TMJ clicking from 4th week (table 3&4). Table 5 reveals the improvements of pain severity between group 1 and group 2 which indicates an early significant improvement in reducing pain started from the 1st week in group 2 (laser group) when compared to group 1. The results of the study suggest that lowenergy laser (LEL) had an effect at the trigger points and had early improvement to increase the pain threshold and decrease muscle tenderness, also had a delay improvement in TMJ clicking and the degree of mouth opening.

DISCUSSION
MPDS is a chronic condition and many patients prefer to avoid drugs and turn to alternative therapy, so LEL was introduced as an alternative non invasive, easy and short term treatment for MPDS. The sex (15 females and 11 males) was in agreement with other previous studies on MPDS. Its prevalence among females and males to be 57.69% and 42.30% respectively. Higher number of females may be attributed to their more cooperation and attention to health compared to males (3). The study reveals a significant early decrease in pain severity and muscle tenderness started from the 1st week in laser group when compared to group 1(table 4&5) among patients with MPDS of the TMJ, this comes in agreement with other studies (7,8) LEL can be used as monotherapy in reducing pain severity (3,9), but it may gives better results if it conjugated with other therapeutic procedures for pain alleviation (10), and in order to maintain these therapeutic effects, the elimination of predisposing factors is essential (3) LEL therapy cant by itself eliminate joint clicking, so more appropriate treatment is recommended. The delay of clicking improvement in TMJ region probably resulted from TMJ abnormal biomechanics as well as induced stresses on capsular and ligament structures leading to local pain in TMJ (3). Although the laser therapy has superiority over NSAIDS by early improvement of pain severity and muscle tenderness but there is no superior improvements after three weeks, so need more investigations about the affectivity with another treatment regimen including different laser wave length, treatment duration, dosage and site of

applications, this comes in agreement with other studies (4, 5, 8). Changes in the range of mouth opening reveal as delay improvement may be related to structural changes in the affected muscles and the creation of muscle taut bonds that prevents these muscles from lengthening (11). The early improvements for pain relief and muscle tenderness in laser irradiated group and continue to the end of observation period, comes in agreement with other studies (2, 7, 8, 12, 13), which might be due to increasing pain threshold by hyper polarization of neuron cell membrane and increasing stimulation threshold along with an increase in the secretion of morphine substances such as encephalin and endorphin which have an analgesic and anti- inflammatory action (14). Considering the theory that trigger points are caused by their inflammatory nature, it can be concluded that LEL radiation leads to decrease edema, inflammation and pain through reducing inflammatory products such as prostaglandin, histamine and kinines (15).

REFERENCES
1. Brosseun L, Welch V, Wells G, et al. low level laser therapy for Osteoarthritis and Rheumatoid arthritis: A meta analysis. J Rheumatol 2000; 27:1961-9. 2. Kiralp MZ, Ari H, Karabek IRI, Dursun H. Comparison of low intensity laser therapy and trigger point injection in the management of myofascial pain syndrome. Pain clinic 2006; 18(1):63-66. 3. Azizi A, Sahebjamee M, Lawaf S, et al. Effects of low-level laser in the treatment of myofascial pain dysfunction syndrome. J Dental Research, Dental clinics, Dental prospects 2007; 1(2). 4. Ali Gur MD, Surac AJ, Cevik R, et al. Efficacy of 904 nm gallium arsenide low level laser therapy in the management of chronic myofascial pain in the neck: A double-blind and randomize-controlled trial-lasers Surg Med 2004 35(3): 229-235. 5. Dundar U, Evcik D, Samli F, et al. The effect of gallium arsenide aluminum laser therapy in the management of cervical myofascial pain syndrome: a double blind, placebo-controlled study. Clin. Rheumat. 2007; 26 (6): 930-934. 6. Altan L, Bing l U, Aykac M, Yurtkura M. Investigation of the effect of Ga-As laser therapy on cervical myofascial pain syndrome. Rheumatology International, 2005; 25 (1): 23-27. 7. Shirani AM, Gutknecht N, Taghizadeh M, Mir M. Low level laser therapy and myofascial pain dysfunction syndrome: a randomized controlled clinical review. Lasers Med Sci. Sep. 2009; 24 (5): 715-20. 8. Marini I, Gatto MR, Bonetti GA. Effects of super pulsed low-level laser therapy on temporomandibular joint. Clin J pain Sep. 2010; 26 (7): 611-6. 9. Cecchereli F, Alafini L, Lo Castro G, et al. Diode laser in cervical myofascial pain: a double-blind study versus placebo. The Clinical Journal of pain 1989; 5 (4): 301-304.

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10. Simunovic Z. Low level laser therapy with trigger points technique: A clinical study on 243 patients. J Clin Laser Med Surg 2009; 14(4). 11. Simons D, Travell J. Myofascial Pain and Dysfuunction. The Triger Point Manual. 2nd ed. Baltimore: Williams& Wilkins 1999; 70:12-13 12. Olavi A, Pekka R, Pertti K, Pekka P. Effects of the infra red laser therapy at treated and non-treated trigger points. Acupunct Electrother Res 1981; 14(1): 9-14.

13. Aral H, Murat B, S leyma G, et al. efficacy of low level laser therapy in myofascial pain syndrome:an algometric and thermographic evaluation. Lasers Surg Med 2003; 33(5): 339-343. 14. Saxen MA. Myofascial pain syndrome: characteristic, diagnosis and treatment. JIDA 1998; 77: 9-12. 15. Cernavin I, Pugatschew A. Laser application in dentistry: a review of the literature. Aust Dent J 1994; 39: 28-32.

Table 1: Demographic data


No. of patients Age Sex No. of trigger points for all patients No. of patients laser irradiated trigger points No. of patients with TMJ clicking No. of clicked joints No. of patients with muscle tenderness Pain severity Degree of mouth opening 26 patients 17-45 years (mean age 31 years) 15 women (57.69%) 11 men (42.30%) 131 trigger points (503.84%) 69 trigger points (530.76%) 25 (96.15%) 48 joints (184.61%) 83 (319.23%) 15 patients with severe pain (57.70%) 11 patients with moderate pain (42.30%) Mean: 27.5mm

Table 2: Pain severity


Group 1 Time Before treatment 1st week 2nd week 3rd week 4th week 2nd month No pain 0 0 0 2 (15.38%) 7 (53.85%) 9 (69.24%) Mild 0 0 2 (15.38%) 6 (46.16%) 4 (30.77%) 2 (15.38%) Moderate 6 (46.16%) 10 (76.92%) 9 (69.24%) 5 (38.46%) 2 (15.38%) 2 (15.38%) Severe 7 (53.85%) 3 (23.08%) 2 (15.38%) 0 0 0 pvalue No pain 0 0.248 (NS) 0.192 (NS) 0.098 (NS) 0.036 (S) 0.039 (S) 1 (7.69%) 3 (23.08%) 6 (46.16%) 9 (69.24%) 10 (76.92%) Group 2 (laser group) Mild 0 4 (30.77%) 5 (38.46%) 5 (38.46%) 2 (15.38%) 2 (15.38%) Moderate 5 (38.46%) 8 (61.54%) 5 (38.46%) 2 (15.38%) 2 (15.38%) 1 (7.7%) Severe 8 (61.54%) 0 0 0 0 0 0.149 (NS) 0.142 (NS) 0.032 (S) 0.031 (S) 0.041 (S) pvalue

(S): significant, (NS): no significant, (HS): highly significant

Table 3: Muscle tenderness, joint clicking, and degree of mouth opening


Group 1 Time Before treatment 1st week 2nd week 3rd week 4th week 2nd month Muscle tenderness 39(300%) 36(276.92%) 31(238.46%) 25(192.30%) 18(138.46%) 10(76.92%) Joint clicking 23(176.92%) 23(176.92%) 21(161.53%) 20(153.84%) 18(138.46%) 15(115.38%) Degree of mouth opening(mm) 28 28 29 30 32 37 Muscle tenderness 44(338.46%) 35(269.23%) 30(230.76%) 27(207.69%) 19(146.15%) 8(61.53%) Group 2 Joint clicking 25(192.30%) 24(184.61%) 24(184.61%) 22(169.23%) 19(146.15%) 17(130.76%) Degree of mouth opening(mm) 27 28 29 31 31 36

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Table 4: chi-square for muscle tenderness and TMJ clicking, t-test for degree of mouth opening, before and after treatment between group 1 and group 2 by time
Group 1 Time 1st week 2nd week 3rd week 4th week 2nd month Muscle tenderness 0.264(NS) 0.263(NS) 0.043(S) 0.038(S) 0.00(HS) Joint clicking 1.00(NS) 0.189(NS) 0.193(NS) 0.043(S) 0.039(S) Degree of mouth opening 1.00(NS) 0.996(NS) 0.768(NS) 0.048(S) 0.043(S) Muscle tenderness 0.043(S) 0.033(S) 0.009(S) 0.00(HS) 0.00(HS) Group 2 Joint clicking 0.893(NS) 0.893(NS) 0.132(NS) 0.043(S) 0.043(S) Degree of mouth opening 0.673(NS) 0.433(NS) 0.048(S) 0.048(S) 0.043(S)

Table 5: chi-square for pain severity between group 1 and group 2 by time
Time Before treatment 1st week 2nd week 3rd week 4th week 2nd month Chi-square 1.00 2.068 2.136 2.268 2.682 2.362 p-value 0.317 0.049 0.047 0.042 0.039 0.041 significant NS S S S S S

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Assessment of salivary elements (Zinc, Copper and Magnesium) among groups of patients with rheumatoid arthritis and chronic periodontitis and its correlation to periodontal health status
Alyamama Mahmood BDS (1) Maha Shukri, BDS, M.Sc. (2)

ABSTRACT
Background: Periodontal diseases are common in the society and some researchers suggestedan association between rheumatoid arthritis (RA) and periodontal diseases. The aims of study were to determine the periodontal health status in patient with RA and chronic periodontitis and compare it with those having chronic periodontitis without RA anddetermine the level of salivary elements Copper(Cu),Zinc( Zn) and Magnesium( Mg) in patients with rheumatoid arthritis and patients have no rheumatoid arthritis (RA) and compare with the control group.And correlate between these salivary elements with the periodontal parameters Plaque index (PLI), gingival index (GI), bleeding on probing (BOP), probing pocketdepth (PPD) and clinical attachment level (CAL). Materials and Methods: In this study, the samples were recruited from patientsreferred todepartment of Rheumatology at Baghdad hospital. Seventy five female patients participated in this study, twenty five of them rheumatoid arthritis patient andhad chronic periodontitis; twenty five were with chronicperiodontitis and have no arthritis; Twenty five patients wereperiodontally and systemically healthy (control group). Patients were with age range 40-50 yearswith no other systemic diseases. The smokers and patients taking medication affecting periodontium status were excluded from the study.Also the patients had normal weight and length. Periodontal parameters were measuredin all groups at four surfaces.Salivary elements (Zn, Cu and Mg) also measured in this study. Results: Patients with RA had higher prevalence of sites presenting dental plaque, a higher rate of gingival inflammation and bleeding on probing,greater probing depth, and greater attachment loss compared with control and high level of Copper and low level of Zinc and Magnesium. Conclusion: The results suggest higher potentiality for moderate to severe periodontitis involvement among RA patients, with higher levels of Copper (Cu), and low level of Zinc (Zn) and Magnesium (Mg). Keywords: chronic periodontitis, rheumatoid arthritis, salivary elements. (J Bagh Coll Dentistry 2012;24(3):87-92).

INTRODUCTION
Periodontitis (PD), the most common oral disease, is a destructive infl ammatory disease of the supporting tissues of the teeth and is caused by group of specific microorganisms; Porphyromonas gingivalis, Prevotellaintermedia, Tannerella forsythia, and Aggregatibacteractinomycetem-comitans (1). The periodontal diseases range from the relatively benign form of gingivitis to aggressive periodontitis. Many of these conditions are not only a threat to the dentition, but may also be a threat to general health. Periodontitis ischaracterized by both connective tissue and alveolar bone destruction due to a chronic inflammation.(2)

(1) (2)

Assistant Lecturer, Department of periodontics, College of Dentistry, University of Al -Mustansiryia Assistant Professor .Department of periodontics, College of Dentistry, University of Baghdad.

Rheumatoid arthritis (RA) is also a chronic destructive inflammatory disease characterized by the accumulation and persistence of an inflammatory infiltrate in the synovial membrane that leads to synovitis and the destruction of the joint architecture resulting in impaired function. Rheumatoid arthritis, a chronic multisystem disease, is also associated with joint connective tissue and bone destruction.(3) The relationship between rheumatoid arthritis and the progression of inflammatory conditions elsewhere in the body, such as periodontitis, is controversial(4).Both periodontitis and RA represent an imbalance between pro-inflammatory cytokinesand antiinflammatory cytokines, which are deemed responsible for tissue damage. (5) Recently, there has been growing evidence suggesting an association between periodontitis and rheumatoid arthritis, as both these conditions are associated with the destruction of bones (6). Still, there is possibility of a common genetic trait predisposing to both these conditions. (5) There is also a considerable amount of evidence indicating that Zn, Cu and Mg may

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contribute in the etiopathogenesis of the rheumatoid arthritis: Zinc (Zn) is a crucial element in a series of cellular functions as normal growth, protein metabolism, membrane stability, and metalloenzyme functions.(7). Zinc, has several other effects on immune response, complement System, lysozomal enzyme release, and macrophage functions. Zn is also indispensible in many steps of the inflammatory reactions. Among these are prostaglandin biosynthesis, stimulation of lymphocytes and immune response. Zn is likewise an important element in collagen tissue formation and bone metabolism. (8). Copper (Cu) is incorporated into the structure of many enzymes and proteins.RA, as a chronic inflammatory disorder, can cause substantial elemental alterations in the body .Inflammation induces consumption of Zn and Cu. (9) Magnesium (Mg) is one of the most abundant cations present in living cells. It is an essential mineral that is needed for a broad variety of physiological functions. Imbalances in magnesium metabolism are common and are associated with different pathological conditions (10) . Many studies suggest that periodontitis may be a risk factor for many systemic diseases which have also been associated with Mg deficiencies (11,12).

MATERIALS AND METHODS


The study population included seventy five female patients, which are dividedinto three groups 1- Group PR (chronic periodontitis/ rheumatoid arthritis):Twenty five diagnosed to have chronic periodontitis disease, and have rheumatoid arthritis. The patients in the RA group were diagnosed according to the Revised Criteria for the classification of Rheumatoid Arthritis of the American College ofRheumatology (13) and also according to the laboratoryinvestigation (ESR, Latex test). A specific exclusion criterion in this group was a female patient never taken any drug used as treatmentto rheumatoid arthritis to prevent any effects of these drugs on periodontal health status. All patients werewith age range (40-50) years old& had normal weight & length according to BMI(Body Mass Index) which its normal value is 18.5-25 and didnt smoke and had no medical condition that would affect their participation in the study. 2- Group P (chronic periodontitis / nonrheumatoid arthritis ):-Twenty five patients diagnosed to have chronic periodontitis and
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didnt haverheumatoidarthritis.chronic periodontitis in patients was defined as the presence of at least four sites with probing pocket depth 4mm with clinical attachment level 1-2mm, this made according to the international classification system for periodontal disease (14). 3- Group H (healthy periodontium / systemically healthy):- Twenty five patients with healthy periodontium. This group represents a base line data. Clinical examination: Oral examination was performed by the same examiner to three groups by periodontal probe on all teeth except third molar and on four surfaces. The collected data include: Plaque index (PLI),Gingival index(GI), Bleeding on probing (BOP), Probing pocket depth (PPD) and Clinical attachment level (CAL). Biochemical Analyses The collection of unstimulated salivary samples was performed to three groups under standard condition following the instructions cited by Tenovuo and Lagerlf (15).Then salivary samples were taken to the laboratory. Samples were centrifuged at 4000 rpm for 15 minutes. The clear supernatant was separated by micropipette and divided into three portions to be stored at (-20 oC) in a deep freeze till being assessed.Frozen saliva were allowed to thaw and come to room temperature before their analysis (16). Thereafter, they were subjected to biochemical analysis. They were determined by Flame Atomic Absorption Spectrophotometer using standardized procedure by air acetylene. The concentration level of each constituent was expressed as (mmol/L) unit.

RESULTS
Clinical Analysis: The mean values of PL.I, GI, PPD and CAL in groups PR and P are shown in Table (1). There was a highly significant difference between these two groups. The number and percentage of bleeding sites in Group PR and Group P.The BOP score 1 was significantly higher in the Group PR than Group P. Salivary elements analysis: Magnesium: Magnesium inGroup PRand Group Pwas lower than GroupH (Table3). Highly significant differences were found between (PR and H Group) and (P and H Group) and no significant differenceswere found between (PR and PGroup) as shown in Table (4). Also

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comparison between the three groups was found highly significant (Table5). Copper: Cu in Group PR and Group P was higher than Group H (Table6). Highly significant differences were found between (PR and H Group) and (P and H Group) and no significant differenceswere found between (PR and PGroup) as shown in Table (7). Also comparison between the three groups was found highly significant (Table 8). Zinc : Zinc in Group PR and Group P was lower than Group H (Table9). Highly significant differences were found between (PR and H Group) and (P and H Group) and no significant differenceswere found between (PR and P Group) as shown in Table (10). Also comparison between the three groups was found highly significant (Table11). Intra- Group Correlation between clinical periodontal parameters and biochemical parameters:There was weak correlation between clinical periodontal parameters (PL.I, GI,BOP, PPD and CAL) and biochemical parameters (Zinc,Copper and Magnesium) as shown in Table (12)

DISCUSSION
Chronic periodontal disease can be considered a potential focus of infection, which worsens the metabolic control of patients with RA.(17). The pathobiology of periodontal disease (PD) and rheumatoid arthritis (RA) is similar, both are inflammatory chronic diseases, with activation of complement, production of cytokines and release of other inflammatory cell products (18,19). The relationship between rheumatoid arthritis (RA) and the progression of inflammatory conditions elsewhere in the body, such as periodontitis, is controversial. (4). Results of this study showed thatthe mean valueof Pl.I of groupPR was significantly higher than that of the group P . This result could be related to thestiffness of hands muscles to achieve good oral hygiene among RA patients. The changes occurring in the life style of RA patients, as hands muscle function reduces and leads to improper oral hygiene mechanism, have been considered as areason for association between RA and periodontitis. This result is in conformity with the work of Ksser et al.(20). There is asignificant increase in the mean values of GI. This elevation of GI reflects a higher inflammation in the Group PR than the group P and could be related to the increase in the plaque as the plaque is the causative factor of

gingival inflammation. This result is agreed with the previous study (20). The percentage of sites with BOP was significantly higher in group PR than group P. The potential altered abilities of RA patients to perform effective oral hygiene could result in an increased BOP that exacerbates the risk for enhanced tissue destruction in periodontitis. Moreover, interesting observations regarding the complexity of the oral and systemic challenge provide unique mechanisms by which dysregulation of host responses could occur (21). The mean value of PPD in RA group was significantly higher compared to chronic periodontitis group. This elevation in the PPD could be related to local and systemic factors. the local factor is the dental plaque which was significantly higher in the RA group and this have influenced PPD in this group. The systemic factor in the RA patients is the defect in the immune system which could result in inflammatory-mediated destruction predisposingto periodontitis due to an unbalanced cytokine expression profile (22). Clinical attachment level refers to the distance from the cementoenameljunction (CEJ) to the location of the inserted probe tip. Thus, loss of fibers attachment expressed at the clinical level the cumulative effect of destructive pathological processes in periodontal together with the protective and destructive effect of the immunological processes.The mean value of CAL were significantly higher in PR group.the local factor and the systemic factor had influenced CAL also in this group.This result is agree with other studies (5,20). Zinc level was decrease in group PR and group P than group H with a highly significant difference between group PR and group H. Inflammation within tissues induces a series of anti-inflammatory responses in which a number of proteinsand enzymes carrying Zn and Cu elements areinvolved. Most notable among these are; metallothioneins (23,24).In this study, we observed decrease in Zn levels in patients with RA. There is considerable evidence from previousstudies that, Zn distribution among the bodycompartments is reorganized by inflammatory process. Zn concentration is determinedby many factors; nutritional status and history ofprevious infections of the patient . Also the previous studies demonstrated decreased Zn levels in RA patients compared with normal subjects,lend support for the idea that reduction of the Znelement is essential in the genesis of the disease (23,24) .

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This study showed that patients with RA have markedly elevated Cu levels compared withnormal subjects; there are a highly significant difference between group PR and group H andgroup P and group H. This result agree with AL-Hadad study in 2005(25) . The Mg level was decreased in group PR and group P than the group H. Magnesium is one of the most abundant cations present in living cells. Itis an essential mineral that is needed for a broad variety of physiologicalfunctions.It may act as an important regulator of cell functions. Its concentration is remarkably constant in healthy subjects. High normal Mg concentrations are protective against various diseases. Imbalances in magnesium metabolism are common and areassociated with different pathological conditions (26). Interactions between and among different steps in thepathogenesis of periodontitis may explain the relationshipbetween periodontal status and the Mg. The line of evidence for biologicallyplausible explanations: In periodontal inflammation, the activation of neutrophilsis an important factor in tissue injury. Neutrophilsinvading periodontal tissues maintain the inflammatoryprocess and participate in tissue destruction manifested byloss of attachment and also by systemic reactions. Magnesium status has a strong relationship withthe immune system, acting as a modulator of the immune response. Activation of neutrophilsis an early effect of hypomagnesaemia, and high Mgconcentrations inhibit free-radical generation . Thus, reduced Mg concentrations areassociated with enhanced inflammatory response tobacterial challenge (27). There was aweak correlation between salivary elements and clinical periodontal parameters. This result may be due to small number of samples.

7.

8.

9. 10. 11.

12.

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14.

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17.

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19.

REFERENCES
1. 2. Saini R. Periodontitis a true infection. J Glob Infect Dis 2009; 1:149-151. DeStefano F, Anda RF, Kahn HS, Williamson DF, Russell CM. Dental disease and risk of coronary heart disease and mortality. Br Med J 1993; 306: 688-691. Weyand CM. New insights into the pathogenesis of rheumatoid arthritis. Rheumatology 2000; 39(Suppl. 1):3-8. Mercado F, Marshall RI, Klestov AC, Bartold PM. Is there a relationship between rheumatoid arthritis and periodontal disease? J Clin Periodontol. 2000; 27(4): 267-72. Eduardo de Paula, Carlos R, Keith LK, Mirian A. Periodontal condition in patients with rheumatoid arthritis. Braz Oral Res 2008; 22(1): 72-7. Depinder KM, Vipinder SG, Usha B. Rheumatoid arthritis and periodontitis: Biological links and the

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emergence of dual purpose therapies. Indian J Dent Res 2009; 20(1): 86-90. Bert L, Vallee, Kenneth H. The Biochemical basis of Zinc physiology: Physiological Reviews 1993; 73: 79-117. Karin LG, Svenson R, Hallgren. Reduced zinc in peripheral blood cells form patients with inflammatory connective tissue diseases. Inflammation 1985; 9: 189-199. Hays W. Principals and Methods of Toxicology. 3rd ed. Philadelphia; 1994. p. 423. Touyz RM. Magnesium in clinical medicine. Front Biosci 2004; 9: 1278-1293. Stalnikowicz R. The significance of routine serum magnesium determination in the ED. Am J Emerg Med 2003; 21: 444-447. Scannapieco FA, Bush RB, Paju S. Associations between periodontal disease and risk for atherosclerosis, cardiovascular disease, and stroke: A systematic review. Ann Periodontol 2003; 8: 3853. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31(3): 315 24. Lang NP, Bartold PM, Cullinam. International classification workshop: Chronic periodontitis. Annals of periodontology 1999; 4: 53. Tenovuo J, Lagerlf F. Saliva. In Textbook of clinical cardiology by Thylstrup A, Fejerskov O. 2nd ed. Munksgaard: Copenhagen; 1994. p.17-43. Goronzy J, Weyand C. Epidemiology, pathology and pathogenesis. In Primer on rheumatic diseases. 11th ed. Atlanta (GA): Arthritis Foundation; 1997. p.155-161. Slots J .Casual or causal relationship between periodontal infection and non-oral disease? J Dent Res 1998; 77: 1764-1765. Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J. International league of associations for rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol 2004; 31: 390-392. Smolik I, Robinson D, El-Gabalawy HS . Periodontitis and rheumatoid arthritis. Epidemiologic, clinical, and immunologic associations. Compend Contin Educ Dent 2009; 30: 188-190. Ksser UR, Gleissner C, Dehne F, Michel A, Bolten WW. Risk for periodontal disease in patients with long standing rheumatoid arthritis. Arthritis Rheum 1997; 40(12): 2248-51. Wegner N, Wait R, Sroka A, Eick S, Nguyen K A, Lundberg K Kinloch A, Venables PJ. Peptidylarginine deiminase from Porphyromonasgingivaliscitrullinates human fibrinogen and alpha-enolase: implications for autoimmunity in rheumatoid arthritis. Arthritis and Rheumatism 2010 . Bartold PM, Marshall RI, Haynes DR. Periodontitis and rheumatoid arthritis. J Periodontol 2005; 76: 2066-2074. Roberts N A. and Robinson PA. Copper chelates of anti rheumatic and anti-inflammatory agents: Their Superoxide Dismutase like activity and stability. Br J Rheumatol 1985; 24: 128-136.

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24. Milanino R, Frigo. Copper and Zinc status in Rheumatoid Arthritis: Studies of plasma erythrocytes, and urine, and their relationship to disease activity markers and pharmacological treatment. Clinical and Experimental Rheumatology 1993; 11: 271-281. 25. Al-Hadad A. Study of the trace elements in patients with rheumatoid arthritis. A thesis submitted in

partial fulfillment for degree of fellowship of Iraqi board for medical specialization in medicine, 2005. 26. Laires MJ, Monteiro CP, Bicho M. Role of cellular magnesium in health and human disease. Front Biosci 2004; 9: 262-276. 27. Mooren FC, Golf SW, Volker K. Effect of magnesium on granulocyte function and on the exercise induced inflammatory response. Magnes Res 2003; 16: 49-58.

Table 1: Mean & SD of Pl.I, GI, PPD & CAL and comparison between PR&P groups
GROUPS PL.I GI PPD CAL Group PR Group P t-test PVALUE SIGN mean SD Mean SD 1.837 0.255 1.149 0.113 7.981 <0.001 HS 1.319 0.076 1.140 0.069 8.641 <0.001 HS 6.065 0.547 4.296 0.479 12.146 <0.001 HS 4.472 0.408 3.684 0.569 5.618 <0.001 HS

Table 2: Number & percentage of bleeding &non bleeding sites among PR &P groups
Score of BOP 0 (no bleeding) 1 (bleeding sites) Group PR No. % 582 30% 1358 70% No. 824 1048 % 44.017% 55.982% Group P Chi-square 79.81 p-value <0.001 Sig HS

Table 3: Mean and standard deviation of Mg ion concentration among three groups
GROUPS Mg mean SD PR Group 0.2212 0.0773 0.2724 0.1242 P Group H Group 0.6652 0.0357

Table 4: Inter groups comparison of Mg ion concentration


Groups t-test PVALUE SIG Group PR and Group P 1.749 0.087 NS Group PR and Group H 26.066 <0.001 HS Group P and Group H 15.193 <0.001 HS

Table 5: Comparison of Mg ion concentration among three groups


Groups P VALUE Anova SIG HS Group PR , P and H <0.001 1.475

Table 6: Mean and standard deviation of Cu ion concentration among three groups
Groups Cu mean SD 0.830 Group PR 4.076 0.787 Group P 3.804 0.249 Group H 2.568

Table 7: Inter group comparison of Cu ion concentration


Groups t-test P VALUE SIG Group PR and Group P 1.189 0.240 NS Group PR and Group H 8 .697 <0.001 HS Group P and Group H 7.484 <0.001 HS

Table 8: Comparison of Cu ion concentration among three groups by Anova .


Groups P VALUE Group PR, P and H <0.001 16.14 Anova SIG HS

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Table 9: Mean and slandered deviation of Znion concentration among three groups
GROUPS Zn mean SD Group PR 2.528 0.795 2.752 0.727 Group P Group H 4.532 0.247

Table 10: Inter group comparison of Zn ion concentration


Groups t-test P VALUE 0.304 Group PR and Group P 1.039 <0.001 Group PR and Group H 2.027 Group P and Group H 1.594 <0.001 SIG NS HS HS

Table 11: Comparison of Zn ion concentration among three groups by Anova


Groups P VALUE Anova SIG HS Group PR, P and H <0.001 30.157

. Table 12: Correlation coefficient(r) between clinical periodontal parameters and biochemical parameters
PL.I Zn Cu GPR Mg Zn GP Cu Mg r p r p r p r p r p r p - 0.172 0.409 -0.3135 0.127 -0.013 0.950 0.220 0.288 0.190 0.360 0.052 0.804 GI - 0.031 0.880 -0.077 0.714 0.352 0.083 0.036 0.862 0.094 0.654 -0.087 0.678 BOP -0.175 0.414 0.360 0.084 0.079 0.712 -0.112 0.603 0.019 0.931 -0.112 0.601 PPD CAL -0.154 -0.371 0.4614 0.067 0.276 0.059 0.180 0.778 0.357 -0.050 0.079 0.811 0.191 0.213 0.358 0.306 -0.245 -0.125 0.236 0.550 0.102 0.209 0.625 0.314

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Inorganic ions level in saliva of patients with chronic periodontitis and healthy subjects
Wasan A. Abid Aun, B.D.S., M.Sc. (1)

ABSTRACT
Background: Chronic periodontitis is an inflammatory disease that extends into the tissues supporting the teeth. Inorganic ions have been most intensely studied as a potential marker for periodontal disease in saliva. The aims of the study were the detection of calcium, magnesium, potassium, and sodium ions levels in saliva of chronic periodontitis patients and healthy subjects and correlate the mean salivary levels of these ions with clinical periodontal parameters [plaque index (PI), gingival index (GI), probing pocket depth (PPD) and clinical attachment level (CAL)]. Materials and methods: The study sample consisted of thirty chronic periodontitis patients of both gender (18 males and 12 females) and thirty healthy control subjects of both gender (15 males and 15 females) with age ranged from 30 to 50 years. Both groups were without any systemic disease. Periodontal parameters used in this study were plaque index (PLI), gingival index (GI), probing pocket depth (PPD) and clinical attachment level (CAL). Unstimulated saliva samples were collected from all subjects and the levels of calcium, magnesium, potassium and sodium in each specimen were analyzed. For each group a statistical analysis was done to estimate the levels of these ions in saliva and to correlate the mean of salivary inorganic levels with clinical periodontal parameters. Results: The present study showed that there was highly significant difference in the levels of salivary inorganic ions (Ca=2, Mg+2, K+1, Na+1) between chronic periodontitis patients and control group with increase in Ca+2, K+1, Na+1 ions levels in saliva of chronic periodontitis and decrease in level of Mg+2 ions in saliva of chronic periodontitis than in control group. There was no correlation between the mean of PLI and Ca+2, Mg+2, K+1 and Na+1 ions in saliva of chronic periodontitis and there was no correlation between the mean of GI and Ca+2, Mg+2 and Na+1 ions and a negative significant correlation with K+1 ions. Concerning PPD and CAL there was no correlation between them and the mean salivary inorganic levels in chronic periodontitis patients. Conclusions: Estimation of these electrolytes or inorganic ions in saliva of chronic periodontitis may be used as potential diagnostic markers of active disease status in periodontal tissues and to predict the effective methods of prevention and treatment. Keyword: chronic periodontitis, inorganic ions, saliva. (J Bagh Coll Dentistry 2012;24(3):93-97).

INTRODUCTION
Periodontal disease is a chronic disease of the oral cavity comprising a group of inflammatory conditions affecting the supporting structures of the dentition (1,2). The natural history of periodontitis follows a discontinuous pattern of exacerbation and remission characterized by disease activity and inactivity (3,4). Periodontitis is a multifactorial disease which is affected by both genetic and environmental factors (5). Clinical parameters such as probing depth, attachment level, gingival index and plaque index, provide information on the severity of periodontitis but they do not measure disease activity, where as microbiological tests, analysis of host response, and genetic analysis have been proposed in an effort to monitor and identify patients at increased risk for periodontitis (4,6). Saliva is a major determinant of the oral environment and serves as an easily available diagnostic and monitoring method for periodontal diseases, more intense saliva research can be observed in recent decades, which leads to a high amount of scientific data presented by numerous engaged researchers of this far-reaching field.
(1)Assistant Lecturer / Department of Dentistry, Al-Yarmouk University College of Dentistry.

Salivary fluid is an exocrine secretion consisting of approximately 99% water and the other 1% is complex of organic and inorganic molecules (7). Saliva containing a variety of electrolytes (calcium, magnesium, potassium, sodium, chloride, bicarbonate and phosphate), some studies have assessed the relationship existed between periodontal disease and those ions (8,9). This study aimed to analyze the salivary levels of calcium, magnesium, potassium and sodium ions in chronic periodontitis and control groups and to correlate the salivary level of these ions with clinical periodontal parameters: plaque index (PLI), gingival index (GI), probing pocket depth (PPD) and clinical attachment level (CAL).

MATERIALS AND METHODS


Human Sample Sample population consisted of sixty male and female, age ranged from 30 to 50 years. Samples collection was started at 20th of February 2011 till May 2011. Patients participating in the present study with chronic periodontitis (no=30, 18 males and 12 females) were recruited from the Clinic of the Department of Periodontics / College of Dentistry/ Baghdad University.

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The control group was taking from the Department of Periodontics (no=30, 15 males and 15 females) with clinically healthy gingiva, no pockets, no bleeding on probing and no evidence of bone loss. Clinical examination Periodontal examination consisted of plaque index (PLI), gingival index (GI), probing pocket depth (PPD) and clinical attachment level (CAL) at 4 sites for all teeth except 3rd molar on (mesial, midvestibular, distal, midlingual), using a calibrated periodontal probe (Michigan O probe). Patients with chronic periodontitis had periodontal pockets equal or greater than 4mm with clinical attachment loss. Inclusion criteria Subjects who had not received any periodontal treatment during the past 3months, those who had not taken any antibiotic therapy during the past 3 months and those who were not suffering from any known systemic diseases or conditions that influence the tissues were included in the study. Subjects were excluded if smokers and pregnant. Collection of saliva samples All participants were instructed not to eat or drink (except water) at least 1 hour prior to donation of saliva, the subject should sit in a relaxed position and samples containing blood should be discarded. Saliva was collected between 9-12 am. After the subject rinse his mouth several times by sterilized water and then wait for 1-2 minutes for water clearance, 5ml of whole unstimulated mixed saliva was collected into polyethylene tubes using a standardized method a (10) . Saliva then centrifuged at 10000 rpm for 10 minutes; this was done within 1hour after collection to eliminate debris and cellular matter, the clear supernatant was separated by micropipette and divided into four portions to be stored at (-20 C) in a deep freeze till being analyzed. Biochemical assay Frozen saliva samples were allowed to thaw and come to room temperature. Therefore, they were subjected to biochemical analysis. The inorganic ions (calcium Ca+2, magnesium Mg+2, potassium K+1, and sodium Na+1) salivary levels were analyzed at the Poisoning Consultation Center / Surgical Specially Hospital. This was done by using Flame Atomic Absorption Spectrophotometer (AAS) using standardized procedure by air- acetylene. The concentration level of each constituent was expressed as (mmol/L) unit. Statistical Analysis
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The data were processed and analyzed using the statistics package for social sciences (SPSS Inc., version 17 for windows XP and excel 2007). Both descriptive and inferential statistics were used. 1. Descriptive Statistics; included mean, standard deviation (SD), range, number (No.), percentage, and statistical tables. 2. Inferential Statistics; included Student t-test. Coefficient of correlation (r). In the statistical evaluation, the following levels of significance are used: P > 0.05 Non-significant (NS) 0.05 ! P > 0.01 * Significant (S) P" 0.01 ** Highly significant (HS)

RESULTS
In this study the mean and standard deviation of plaque and gingival index in chronic periodontitis patients were (1.340.44, 1.230.26) respectively and in control group were (0.560.24, 0.690.16) respectively as shown in table 1. There was highly significant difference in mean PLI & GI (P<0.01). The mean and mean percentage of total sites with standard deviation of probing pocket depth and clinical attachment level with different scales (0. 1, 2, 3) are shown in table 2 and 3 respectively. Descriptive statistics of inorganic ions (Ca+2, Mg+2, K+1, and Na+1) salivary levels (mmol/L); mean and standard deviation for each group are shown in table 4. The mean salivary levels of these ions in chronic periodontitis patients were (1.940.42, 0.270.06, 8.751.37, 17.162.69 ) in mmol/L respectively while in control group were (1.230.30, 0.300.07, 7.370.86, 11.931.18) in mmol/L respectively. The result was highly significant difference in the mean of Ca+2, K+1, and Na+1 ions levels (P<0.01) with increase in levels of these ions in saliva of chronic periodontitis patients. Concerning Mg+2 ions there was significant difference in the mean of Mg+2 ions level (P<0.05) with increase in Mg+2 ions level in saliva of control group as shown in this table. The statistical analysis using the correlation coefficient (r) between the salivary level of inorganic ions with PLI and GI in chronic periodontitis patients was seen in table 5. There was no significant correlation between the mean of PLI and the mean of (Ca+2, Mg+2, K+1 and Na+1) ions salivary levels. Concerning GI a non significant correlation with the mean of (Ca+2, Mg+2, and Na+1) ions salivary level and a significant negative correlation were found between GI and K+1 ion. Table 6 shows the

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correlation between PPD and CAL with different scales (0, 1, 2, 3) and with the mean of inorganic ions salivary levels, the result was no significant correlation found between them as shown in this table.

DISCUSSION
In this study there was highly significant difference in PLI and GI between chronic periodontitis patients and control group, this is obvious since the accumulation of plaque is the primary cause of periodontal disease and chronic periodontitis is a chronic inflammation of the gingiva and connective tissue (2). Concerning inorganic ions, there was highly significant difference in Ca+2 ions levels between chronic periodontitis patients and control group with increase in Ca+2 level in chronic periodontitis, this is in agreement with Acharya et al who found that periodontal disease is associated with higher salivary calcium levels than that in periodontal health, indicating that the calcium level of saliva could possibly be a risk factor for development of periodontal diseases(11) and disagree with Omar who found that there was no significant difference in salivary levels of Ca+2 ions between chronic periodontitis and healthy periodontium group (12). Calcium (Ca+2) is the ion that has been most intensely studied as a potential marker for periodontal disease in saliva. Sewon et al. (17) reported a higher concentration of Ca+2 detected in whole stimulated saliva from the periodontitis patients. The authors concluded that an elevated Ca+2 concentrations in saliva were characteristic of patients with periodontitis. Nevertheless, the importance of the salivary Ca+2 concentrations in relationship to progression of periodontal disease is not defined. Considering the distribution of Ca+2, this ion appears to hold only limited promise as a marker for periodontal disease (6). In this study there was significant difference in salivary levels of Mg+2 ions between chronic periodontitis and control groups with increase in Mg+2 ions salivary levels in control group these results agree with Meisel et al. who found that magnesium deficiency is associated with periodontal disease (13) and disagree with Omar who found that there was no significant difference in salivary Mg+2 ions concentration between chronic periodontitis and healthy periodontium groups (12). Numerous studies show that magnesium may be of particular importance for preventing periodontal disease, these findings arent overly surprisingmagnesium is, after all, an important structural component of teeth and bone. But the unique importance of magnesium for dental health may involve far
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more than just magnesiums role as a structural element of mineralized tissue. Evidence shows that magnesium has the unique ability to reduce inflammation-including the inflammation caused by bacterial toxins. Periodontal disease, of course, is characterized by chronic inflammation resulting from bacteria in the oral cavity. But a similar sort of inflammation is increasingly thought to be a common thread running through all disorders of aging. As magnesium seems to combat inflammation at the most fundamental cellular level, its not surprising to see that magnesium may be a first line of defense against not only periodontal disease, but all inflammatory and agerelated disorders (13). In this study there was highly significant difference in salivary level of both Na+1 and K+1 ions between chronic periodontitis and control groups with increase in their level in chronic periodontitis patients. This was in agreement with Omar who found high significant differences between chronic periodontitis and healthy periodontium groups (12) and disagrees with Al-Bahadli who found non significant differences between both groups (14). High Na+ level may be due to the presence of large quantity of Na+ enters into immediate exchange with that in the remainder of extracellular spaces. It is conceivable that with alveolar bone destruction. Increased quantities of Na+ may be made available to the extracellular compartment and saliva (17). The increased K+ is probably derived from intracellular sources in the inflamed tissues and from degenerating epithelial and connective tissue (17). The present study shows that there was no correlation in chronic periodontitis patients with PLI and Ca+2, Mg+2, K+1 and Na+1 ions level in saliva. Concerning GI, there was no relation between salivary levels of Ca+2, Mg+2 and Na+1 ions in chronic periodontitis patients and GI and there was a negative correlation between GI and K+1 ions in the same group. The explanation for these results needs further studies. There was no significant correlation found between salivary levels of Ca+2, Mg+2, K+1 and Na+1 with PPD with different scales (0, 1, 2) and CAL with different scales (0,1, 2, 3) these results agree with Ebru and Ali whom approved a non significant correlation between PPD and the mean of salivary minerals(15) and disagree with Ebru and Ali who found a positive correlation between salivary minerals and CAL (15) .

REFERENCES
1. Iva. A diagnostic tool for periodontal disease: Current state and future directions. Periodontol 2000 2009; 50:52-64.

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2. Armitage GC. Development of a classification system for periodontal diseases and conditions. Ann Periodontol 1999; 4:1-6. 3. Offenbacher S. Periodontal diseases: Pathogenesis. Ann Periodontol 1996; 1: 821-78. 4. Sahingur SE, Cohen RE. Analysis of host responses and risk for disease progression. Periodontol 2000 2004; 34:57-83. 5. Genco RG. Current view of risk factors for periodontal diseases. J Periodontol 1996; 67:1041-9. 6. Kaufman I, Lamster IB, Analysis of saliva for periodontal diagnosis: A review. J Clin Periodntol 2000; 27:453-65. 7. Ahmadi MF, Davoodi P, Dalband M, Hendi SS. Saliva as a mirror of the body health. DJH 2010; 1(2):1-15. 8. Berkovitz BKB, Holland GR, Moxham BJ. Oral anatomy, histology and embryology. 3rd ed. NewYork: Mosby; 2002. 9. Ferraris MEG, Munz AC. Histologia embriologia bucodental. 2nd ed. Rio de Janeiro: Guanabara Koogan; 2006. 10. Thylstrup A, Fejerskov O: Textbook of Clinical Cariology. 2nd ed., Munksgaard, 1996; 17-43. 11. Acharya A, Kharadi MD, Dhavale R, Deshmukh VL, Sontakke AN. High salivary calcium level associated with periodontal disease in Indian subjects- a pilot study, Oral Health Dent 2011; 9(2): 195-200.

12. Ali OH. Detection of salivary flow rate and minerals in smokers and non smokers with chronic periodontitis (Clinical and Biochemical study). A master thesis, Department of Periodontics, College of Dentistry, University of Baghdad, 2011. 13. Meisel P, Schwahn C, Luedemann J, John U, Kroemer HK, Kocher T. Magnesium deficiency is associated with periodontal disease. J Dent Res 2005; 84(10):037-41. 14. Al-Bahadli MA. Quantitative determination of essential elements in saliva associated with different severities of periodontal disease and its correlation with histopathological picture. A master thesis, Department of Periodontics, College of Dentistry, University of Baghdad, 2007. 15. Ebru E, Ali E. The detection of salivary minerals in smokers and non smokers with chronic periodontitis by inductively coupled plasma atomic emission. J Periodontol 2006; 77: 990-5. 16. Sewon L, Karjalainen SM, Sainio M. Seppa O. Calcium and other salivary factors in periodontitis affected subjects prior to treatment. J Clin Periodontol1995; 22:267-70. 17. Koregol AC, More SP, Nainegali S, Kalburgi N, Verma S. Analysis of inorganic ions in gingival crevicular fluid as indicators of periodontal disease activity: A clinico-biochemical study. Contemp Clin Dent 2011; 2(4):278-82.

Table 1: Mean and SD (Range) of PLI and GI in Chronic periodontitis and control groups
Mean PLI Mean GI Chronic periodontitis 1.340.44 (0.3-3.0) 1.230.26 (1.0-2.0)
**HS

Control 0.560.24 (0.1-0.8) 0.690.16 (0.4-1.1)

T;df;P 0.0001** 0.0001**

Table 2: Mean and SD of sites according to different probing pocket depth scales
Chronic periodontitis PPD scale0 (1-3mm) PPD scale1(4-5mm) PPD scale2( 6mm) PPD total PD% Affected MeanSD(Range) 69.8717.36 (26.0-102.0 ) 13.8011.36 (3.0-49.0) 3.806.08 (0-32.0) 87.4712.37 (64.0-108.0) 19.9316.78 (4.3-71.7)

Table 3: Mean and SD (Range) of sites according to different clinical attachment level scales
Chronic periodontitis 48.6723.80 (0-89.0) CAL scale0(no attachment loss) 12.906.74 (0-25.0) CAL scale1(mild 1-2mm) 13.377.86 (1.0-29.0) CAL scale2(moderate 3-4mm) 11.3012.02 (0-45.0) CAL scale3(severe 5mm) 86.2315.76 (31.0-108.0) CAL total 45.4223.06 (10.7-100.0) CAL% Affected

Table 4: Mean and SD of salivary inorganic ions in study groups


Ca++ (mmol/L) Mg++ (mmol/L) K+ (mmol/L) Na+ (mmol/L) Chronic periodontitis 1.940.42 0.270.06 8.751.37 17.162.69
**HS *S

Control 1.230.30 0.300.07 7.370.86 11.931.18

T;df;P 0.0001** 0.042* 0.0001** 0.0001**

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Table 5: Correlation coefficient of salivary level of inorganic ions with mean PLI and GI in chronic periodontitis group
Ca++ (mmol/L) Mg++ (mmol/L) K+ (mmol/L) Na+ (mmol/L) Chronic periodontitis Mean PLI Mean GI r P r P 0.249 0.185 0.025 0.897 0.135 0.476 -0.268 0.152 -0.251 0.182 -0.534** 0.002 -0.220 0.244 -0.151 0.427

**Highly significant correlation at 0.05 of significance

Table 6: Correlation coefficient of salivary level of inorganic ions with PPD and CAL
r P r P r P r P r P r P r P r P r P Chronic periodontitis Ca++ (mmol/L) Mg++ (mmol/L) K+ (mmol/L) Na+ (mmol/L) -0.313 -0.026 0.059 -0.241 0.092 0.892 0.756 0.200 0.305 0.261 -0.008 -0.014 0.101 0.163 0.968 0.942 0.504 0.160 -0.335 0.349 0.209 0.734 0.843 0.828 0.015 0.251 0.047 -0.262 0.938 0.181 0.805 0.162 -0.138 0.090 0.025 -0.215 0.467 0.635 0.896 0.253 0.121 0.029 0.178 -0.140 0.524 0.880 0.346 0.460 -0.028 0.011 0.127 -0.130 0.885 0.955 0.504 0.494 0.224 0.160 0.013 0.145 0.234 0.400 0.944 0.445 0.001 0.276 0.187 -0.339 0.997 0.140 0.321 0.067
Non significant correlation at 0.05 level of significance

PD scale0 PD scale1 PD scale2 PD total CAL scale0 CAL scale1 CAL scale2 CAL scale3 CAL total

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The efficiency and stability of maxillary expansion with Quadhelix; a longitudinal study
Anfal Abdul- Majeed Al-Ani, B.D.S., M.Sc. (1)

ABSTRACT
Background: Transverse maxillary deficiencies constitute a routine clinical problem in orthodontics, Quadhelixis one of the slow maxillary expanders which used to solve these problems. The purpose of this study was to investigate the long-term clinical responses of Quadhelixas slow maxillary expander; a treatment performed for early permanent dentition in Class I malocclusion. Subjects and methods: A study sample of 30 patients (13 males and 17 females) were selected on the criteria of having Class I permanent dentition with transverse maxillary deficiency, and mild or no crowding mandibular dental arch, treated with Quadhelix expander without extraction, with active mechanotherapy for teeth alignment (as needed). The mean age of the sample was 12 years and 2 months for girls and 12 years and 11 months for boys at treatment initiation. Cast and cephalometric measurements outcomes were evaluated at pretreatment phase A0, postactiveexpansion phase A1, post-retentive phaseA2 (one year after A1), and at the end of long term follow-up A3(three years after A1). The results were compared to a control group of 30 subjects (15 males and 15 females) of Class I with normal occlusions of the same age group. Simple training to withdraw bad oral habitswas carried out all over the study. Results: Casts and cephalometric x-rays measurements were quantified and compared among phase A0, A1, A2, and A3. Using Students t-test, the study group demonstrated a significant increase in the arch width values during the activeexpanding phase (P, .001) and less significant increaseduring the long-term follow-up (P, .01). Stable arch length and no increase in the lower facial height were beneficial results. Conclusions: The long-term clinical response demonstrated the efficiency and stability of this type of treatment in achieving maxillary arch width with no threatening of mandibular downward rotation. The follow-up evaluation confirmed the validity of overtreatment. Key words: Quadhelix, transverse maxillary deficiencies. (J Bagh Coll Dentistry 2012;24(3):98-105).

INTRODUCTION
One of the routine clinical problems in orthodontics is the transverse maxillary deficiencies.It can be manifested with Class I, II and III. Itmay give rise to several clinical manifestations such as maxillary hypoplasia, asymmetrical facial growth, positional and functional mandibular deviation, adverse periodontalresponses and other functional problems,in addition to itsbad esthetic appearance, 1-5so expansion to normalize the constricted maxillary arch, is required. Screws with rapid effect on the mid palatine suture could have some drawbacksand perceptible changes in the sagittal and vertical facial planes such as, downward maxillary displacement and extrusion of the supporting teeth, leading to downward and backward mandibular rotation,increasing; in the inclination of the mandibular plane, inthe lower anterior facial height, and in facial convexity resulting in an evident bite opening in the anterior region.6-10 Some orthodontists have advised against performing rapid expanders in patients with predominantly vertical growth patterns and convex facial profiles9,11to prevent worsening of the malocclusion.
(1)Assistant Lecturer, Department of POP, College of Dentistry, Al-Mustansyria University

Other clinicians have recommended the simultaneous use of other appliances to minimize the undesirable antero-posterior and vertical effects.12 Hicks13revealed that slow expanders achieve more physiological reorganization of the maxilla in the three planes of space, exhibiting substantial skeletal changes in children, and providing more stability and less relapse possibilities than rapid expanders. The Quadhelix considered as a slow tissue born expanding appliance, a technique which is preferable to many, 13-16for better muscles adaptation and less relapse.Ricketts14had developed it in1975, fromPorters W arch, adding four loops to theappliance, increases the wire length from 40 to 50mm, to be more flexible and to soften theforcefor better control for the molar rotations and molar torqueing, andalso can be adjusted to expand the molars and anterior teeth differentially.Many authors have written that the Quad-Helix appliance can promote skeletal changes on maxillarybone in children.13-16These features make the Quad-Helix anextremely versatile appliance.15However, a longitudinal study of patients with Class I dentitions and transverse maxillary deficiencies expanded with Quadhelix as a slow expander, is an opportunity to clarify the action and the drawbacks of this appliance. This investigation was planned to evaluate dental arch width and length changes and also the

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vertical changes in serial casts andCephalometric x-rays as clinical response to Quadhelixexpanding treatment performed during the early permanent dentition.

MATERIALS AND METHODS


This longitudinal study is a retrospective clinical trial forconsecutively treated 30 (13 males - 17 females) Caucasian patients from those who attended Al-Dawoodi health center for dental care in Baghdad, Al-Karkh, from May 2005 - August 2006. Thepatients were selectedon the criteria thatthey all haveClass I malocclusion with transverse maxillary deficiencies, mild or no crowding mandibular arches, no deciduous teeth(all normally exfoliated), incomplete erupted canines, premolarsand 2ndmolars which were not fully rooted formed and their apices were not

closed yet.The mean age for the study group at phase A0for girlswas12 years and 2 (4-5 months) and for boys was13 years (4-5 months). A control group of 30 subjects (15 males- 15 females) of Class I, with normal occlusions, selectedfrom Al-Mutamaizeen secondary schools for boys and for girls, at the same age group, for the growth factor exclusion. Recordswere registered for the study groupincludinginitial casts and routine x-rays (Fig.1). Castand cephalometricx-ray were recordedat pre-treatment phase (A0), at the end of the active expansion phase(A1), after one yearat the end ofthe retentive phase (A2), and after three years from A1 (A3) (2 years period of follow up).Records were obtained for the control group at about the same intervals. The patients recordsat each phase were completed within the same clinic.

Fig.1: Cephalometric and panoramic routine x-rays registration records.


In phase A0, all the permanent teeth (with theexception of the wisdom teeth) were either visible in the oral cavity or about to be erupted. Canines, premolars and the 2ndmolars were in an incomplete eruption, their root apices were not closed yet.

Fig.2: A-Maxillary and mandibular Class I normal occlusion: maxillary and mandibular intermolar width; measurements on dental casts. B- Expansion needed calculated as the difference between them (-x mm).
In normal occlusion the disto-buccal cusp of the 1st mandibular molar occludes in the central fossa of the 1stmaxillary molar, the maxillary and mandibular intermolar widths are equal (Fig.2 Orthodontics, Pedodontics and Preventive Dentistry 99

A); negative difference indicated narrower maxillary than mandibular width (Fig.2 -B).16 The amount of correction for each patient was achieveddirectly on dental casts by dial vernier calipers to the nearest 0.01 mm, for the maxillary

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and the mandibular intermolar widths. The deference between the 1stand2ndmeasurements

indicates the total expansion needed, adding to it an overcorrection of 3 mm for each side.

Fig. 3: Dental linear measurements on the cast, in the maxilla (a+b),(c),(d), and the mandible, (e) and (f). (a+b) = Arch length; the sum of (a) + (b). (c)= Arch width (occlusal value OV). (d) =Arch width (lingual value LV). (e)= Arch width (occlusal value OV). (f) =Arch width (lingual value LV).
Linear measurements were made directly on the castsas some authors recommendation:17-19 1. Arch length is the sum of the measurements from thelabial contact point on the mesial aspect of theupper right central incisor to the mesial contact point of the 1st upper right permanent molar (a), plus the same measurementof the leftarch teeth (b). 2. Arch width at the occlusal level between the mesiobuccal cusp points of the right and left upper molars (occlusal value OV) (c). 3. Arch width at the cervical level measured at the lingual groove with the cervical gingival margin of the right and left upper1st permanent molars (lingual value LV)(d). 4. Mandibular arch width OV (e) is the measurement between the right and left lower 1st molars central fossae. 5. Mandibular arch width LV (f) is the measurement between the right and left lower 1st molarslingual grooves at the cervical level (Fig. 3).

Fig.4: A-The cephalometricalangular measurements NS-Man, Max-Man andCo-Go-Me (degrees). B-Linear measurements ANS-Gn and Co-Go (mm).
The angular and linear cephalometricrecords whichtraced on the cephalometric films were: NSMan, Max-Man and Co-Go-Me angular measurements (degrees)(Fig.4 -A),and ANS-Gn and Co-Go linearmeasurements (mm) for the study and control group during the different studied phases(Fig.4 -B). The study group was treated by expanding the maxilla with the tissue-borne slow expander Quadhelix (Fig.5)with no teeth extraction. A subsequent comprehensive orthodontic treatment was implemented for teeth alignment when needed. Quad-helix appliance (Fig.5) was constructed from 0.036 round stainless steel wire, and

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stainless steel bands were fitted to the maxillary permanent 1stmolars, soldered with silver solder. The appliances were used to achieve transpalatal expansion in the anterior and posterior regions. The Quadhelix was activated for transpalatal expansion by adjusting both of the lingual arms to

give anterior expansion while the anterior midpalatal body wire was activated for posterior expansion. Each expansion adjustment was measured with a Boley gauge to allow equal activation for both the right and left sides.

Fig.5: Quadhelixwith anterior coils, posterior U-loopsand stainless steel bands fitted to the 1st upper molars.
The expander appliance was activated immediately after adjustment inside the mouth, before cementation. The subjects were seen at two weeksintervals,8-10times.At each visit the appliance was re-adapted, to correct the arch shaping.Additional activation, the clinicians can be done by using a three jaw plier inside the mouth. The duration of active treatment of the Quadhelix varied from 4 to 5 months, ends the (A1)phase.The appliance then stabilized in situ, holding the archpassivelyfor 6 months, during which fixed appliance could be applied (if needed),withoutinterference with the expander. After the removal of the expander,a passive, removable acrylic retainer was inserted within 48 hour for another 6 months, collectingone yearretentive period (A2). Simple training to withdrawbad oro-facial over-activitiesof tongue, lips and checks (Fig. 6) was carried out all over thetreatment and follow-up period forthe studygroup.

Fig. 6: Soft tissue bad habits of tongue lips and checks. independently on two separate occasionswith a Statistical analysis: three weeks interval.The differencesbetween the Student`st-test was used to detect and evaluate independent repeated measurementsof each of the alterations between thephases (A0-A1), (A0-A2), 10 individual, ondifferent phases of the study, (A1-A3), and (A1-A3)(table 1).Thestatistical were calculated. significance of intergroup difference was assessed in (table 2)and the data analysis of RESULTS cephalometricmeasurements were shown in (table The preliminary error tests were made.With 3). Student`s t-test; the mean difference for cast Method error: measurements was 0.175 mm, while Dental casts and cephalometric x-rays of 10 forcephalometric angular measurements ranged patients,before and after active treatment with from 0.32 to 0.65 , and for linear measurements Quadhelix and also after retentive and follow-up from 0.53 to 0.74 mm, which was considered not phases,were used to investigate the method significant (P, .05).A descriptive sex analysis error.These measurementswere recorded twice initially was made and theresults of the Students
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t-test revealed nostatistically significant sex differences when the observedvariables in different phases were considered. Therefore; further analyses were performed on the group as a whole. Descriptive statistics for the alterations ofthe study and control groups during the studied time intervals are shown intable 1,2&3.The mean increase in the maxillary arch width for the

occlusal value OV (c)of the study group, at (A1) was about 6 mm, which reduced to 3 mm at(A3), and about 4 mm for the lingual value (d), which reduced to 3 mm at(A3)(table 1).Generally the means for the study group were less than those of the control group at A0, while they get over them at A1 phase, and reduce again to resemble them with slight lower rates atA2 and A3 phases.

Table 1: The descriptive statistics of the maxillary and mandibular arch width and length alterations for the study and control groups during the studied time intervals
Phase Regon AL (a+b) OV (c) LV (d) OV (e) LV (f) AL (a+b) OV (c) LV (d) OV (e) LV (f) AL (a+b) OV (c) LV (d) OV (e) LV (f) AL (a+b) OV (c) LV (d) OV (e) LV (f) Mean 70.15 48.25 32.5 34.5 28.3 73.65 54.2 36.7 37.8 31.8 72.25 52.5 35.75 36.6 30.6 71.7 51.25 35.55 36.5 30.2 Study Group SD Min Max 3.84 62.5 79.2 2.82 42.85 54.35 2.27 27.5 36.75 2.65 29.6 39.25 1.92 26.7 34.5 3.08 67.95 78.5 2.41 47. 5 59.5 2.35 32.75 40.25 2.55 33.5 44.5 2.27 29.75 36,5 3.16 65.75 81.35 2.57 48.25 58.75 2.24 31.5 39.5 2.31 32 42.25 2.11 28.5 35.75 3.26 60.5 78.75 2.55 47.5 56.5 2.27 31.15 40.15 2.31 31.75 41.5 2.07 27.75 35.25 Median 69.75 47.5 32.5 35 28 73.5 55.25 37.25 38 32 72.5 53 35.5 37 30.5 71.5 51.5 35.5 36 30 Mean 72.55 53.25 35.5 37.5 30.8 72.25 53.15 35.7 37.6 31.2 72.1 52.75 36.15 37.75 31.6 71.95 52.55 36.55 37.3 31.7 Control Group SD Min Max 3.62 64.5 78.6 2.93 46.15 61.75 2.88 31.5 38.5 2.96 33.35 42.5 2.18 28.2 33.25 2.95 66.5 80.5 2.23 49.35 60.25 2.19 30. 5 42.5 2.34 34 43 2.22 28.5 33.5 3.1 64.25 80.35 2.61 47.5 59.25 2.33 32.25 41.5 2.36 34.5 43.2 2.23 28.7 33.75 3.36 62.25 80.15 2.78 49.5 58.15 2.68 33.5 42.5 2.34 35 43.5 2.24 29 36.5 Median 72.5 52.5 32.5 38 31 72.5 53.5 37.25 38 31.5 72 52.5 36.5 37.5 32 71.5 52.5 36.5 37 32

A0

A1

A2

A3

Tipping of the anchor teeth is the amount of difference between theocclusal points versus the lingual points, its mean for the study group atA0was 15.75 mm (48.25 -32.5), resembling its value (15.7 mm) at A3 , very close to the control group at A3 (16 mm) (table 1).The meantippingof the 1st permanent molar for the study group atA1,A2andA2was 1.75, 1, and - 0.05

mmrespectively. Arch lengths; tend to return to the initial values at the end of the patients` watching time. Other cast measurementsof the study group during (A0- A1)(A0- A2)(table 2) demonstrated a significant increase (P, .001) and continued to besignificant(P, .01)during (A1A2)(A1-A3).

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Table 2: Descriptive statistics of the cast values; mean, standard deviation (SD) and significance (P).
Region AL(a+b) OV (c) LV (d) OV (e) LV (f) AL(a+b) OV (c) LV (d) OV (e) LV (f) Mean 3.5 5.95 4.2 3.3 3.5 -0.3 -0.1 0.2 0.1 0.4 A0-A1 SD 2.42 2.82 1.86 2.32 2.55 0.89 0.37 0.55 0.21 0.94 P *** *** *** *** *** NS NS NS NS NS Mean 2.62 4.25 3.25 2.1 2.3 -0.5 -0.6 0.65 0.1 0. 8 A0-A2 SD 2.1 2.14 1.99 1.57 1.65 1.09 1.17 1.21 0.23 1.25 P ** *** *** *** *** NS NS * NS NS Mean -1.4 -1.7 -0.95 -1.2 -1.2 -0.15 -0.4 0.45 0.3 0.4 A1-A2 SD 2.05 1.78 1.51 1.63 1.55 0.34 0.91 0.95 0.75 0.93 P NS ** ** ** ** NS NS * NS NS Mean -1.25 -2.95 -1.15 -1.3 -1.6 -0.15 -0.6 0.85 -0.4 0.5 A1-A3 SD 2.09 2.27 1.78 1.68 1.87 0.41 0.98 1.34 0.92 1.07 P NS ** ** ** ** NS NS * NS NS

Control Group Study Group

NSNot significant* Significant(P, .05)** Significant (P, .01). *** Significant (P, .001).

Table 3 reveals a slight increase in the lower facial height of the study group compared with the control group duringA0 and A1 inangular and

linear measurement, which were gradually approximate the normal ranges during the later phases.

Table 3: The descriptive statistics of the cephalometric angular and linear measurementsfor the study and control group during thestudied phases
Measurements A0 NS-Man Max-Man Co-Go-Me NS-Man Max-Man Co-Go-Me NS-Man Max-Man Co-Go-Me NS-Man Max-Man Co-Go-Me ANS-Gn Co-Go ANS-Gn Co-Go ANS-Gn Co-Go ANS-Gn Co-Go Mean 31.5 22.8 121.9 32.2 23.82 122.4 32.9 24.63 123.2 33.78 25.85 123.6 60.72 52.16 62.34 54.96 62.88 55.23 63.73 55.96 Study Group SD SE Median 5.38 1.03 32 5.32 1.01 23 6.87 1.25 123 5.45 1.03 32 5.48 1 24 5.23 0.96 122 5.67 1.08 32 5.86 1.16 25 5.17 0.98 123 5.94 1.15 33 6.26 1.22 26 6.53 1.19 124 4.47 0.79 61 4.32 0.79 52.5 4.88 0.93 62.5 6.41 1.07 55 5.21 0.88 63.5 6.23 0.88 55.5 6.55 1.02 64 6.64 1.17 56 Mean 30.78 21.85 120.4 31.78 22.85 121.63 32.31 23.8 122.45 32.9 24.9 123.32 59.75 52.62 60.77 54.58 63.86 55.76 62.6 56.2 Control Group SD SE Median 4.94 0.9 32 5.2 0.82 22 5.17 0.94 120 4.94 0.98 32 5.35 0.91 23 6.53 0.97 122 5.24 1.02 32 5.45 1.05 24 6.67 1.08 123 5.77 1.07 33 5.63 0.98 25 6.35 1.1 124 4.15 0.76 60 4.67 0.85 53 4.91 1.01 61 4.78 0.95 55 5.32 1.03 62 5.21 0.99 56.5 5.89 1.06 63 5.88 1.03 56.5

Angular measurement (degree)

A1 A2

A3 A0 Linear measurement (mm) A1 A2 A3

DISCUSSION
Maxillary expansion appliances have been used to correct maxillary transverse deficiency. The expansion treatment modalities which used today are: The rapid maxillary expansion (RME) and the slow maxillary expansion (SME), a controversy regarding the use of their appliances exists. Although the objective of both appliances is to achieve inter-maxillary expansion, the actual effect, the design and the type of activation are
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different. Clinicians should not assume that the skeletal and dental effects of the two appliances are equivalent, and they should consider the two treatment modalities separately.19 RME has been used extensively.20-22 Some limitations associated with it have been reported, including bite opening, relapse, microtrauma of the TM joint and the mid-palatal suture, root resorption, tissue impingement and pain, and excessive tipping of anchorage teeth.21-25 SME procedures produce less tissue resistance around the circum-maxillary structures and

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therefore, improve bone formation in the intermaxillary sutures.16, 24-27 Quadhelix; the most frequently used SME technique, is preferable to many,for better muscle adaptation and less relapse.12-15,27Although itstreatment involve an average time of4-5 months, normal growth patterns can produce some dental and skeletal changes during that time.When the reported treatment changes were small, with significant standard deviations, the importance of the use of control group increased.19 Thus, it was recommendedto include a control group. The needed correction of the maxillary arch width was determined, adding an overcorrection of 3mmeach side, to controlrelapse potency. Hicks13 reported the amount of relapseis related to the retention procedure after expansion, he concluded that it would benecessary to use fixed retention for at least two months.Spillane and McNamara26 evaluated the stability of maxillary expansion in patientstreated during mixed dentition, recommended the use of a fixed retainer for 5 months, then a retainer for one year or more after the expander removal. In the current study table 1& 2revealsignificant increasein arch width at the end of A1 (P, .001) followed bya significant reductionduring the retentive(A2)(P, .001)and the follow-up periods (A3)(P, .01),but no case of relapse or a posterior crossbite atA3 was noted.This probably was due to the overcorrection and to the simple trainingthat was performed to withdraw the bad oro-facial muscles overactivities, sharing in the correction of forces affecting the maxillary width stability. Noting that all the study group individuals who complain from arch widthdeficiency, showed one or more oro-facial bad habits, which were simply instructed to be withdrawn and succeeded to be stopped during the patients` watching time. During the stabilization period of retention, andbecause of the flexibility of Quadhelix, there was reduction in the maxillary 1st molars lateral tipping, torqueing the posterior teeth, up righting them in position, reducing the relapse tendency and preserving the arch length.19,28,29 The mandibular width measurement showed alsoan increase. The slow correction the narrow maxilla fosters and gives enough time for this increase and releases the mandible to a normal transverse growth,this was confirmed by many researches.19,28 Table 3 revealed no effect on the lower facial height as angular and linear cephalometric measurements, this can be explained because of its slow and continuous action,13,15,16,18 unlike the
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rapid expanders, recent studies reveal their ability to increase the lower facial heigh,22-24giving an advantage for Quadhelix over the rapid expander.15, 19,28 As conclusion; Quadhelix is able to induce stable favorable changesin the width of the dental arches, with no increase in the lower facial height .The long-term clinical response to Quadhelix demonstrates a significant increase in the transpalatal width, confirming the validity of: overcorrection, the training to withdraw bad oral habits, and the one year retention periods; releasing the mandible to its normal growth.

REFERENCES
1. Sayin MO, Turkkahraman H. Comparison of dental arch and alveolar widths of patients with Class II, division I malocclusion and subjects with Class I ideal occlusion. Angle Orthod 2004; 74:356-360. 2. Bartzela T, Jonas I. Long-term stability of unilateral posterior crossbite correction. Angle Orthod 2007; 77(2): 237-43. 3. Petren S, Bondemark L, Soderfeldt B. A systematic review concerning early orthodontic treatment of unilateral posterior crossbite. Angle Orthod 2003; 73(5): 588-96. 4. Schiffman PH, Tuncay OC. Maxillary expansion: a meta-analysis. Clin Orthod Res 2001; 4(2): 86-96 5. Lux CJ, Conradt C, Burden D, Komposch G. Dental arch widths and mandibular-maxillary base widths in Class II malocclusions between early mixed dentition and permanent dentition. Angle Orthod 2003; 73: 674685. 6. Bigby M, Williams H. Appraising systematic reviews and metaanalyses. Arch Dermatol 2003; 139: 795 798. 7. Karaman AI. The effects of nitanium maxillary expander appliances on dentofacial structures. Angle Orthod 2002; 72(4): 344-54. 8. Chaconas SJ, Caputo AA. Observation of orthopedic force distribution produced by maxillary orthodontic appliances. Am J Orthod 1982; 82(6): 492-501. 9. Brin I, Ben-Bassat Y, Blustein Y. Skeletal and functional effects of treatment for unilateral posterior crossbite. Am J Orthod Dentofac Orthop 1996; 109 (2): 173-9. 10. Kutin G, Hawes RR. Posterior crossbites in the deciduous and mixed dentition. Am J Orthod 1969; 56: 491-504. 11. Ladner PT, Muhl ZF. Changes concurrent with orthodontic treatment when maxillary expansion is a primary goal. Am J Orthod Dentofac Orthop 1995; 108(2): 184-93. 12. Duarte MSO. Quad-helix appliance and its modalities. R Dental Press Ortodon Ortop Facial 2006; 11(2): 128-156. 13. Hicks EP. Slow maxillary expansion: a clinical study of the skeletal versus dental response to low magnitude force. Am J Orthod 1978; 73(2): 121-41. 14. Ricketts, R M. Logic and Keys to Bio Philosophy and Treatment Mechanics. 1986; American Institute for Bioprogresive education, Scottsdale AZ, p.98. 15. Boysen B, La Cour K, Athanasiou AE, et al. Threedimensional evaluation of dentoskeletal changes after

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16. 17.

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posterior cross-bite correction by quad-helix or removable appliances. Br J Orthod 1992; 19(2): 97107. Proffit WR, Fields HW, Sarver DM. Contemporary orthodontics. 4th ed. St. Louis: Mosby Elsevier; 2007. McDougall PD, McNamara JA Jr, Dierkes M. Arch width development in Class II patients treated with Frankel appliance. Am J Orthod Dentofac Orthop 1982; 1022. Lima AL, Filho RL, Bolognese AM. Long-term clinical outcome of rapid maxillary expansion as the only treatment performed in Class I malocclusion. Angle Orthod 2005; 75: 416420. Lagravere MO, Major PW, Flores-Mir C. Skeletal and dental changes with fixed slow maxillary expansion treatment; A systematic review JADA 2005; 136: 19499. Harrison JE, Ashby D. Orthodontic treatment for posterior crossbites. Cochrane Database Syst Rev 2001;1, CD000979. McNamara JA Jr, Baccetti T, Franchi L, Herberger TA. Rapidmaxillary expansion followed by fixed appliances: a long-term evaluationof changes in arch dimensions. Angle Orthod 2003; 73(4): 344-53. Chang JY, McNamara JA Jr, Herberger TA. A longitudinal study of skeletal side effects induced by rapid maxillary expansion. Am J Orthod Dentofac Orthop 1997; 112(3): 330-7. Ciambotti C, Ngan P, Durkee M, Kohli K, Kim H. A comparison of dental and dentoalveolar changes between rapid palatal expansion and nickel-titanium palatal expansion appliances. Am J Orthod Dentofac Orthop 2001; 119: 1120. Garib DG, Henriques JFC, Carvalho PEG, Gomes S C. Longitudinal effects of rapid maxillary expansion;a retrospective cephalometric study. Angle Orthod 2007; 77(3): 442-8. Akkaya S, Lorenzon S, Ucem TT. Comparison of dental arch and arch perimeter changes between bonded rapid and slow maxillary expansion procedures. Eur J Orthod 1998; 20(3):255-61. Spillane LM, McNamara JA Jr. Maxillary adaptations following expansion in the mixed dentition. Semin Orthod 1995; 1: 176187. Henry RJ. Slow maxillary expansion: a review of quad-helix therapy during the transitional dentition. ASDC J Dent Child 1993; 60(4): 408-13. Iseri H, Ozsoy S. Semirapid maxillary expansiona study of longterm transverse effects in older adolescents and adults. Angle Orthod 2004; 74:7178. McDonald JL, Shofer FS, Ghafari J. Effect of molar rotation on arch length. Clin Orthod Res 2001; 4(2): 79-85.

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Effect of casein phosphopeptide

Effect of casein phosphopeptide-amorphous calcium phosphate on surface roughness of a silorane-based and methacrylate-based composite resin (In vitro comparative study)
Baydaa Hussein, B.D.S., M.Sc. (1)

ABSTRACT
Background: When using a fluoridated agent for caries-preventive intervention, the clinician should be careful not to allow the agent to come into contact with the composite restorations since topically applied fluorides were found to induce adverse effects on the morphologic characteristics and composition of composite restorations. A new remineralizing agent "MI Paste" and its fluoridated form "MI Paste Plus" based on Recaldent technology (CPP-ACP) were developed. This study was conducted with the aim of assessing the effect of these new remineralizing agents on surface roughness of two types of composite resin materials. Materials and Method: Cylindrical specimens 12mm in diameter and 2mm in height were prepared from two types of composite resin materials: Filtek P90 (a silorane-based composite material) and Filtek Z350 XT (a methacrylatebased nanofill composite material). Each specimen was cured against a celluloid strip in a specially designed cylindrical mold using a QTH light curing unit for 40 seconds. Forty specimens were prepared for each composite type and subdivided into four subgroups of ten specimens each: Subgroup 1: without treatment, dry-stored in an incubator at 37 C for one week (control subgroup), Subgroup 2: without treatment, stored in deionized water in an incubator at 37 C for one week, Subgroup 3: treated with MI Paste once daily for one week, and Subgroup 4: treated with MI Paste Plus once daily for one week. Surface roughness of the specimens was obtained with a surface profile testing machine, which used the roughness average (Ra) to assess surface changes. Several measurements were taken for each specimen and the mean value of these measurements on one specimen was regarded as the Ra of that specimen. The mean Ra value of each subgroup was then calculated. Results: The results of this study showed statistically non-significant differences among the different subgroups of Filtek P90 composite resin material. Concerning Filtek Z350 XT composite resin material, the results showed a statistically highly significant difference in surface roughness between the subgroup stored in deionized water and the control one, with statistically non-significant difference between the subgroups treated with MI Paste and MI Paste Plus and the control subgroup. Comparison of significance between the corresponding subgroups of both composite types revealed statistically non-significant differences except for subgroup 2 which showed a statistically significant higher surface roughness in Filtek Z350 XT than Filtek P90. Conclusions: The daily application of the MI Paste and MI Paste Plus for one week had non significant effect on surface roughness of the silorane-based composite resin material Filtek P90. On the other hand, the application of these agents caused surface smoothening of the nanofilled methacrylate-based composite resin material Filtek Z350 XT. Key words: MI Paste, MI Paste Plus, surface roughness, silorane, Filtek P90, Filtek Z350 XT. (J Bagh Coll Dentistry 2012;24(3):106-112).

INTRODUCTION
Composites are widely used in dentistry due to their excellent esthetic, strength, moderate cost compared to that of ceramics, and ability to micromechanically bond with the adequate tooth structure(1). Composite materials have improved greatly since their introduction and there is a general shifting away from amalgams toward composite resins (2). Composites consist of fillers embedded in a chemically-reactive organic resin matrix. Improvements in the composite materials were achieved, to a great extent, by optimizing the fillers (3). One of the most important advances of the last few years in the field of filler technology is the application of nanotechnology to dental composites (4).
(1) Lecturer, Department of Paedodontic and Preventive Dentistry, College of Dentistry, University of Baghdad.

The nanotechnology is aimed to improve the physical and mechanical properties of the composite restoratives. Moreover, inclusion of smaller filler particles as nano-size in the final formulation of the composites results in reduction of composite's shrinkage and improving their total mechanical properties(5). However, the shrinkage intrinsic to the methacrylate resin has remained a major challenge. Therefore, exchanging the resin seems the most promising pathway to solve the shrinkage problem (3). With the same objective of reducing the polymerization shrinkage, different high molecular weight matrix resin compositions have been employed. One of theses new matrix resin systems is silorane. Siloranes are a totally new class of compounds for use in dentistry. The name silorane derives from its chemical building blocks siloxanes and oxiranes. The combination of the two chemical building blocks of siloxanes and oxiranes provides a biocompatible, hydrophobic

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and low-shrinking silorane. This innovative resin matrix represents the major difference of siloranebased materials compared to conventional methacrylates (6). Application of fluorides agents to the tooth surface is effective in preventing dental caries. The most commonly used professionally applied fluoride agents are fluoride varnishes and acidulated phosphate fluoride gel (APF)(7). APF agents have been used in caries-preventive interventions for more than two decades, and their effectiveness is widely known. In recent years, APF agents have been used by dentists to treat adults as well as infants and young children because of effectiveness and convenience. However, APF agents have been shown to have several practical disadvantages, one being that the hydrofluoric acid generated in APF agents is known to dissolve porcelain surfaces and negatively affect glass-ionomer cement and composite materials. Furthermore, the color of some composite materials is reportedly influenced by repeated use of a fluoride varnish(8). On the other hand, topically applied stannous fluoride and sodium fluoride gels have been found to induce adverse effects on the surface roughness and morphologic characteristics of composite materials(9). An increase in the surface roughness and discoloration has been used as a criterion to assess and predict the clinical deterioration of restorations constructed with different types of materials (10). Surface roughness increase of composite resins has many negative effects, such as susceptibility to staining, recurrent caries, plaque retention, and increase the colonization and adhesion of bacteria on composite resins. The smoother the resin composite, the more comfortable it may be to the patient (11). The mean critical value of surface roughness for bacterial colonization of several dental materials has been measured as 0.2m. Above this value, increased bacterial colonization and dental plaque accumulation may occur and thus favoring the development of both caries and periodontal inflammation on one hand and could damage the restorative material surface from the other hand (10) . A new remineralizing agent named GC Tooth Mousse or MI Paste * based on Recaldent technology (CPP-ACP) was developed to capitalize the anti-caries properties of milk. The anticariogenic properties of milk and its products have been attributed to direct chemical effect of phosphoprotein casein and calcium phosphate. It has been suggested that casein phosphopeptides (CPP) have the ability to stabilize calcium

phosphate in solution by binding amorphous calcium phosphate (ACP) with their multiple phosphoserine residues, thereby allowing the formation of small CPP-ACP clusters. CPP-ACP prevents tooth demineralization and enhances remineralization (12). An analysis of the chemistry of demineralization and remineralization indicates that a major source of mineral loss in the caries process is the destruction of apatite with the creation of water as a by-product, and the leakage of a neutral species calcium hydrogen phosphate across a porous enamel surface. When placed on the surface of a tooth, CPP-ACP interacts with hydrogen ions and forms the same species calcium hydrogen phosphate which, under a diffusion gradient, can enter into the tooth, react with and consume the water to produce enamel mineral, thereby removing subsurface mineral defects. It has too many applications in dentistry such as the treatment of white spot lesions, treatment of tooth sensitivity, after bleaching and after removal of orthodontic appliances, caries stabilization, root surface caries, fluorosis, and to protect the very young child (13). This study was conducted with the aim of assessing the effect of these new remineralizing agents on surface roughness of a silorane-based and methacrylate-based composite resin materials.

MATERIALS AND METHOD


Two types of composite resin were used in this study: (1) Filtek P90 (a silorane-based low shrink posterior composite, shade A2) (3M ESPE, USA) and (2) Filtek Z350 XT (a methacrylatebased nanofill composite, shade A2 body) (3M ESPE, USA). Filtek P90 is a light-curing, radioopaque, silorane-based composite for the posterior area. It contains 55% volume (76% weight) inorganic fillers of quartz and yttrium fluoride with a particle size between 0.1 and 2 m (mean 0.47 m). It contains a hydrophobic resin matrix composed of siloxane and oxirane (14). Filtek Z350 XT is a universal restorative material designed for use in anterior and posterior restorations. The fillers are a combination of a non-agglomerated/non-aggregated 4-11 nm zirconia filler and an aggregated zirconia/silica cluster filler (comprised of 20 nm silica and 4-11 nm zirconia particles). The inorganic filler loading is about 63.3% by volume (78.5% by weight). It contains bis-GMA, UDMA, TEGDMA, PEGDMA and bis-EMA resins (15).
*GC MI PasteTM / GC MI Paste PlusTM and GC Tooth Mousse / GC Tooth Mousse Plus are synonymous. In the United States, it is called MI Paste or MI Paste Plus. In Australia, it is called Tooth Mousse or Tooth Mousse Plus.

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Two remineralizing agents were used in this study: GC MI PasteTM and GC MI Paste PlusTM (GC America Inc, USA). MI Paste and MI Paste Plus contain the same ingredients except that the MI Paste Plus contains 0.2% sodium fluoride (900 ppm). The ingredients are: pure water, glycerol, CPP-ACP, D-sorbitol, CMC-Na, Propylene glycol, Silicone dioxide, Titanium dioxide, Xylitol, Phosphoric acid, Flavoring, Zinc oxide, Sodium saccharin, Ethyl p-hydroxybenzoate, Propyl p-hydroxybenzoate, and Butyl phydroxybenzoate (16). Sample Preparation A specially designed cylindrical mold (12mm diameter and 2mm height) was used to prepare cylindrical composite specimens. A celluloid strip was placed on a flat glass slide on top of a white background. The mold was then placed on it and slightly overfilled in one increment with one of the composite materials and a second celluloid strip was then placed on top of the mold and overlaid with another glass slide with the application of 100gm load to extrude excess material. The top slide was then removed and the composite resin light-cured with Coltolux 5 conventional quartz-tungsten-halogen (QTH) light curing unit (Coltene Whaledent, France) for 40 seconds following the manufacturer's instructions. The tip of the light curing unit was placed in direct contact with the overlaid celluloid strip. After light curing, the overlaid celluloid strip was removed and the specimen was taken from the mold. No polishing of the specimens was done. Sample Grouping For each type of composite, 40 specimens were prepared and divided into 4 subgroups of ten specimens each: -Subgroup 1: without treatment, dry-stored in an incubator at 37!C for one week. -Subgroup 2: without treatment, stored in deionized water in an incubator at 37!C for one week. -Subgroup 3: treated with MI Paste once daily for one week, stored in deionized water in an incubator at 37!C. -Subgroup 4: treated with MI Paste Plus once daily for one week, stored in deionized water in an incubator at 37!C. Subgroup 1 represented a control group which provided baseline measurements with which surface roughness of other subgroups would be compared. Subgroup 2 represented a negative control group to exclude the effect of deionized water on surface roughness as subgroup 3 and subgroup 4 were stored in deionized water.

The method of application of MI Paste and MI Paste Plus was as follows: Eight milligrams were freshly squeezed from the tube and placed on the top surface of the composite specimens for four minutes. Then each specimen was thoroughly rinsed with deionized water for two minutes. The specimens were then restored in deionized water for the next day at a temperature of 37!C in an incubator. This procedure was repeated daily for one week (17). . Surface Roughness Measurement Surface roughness of the specimens was obtained with a surface profile testing machine (Hand-Held Roughness Tester, TR200, TimeGroup Inc. China). The roughness average (Ra) was used to assess surface changes, which is the arithmetic mean of roughness profile within a measuring length. Several measurements were taken for each specimen and the mean value of these measurements on one specimen was regarded as the Ra of that specimen. The Ra values were automatically calculated by the profilometer. The mean Ra value of each subgroup was then calculated (18).

RESULTS
The descriptive statistics (mean and standard deviation) of surface roughness (Ra value in m) of the different subgroups of Filtek P90 and Filtek Z350 XT composite resin materials and the comparison of significance of the different subgroups of each composite resin material by One-way ANOVA test are presented in Table 1. From this, it can be seen that the mean Ra values of all subgroups of Filtek P90 composite resin material were higher than the mean Ra values of their corresponding subgroups of Filtek Z350 XT composite resin material except for the subgroup in which the composite resin specimens were stored in deionized water, which showed a higher mean Ra value in Filtek Z350 XT than Filtek P90 composite resin material. Comparison of significance among the different subgroups of Filtek P90 and Filtek Z350 XT composite resin materials by One-way ANOVA test revealed statistically non-significant differences among the different subgroups of Filtek P90 composite resin material (p>0.05), and a statistically highly significant difference among the different subgroups of Filtek Z350 XT composite resin material (p<0.01). Further analysis among the different subgroups of Filtek Z350 XT composite resin material using LSD (Least Significant Difference) test revealed a statistically highly significant difference in surface roughness between the subgroup stored in deionized water and the

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untreated control subgroup (p<0.01), while statistically non-significant difference between the subgroups treated with MI Paste and MI Paste Plus and the untreated control subgroup (p>0.05) as shown in Table 2. In addition, there was a statistically highly significant difference in surface roughness between the subgroup stored in deionized water and the subgroups treated with MI Paste and MI Paste Plus (p<0.01), with statistically non-significant difference between the latter two subgroups (p>0.05). Comparison of significance between the different subgroups of Filtek P90 composite resin material and their corresponding subgroups of Filtek Z350 XT composite resin material using student t-test (Table 3) revealed statistically non-significant differences (p>0.05) except for the subgroup stored in deionized water, which showed a statistically significant higher surface roughness in Filtek Z350 XT than Filtek P90 (p>0.01).

DISCUSSION
Topical fluoride treatment has been advocated for patients with a high incidence of caries, and daily applications were shown to reduce the caries index and arrest existing lesions (19). However, topically applied fluorides were found to induce adverse effects on the morphologic characteristics and composition of composite restorations(9), and thus it is recommended when using a fluoridated agent for caries-preventive intervention that the clinician should be careful not to allow the agent to come into contact with the composite restorations (8).. Due to the above mentioned detrimental effects of topically applied fluorides on composite materials and with the introduction of the new remineralizing agent "MI Paste" and its fluoridated form "MI Paste Plus" based on Recaldent technology (CPP-ACP), this study was conducted to assess the effect of these new remineralizing agents on surface roughness of a silorane-based and methacrylate-based composite resin materials. MI Paste Plus offers the same benefits of regular MI Paste, enhanced with 0.2% sodium fluoride to further promote remineralization and protect teeth from caries development. MI Paste Plus is the only product that provides the correct bio-available ratio of: 5-calcium, 3-phosphate, 1fluoride, the same ratio that is found in healthy enamel. For children under the age of 6 years, it is recommended to use MI Paste which does not contain fluoride, while MI Paste Plus is recommended for patients 6 years and older (13).

The selection of Filtek P90 and Filtek Z350 XT composite resin materials for use in this study is due to their different filler and resin matrix formulations. and each one represents one of the recent developments in composite technology: Filtek P90 which is a siloranebased low-shrinking material (14), and Filtek Z350 XT which is one of the newest products based on nanotechnology (15). The surface roughness property of any material is the result of an interaction of multiple factors. Some of them are intrinsic that are related to the material itself, such as the filler (type, shape, size and distribution of the particles), the type of resinous matrix as well as the ultimate degree of cure reached, and the efficiency at the filler/matrix interface. Other factors are extrinsic that are associated with the type of polishing system used (20). In this study, the composite specimens were cured against a celluloid strip and were not polished in order to exclude the effect of the extrinsic factors on the surface roughness as the tested composite resins differ in their inorganic component (the type, size, and amount of filler loading) which affects their polishability. In addition, it has been shown that the experimental finishing and polishing procedures are inherently destructive to the restoration and may lead to micro-crack formation. Moreover, it has been shown that the smoothest surface was obtained when the composite resin is cured against a clear matrix(21). Deionized distilled water was used as a storage medium because it simulates the wet oral environment provided by saliva and water. Saliva is a dilute fluid comprising 99% water and the concentration of the dissolved solids (organic and inorganic) are characterized by wide variations, both between individuals and within a single individual. Due to these variations, water was used as a storage medium in this study. One week storage of the specimens in deionized water was done in this study since it has been found that the maximum amount of water absorption occurs in the first week(22). The increase in surface roughness of Filtek Z350 XT composite resin material after one week of storage in deionized water as compared with the control could be attributed to water sorption. Water could cause softening of the polymer component by swelling the network and reducing the frictional forces between polymeric chains, resulting in hydrolytic degradation of the resin matrix with micro-cracks formation and exposure of the filler particles(23). It has been found that the polymerization shrinkage of the methacrylate-

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based composite materials and water sorption with the diffusion of moisture through the resin component lead to the initiation and propagation of micro-cracks in the resin matrix(24). Filtek Z350 XT contains bis-GMA, UDMA, TEGDMA, PEGDMA and bis-EMA resins(15). Except for bisEMA, which is an ethoxylated version of bisGMA, other molecules (bis-GMA, UDMA, and TEGDMA) have hydroxyl groups which promote water sorption. Moreover, the incorporation of TEGDMA in Filtek Z350 XT composite material resulted in an increase in water uptake as this monomer presents higher hydrophilicity when compared with bis-GMA and UDMA(25). This finding is in agreement with the results of Yesilyurt et al.(2009)(25) and Catelan et al. (2010)(23) who observed a significant softening of Filtek Z350 XT after immersion in deionized water for 7 days and 28 days, respectively. On the other hand, Yap et al. (2001)(26) reported that zirconia glass fillers were also susceptible to aqueous attack. Filtek Z350 XT contained zirconia/silica fillers which could be another cause for the increased roughness after water immersion. However, this disagrees with Nayif et al. (2004)(27) who claimed that deionized distilled water is the least solution that causes filler debonding in comparison with the oral environment. When MI Paste and MI Paste Plus were applied to Filtek Z350 XT composite resin, it was found that the surface roughness was decreased as compared to composite resin specimens that were stored in deionized water, a decrease to the limit that it became statistically non-significant when compared with the untreated composite resin specimens of the control subgroup. This finding would suggest that the MI Paste and MI Paste Plus might have chemically attacked the inorganic filler particles, counteracting the water sorption effect of deionized water which acted on the resin matrix, resulting in surface smoothening. This effect could be attributed to the presence of phosphoric acid in the chemical composition of the MI Paste and MI Paste Plus, which might caused etching of the filler particles exposed after disintegration of the superficial resin layer as a result of water sorption, and the presence of silicone dioxide and titanium oxide which act as abrasives. Phosphoric acid, silicone dioxide, and titanium oxide can attack the inorganic particles, and when these are very small, as in the nanofiller composite resin, the size of the defects created are practically imperceptible, resulting in surface smoothening. Another possible cause for such a surface smoothening effect could be attributed to that

when MI Paste and MI Paste Plus were applied, they were retained in the micro-porosities created by water sorption resulting in surface smoothening(28). However, this finding disagrees with the results of Prabhakar et al.(2009)(7) who found that the application of GC Tooth Mousse causes minimal changes in color and surface roughness of composite and glass ionomer cement. Such contrary in results could be attributed to the difference in the composite resin type used which was Z250, a microhybrid composite resin, which has larger filler particles than Filtek Z350 XT composite, so the size of the defects created after the application of MI Paste and MI Paste Plus would be relatively larger as compared with the nanofilled composite resin Filtek Z350 XT used in this study. The statistically non-significant difference in surface roughness of Filtek Z350 XT composite resin specimens treated with MI Paste and those treated with the MI Paste Plus despite the fluoride content of the latter, whose surface roughening effect on composite resin is well documented, could be attributed to that the MI Paste Plus contains sodium fluoride which is neutral (pH=7) and its concentration is lower than that used in the sodium fluoride gel intended for professional application. The concentration of sodium fluoride in the MI Paste Plus is 0.2% (900 ppm) which is the equal to fluoride concentration in a tooth paste, while its concentration in the gel intended for professional application is 2%. Concerning Filtek P90 composite resin, the statistically non-significant differences in surface roughness after one week of immersion in deionized water and after application of the MI Paste and MI Paste Plus as compared with the surface roughness of the control subgroup could be related to its novel chemistry. Siloranes are a totally new class of compounds for the use in dentistry. The name silorane derives from its chemical building blocks siloxanes and oxiranes. Siloxanes are well known by their distinct hydrophobicity. By incorporating the siloxanes into the dental silorane resin, this property was transferred to the Filtek P90 composite. The oxirane polymers are known for their low shrinkage and the outstanding stability toward many physical and chemo-physical forces and influences. The combination of the two chemical building blocks of siloxanes and oxiranes provides the biocompatible, hydrophobic and lowshrinking silorane base of Filtek P90 Low Shrink Posterior Restorative. This innovative resin matrix represents the major difference of Filtek P90 restorative compared to conventional methacrylates(6). While Filtek Z350 XT resin

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matrix is composed of molecules with hydroxyl groups which promote water sorption, Filtek P90 composite has 3,4-epoxycyclohexylcyclopolymethylsiloxane. This backbone imparts hydrophobicity, thereby curtailing water sorption(25). On the other hand, in addition to the hydrophobicity of the silorane resin, the difference in filler composition could be another contributing factor for the statistically nonsignificant difference in surface roughness of Filtek P90 after storage in deionized water as reported by Yap et al. (2001)(26). Filtek P90 contains quartz fillers which are more resistant to aqueous attack than the zirconia/silica fillers of Filtek Z350 XT composite material. The lower surface roughness of Filtek Z350 XT at baseline as compared with Filtek P90, although statistically not significant, is due to the smaller mean particle size of Filtek Z350 XT which is classified as a nanofilled composite, while Filtek P90 is classified as a microhybrid composite with larger mean particle size of 0.47 m. However, the statistically non-significant difference in surface roughness could be attributed to the lower filler volume of Filtek P90 as compared with the Filtek Z350 XT. Moreover, both materials were cured against a celluloid strip which produced a smooth surface. However, this finding disagrees with the results of Jehad (2011)(9), who found that the surface roughness of Filtek P90 composite resin specimens was lower than that of Filtek Z350 XT composite resin specimens. Such a difference in results could be attributed to that the composite resin specimens were polished prior to surface roughness measurement, while in our study no polishing was done. An interesting finding in this study is that the average surface roughness of both materials in all circumstances didn't exceed the critical threshold value of 0.2m, which allows plaque accumulation. Surface roughness may be clinically relevant only when the Ra values were above this critical threshold. Thus it can be stated that the application of the MI Paste or MI Paste Plus as a remineralizing agent has no detrimental effect on surface roughness of silorane-based composite material. On the other hand, these agents could cause a surface smoothening effect of nanofilled methacrylate-based composite resin material, so when the MI Paste or MI Paste Plus would be used for any of the intended indications for their use the clinician could allow these agents to come into contact with the composite restorations in contrary to topically applied fluorides which were found to induce adverse

effects on the morphologic characteristics and composition of composite restoration As a conclusions, the daily application of the MI Paste and MI Paste Plus for one week had non significant effect on surface roughness of the silorane-based composite resin material Filtek P90. On the other hand, the application of these agents caused surface smoothening of the nanofilled methacrylate-based composite resin material Filtek Z350 XT.

REFERENCES
1. Hunan LU, Roeder LB, Lei L, Powers JM. Effect of surface roughness on stain resistance of dental resin composites. J Esthet Restore Dent 2005;17(2):102-9. 2. Shenoy A. Is it the end of the road for dental amalgam. A critical review? J Conserv Dent 2008;11:99107. 3. Filtek LS Low Shrink Posterior Restorative. Technical Product Profile. 3M ESPE. 4. Beun S, Glorieux T, Devaux J, Vreven J, Leloup G. Characterization of nanofilled compared to universal and microfilled composites. Dent mater 2007; 23 (1): 51-9. 5. Condon JR, Ferracane JL. Reduced polymerization stress through non-bonded nanofiller particles. Biomater 2002; 23(18): 3807-15.[IVSL]. 6. Weinmann W, Thalacker C, Guggenberger R. Siloranes in dental composites. Dent Mater 2005;21: 68-74. 7. Prabhakar AR, Mahantesh TB, Vishwas TD, Aparna Kabaded. Effect of surface treatment with remineralizing on color the stability and roughness of esthetic restorative materials. Rev Cln Pesq Odontol. 2009; 5(1): 19-27. 8. Tanoue N, Soeno K, Kawasaki K, Atsuta M. Influence of acidulated phosphate fluoride solution on the color stability of indirect composites. J Prosthet Dent 2004; 92: 343-7. 9. Jehad RH. Comparison of the effect of stannous fluoride and sodium fluoride on surface roughness of Silorane and methacrylate based restorative material using light polarizing microscope. J Bagh College Dentistry 2011; 23(2): 31-5. 10. Yip KH-K, Peng D, Smales RG. Effect of APF gel on the physical structure of compomers and glass ionomer cements. Oper Dent 2001;26(3):231-8. 11. Karkmaz Y, Ozel E, Attar N, Aksoy G. The influence of one-step polishing systems on the surface roughness and microhardness of nanocomposites. Oper Dent 2008;33:44-50. 12. Oshiro M, Yamaguchi K, Takamizawa T, Inage H, Watanabe T, Irokawa A. Effect of CPP-ACP paste on tooth mineralization: an FE-SEM study. J Oral Science 2007;49(2):115-120. 13. MI Paste MI Paste Plus Brochure. Anthology of Applications. Professor Laurie Walsh / University of Queensland, Australia. GC America Inc. 3737 W. 127th St. Alsip, IL 60803 www.gcamerica.com Copyright 2007. 14. Leaflet of Filtek P90 Low Shrink Posterior Restorative. 3M ESPE, USA and 3M ESPE AG, Germany. 15. Leaflet of Filtek Z350 XT Universal Restorative. 3M ESPE, USA.

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16. Leaflet of MI Paste and MI Paste Plus. GC America Inc, USA. 17. Fahad AH. Effect of Casein PhosphopeptideAmorphous Calcium Phosphate on the microhardness and microscopic features of the sound enamel and initial caries-like lesion of permanent teeth, compared to fluoridated agents. A Master Thesis 2010. College of Dentistry, University of Baghdad. 18. Badra VV, Faraori JJ, Ramos RP, Palma-Dibb RG. Influence of different beverages on the microhardness and surface roughness of resin composites. Oper Dent 2005; 30(2): 309-14. 19. Dreizen S, Daly T, Drane JB. Prevention of xerostomia-related dental caries in irradiated cancer patients. J Dent Res 1997;56:99-104. 20. Marghalani HY. Effect of finishing/polishing systems on the surface roughness of novel posterior composites. J Esthet Rest Dent 2010; 22(2): 127-38. 21. Filho NH, d'Azevedo MT, Nagem HD, Marsola FP. Surface roughness of composite resins after finishing and polishing. Braz Dent J 2003;14(1):37-41. 22. Huang C, Cheung G. Hygroscopic expansion of compomer and a composite on artificial gap reduction. J Dent 2002; 30: 11-9.

23. Catelan A, Briso ALF, Sundfeld RH, Dos Santos PH. Effect of artificial aging on the roughness and microhardness of sealed composites. J Esthet Rest Dent 2010;22(5):324-31. 24. Lee SY, Greener EH, Menis DL. Detection of leached moieties from dental composites in fluids simulating food and saliva. Dent Mater 1995;11:348-53. 25. Yesilyurt C, Yoldas O, Altintas SH, Kusgos A. Effect of food-simulting liquids on the mechanical properties of a silorane-based dental composite. Dent Mater J 2009:28(3):362-67. 26. Yap AU, Tan SH, Wee SS, Lee CW, Lim EL, Zeng Y. Chemical degradation of composite restoratives. J Oral Rehabil 2001;28:1015-21. 27. Nayif MM, Suliman AA, Ismael SA. Evaluation of composite resin surface roughness after two years of storage in deionized water. Iraqi J Oral and Dent Sc 2004;3(1):8-12. 28. Botta AC, Mollica FB, Ribeiro CF, Mximo deAraujo MA, Di Nicol R, Balducci I. Influence of topical acidulated phosphate fluoride on surface roughness of human enamel and different restorative materials. Rev odonto cinc 2010;25(1):83-7.

Table 1: Surface roughness (m) (mean standard deviation ) of different subgroups of Filtek P90 and Filtek Z350 XT composite resin materials
Mean Filtek P90 S.D Mean Filtek Z350 XT S.D Without Treatment Deionized Water MI Paste MI Paste Plus F Significance 0.0568 0.0572 0.0566 0.0570 0.013 .998 (NS) 0.0065 0.0064 0.0081 0.0071 0.0525 0.0655 0.0530 0.0536 6.218 .002 (HS) 0.0073 0.0079 0.0078 0.0084

Table 2: LSD test for comparison of surface roughness among the different subgroups of Filtek Z350 XT composite resin material
Filtek Z350 XT Without Treatment Deionized Water 0.001 (HS) 0.885 (NS) 0.758 (NS) 0.001 (HS) 0.002 (HS) 0.876 (NS) MI Paste MI Paste Plus

Table 3: Student t-test comparison between different subgroups of Filtek P90 composite resin material and their corresponding subgroups of Filtek Z350 XT composite resin material
Subgroups t Significance P90 (Without Treatment) Z350 XT (Without Treatment) 1.240 .246 (NS) -2.300 .047 (S) P90 (Deionized Water) Z350 XT (Deionized Water) 1.243 .245 (NS) P90 (MI Paste) Z350 XT (MI Paste) 0.848 .419 (NS) P90 (MI Paste Plus) Z350 XT (MI Paste Plus)

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Effect of repeated pregnancies

Effect of repeated pregnancies on periodontal health status


Baydaa A. Yas, B.D.S., M.Sc., Ph.D. (1)

ABSTRACT
Background: Pregnant women are particularly susceptible to gum and periodontal disease. The aim of this study was to assess the effect of repeated pregnancies on the periodontal health status. Materials and methods: This study evaluated the oral hygiene and periodontal status (plaque index, calculus index, gingival index and probing pocket depth) of 224 pregnant women in Baghdad governorate (112 primigravidae and 112 multigravidae), in relation to socio-demographic and clinical variables. The age range was 20-25 years. Results: No significant differences were found in oral hygiene and gingival health condition between primigravidae and multigravidae women. Also probing pocket depth whether present or absent revealed equal percentage in both. The majority of the pregnant women was of urban residence and had lower educational level. Higher percentage of them visits the dental clinic for relieving pain only. Conclusion: Multiple pregnancies had unpronounced influence on the periodontal health status. Future studies should include the clinical attachment level measurement. Key words: Primigravidae, multigravidae, periodontal health. (J Bagh Coll Dentistry 2012;24(3):113-115).

INTRODUCTION
Pregnancy is a delicate process involving complex physical and physiological changes (1). Pregnant women do experience metabolic changes which could, sometimes, appreciably alter their oral metabolism. These include alterations in hormone levels, microbial strains present in the oral cavity, immune response, and cellular metabolism (2, 3, 4).The increase in estrogen and progesterone levels result in change in vascular permeability that lead to gingival swelling and an increase in crevicular fluid levels. In addition, production of prostaglandins is stimulated, possibly generating an increase in gingival inflammation (5, 6) . Repeated pregnancies have been shown to have an effect on the periodontal health. Different opinions exist; some found that multigravidae women had significantly higher gingival index and probing pocket depth scores compared with primigravidae women (7). While others reported that neither the period of pregnancy, nor the parity have an influence in the importance of the periodontal hurts (8). No Iraqi studies could be found related to this subject. The aim of this study was to evaluate the periodontal health status in relation to the number of pregnancies and a series of socio-demographic and clinical variables.

Those women were chosen at random (simple random sampling) from the Maternal and Child Health Care Centers distributed in the various geographical areas within Baghdad governorate. Prior to clinical examination women were interviewed about their socio-demographic (i.e. age, area of residency, and the educational level) and clinical background (i.e. trimester and frequency of dental visits during pregnancy). The educational level was divided into two groups: lower educational level (i.e. Illiterate, primary, intermediate and secondary school) and higher educational level (i.e. Diploma, college and postgraduate). All pregnant women were examined in a dental chair using a dental mirror, dental explorer (No.00, Derfla, West Germany), and Williams periodontal probe. Plaque index system (PlI) (9), calculus index system (CalI) (10) and gingival index system (GI) (11) were used for recording the oral cleanliness and gingival health condition respectively. Also probing pocket depth (PPD) was measured (12). Six Ramfjord teeth were examined. Data analyses were conducted by the application of the Statistical Package for Social Science (SSPS version 11). The Student's t-test and one-way ANOVA were applied. Probability was accepted at levels of 5%.

RESULTS
Socio-demographic and clinical descriptions of the sample were reported in Table (1). The majority of the pregnant women were of urban residence. Also a higher percentage of them had lower educational level and only small proportion had higher educational level. More than half of primigravidae and multigravidae women were in their second trimester of pregnancy. Also higher percentage of both primigravidae and multigravidae women visit the dental clinic only for relieving pain. Table (2) revealed statistically

MATERIALS AND METHODS


The sample consisted of 224 pregnant women with an age range of 20-25 years. The sample was equally divided into 112 primigravidae women (i.e. first pregnancy) and 112 multigravidae women (i.e. second pregnancy or more).

(1) Lecturer. Department of Paedodontic and Preventive Dentistry, College of Dentistry, University of Baghdad.

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no significant differences in oral hygiene and gingival health condition as measured by PlI, CalI and GI between primigravidae and multigravidae women (P>0.05). Similarly, no significant differences in oral hygiene and gingival health condition were found among different trimesters of pregnancy (P>0.05), (Table 3). For both primigravidae and multigravidae women, the percentage of probing pocket depth equal to 4 mm or more (score 1) was very low, while the majority of them had probing pocket depth that is less than 4 mm (score 0). Also probing pocket depth whether present (score 1) or absent (score 0) revealed equal percentages in both (Table 4).

some effect on the physiology of gingival tissues so that the inflammatory response of the host to bacterial plaque agents increases. In summary repeated pregnancies had undefined effect on the periodontal health status. Further studies are needed that should include the clinical attachment level measurement.

REFERENCES
1. Gajendra S, Kumar JV. Oral health and pregnancy: a review. NY State Dent J 2004; 70(1):40-4. 2. Sooriyamoorthy M, Gower DB. Hormonal influence of gingival tissue: relationship to periodontal disease. J Clin Periodontol 1989; 16:201-8. 3. Raber-Durlacher JE, Zeijlemaker WP, Meinesz AAP, Abraham-Inpijn L. CD4 to CD8 ratio and in vitro lymphoproliferative responses during experimental gingivitis in pregnancy and postpartum. J Periodontal 1991; 62: 663-7 (IVSL). 4. Amar S, Chung KM. Influence of hormonal variation on the periodontium in women. Periodontol 2000 1994; 6: 79-87. 5. Zachariasen RD. The effect of elevated ovarian hormones on periodontal health: Oral contraceptives and pregnancy. Women Health 1993; 20: 21-30 (IVSL). 6. Mariotti A. Sex steroid hormones and cell dynamics in the periodontium. Crit Rev Oral Biol Med 1994; 5: 273. 7. Taani DQ, Habashneh R, Hammad MM,Batieha A. The periodontal status of pregnant women and its relationship with sociodemographic and clinical variables. J Oral Rehab 2003; 30: 440-5. 8. Ahnoux A, Aoussi EL, Anongba DS, Kone D, el Radi T, Brou E. Pregnancy and periodontal health. Study of 133 pregnant women. Odontostomatol Trop 2003; 26 (102): 37-40. 9. Silness J, Le H. Periodontal disease in periodontal disease in pregnancy . Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964; 22: 121-35. 10. Ramfjord SP. Indices for prevalence and incidence of periodontal disease. J Periodontol 1959; 30: 51-9. 11. Le H, Silness J. Periodontal disease in pregnancy !. Prevalence and severity. Acta Odontol Scand 1963; 21: 533-51. 12. Salameh RM. The periodontal status during pregnancy and intake of contraceptives. Master Thesis, College of Dentistry, University of Baghdad. 2000. 13. Suliaman AW. Oral health status and cariogenic microflora during pregnancy. Master Thesis, College of Dentistry, University of Baghdad. 1995. 14. Scheutz F, Baelum V, Matee MI, Mwangosi I. Motherhood and dental disease. Community Dent Health 2002; 19 (2): 67-72. 15. Durdyniiazov MK, Alimskii AV. The social hygiene aspects of oral morbidity in multiparous women. Stomatologiia 1993; 72(1):60-5. 16. Khader YS, Rice JC, Lefante JJ. Factors associated with periodontal disease in dental teaching clinic population in northern Jordan. J Periodontol 2003; 74 (11):1610-7. 17. Machuca G, Khoshfeiz O, Lacalle JR, Machuca C, Bullon P. The influence of general health and sociocultural variables on the periodontal condition of pregnant women. J periodontol 1999; 70:779-85.

DISCUSSION
Result of this study clearly demonstrates that dental plaque and calculus accumulations were almost similar in primigravidae and multigravidae women, the differences were statistically not significant between the two groups. This may be attributed to the lower educational level that those women had which might lead to poor dental attendance and negligence of their oral hygiene. Concerning gingival inflammation, no significant difference could be found between the two groups. This may be related to the transient nature of pregnancy-associated gingivitis that is reversed after delivery (13). Also probing pocket depth scores were similar in both primigravidae and multigravidae women, this could be explained by the fact that the oral cleanliness and gingival health were almost similar in both groups. This result agree with Ahnoux et al study (8) in Ivory Coast, but disagree with Scheutz et al (14) and Durdyniiazov and Alimskii (15) studies in Tanzania and Turkmenistan respectively, who reported an association between the number of children and periodontal destruction. Results also revealed that the majority of the pregnant women had probing pocket depth that is less than 4mm, while the minority had probing pocket depth equal to 4mm or more and this could be explained that in the current investigation pregnant women examined were young since the age is one of the risk factors or indicators of the periodontal disease (16). Unfortunately, the clinical attachment level was not included, as it might provide more precise information. Data collected in this study showed no significant difference in oral hygiene and gingival health condition among various trimesters of pregnancy, this result was in accordance to that of Ahnoux et al study (8), but in reverse to Machuca et al study (17), as they reported that the trimester of pregnancy, in some circumstances, may have
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Table 1: Distribution of primigravidae and multigravidae women according to sociodemographic and clinical variables.
Variable Socio-demographic variables Age group 20-22 years 23-25 years Total Place of residence Urban Periurban Total Educational level Low level High level Total Clinical variables Trimester First Second Third Total Frequency of dental visits every three months Once a month On pain No need Total Primigravidae Multigravidae No. % No. %

66 46 112 84 28 112 97 15 112

58.93 41.07 100 75 25 100 86.61 13.39 100

43 69 112 80 32 112 103 9 112

38.39 61.61 100 71.43 28.57 100 91.96 8.04 100

17 69 26 112 3 12 54 43 112

15.18 61.61 23.21 100 2.68 10.71 48.21 38.39 100

17 61 34 112 1 9 60 42 112

15.18 54.46 30.36 100 0.89 8.04 53.57 37.5 100

Table 2: Oral variables (PlI, CalI, and GI) of primigravidae and multigravidae women.
Oral variables PlI CalI GI primigravidae multigravidae Mean SD Mean SD 0.86 0.35 0.92 0.39 1.91 0.43 1.89 0.39 0.19 0.23 0.19 0.30

Table 3: Oral variables (PlI, CalI, and GI) according to the trimester of pregnancy.
Trimester PlI CalI GI Mean SD Mean SD Mean SD 0.86 0.31 1.81 0.39 0.16 0.22 First Second 0.91 0.40 1.94 0.43 0.19 0.29 0.87 0.35 1.86 0.38 0.21 0.25 third

Table 4: Distribution of Primigravidae and Multigravidae women (Number and Percentage) according to clinical pocket depth score.
Score 0 1 Primigravidae Multigravidae No. % No. % 110 98.21 110 98.21 2 1.79 2 1.79

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Effect of thymus vulgaris

Effect of thymus vulgaris extract on streptococci and mutans streptococci, in comparison to chlorhexidine gluconate (in vivo study)
Eman A. Al-Timimi, B.D.S, M.Sc (1) Mohammed AL-Casey B.D.S., M. P.H., M.S.P.H (2)

ABSTRACT
Background: Thymus vulgaris L. (Thyme) is an aromatic plant which has been used since ancient times for its culinary and medicinal effects almost everywhere in the world. In Medicine, it is used as antispasmolytic, antibacterial, antifungal, expectorant, and antiseptic. Externally, it is used in the treatment of tonsillitis, gum diseases, and fungal infections. The aim of this study was to test the effects of Thymus Vulgaris extract on the viable count of Salivary Streptococci and Mutans Streptococci and on salivary flow rate and pH in comparison to Chlorhexidine Gluconate 0.2% and de-ionized water in vivo. Materials and method: Thymus vulgaris extract was prepared and different concentrations of Thyme extract were prepared and estimated in gm /100 ml of De-ionized water. Chlorhexidine gluconate 0.2% used as a control positive; while deionized water was used as a control negative. Stimulated saliva was collected from 15 volunteers, divided into three groups each group rinsed with one of the tested agents (Chlorhexidine, Thyme extract and De-ionized water) for one minute. The counts of bacteria were recorded at different time point (before rinsing, one minute after rinsing, 15 minutes, 30 minutes and one hour). Colonies were counted using colony counter. Salivary pH and volume of saliva were measured also. Results: Thymus Vulgaris extract had a significant antimicrobial activity against Streptococci and Mutans Streptococci in the following time points (15 minutes and 30 minute) and a highly significant reduction in the counts of salivary streptococci and Mutans Streptococci (P<0.001) had been found after one hour, but Chlorhexidine is still more effective than other agents in reduction of salivary counts of these two types of bacteria. Salivary flow rates and pH was measured for the three agents before and after rinsing for five time intervals. Immediately after rinsing, salivary flow rates and pH increased for the three mouth rinses. The increase in Salivary flow rates and pH continued after half an hour and then started to decrease after one hour for Chlorhexidine and Thymus vulgaris extract. Chlorhexidine had the highest increase in salivary flow rates and pH followed by Thymus Vulgaris then De-ionized water. Conclusion: Thymus Vulgaris extract was effective against both Streptococci and Mutans Streptococci and Chlorhexidine is still more effective than other agents. Keyword: Mutans Streptococci, Thymus Vulgaris extracts, Chlorhexidine. (J Bagh Coll Dentistry 2012;24(3):116-121).

INTRODUCTION
Dental caries is an important Dental Public Health Problem and is also the most prevalent oral disease among people in the world. Dental caries is a chronic infectious disease initiated through a series complex, chemical and microbial reactions associated with dental plaque biofilms which results in localized demineralization of calcified tissues of teeth and dissolution of the organic component (1). The mouth contains a wide variety of oral bacteria, but only a few specific species of bacteria are believed to cause dental caries; Streptococcus Mutans and Lactobacilli among them (2). S. Mutans has been identified as the principal cariogenic bacterium for caries initiation (3) . Treatment of dental caries and other plaque related diseases need a lot of cost as well as manpower.
(1) (2) MSc Preventive dentistry, Department of Preventive dentistry, College of Dentistry, Baghdad University Professor, Department of Preventive dentistry, College of Dentistry, Baghdad University.

Prevention, including the use of chemical therapies, is more cost effective as the patient shifts from a high-risk to a low-risk level (4). The elimination of cariogenic bacteria from the oral cavity using antibacterial agents is one of the primary strategies for the prevention of dental caries (5). Chlorhexidine is a potent antimicrobial agent particularly against Mutans Streptococci (6). However, local side effects such as tooth staining, taste altering and desquamation of oral mucosa have limited the long-term use of CHX; this has led to continuous and extensive investigations, seeking alternative agents. Alternative agents based on herbal extracts are therefore of particular interest (7). Thymus Vulgaris L. (Thyme), locally knownzaatar is widely used in folk medicine (8) .The aromatic and medicinal properties of the genus Thymus has made it one of the most popular plants all over the world. Recent studies have shown that Thymus species have strong antibacterial, antifungal, antiviral and antiparasitic effects (9). The antimicrobial properties of herbs rich in volatile oil (like Thymus Vulgaris) are well known since thousand years (10).

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The Iraqi flora is rich in plants unsubmitted to any previous study. The possibility of finding new antimicrobial agents is still widely ahead, and since there in no previous Iraqi studies to evaluate in vitro and in vivo the antimicrobial activity of an extract prepared from Thymus Vulgaris on Streptococcus Mutans and to show the possibility of their application for the prevention of dental caries in human; this study was designed.

MATERIALS AND METHODS


Preparation of Thymus Vulgaris extract The preparation of ethanolic extract of Thymus Vulgaris carried out according to the method described by Nweze et al (11), which involved the maceration of 200 gram of plant in one liter of absolute ethanol (ethanol 99.8%). The container was sealed with paper foil to prevent loss of volatile solvent and left at room temperature for 24 hours. At the end of this period, the contents were filtered using filter paper (No.1) into a beaker. The filtered solution then concentrated by evaporating the solvent in a hot air oven at 40C for 24 hr. The extract powder was then weighed, kept in sterile bottles, labeled accordingly and stored in the refrigerator while the test lasted. Study sample Volunteers involved in this study were female students in the College of Dentistry-University of Baghdad, of an age ranging between (20-24 years). The total number of volunteers were 15 and they were divided into 3 groups (each group was made up of 5 volunteers), the first group was the experimental group and they used Thyme extract mouth rinse 5%, while the second group used CHX 0.2% mouth rinse as control positive and the third group used de-ionized water mouth rinses as control negative. All the volunteer participated in this experiment were healthy looking female with no signs or symptoms of any systematic disease, did not receive any antimicrobial agents during the last two weeks prior to the study, not wearing any fixed or removable prosthesis or orthodontic appliance Breakfast was taken by volunteers about 2 hours prior to salivary sample collection. Mouth washing and sample collection was carried out at 9.00 a.m. Subjects were asked to stop brushing and other oral hygiene practices for that morning (12) . Experimental Procedure 1-10 ml of 5% ethanolic Thymus Vulgaris extract, De-ionized water, and Chlorhexidine gluconate 0.2% mouthwash were prepared. 2-Stimulated saliva was collected in sterile screwcapped bottles, by chewing a piece of Arabic gum

0.5 gm for one minute (13), each participant was asked to rinse with aqueous solution for 1 minute, then expectorate, stimulated salivary samples were recollected after 1 minute, 15 minutes, 30 minutes, and 1 hour, during this time volunteers were asked not to eat or drink anything except water. Within less than 15 minutes, the pH of saliva was measured by the digital pH meter; also the volume of saliva was measured also. 3-Sample of saliva were processed immediately, they were dispersed for 1 minute by vortex mixer, then 0.1 ml of saliva transferred to 0.9 ml of Phosphate buffer saline, tenfold dilutions were performed. From the dilution 10-3, 0.1 ml was taken and spread in duplicate on the surface of Mitis Salivarius Agar (MSA) and Mitis Salivarius Bacitracin Agar (MSBA) plates, then incubated anaerobically for 48 hr. at 37 oC, and aerobically for 24 hr. at room temperature. Following identifications, colonies of Streptococci on MSA and Mutans Streptococci on MSB agar plates were counted by the use of colony counter. The counts were expressed as the colony forming unit taking in consideration dilution factor x 10 (to be) / ml of saliva (CFU/ml), taking also the pH and volume of saliva. Information regarding the taste of solution was recorded in addition to any discomfort following the rinse and after 24 hours.

RESULTS
The weight of the final extracts powder was 18.40 gm .The extract powder was dark green in color with very distinguishing odor. Different concentrations were prepared by dissolving the powder in gm/100ml of de-ionized water. Counts of bacteria were estimated before and after single rinse with selected agents in five time points (before rinsing, one minute after rinsing , 15 minute, 30 minute and one hour) ,Table (1). For all agents a reduction in the viability counts of Streptococci were observed one minute immediately after rinsing. For Thymus Vulgaris extract, the reduction in the counts of bacteria continued after half an hour, but there were increase in the counts after one hour; while for deionized water the increase started after 30 minutes and continued. CHX reduced the counts of bacteria for all the time intervals. CHX had the maximum reduction in Streptococci counts followed by ethanolic thyme extract (5%), while the de-ionized water had the least reduction of bacterial counts among the tested agents. The difference between the counts of streptococci for the three mouth rinses was examined by ANOVA test which is shown in (Table 2), no significant difference were found in counts of bacteria between the three agents before and immediately

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one minute after rinsing while a significant reduction was recorded after 15 minutes . A

highly significant difference was found in the following time points (30 minutes and one hour).

Table 1: Mean counts of salivary Streptococci x103 and Standard Deviation of three mouthwash in vivo.
Time Before rinsing After 1 min After15mins After 1/2 hr. After 1 hour Thymus Vulgaris CHX mouthwash mouthwash Mean SD Mean SD 401.8 393.4 383.2 378.2 384.6 8.012 5.128 4.438 5.585 9.633 405.4 388.6 369.6 360.4 356.4 3.286 7.536 12.421 13.069 11.126 D.W. mouthwash Mean SD 399.2 391.4 386.4 390.6 396.8 7.328 4.219 6.024 4.615 3.962

Table 2: One way ANOVA test among viable count of Streptococci x10 3for the three mouth washes at 5 time intervals in vivo.
Mouthwashes Time F- test p-value Description 1.130 0.355 NS Base line Thymus Vulgaris mouthwash NS 1 min 0.864 0.446 S CHX mouthwash 15 mins 5.676 0.018 HS 1/2 hr. 15.480 0.000 D.W. mouthwash HS 1 hr. 27.725 0.000
d.f = 2

Counts of Mutans Streptococci bacteria were estimated before and after single rinse with 5% Thymus vulgaris extract, CHX Gluconate and Deionized water at each time interval (before rinsing, one minute, 15 minute, 30 minute and one hour), Table (3). For all agents a reduction in the viability counts of Mutans Streptococci were observed one minute immediately after rinsing. The reduction in the counts of Mutans Streptococci bacteria for Thyme extract continued after half an hour, but there was an increase in the counts after one hour; while for deionized water the increase in the bacterial count started after 30 minutes and continued. CHX reduced the counts of bacteria for all the time intervals. CHX had the maximum reduction in Mutans Streptococci counts followed by ethanolic Thyme extract (5%). The difference between the counts of Mutans streptococci for the three mouth rinses was examined by ANOVA test showed in Table (4), no significant differences were found in counts of bacteria between the three agents before and immediately one minute after rinsing while a significant reduction was recorded after 15

minutes and 30 minutes. A highly significant difference was found after one hour. Salivary flow rate was measured before and after single rinse with three mouth rinses (Thymus Vulgaris extract, CHX and D.W.) as shown in Table (5). Salivary flow rate was increased immediately after rinsing for the three mouth rinses; Deionized water showed slight increase in the flow rate from the base line, which continued for 15 minutes, after that the flow rate, was decreased gradually. Rinsing with CHX or thyme extract resulted in a marked increase in the mean values of salivary flow rate immediately, which continued for half an hour, and then the flow rate gradually decreased but remained higher than the baseline value. The difference between the salivary flow rates for the three mouth rinses was examined by ANOVA test showed in Table (6), statistically no significant difference was found between the three agents before rinsing and immediately after rinsing, while a significant difference was found in the following time points between the three agents.

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Table 3: Mean counts of Mutans Streptococci x103 and standard deviation of three mouthwashes in vivo.
Thymus Vulgaris mouthwash CHX mouthwash D.W. mouthwash Mean SD Mean SD Mean SD 270.2 14.498 287.2 27.878 257.4 21.870 Before rinsing 249.6 22.097 234.8 18.226 241.4 16.682 After1 min 236.8 13.217 212.8 18.212 235.2 13.367 After15mins 226.8 10.616 202.4 18.063 246.6 11.928 After 1/2 hr. 240.4 5.727 211.4 15.126 263.4 17.096 After1hour Time

Table 4: One way ANOVA test among viable count of Mutans Streptococci x10 3for the three mouthwashes at 5 time intervals in vivo.
Mouthwashes Time F- test p-value Description NS ThymusVulgaris mouthwash Base line 2.326 0.14 0.750 0.493 NS 1 minute 3.942 0.048 S CHX mouthwash 15 minute 12.649 0.001 S 1/2 hr. D.W. mouthwash 18.388 0.000 HS 1 hr.
d.f = 2

Table 5: Mean and Standard Deviation of salivary flow rates before and after rinsing with Thymus Vulgaris extract, CHX and D.W. mouthwashes.
Time Before rinsing After 1 min After15mins After 1/2 hr. After 1 hour Thymus Vulgaris mouthwash Mean SD 401.8 8.012 393.4 5.128 383.2 4.438 378.2 5.585 384.6 9.633 CHX mouthwash Mean 405.4 388.6 369.6 360.4 356.4 SD 3.286 7.536 12.421 13.069 11.126 D.W. mouthwash Mean 399.2 391.4 386.4 390.6 396.8 SD 7.328 4.219 6.024 4.615 3.962

Table 6: One way ANOVA test of salivary flow rates for Thymus Vulgaris extract, CHX and D.W. mouthwashes by time.
Mouthwashes Time F- test p-value Description NS Thymus Vulgaris mouthwash Base line 0.669 0.53 NS 1 min 3.519 0.063 S CHX mouthwash 15 mins 6.756 0.011 S 1/2 hr. 8.962 0.004 D.W. mouthwash S 1 hr. 4.995 0.026

The salivary pH for the three mouth rinses was measured. Immediately following rinsing with three mouthwashes, the pH value increased markedly. For Thyme extract 5% and CHX 0.2%, immediately one minute after rinsing a slight increase in the pH of saliva were found which continue to increase after half an hour then gradually decreased after one hour but still higher than the base line, while rinsing with de-ionized water resulted in slightly increase in the pH, which decreased after 30 minutes and by one hour

it reached lower than the base line value, Table(7). The difference between the salivary pH for the three mouth rinses was examined by ANOVA test which is shown in Table (8), statistically no significant difference was found between the three agents before rinsing with a significant difference were seen immediately after rinsing and after 15 minutes, while a highly significant difference was found in the following time point.

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Table 7: Mean and standard deviation of PH before and after Thyme, CHX and D.W. mouthwashes.
Time Before rinsing After1 min After 15 mins After 1/2 hr. After 1 hour Thymus Vulgaris mouthwash Mean SD 7.21 0.033 7.32 0.018 7.4 0.033 7.44 0.441 7.29 0.036 CHX mouthwash Mean 7.25 7.4 7.51 7.55 7.47 SD 0.024 0.061 0.055 0.087 0.134 D.W. mouthwash Mean 7.2 7.3 7.35 7.25 7.18 SD 0.056 0.059 0.124 0.093 0.033

Table 8: One way ANOVA test of PH for Thymus Vulgaris extract, CHX and D.W. mouthwashes by time. Mouthwashes Time F- test p-value Description
ThymusVulgaris mouthwash CHX mouthwash D.W. mouthwash Base line 1 min 15 mins 1/2 hr. 1 hr. 2.016 5.384 5.675 25.457 15.702 0.176 0.021 0.018 0.000 0.000 NS S S HS HS

DISCUSSION
Results of this study revealed that 5% Thymus Vulgaris extract had significant antimicrobial activity against Streptococci and Mutans Streptococci as it can reduce the viable count of the bacteria profoundly in comparison to deionized water after half an hour. Much of the antimicrobial activity in Thymus genus appears to be associated with phenolic compounds (Thymol & Carvacrol) which make up a large percentage of Thyme constituents (14, 15, 16, 17, 18). The reduction in bacterial population trend continued until an hour for CHX; this could be due to CHX having a prolonged bacteriostatic action and ability to absorb onto the pellicle coated enamel surface and dental plaque during rinsing. For Thymus Vulgaris extract and CHX, salivary flow rates continue to increase after half an hour then began to drop down slowly but still more than the base line after 1 hour of rinsing. The possible explanation is that any mechanical stimulation in form of mouth rinses can increase the salivary flow rates or the stimulation of chemoreceptor in the taste buds gives rise to increased salivary secretion (19). The mechanism behind the elevated PH with Thymus Vulgaris mouthrinse could be due to a buffering capacity of the Thymus Vulgaris extract, salivary stimulation due to Thyme taste, and/or antibacterial activity against acidproducing bacteria. The possible explanation of the increase in PH in general depended on the following facts:-

1-The salivary pH is highly dependent on the salivary flow rate (20). The increase in salivary flow rate lead to increase in Salivary buffering capacity which is important in maintaining a pH level in saliva and plaque. Salivary stimulation leads to increase in the concentration of bicarbonate in the saliva entering the mouth. This bicarbonate raises the pH of the saliva, and greatly increases its buffering power; the saliva is therefore much more effective in neutralizing and buffering food acids and acids arising in plaque from the fermentation of carbohydrate by microorganisms. At the same time, remineralizing ions (phosphate, calcium) the of saliva changes as a result of the rise in pH (21, 22). 2-One of the important functions of saliva is maintenance of oral hard tissue integrity by providing mechanical cleansing and antimicrobial actions. Saliva dilutes and eliminates dietary sugars and acids as well as oral microorganisms from the mouth by a process referred to as oral clearance (19).

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14. Consentino S, Tuberoso C, Pisano B, Satta M, Arzedi E, Palmas F. In vitro antimicrobial activity and chemical composition of sardinian Thymus essential oils. Lett. Appl. Microbiol 1999; 29: 1305. 15. Davidson P M, Naidu A. Phyto-phenol. In: Naidu, AS. (Ed.), Natural Food Antimicrobial Systems. CRC Press, Boca Raton, Florida 2000: 26594. 16. Skocibusic M, Bezic N, Dunkic V. Phytochemical composition and antimicrobial activities of essential oils from Saturejasubspicata Vis. Growing in Croatia. Food Chem J. 2006; 96: 208. 17. Rota M, Herrera A, Martinez R, Sotomayor J , Jordan M. Antimicrobial activity and chemical composition of Thymus vulgaris, Thymus zygis and Thymus hyemalis essential oils. Food Cont 2007; 19: 6817. 18. Al-Saimary I, Bakr S, Khudaier B, Abass Y. Efficiency of antibacterial agents extracted from Thymus vulgaris L. (Lamiaceae). The Int J of Nutri and Wellness 2007; 4 (1). 19. Pedersen A, Bardow A, Jensen S , Nauntofte B. Saliva and gastrointestinal functions of taste, mastication, swallowing and digestion. Oral Dis. 2002; 8(3): 117-29. 20. Svensson P, Nauntofte B, Timothy S, Bardow A, Pedersen A, Nauntofte B. Clinical Oral Physiology. Copenhagen: Quintessence Publishing Co Ltd. 2004, p.17-51. 21. Bardow A, Moe D, Nyvad B, Nauntofte B. The buffer capacity and buffer systems of human whole saliva measured without loss of CO2. Arch Oral Biol 2000; 45; 1-12. 22. Rantonen P. Salivary flow and composition in healthy and diseased adults. Thesis submitted to University of Helsinki, Institute of Dentistry. 2003.

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Pain intensity and control

Pain intensity and control with fixed orthodontic appliance therapy (A clinical comparative study on Iraqi sample)
Hayder Fadhil Saloom, B.D.S., M.Sc. (1)

ABSTRACT
Background: The purpose of this prospective, randomized clinical trial was to investigate the level and intensity of patients` pain and discomfort, and to compare between the use of Bite Wafer and Paracetamol in reducing pain and discomfort associated with initial orthodontic tooth movement in both adolescents and adults. Sample: 110 subjects with two age groups, 52 adolescents with age range from 12 to less than 18 years and 58 adults with age range 18-24 years, successfully completing the study. For each subject fixed orthodontic appliance (Roth 0.022) was bonded and round 0.014 NiTi arch wire was ligated with elastic ligature. The subjects in the Bite Wafer group were instructed to chew on it whenever they feel pain for the next 7 days, and document the time and effectiveness of it in the questionnaire. The Paracetamol group subjects instructed to use Paracetamol 500mg to relive pain and record times and effectiveness of its use in questionnaire. Results: The peak of pain was occurred in the first day and declined gradually till totally disappeared at the sixth day after initial arch wire placement. A marked reduction of pain intensity was noticed in both adolescents and adults groups, using Bite Wafer, from the first to the sixth day which is much higher than Paracetamol group especially in adolescents. No gender differences (P>0.05) was noticed in this study. Conclusion: Although both Bite Wafer and Paracetamol reduced pain gradually, Bite Wafer reduced pain more obviously and safely in comparison to Paracetamol especially in adolescents. Key words: pain, Wafer, orthodontic. (J Bagh Coll Dentistry 2012;24(3):122-128).

INTRODUCTION
Fear of pain is a key element in deterring patients from seeking orthodontic treatment1. Orthodontic therapy was reported to be painful for 90% of patients, with some 30% contemplating terminating their treatment early because of discomfort2. Pain and discomfort after appliance adjustment had been shown to be higher than after dental extraction3. Patients in orthodontic treatment often describe the discomfort as pressure, tension, ache, or soreness of the teeth4. The pain intensity usually increases gradually from 2 hours after application of orthodontic force to a peak at 24 hours, with pain resolution by the seventh day5-7. Pain-free orthodontics is not often considered, and often the orthodontist ignores the patient's discomfort8. However, it is increasingly less acceptable for the patients to suffer from pain or discomfort9. In 2007 Proffit et al., identified an immediate and delayed pain response; the immediate pain related to the initial compression of the periodontal ligament immediately after placement of the archwire. Whereas, the delayed response started a few hours later, was termed hyperalgesia of the periodontal ligament. Orthodontic treatment provides the type of chronic periodontal insult that encourages a continual and increased production of prostoglandins10, which have been shown to cause hyperalgesia, an increased sensitivity to noxious agents such as histamine,
(1)Assistant professor. Department of Orthodontics. College of Dentistry. University of Baghdad.

bradykinin, serotonin, acetylcholine, and substance P. There are indication that perceptios of pain are due to changes in blood flow in the periodontal ligament and are correlated with the presence of substances as prostaglandins and substance P11. Pain management: the most frequently recommended treatments for pain are over-thecounter analgesics (OTC), often a nonsteroidal anti-inflammatory drug (NSAID) that works by inhibiting prostaglandin synthesis. Previous studies assessing the efficacy of analgesics for pain management have focused on medications administered immediately before12,13 or before and immediately after the orthodontic procedures14,15. All of these studies reported that analgesics were effective for pain management. The overuse and potential side effects of OTC analgesics have been raised as a concern, particularly for children16,17, because of these concerns, non-analgesic pain management approaches such as chewing gum or chewing on a plastic wafer have been recommended to loosen the tightly grouped fibers around the nerves and blood vessels, restoring normal vascular and lymphatic circulation, thus preventing or relieving inflammation and edema10. Although several studies have indicated that the rhythmic chewing of either Aspergum (Insight Pharmaceuticals, Langhorne, Pa)18, a gum containing aspirin, or a bite wafer (BW)19 reduced the pain and discomfort after archwire placement. No comparison of the effectiveness of either of these non-pharmacologic approaches and OTC medications has been researched in Iraq.

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This study aimed to investigate the level and intensity of patients` pain and discomfort following insertion of fixed orthodontic appliance and to compare the effectiveness of Bite Wafer as a non-pharmacologic approach for pain management of orthodontic treatment, with Paracetamol analgesics during the first week after initial archwire placement.

MATERIALS AND METHODS


This study started with 160 patients; 12-24 years old divided into two age groups, group A 12 to less than18 years old and group B 18 up to 24 years old. Within both A and B groups, patients are randomly allocated into 1 of 2 groups: the Bite Wafer group and the Paracetamol analgesic medication group. They are scheduled to begin the orthodontic treatment (banding and bonding of at least 10 teeth and archwire placement in at least single arch) 20. Readjusted edgewise (Roth prescription) brackets would be used with 0.022in slot; and 0.014-in Nickel Titanium archwire was used as the first archwire with all the teeth engaged with ligature elastics. The pain management instructions were given to each group as follow: *Bite Wafer group: instruct them to use a Bite Wafer (Fig 1) as a measure to relive pain or discomfort and never use any medication to control pain. Ask them to chew on the Wafer, under supervision, for 1012 minutes within an hour after placement of archwire, also tell them to document how many times the Wafer had been used, and its effectiveness for discomfort relief at the end of each day for seven days21.Additional Wafers are given to the patients to chew them as much as they want whenever they feel discomfort or pain. * Paracetamol group: for this group 500 mg paracetamol tablet, are given and ask them to take it whenever they need to relive pain or discomfort. They also asked to document the number of tablets that required for pain relief 22. Before placement of the appliance, a preoperative Visual Analog Scale (VAS) score was taken to exclude any pre-existing painful condition. Immediately after placement of a fixed appliance, the patient completes the VAS questionnaires at the clinic. The VAS (Fig.2) consisted of an unmarked horizontal line with a descriptive terminology, e.g. not hurting, no discomfort, no pain, hurting a whole lot, very uncomfortable, severe pain, and pictorially, with a happy face and sad face. The VAS was translated to simple Arabic words to simplify the scoring for the patients. Each patient was asked to put a mark on the line that best corresponded to the level of pain
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he/she felt at that moment. If there is no pain, the patient would mark the end of the line by happy face. If there is some pain, the patient would mark the line somewhere in the middle, depending on the severity. The patients were asked to complete the questionnaire to rate their pain and its intensity at 7 times after archwire placement: the bed time of the same day, bed time of the 2nd, 3rd, 4th, 5th,6th and 7th days later. At the end of the week, the patient should return the questionnaire for analysis. The data will be collected for drawing a curve of pain intensity and amount of pain reduction or increment for each group. Statistical analyses: Data were collected and analyzed using SPSS software version 19 for windows XP Chicago, USA. The following statistics were used: Descriptive statistics include sample distribution within each age group for both genders, with means, medians, standard deviations, and P values using nonparametric Mann-Whitney U test to compare between genders, two age groups and two different methods of pain control. In statistical evaluation, the following levels of significance are used: P > 0.05 NS Non-significant 0.05 P > 0.01 * Significant 0.01 P ** Highly significant

RESULTS
The study started with 160 subjects, 80 adults and 80 adolescents, 28 persons were dropped from adolescents group and 22 from adults group resulting in 110 subjects, 52 adolescents and 58 adults successfully completing the survey, including both genders. Descriptive statistics including number of participants (N), means, medians and standard deviations of pain intensity, according to VAS, after pain management in both Bite Wafer and Paracetamol groups for both adolescents and adults groups through the whole study period are shown in Tables 1,2 and 3. Moreover, the sixth and seventh days showed zero findings for all variables that cannot be encountered statistically. The Mann-Whitney U test showed no significant differences (P>0.05) between males and females at any variable; therefore, the findings were evaluated with no gender discrimination, and the data for males and females were combined for analyses (Table 1). The peak of pain was occurred in the first day and little bit higher in adolescents than in adults and declined gradually till totally disappeared at the sixth day after initial arch wire placement, (Figures 3-6).

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In comparison of age groups, in Bite Wafer group a highly significant difference (P<0.01) was found only in the first day and a non significant difference (p>0.05) in all other days, which means both age groups got approximately the same benefit from Bite Wafer in pain reduction, whereas in group using Paracetamol, a highly significant difference (P<0.01) was found between adolescents and adults in all five days, which means pain reduction is significantly higher in adults using Paracetamol than in adolescents (Table 2). In comparison of pain management regimens, Bite Wafer and Paracetamol groups, MannWhitney U test showed a highly significant difference (P<0.01) which mean more pain reduction in Bite Wafer group than in Paracetamol group (Table 3). In comparison of pain intensity before and after pain management procedure, a marked reduction of pain intensity was noticed in both adolescents and adults, using Bite Wafer, from the first to the sixth day (Figure 3 and 4), whereas, in group using Paracetamol, adolescents showed a fluctuant pain intensity level which become little bit increased in the first, second and fourth days and little bit decreased in the third, fifth and sixth days. On the other hand, adults in Paracetamol group showed more similar pattern to Bite Wafer group in gradual pain reduction than adolescents from the first to the sixth day of study (Figure 5 and 6).

DISCUSSION
Since the data in this study were collected by questionnaires, a firm instruction to the participants and/or parents has been given and even training how to use the Bite Wafer has been done in the first visit to reduce the subjectivity as much as possible. Also the questionnaire was translated to simple Arabic words to simplify the selection of the most appropriate score in the visual analog scale (VAS). Although there is evidence to suggest higher levels of pain reported by females10, this study showed no statistically significant difference between males and females (P>0.05) in all records and this come in accordance with several other studies 6,15,21. Pain intensity for both Bite Wafer and Paracetamol groups in both adolescents and adults followed a similar pattern, pain peaked in the first day and decreased over the rest of the week and this come in coincidence with findings of other previous studies 6,21, which demonstrated a gradual declination of pain levels during the week after placement of orthodontic appliances.
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Bite Wafer reduced the pain to a minimal level in both adolescents and adults through the recording period (Figures 3 and 4), the pain reported by Paracetamol group was slightly higher than Bite Wafer group in adults, whereas in adolescents it was much higher and sometimes pain intensity increased, this could be explained by the frequency of analgesic intake, as shown in Figure 7 and 8, the adults used the Bite Wafer more frequently than analgesic resulting in more pain reduction in Bite Wafer group than in Paracetamol group (Figure 4 and 6), also the adolescents used the Bite Wafer more than analgesic thats why pain reduced markedly with Bite Wafer (Figures 3 and 5) whereas pain sometime increased in Paracetamol group especially in adolescents this is because less analgesic intake was noticed by adolescents in comparison to adults (Figure 8) which could be related to the afraid of the side effects of drugs, since some drugs may cause gastric or duodenal ulceration, bleeding disorders, asthma, renal insufficiency and drug allergy, that make persons afraid of freely use analgesics. In contrast, the effect of Bite Wafer in pain relief comes from the acceleration of blood flow into and around the periodontal membrane, which in turn reduces the edema caused by the trauma of orthodontic forces, this agree with findings of many studies10,15 and disagree with others in which more pain was reported with Bite Wafer22. As a conclusion, It seems reasonable for orthodontists to offer Bite Wafers to patients as another possible way to alleviate the pain and discomfort during the leveling and alignment steps of orthodontic treatment with fixed appliances and further studies were suggested to investigate its effect on the orthodontic tooth movement.

REFERENCES
1. 2. Oliver RG, Knapman YM. Attitudes to orthodontic treatment. Br J Orthod 1985; 12: 179-88. Lew KK. Attitudes and perception of adults toward orthodontic treatment. Community Dent Oral Epidemiol 1993; 21: 31-5. Jones ML, Chan C. Pain in the early stages of orthodontic treatment. J Clin Orthod 1992; 26: 311-3. Ngan P, Kess B, Wilson S. Perception of discomfort by patients undergoing orthodontic treatment. Am J Orthod 1989; 96: 47-55. Bernhardt MK, Southard KA, Batterson KD, Logan HL, Baker KA, Jakob son JR. The effect of preemptive and/or operative ibuprofen therapy for orthodontic pain. Am J Orthod Dentofacial Orthop 2001; 120: 20-7. Erdic AM, Dincer B. Perception of pain during orthodontic treatment with fixed appliances. Eur J Orthod 2004; 26: 79-85.

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Scheurer PA, Firestone AR, Burgin WB. Perception of pain as a result of orthodontic treatment with fixed appliances. Eur J Orthod 1996; 18: 49-57. Blechman AM. Pain-free and mobility-free orthodontics. Am J Orthod Dentofacial Orthop 1998; 113: 397-83. Azerad J. Pain mechanisms and their current treatment. Rev Orthop Dento-Facial 1999; 33:43-73. Proffit WR, Fields HW, Sarver DM. Biological basis of orthodontic therapy. In: Proffit WR, Fields HW, Sarver DM, editors. Contemporary orthodontics. 4th ed. St Louis: Mosby; 2007. Norevall LI, Forsgren S, Mattson L.Expression of neuropepetides (CGRP, ssubstance P) during and after orthodontic tooth movement in the rat. Eur J Orthod 1995; 17:311-325. Bird SE, Williams K, Kula K. Preoperative acetaminophen vs ibuprofen for control of pain after orthodontic separator placement. Am J Orthod Dentofac Orthop 2007; 132:504-10. Polat O, Karaman AI, Durmus E. Effects of preoperative ibuprofen and naproxen sodium on orthodontic pain. Angle Orthod 2005; 75: 791-6. IVSL. Polat O, Karaman AI. Pain control during fixed orthodontic appliance therapy. Angle Orthod 2005; 75:214-9. IVSL. Bradley RL, Ellis PE, Thomas P, Bellis H, Ireland AJ, Sandy JR. A randomized clinical trial comparing

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the efficacy of ibuprofen and paracetomal in the control of orthodontic pain. Am J Orthod Dentofac Orthop 2007; 132:511-7. Goerge M, Selvarajan S, Indumatti C. Drug therapy for trigeminal neuralgia. E-Journal of Dentistry 2011; 1(2):28-31. IVSL. Bloom BS. Over the counter NSAIDs and GI side effects (abstract). Int Soc Technol Assess Health Care 2001; 17. White LW. Pain and cooperation in orthodontic treatment. J Clin Orthod 1984; 18:572-5. Hwang JY, Tee CH, Huang AT, Taft L. Effectiveness of thera-bite wafers in reducing pain. J Clin Orthod 1994; 28:291-2. Reza S, larry J, Oesterle W, Craig S, Sheldon MN. Comparison of the efficacy of ibuprofen and acetaminophen in controlling pain after orthodontic tooth movement. Am J Orthod Dentofac Orthop 2009; 135:516-21. Murdock S, Phillips C, Khondker Z, Garland H. Treatment of pain after initial archwire placement: A noninferiority clinical trial comparing over-thecounter analgesic and bite-wafer use. Am J Orthod Dentofac Orthop 2010; 137(3): 316-23. Jassim TA. Pharmacological control of orthodontic pain. A master thesis, Orthodontic department, college of dentistry, university of Baghdad, 2006.

Figure 1: Bite Wafer

Figure 2: Visual Analog Scale (VAS)

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Table 1: Gender difference in pain intensity (VAS) after pain management regimes in seven days
Time (day) 1st 2nd 3rd 4th 5th 6th 7th Adolescent using Bite Wafer Females (N=14) Mann-Whitney U test Mean Median SD Mann-Whitney U p value 1.0 1.0 0.7 61.5 0.243 0.6 1.0 0.5 76.5 0.687 0.4 0.0 0.5 69.0 0.448 0.1 0.0 0.3 58.5 0.186 0.0 0.0 0.0 84.5 1.000 0.0 0.0 0.0 0.0 0.0 0.0 Adult using Bite Wafer Males (N=13) Females (N=15) Mann-Whitney U test Mean Median SD Mean Median SD Mann-Whitney U p value 0.8 1.0 0.7 0.5 0.0 0.6 84.0 0.377 0.6 1.0 0.6 0.5 0.0 0.7 85.5 0.400 0.6 1.0 0.5 0.5 1.0 0.5 101.0 0.880 0.1 0.0 0.3 0.2 0.0 0.4 91.5 0.561 0.0 0.0 0.0 0.0 0.0 0.0 105.0 1.000 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Adolescent using Paracetamol Males (N=13) Females (12) Mann-Whitney U test Mean Median SD Mean Median SD Mann-Whitney U p value 6.4 7.0 2.4 7.4 7.0 1.8 78.0 0.387 6.5 7.0 2.7 6.2 6.0 1.0 67.5 0.170 2.2 2.0 2.0 2.4 2.0 0.9 87.5 0.650 2.8 2.0 0.9 2.4 3.0 1.3 83.5 0.525 0.5 0.0 0.8 1.0 1.0 0.8 59.0 0.080 0.2 0.0 0.4 0.2 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 Adult using Paracetamol Males (N=15) Females (N=15) Mann-Whitney U test Mean Median SD Mean Median SD Mann-Whitney U p value 2.6 1.5 2.3 1.7 2.0 0.8 87.0 0.905 1.9 1.5 1.5 1.7 2.0 0.7 82.0 0.719 1.0 1.0 0.9 1.1 1.0 0.7 66.5 0.256 1.4 1.0 0.8 1.3 1.0 0.6 79.0 0.614 0.2 0.0 0.4 0.3 0.0 0.5 73.5 0.427 0.0 0.0 0.0 0.1 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 P>0.05 = No significant difference Males ( N=13) Mean Median SD 1.5 1.0 0.9 0.8 1.0 0.7 0.6 1.0 0.7 0.4 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Time (day) 1st 2nd 3rd 4th 5th 6th 7th

Time (day) 1st 2nd 3rd 4th 5th 6th 7th

Time (day) 1st 2nd 3rd 4th 5th 6th 7th

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Table 2: Age difference in pain intensity (VAS) after pain management regimes in seven days
Adolescent (N=27) Mean Median SD 1.2 1.0 0.8 0.7 1.0 0.6 0.5 0.0 0.6 0.2 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Adolescent (25) Mean Median SD 6.9 7.0 2.1 6.3 6.5 2.0 2.3 2.0 1.5 2.6 2.5 1.1 0.7 0.5 0.8 0.0 0.0 0.0 0.0 0.0 0.0 Bite Wafer Adult (N=28) Mean Median 0.7 1.0 0.6 0.0 0.6 1.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Paracetamol Adult (N=30) Mean Median 2.2 2.0 1.8 2.0 1.1 1.0 1.4 1.0 0.2 0.0 0.0 0.0 0.0 0.0 Mann-Whitney U test Mann-Whitney U P value 232.0 0.008 325.0 0.330 351.0 0.616 342.0 0.377 377.0 1.000

Time (day) 1st 2nd 3rd 4th 5th 6th 7th

SD 0.7 0.7 0.5 0.4 0.0 0.0 0.0

Time (day) 1st 2nd 3rd 4th 5th 6th 7th

SD 1.8 1.2 0.8 0.7 0.4 0.0 0.0

Mann-Whitney U test Mann-Whitney U P value 23.0 0.000 48.0 0.000 162.0 0.000 124.0 0.000 213.0 0.002

P>0.05 = No significant difference

Table 3: Management regimes difference of pain intensity (VAS) for both age groups in seven days
Adolescent Paracetamol (N=25) Mean Median SD 7.3 7.0 1.7 5.7 6.0 2.2 1.7 2.0 1.0 2.5 3.0 1.1 0.9 1.0 0.7 0.0 0.0 0.0 0.0 0.0 0.0 Adult Paracetamol (N=30) Mean Median SD 1.8 2.0 0.8 1.9 2.0 0.9 1.2 1.0 0.9 1.4 1.0 0.6 0.3 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0

Time (day) 1st 2nd 3rd 4th 5th 6th 7th

Bite wafer (N=27) Mean Median SD 1.2 1.0 0.8 0.7 1.0 0.6 0.5 0.0 0.6 0.2 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Bite wafer (N=28) Mean Median SD 0.7 1.0 0.7 0.6 0.0 0.7 0.6 1.0 0.5 0.1 0.0 0.4 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

Mann-Whitney U test Mann-Whitney U p level 0.0 0.000 30.0 0.000 86.5 0.000 22.0 0.000 78.0 0.000

Time (day) 1st 2nd 3rd 4th 5th 6th 7th

Mann-Whitney U test Mann-Whitney U p level 80.0 0.000 66.5 0.000 154.0 0.008 34.5 0.000 188.5 0.006

P>0.05 = No significant difference

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7 6 5 4 3 2 1 0 1st 2nd 3rd 4th 5th

B A

7 6 5 4 3 2 1 0 1st 2nd 3rd 4th 5th

Pain intensity (VAS)

Pain intensity (VAS)

B A

6th

6th

Time (day)

Time (day)

Figure 3: Pain intensity before(B) and after(A) using Bite Wafer in adolescents

Figure 4: Pain intensity before(B) and after(A) using Bite Wafer in adults

7 6 5 4 3 2 1 0 1st 2nd 3rd 4th 5th

B A

7 6 5 4 3 2 1 0 1st 2nd 3rd 4th 5th

Pain intensity (VAS)

Pain intensity (VAS)

B A

6th

6th

Time (day)

Time (day)

Figure 5: Pain intensity before(B) and after(A) using paracetamol in adolescents

Figure 6: Pain intensity before(B) and after(A) using paracetamol in adults

3.0 Frequency of Bite Wafer Frequency of paracetamol 2.5 2.0 1.5 1.0 0.5 0.0 1st 2nd 3th 4th 5th Adolescent Adult

3.0 2.5 2.0 1.5 1.0 0.5 0.0 1st 2nd 3rd 4th 5th Adolescent Adult

Time (day)

Time (day)

Figure 7: Frequency of using Bite Wafer by adolescents and adults

Figure 8: Frequency of using paracetamol by adolescents and adults

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Salivary vitamins and total proteins, in relation to cariesexperience and gingival health, according to nutritional status of a group of five-year old children
Nada J. Radhi B.D.S., M.Sc., Ph.D. (1)

ABSTRACT
Background: Malnutrition influences the development of the teeth and the formation, function and secretion of the salivary glands, which in turn influence susceptibility to dental caries and gingival disease. The aims of this study were to assess the salivary antioxidants (vitamin A, C and E) levels as well as total protein and their relation to caries severity and gingival health status among mal- and well-nourished children. Materials and Methods: The sample consisted of 60 children and they divided according to nutritional status (30 malnourished and 30 well nourished). The 2000 Centers for Disease Control and Prevention (CDC) growth charts was used for assessment of nutritional status (height for age). The age was five years old. Caries severity (d1-4s) was assessed according to Muhlemann (1976). Dental plaque recorded following the criteria described by Sillness and Le (1964). The gingival index (GI) was used according to Le and Sillness criteria (1963). Stimulated whole saliva samples were collected and chemically analyzed by using colorimetric method to determine the salivary antioxidants (vitamin A, C and E) and total protein. All data were analyzed using SPSS version 18. Results: Results recorded a higher mean value of dmfs among malnourished in comparison to well nourished with statistically highly significant difference (P< 0.001). According to grades of lesion severity, d4 was significantly the higher among malnourished children (P< 0.001). Strong highly significant correlations were noticed between ds, dmfs and PI among malnourished and well nourished children. Significantly lower values of vitamins and total protein were noticed among malnourished children compared to well nourished (P< 0.001). Negative highly significant correlations were found with all vitamins among malnourished children regarding caries-experience and GI. Conclusion: Childhood chronic malnutrition (stunting) is associated with salivary hypofunction. This may act as a risk factor for dental caries and gingival disease in the target group. Key Words: vitamin A, C, E, total protein, dental caries, gingival disease. (J Bagh Coll Dentistry 2012;24(3):129-136).

INTRODUCTION
Anthropometry can be used to assess nutritional status at both the individual level and the population level and assist to classify person as mal or well nourished in relation to specific level of indicator (1). Height for age indicator provides an excellent index of long term cumulative inadequacies of nutrition (chronic under nutrition: shortness or stunting), which is frequently associated with poor overall economic conditions and/or repeated exposure to adverse conditions (2). Deficiencies of specific nutrients do influence the development of the teeth and the formation, function and secretion of the salivary glands, which in turn influence susceptibility to dental caries and gingival disease (3). The index age (5 years) is considered as a critical human life stage which has recorded the past and present history of malnutrition and oral health conditions (4, 5). Several studies reported that malnourished children were more susceptible to be affected by dental caries and gingival disease (6-12).

Saliva is important for the health of oral soft and hard tissues. The complexity of physical and chemical composition of salivary secretions performs a considerable number of protective functions through buffer capacity, minerals, total protein and antioxidants (proteins and vitamins) (13-15) . Salivary antioxidants were found to reduce the susceptibility to dental caries and periodontal disease (16-19). As far as it is known, no Iraqi study was found to determine the relation between salivary vitamins, total proteins and dental caries as well as gingival disease among a group of mal and well nourished children aged 5 years old. Therefore, this study was conducted.

SUBJECTS AND METHODS


The sample consisted of 60 children with age 5 years old from kindergartens in Baghdad. The selected sample divided according to nutritional status (30 malnourished and 30 well nourished children). All childs had deciduous teeth, no permanent teeth erupted. Carious tooth was diagnosed according to criteria suggested by Muhlemann, this allowed recording decayed lesion of primary teeth by severity (d1-4mfs) (20). Plane mouth mirror and sickle-shaped dental explorer were used. Dental plaque (PI) recorded (21) following the criteria described by Sillness and Le. For the assessment of gingival health

(1) Lecturer, Department of Pediatric and Preventive dentistry, College of Dentistry, University of Baghdad.

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condition, the gingival index (GI) of Le and Sillness used (22), the whole teeth were examined and four surfaces of each tooth were scored. Nutritional status was assessed by the indicator: height for age (HA) (23) by using height measuring board concerning the date of birth. The value of nutritional indicators was compared with the international reference values that defined by the 2000 Centers for Disease Control and Prevention (CDC) growth charts (24). The cut-off point (HA) to define malnutrition was -2 SD (25). Each child was asked to chew a piece of Arabic gum (0.5-0.7 gm) until 5 ml of whole stimulated saliva was collected in a sterile capped bottle using a standardized method (26). Salivary vitamin E (mg/dl) was determined by using a colorimetric method (27). This method depends on Emmerie-Engel reaction (oxidationreduction reaction). Level of vitamin C (mg/dl) in saliva was determined photometrically with 2, 4-dinitrophenyl hydrazine (DNPH) to form red bis-hydrazone (28). The optical density differences between irradiated and non-irradiated saliva extracts can therefore be used to estimate vitamin A concentration in saliva (29). Total protein (mg/dl) determined by colorimetric method. A ready kit was used by Labkit, Nau J. Protein react in acid solution with pirogallol red and molybdate to form a colored complex. The intensity of the color formed is proportional to the protein concentration in the sample (30). SPSS version 18 (Statistical Package for Social Sciences) was used for statistical analyses. The variables were described by mean and SD and the parametric statistical tests of significance were used. The independent samples t-test was used to test the statistical significance of difference in mean between groups of study. The linear correlation between two quantitative variables is measured by Spearman's rank linear correlation coefficient, while multiple linear regressions were used to assess independent effect of explanatory variables on dependent quantitative variable. The confidence limit was accepted at 95%.

RESULTS
The relative frequency of caries free children among well nourished and malnourished groups is seen in Table (1). A higher percentage of caries-free children were recorded among well nourished compared to malnourished children. Table (2) represents the caries-experience (dmfs) of primary teeth among malnourished and well nourished children. A higher mean value of decayed surface (ds) was noticed among
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malnourished children compared to well nourished with statistically highly significant difference (t= 5.497, df= 58, P< 0.001). A statistically significant difference was recorded between two groups regarding missing surface (ms), as a higher value was noticed among malnourished group (t= 2.175, df= 58, P< 0.05). A higher mean value of filling surface (fs) was recorded among malnourished with no statistical significant difference (P> 0.05). A higher mean value of dmfs was reported among malnourished in comparison to well nourished children with statistically highly significant difference (t= 6.349, df= 58, P< 0.001). Table (3) illustrates the grades of caries severity for primary teeth among study groups. A higher mean values of d1, d2, d3 and d4 were recorded among malnourished children in comparison to well nourished, a statistically significant and highly significant differences were noticed regarding d2, d3 and d4 (d2: t= 2.336, df= 58, P< 0.05; d3: t= 2.473, df= 58, P< 0.05; d4: t= 4.104, df= 58, P< 0.001). Table (4) demonstrates the mean values of plaque and gingival indices among malnourished and well nourished children. A higher mean values of plaque and gingival indices were recorded among malnourished children in comparison to well nourished, differences were statistically highly significant (PI: t= 7.340, df= 58, P< 0.001; GI: t= 7.826, df= 58, P< 0.001). The correlation coefficient between caries experience of primary teeth and dental plaque in both groups is seen in Table (5). Positive strong highly significant correlations were noticed between ds, dmfs and PI among malnourished and well nourished children. A positive strong highly significant correlation was recorded between plaque and gingival indices in both groups (r = 0.99, P< 0.001; r = 0.97, P< 0.001) respectively. The mean values of salivary vitamins and total protein among malnourished and well nourished children are seen in Table (6). A lower mean values of salivary vitamins (A, C and E) and total protein were recorded among malnourished children in comparison to well nourished children with statistically highly significant differences (vitamin A: t= 11.658, df= 58, P< 0.001; vitamin C: t= 5.197, df= 58, P< 0.001; vitamin E: t= 21.460, df= 58, P< 0.001; total protein: t= 14.391, df= 58, P< 0.001). Table (7) illustrates the correlation coefficient between caries-experience (ds, dmfs) and salivary vitamins and total protein in both groups. Results revealed strong negative highly significant correlations between salivary

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vitamins and caries-experience in malnourished children. Regarding total protein, a positive weak significant correlation was noticed with ds, while it was not significant with dmfs. In well nourished children the direction of correlation was varied between negative and positive with higher significance regarding ds, while it was non significant correlation with dmfs. Table (8) illustrates the correlation coefficient between grades of caries severity and salivary vitamins and total protein among both groups. In malnourished children a positive highly significant correlations were recorded between d1 and vitamin A, d4 and total protein. While negative highly significant correlations were noticed for vitamins A, C, E with d4. Among well nourished children a positive highly significant correlations were recorded between d3, d4 and vitamin A, C while negative correlation with vitamin E and total protein. Table (9) demonstrates the correlation between PI, GI and salivary vitamins and total protein among both groups. In mal nourished children a positive highly significant correlations were seen between PI, GI and total protein, while negative highly significant correlations were found with all vitamins. In well nourished children a positive highly significant correlations were seen between GI and vitamins A and C, while negative highly significant correlations were found with vitamin E and total protein. Table (10) demonstrates the multiple linear regressions of dmfs with salivary constituents and plaque index. Vitamin A and PI were the most important factors in predicting dmfs, for each one unit increased in vitamin A the dmfs is expected to significantly decrease by 447.116, after adjusting other explanatory variables included in the model. While for each one unit increased in PI the dmfs is expected to significantly increase by mean of 12.260. The model was statistically significant and being able to explain 87 % of observed variation in the outcome variable (dmfs). Table (11) represents the multiple linear regressions of GI with salivary constituents and plaque index. For each one unit increased in vitamin A and PI, the GI increase by 10.7 and 0.94 respectively. The model was statistically significant and being able to explain 99% of observed variation in the outcome variable (GI).

DISCUSSION
Protein-energy malnutrition occurs when there are deficiencies in protein, energy foods or both, relative to a bodys needs. Studies suggest that caries of the primary dentition is associated
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with early childhood malnutrition (7-9, 31). In the present study a higher mean value of dmfs was recorded among malnourished in comparison to well nourished children with statistically highly significant difference. Mal- nutrition may influence the development of the teeth (organic and inorganic composition) (3, 32) and this may explain the higher mean values of d1, d2, d3 and d4 among malnourished children. Protein-energy malnutrition (PEM) appears to have multiple effects on the oral tissues and subsequent oral disease development. During childhood, malnutrition may restrict organ development as salivary glands leading to a diminished metabolic capacity involving concomitant deficiencies of antioxidant nutrients (32-35) . This may explain the finding of present study as a lower mean values of salivary vitamins (A, C and E) and total protein were recorded among malnourished children compared to well nourished with highly significant differences. This was also found by other limited studies (33, 36-38) and animal study concerning vitamin A (39). Although lower mean values of salivary vitamins were noticed among malnourished children, strong negative highly significant correlations were recorded between vitamins A, C, E and caries-experience (d4, ds, dmfs) of primary teeth. While a positive highly significant correlations was seen with cariesexperience (d3, d4, ds) among well nourished. Salivary antioxidant system serves as an important ingredient in building resistance and might reduce the susceptibility to dental caries because of its free radical scavenging action (40). These vitamins were found adversely affect the oxidative carbohydrate metabolism within the plaque and this will affect the oxidationreduction balance within the cell of microorganism thereby affecting bacteria metabolism and energy generation leading to cell death (41). These findings were also recorded by previous Iraqi studies among healthy adult and elderly individuals concerning vitamin A, C and E (18, 19). The present study revealed a positive non significant correlation between d1 and vitamins among malnourished children; this may be explaining the protective role of these vitamins against oxidative stress (42). Salivary glandular hypofunction and saliva compositional changes may be mechanisms through which malnutrition is associated with caries. Regarding salivary total protein, a positive significant correlation with ds and d4 was seen among malnourished while it was negative highly significant correlation among well nourished children and this was found in other studies (18, 43,

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. This may explain the anticaries effect of total protein as increased concentration may give a protective role. In humans, after eruption of teeth there is no direct effect of protein on tooth susceptibility to caries, theoretically protein adsorbs on tooth surface and could decreases dental caries risk, but precise evidence is lacking (45) . Dental plaque is a complex microbial community growing as a biofilm on enamel surfaces. The etiology of both dental caries (tooth decay) and various forms of periodontal disease has long been recognized to be related to bacterial accumulations and plaque composition (32) and this finding was confirmed by studies (46, 47) as well as this present study; as a positive strong highly significant correlation was recorded among both groups. Gingival index showed a higher mean value among malnourished children in the present study and this was also reported by other studies (48, 49) . Vitamin requirements vary from species to species and are influenced by age, gender, and physiological conditions such as pregnancy, breast-feeding, physical exercise, and nutrition (50) . Periodontal disease progresses more rapidly in undernourished populations, the important role of nutrition in maintaining an adequate host immune response may explain this observation (51) . Although a lower mean of plaque and gingival index was seen among malnourished children, strong negative highly significant correlations were noticed with salivary vitamins. Other studies reported the same findings (38, 52, 53). The contents of saliva are likely influenced by nutrients consumed daily, with consequences to oral health (50, 54, 55). Low salivary antioxidant levels could be the result of periodontal inflammation or could be a risk factor for periodontitis, Hypothetical associations between gingivitis and vitamins are supported by the observations that additional vitamins are required during infectious diseases, due to increased oxidative stress. It appears that water soluble antioxidant nutrients reduced vitamin C may be initially consumed, followed by lipid soluble antioxidants E. Also, it has been reported that vitamin C regenerates vitamin E by nonenzymatic mechanisms (56). These results can also explained as the periodontal disease is associated with reduced levels of salivary antioxidant and this may lead to increased oxidative damage within the oral cavity (57). protective effects of vitamin C in maintaining tissue homeostasis include its key function in collagen synthesis and therefore maintenance of the structural integrity of the connective tissues
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as well as its role as a radical scavenger, so lower vitamin C level is associated primarily with defective collagen synthesis, causing tissue dysfunction such as impaired wound healing and ruptured capillaries because of insufficient support of the capillary walls by the connective tissues (58). Total protein was found to correlate positively and strongly highly significant with plaque index and gingival index among malnourished children and only with GI among well nourished. This was also found in other study (18). This may provide protection against Reactive Oxygen Species (ROS) induced damage of periodontal tissue (51, 59). This study suggests that malnutrition act as a risk factor for dental caries and gingival disease.

REFERENCES
Gershwin ME, German JB, Keen CL. Nutrition and immunology. Human Press, USA. 2000, PP:3-13. 2. Gorstein J, Sullivan K, Yip R, De Onis M, Trowbridge F, Fajans P, Clugston G. Issues in the assessment of nutrition status using anthropometry. Bull WHO 1994; 72(2):273-83. 3. Moynihan P. Diet and dental caries. In: The prevention of oral disease by Murray JJ, Nunn JH, Steele JG. 4th ed. Oxford, Italy. 2003, PP:9-34. 4. WHO: Oral health surveys basic methods 4th ed. World Health Organization. Geneva, Switzerland, 1997. 5. Lingstrm P, Moynihan P. Nutrition, saliva and oral health. Nutrition 2003; 19:567-9. 6. Alvarez JO, Caceda J, Woolley TW, Carley KW, Baiocchi N, Caravedo L, Navia JM. A longitudinal study of dental caries in the primary teeth of children who suffered from infant malnutrition. J Dent Res 1993; 72(12):1573-6. 7. Al-Obaidi WS.: Oral health status in relation to nutritional status among kindergarten children in Baghdad-Iraq. M. Sc. Thesis, College of Dentistry, University of Baghdad, 1995. 8. Diab BS. Nutritional status in relation to oral health condition among 6-10 years primary school Iraqi children in the middle region of Iraq. Ph.D. Thesis, College of Dentistry, University of Baghdad, 2003. 9. Psoter WJ, Reid BC, Katz RV.: Malnutrition and dental caries: A review of literature. Caries Res 2005; 39(6):441-7. [IVSL] 10. Droosh MK. Protein-energy malnutrition in relation to oral health condition among 6 and 9 year old primary school children in Sulaimania city in Iraq. M. Sc. Thesis, College of Dentistry, University of Baghdad, 2007. 11. Al-Janabi WHE. Dental caries, enamel defect and malocclusion of primary dentition in relation to nutritional status among kindergarten children in Hilla city. M. Sc. Thesis, College of Dentistry, University of Baghdad-Iraq, 2008. 12. Hasan ZS. The effect of nutritional status on dental health. Salivary physicochemical characteristics and odontometric measurements among five years old kindergarten children and fifteen years old students. Ph.D. Thesis, College of Dentistry, University of Baghdad, 2010. 1.

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13. Whelton H. Introduction: The anatomy and physiology of salivary glands. In: Saliva and oral health by Edgar WM and O'Mullane DM. Chapter one. 2nd ed. British Dental Association, London, 1996, PP: 1-8. 14. Lenander-Lumikari M, Loimaranta V. Saliva and dental caries. Adv Dent Res 2000; 14:40-47. 15. Gorelik S, Kohen R, Ligumsky M, Kanner J. Saliva plays a dual role in oxidation process in stomach medium. Arch Biochem Biophys 2007; 458(2):236243. 16. Marquis RE. Oxygen metabolism, oxidative stress and acid-base physiology of dental plaque biofilms. J Ind Microbiol 1995; 15(3):198-207. 17. Staudte H. Vitamin C and periodontal disease. Dental Abstracts 2006; 51 (3): 174-175. 18. Yas B. Salivary antioxidants and physicochemical characteristics related to oral health status among a group of old adults. Ph.D. Thesis, College of Dentistry, University of Baghdad, 2009. 19. Kanan B. Oral health condition and salivary lipid soluble vitamins among group of women aged 30-39 years with breast cancer. Ms.C. Thesis, College of Dentistry, University of Baghdad, 2011. 20. Muhlemann HR.: Oral epidemiology-caries. In: Introduction to oral preventive medicine. Buch-und Zeitschriften- Verlag, Die Quintessenze, 1976 (Translated in English). 21. Silness J, Le H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964; 22:121-135. 22. Le H and Silness J. Periodontal disease in pregnancy. I. Acta Odontol Scand 1963; 21:533-51. 23. Trowbridge FL. Evaluating nutritional status of infant and children. In Paige D.M. eds. Clinical nutrition. 2nd ed. The C.V. Mosby Comp. St Louis. Washington D.C. Toronto, 1988, PP:119-36. 24. Kuczmarski RJ, Ogden CL, Grummer-Strawn LM. CDC growth charts: United States. Advance data from vital and health statistics; no. 314. Hyattsville, Maryland: National Center for Health Statistics, 2000. 25. Dipley MJ, Staehling N, Nieburg P, Trowbiidge FL. Interpretation of Z-score anthropometric indicators derived from the international growth reference. Am J Clin Nutr 1987; 46: 749-62. 26. Tenovuo J and Lagerlf F.: Saliva In: Textbook of clinical cariology edt. By Thylstrup A and Fejerskov O. 2nd ed. Munksgaard, Copenhagen, Denmark; 1996, PP:17-44. 27. Varley H. Practical clinical biochemistry. 4th ed. The white friars press limited. London and Tonbridge, Great Britain, 1967. 28. Colowick SP, Kaplan NO. Methods in enzymology. Vol. 62, part D, Academic press, USA, 7, 1979. 29. Wooton ID. Microanalysis in medical biochemistry. 5th edition; Churchill living stone, Edinburgh & London, 1974. 30. Orsonneau JL, Douet P, Massoubre C, Lustenberger P, Benard S. An improved pyrogallol redmolybdate method for determining total urinary protein. Clin Chem 1989; 35: 2233-6. 31. Radhi NJ. Oral health status in relation to nutritional analysis and salivary constituents among a group of children with Down's Syndrome in comparison to normal children. Ph.D. Thesis, College of Dentistry, University of Baghdad, 2009.

32. Thylstrup A and Fejerskov O. Textbook of clinical cariology. 2nd ed. Munksgaard, Denmark; 1996. 33. Ohmori K, Ebihara S, Kuriyama S, Ugajin T, Ogata M, Hozawa A, Mutasui T, Tsubono Y, Arai H, Sasaki H, Tsugi I. The relationship between body mass index and a plasma lipid peroxidation biomarker in an older, healthy asian community. Ann-Epidemiology 2005; 15 (1):80-4. 34. Psoter WJ, Gebrian B, Prophete S, Reid BC, Katz RV. Effect of early childhood malnutrition on tooth eruption patterns in Haitian adolescents. Comm Dent Oral Epi. 2007 in press. 35. Psoter WJ, Spielman AL, Gebrian B, St Jean R, Katz RV.: Effect of childhood malnutrition on salivary flow and pH. Arch Oral Biol 2008, 53(3):231-7. [IVSL] 36. Johansson I, Lumikari M, Saellstrom AK. Saliva composition in Indian children with chronic protein energy malnutrition. JDR 1994; 73(1):11-19. 37. Pannunzio E, Amancio O, Vitalle M, De Souza D, Mendes F, Nicolau J: analysis of the stimulated whole saliva in overweight and obese school children. Rev Assoc Med Bras 2010; 56(1): 32-6. 38. Witwit SS. Salivary antioxidants and nutritional status among chronic periodontitis patients. MS.C. Thesis, College of Dentistry, University of Baghdad, 2010. 39. Salley JJ, Bryson WF and Eshleman JR. The effect of chronic vitamin A deficiency on dental caries in the Syrian. J Dent Res 1959; 38: 1038-43. 40. Simonff M, Sergeang C, Gamier N, Moretto P, Vlabador Y, Simonoff G, Conri C. Antioxidant status (selenium), vitamins A and E and aging. EXE 1992; 62:368-97. 41. Marques RE.: Oxygen metabolism, oxidative stress and acid-base physiology of dental plaque biofilms. J Ind Microbiol 1995; 15(3):198-207. 42. Herbert V. Prooxidant effects of antioxidant vitamins. Introduction. J Nutr 1996; 126:197S-200S. 43. Cheatham C, Michalic S, Laven G. Immunoglobulin in saliva of protein-calorie malnourished hospitalized adults. Nutrition Res 2006; 4(1):33-41. 44. Tulunoglu O, Demirtas I, Tulunoglu I. Total antioxidant levels of saliva in children related to caries, age, and gender. Int J Pediatr Dent 2006; 16(3):186-91. 45. Hay DI, Bowen WH. The functions of salivary proteins. In: Saliva and oral health by Edgar WM and O'Mullane DM. Chapter one. 2nd ed. British Dental Association, London, 1996, PP:105-122. 46. El-Samarrai SK.: Major and trace elements of permanent teeth and saliva among a group of adolescent, in relation to dental caries, gingivitis and mutans streptococci. Ph.D. Thesis, College of Dentistry, University of Baghdad, 2001. 47. Rozkiewicz D, Daniluk T, Zaremba ML, CylwikRokicka D, uczaj-Cepowicz E, Milewska R, Marczuk-Kolada G, Stokowska W. Bacterial composition in the supragingival plaques of children with and without dental caries. Advances Medical Sciences 2006; 51(1):182-6. 48. Abrams, Romberq. Gingivitis in children with malnutrition. J Clin Pediatr Dent. 1999;23(3):18994. 49. Dasnash. The relation between protein energy malnutrition and gingival status in children. East Mediterr Health J.2000; 6(2-3):507-10.

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50. Mangan DF. Nutrition and oral infectious diseases: Connections and future research. Compend Cont Educ Dent 2002; 23(5):416-422. 51. Enwonwu CO.: Interface of malnutrition and periodontal diseases. Am J Clin Nutr 1995, 61(1):430-436. 52. Slade EW Jr, Bartuska D, Rose LF, Cohen DW. Vitamin E and periodontal disease. J Periodontol, 1976; 47(6):352-4. 53. Chapple I L C, Matthews JB. The role of reactive oxygen and antioxidant species in periodontal tissue destruction. Periodontol 2007; 43: 160232. 54. Jentsch H, Beetke E, Gcke R. Salivary analyses and caries increment over 4 years: an approach by cluster analysis. Clin Oral Invest 2004; 8:156-160. [IVSL]

55. Raymond G, Schipper a, Erika Silletti a, Monique H, Vingerhoeds. Saliva as research material: biochemical, physiochemical, and practical aspects. Archives of Oral Biology 2007;( 52):1114-35. 56. Cohen AC. Partners in defense, vitamin E and C. J Physiol Pharmacol 1993; 71: 725-31. 57. Sculley DV and Langley-Evans SC. Salivary antioxidants and periodontal disease status. Proceedings of the Nutrition Society 2003; 61(1): 137143. 58. Balwant R. Salivary Levels Vitamin E and C in Different Histological Grading of Oral Cancer. Pesq Bras. Odontoped Clin Integr 2008; 8(1): 123-5. 59. Rudney J, Larson C. Saliva protein binding to streptococcal layers placed at different oral sites in 48 persons. J Dent Res. 1996; 75(10):1789-97.

Table 1: The Relative Frequency of Caries Free Children by Gender


Malnourished Well nourished Caries free No. % No. % (0/15) 0.0 (4/15) 26.7 Males (0/15) 0.0 (3/15) 20.0 Females (0/30) 0.0 (7/30) 23.3 Total

Table 2: Caries Experience (dmfs) of Primary Teeth among Malnourished and Well nourished Children
ds ms fs dmfs Mean SD Mean SD Mean SD Mean SD Malnourished 20.3612.79 1.433.19 0.270.87 22.0612.08 Well nourished 6.136.12 0.130.73 0.130.51 6.396.05 Groups

Table 3: Decayed Surfaces of Primary Teeth by Grades of Lesion Severity (d1-4) of Malnourished and Well nourished Children
d2 d3 d4 d1 Mean SD Mean SD Mean SD Mean SD Malnourished 1.231.85 1.801.86 4.363.77 12.9613.69 Well nourished 0.901.77 0.831.29 2.073.42 2.333.73 Groups

Table 4: Plaque and Gingival Indices of Malnourished and Well nourished Children
Mal nourished Well nourished Groups PI GI Mean SD 1.29 0.67 1.24 0.67 Mean SD 0.31 0.29 0.23 0.22

Table 5: Correlation Coefficient between Plaque Index and Caries Experience of Primary Teeth in Malnourished and Well nourished Children
ds dmfs r P r P Mal nourished 0.91 <0.001* 0.90 <0.001* Well nourished 0.87 <0.001* 0.86 <0.001* Groups
* Highly Significant

Table 6: Mean Values of Salivary Vitamins (mg/dl) and Total protein (mg/dl) among Malnourished and Well nourished Children
Vitamin A Vitamin C Vitamin E Total protein Mean SD Mean SD Mean SD Mean SD Malnourished 0.0130.003 0.0200.005 0.0250.036 0.1990.175 Well nourished 0.0280.006 0.0390.004 0.0450.004 0.3980.074 Groups

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Table 7: Correlation Coefficient between Caries Experience (ds, dmfs) of Primary Teeth and Salivary Vitamins (mg/dl) and Total protein (mg/dl) in Malnourished and Well nourished Children
ds dmfs r P r P Vitamin A -0.74 <0.001** -0.76 <0.001** Vitamin C -0.80 <0.001** -0.85 <0.001** Malnourished Vitamin E -0.80 <0.001** -0.79 <0.001** 0.08 Total protein 0.37 0.04* 0.32 Vitamin A 0.87 <0.001** -0.03 0.87 Vitamin C 0.91 <0.001** -0.02 0.88 Well nourished Vitamin E -0.86 <0.001** -0.08 0.64 Total protein -0.81 <0.001** 0.006 0.97 Groups Variables
* Significant, ** Highly Significant

Table 8: Correlation Coefficient between Caries Experience (Grades of Caries Severity) of Primary Teeth and Salivary Vitamins (mg/dl) and Total protein (mg/dl) among Malnourished and Well nourished Children
d1 d2 d3 d4 r P r P r P r P Vitamin A 0.50 0.004** -0.13 0.44 -0.25 0.18 -0.67 <0.001** Vitamin C 0.29 0.11 -0.20 0.91 0.01 0.93 -0.76 <0.001** Malnourished Vitamin E 0.23 0.20 -0.14 0.43 -0.03 0.86 -0.73 <0.001** Total protein -0.03 0.86 -0.14 0.46 -0.10 0.56 0.40 0.02* Vitamin A 0.10 0.58 0.14 0.43 0.56 0.001** 0.81 <0.001** Vitamin C 0.11 0.55 0.13 0.46 0.62 <0.001** 0.82 <0.001** Well nourished Vitamin E -0.20 0.28 -0.20 0.28 -0.63 <0.001** -0.64 <0.001** Total protein -0.17 0.35 -0.28 0.13 -0.53 0.002** -0.67 <0.001** Groups Variables
* Significant, ** Highly Significant

Table 9: Correlation Coefficient between Plaque Index, Gingival Index and Salivary Vitamins and Total protein In Malnourished and Well nourished Children
PI GI r P r P Vitamin A -0.63 <0.001* -0.62 <0.001* Vitamin C -0.81 <0.001* -0.79 <0.001* Malnourished Vitamin E -0.77 <0.001* -0. 77 <0.001* Total protein 0.49 0.005* 0.48 0.006* Vitamin A -0.08 0.67 0.88 <0.001* Vitamin C -0.06 0.73 0.91 <0.001* Well nourished Vitamin E -0.12 0.51 -0.89 <0.001* Total protein 0.07 0.69 -0.82 <0.001* Groups Variables
* Highly Significant

Table 10: Multiple Linear Regressions of dmfs with Salivary Constituents and Plaque Index
Variables Partial Regression Coefficient Standardized Coefficient P-Value -447.116 -0.301 0.022* Vitamin A -288.393 -0.243 0.190 Vitamin C 249.187 0.219 0.164 Vitamin E 10.197 0.094 0.307 Total protein 12.260 0.705 0.000** PI
P (model) < 0.001 R2= 0.87 *Significant, **Highly significant

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Table 11: Multiple Linear Regressions of Gingival Index with Salivary Vriables and Plaque Index
Variables Partial Regression Coefficient Standardized Coefficient 10.752 0.126 Vitamin A -2.564 -0.038 Vitamin C -10.147 -0.155 Vitamin E 0.108 0.017 Total protein 0.947 0.946 PI
P (model) < 0.001 R2= 0.99 *Highly significant

P-Value 0.000* 0.408 0.000* 0.444 0.000*

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Orthodontic bracket failure

Orthodontic bracket failure rate; A comparative clinical study between light cured and chemically cured (no mix) bonding systems
Natheer A. Rasheed, B.D.S., M.Sc. (1)

ABSTRACT
Background: The purpose of this in vivo study was to investigate the difference in brackets failure rate between light cured and chemical cured (no mix) composites used in brackets bonding procedures. Materials and methods :A total of 729 Roth 0.22 stainless steel brackets were bonded to 40 patients requiring orthodontic treatment in the form of 3 groups ;light cured 237 ,no mix 240 ,control(split mouth design)249. Results: The results showed non significant difference in brackets failure rate between the groups and the majority of the failed brackets were in the posterior region. Conclusion: Clinically, the light cured bonding system has a comparable strength to no mix. Key words: Light cure composite, no mix, bonding. (J Bagh Coll Dentistry 2012;24(3):137-139).

INTRODUCTION
Orthodontic treatment with brackets generally takes approximately 2 years. Bond failure of brackets during this period retards treatment and is costly in terms of time, material, and patient inconvenience.. Hypothetically, in-vivo testing in controlled trials is the best way to test the effectiveness of a bonding system and any detrimental effects on the enamel 1. Bond failure during orthodontic treatment is relatively frequent and undesirable. The time taken to clean, prepare, and bond new bracket can be disruptive in a busy practice and it might also lengthen the overall treatment time2. To overcome bond failure, good bond strength must be achieved which depends on; The nature of enamel surface, Enamel conditioning procedures, the etching time , The shape and the design of the bracket base and The type of adhesive used 3 which should be; Dimensionally stable, Quite fluid, has Excellent inherent strength and Easy to use clinically 4. The chemically cured composite (self curing acrylic) was the first system developed for orthodontic bracket bonding 5,The polymerization of chemically cured composites starts immediately after mixing, limiting the working time, the number and accuracy of bracket placement with each mix 6. Auto cured (chemically activated) systems generates a small amounts of heat during curing 7. And The liquid activator of no-mix system is toxic, and allergic reaction has been reported in patients, dental assistants and doctors when such adhesives were used 8-10 .

A visible-light curing system was suggested in 1979 by Douglas et al, to be used in the restorative dentistry and orthodontics, to activate polymerization of filled and unfilled resins 11. Sonis (12) and Hamula (13) stated that the light cured adhesives offer a number of significant advantages over chemically cured ones, unlimited working time during bracket placement, less patient discomfort because of accelerated setting time. Significant less chair time, arch wire can be placed immediately without having to wait 8-10 minutes for the adhesive to bond completely, Bracket placement and flash removal are much easier with the light activated composites, and Easier clean-up. Read 14 stated that an immediate force could be placed on the bracket after curing because the entire adhesive under the bracket would be polymerized after the initial exposure to the visible light. However, when HelvatjoglouAntoniadi et al 15 evaluated the degree of polymerization of both visible light and chemically cured adhesives for setting times from 10 minutes to 12 months, they reported a continued hardening or progressive polymerization throughout this period.

MATERIALS AND METHODS


The subjects of this study were a 40 patients requiring treatment with fixed orthodontic appliances meeting the following criteria: the planned mechano-therapies are as similar as possible; their teeth are free of caries, reconstructions or enamel disorders at the facial surface, no undesirable antagonistic contacts between teeth and brackets. A total of 729 Roth 0.22 (ortho classic) brackets were bonded. for 14 patients; a split mouth design were used in which 127 brackets were bonded by chemically cured composite (no

(1)Lecturer. Department of POP. College of Dentistry, University of Al-Anbar.

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mix) and 122 brackets were bonded by light cured composite and it is considered as the control group, for 13 patients; 237 brackets were bonded totally by light cured composite and for the rest 13 patients; 240 brackets were bonded totally by chemical cured composite (no mix). Method: prophylaxis of teeth with non fluoridated pomus followed by etching with 37% phosphoric acid (resilience) liquid etchant from ortho technology, for 30 seconds (al umar2001), then the teeth washed by water spray for 10 seconds and dried . Bonding by no mix: a thin layer of a (resilience) liquid activator was applied on the facial surface of the teeth and on bracket base. (Resilience) no mix bracket adhesive was applied to the bracket base which was then placed on the tooth surface and pressed firmly, excess resin was removed around the bracket with a probe. Bonding with light cure :a thin layer of( resilience ) sealant resin were applied on the teeth and exposed for 10 seconds per tooth to a visible light source (wood pecker ) led type from (Guilin wood pecker medical instruments ),wave length;480nm and intensity of 850-1000mW/ cm .a (resilience ) light cured bracket adhesive was applied to the base of the bracket ,which was then applied on the tooth surface and pressed firmly,

excess resin was removed ,and it was polymerized by a (wood pecker ) light source (10 seconds for each bracket-adhesive interface: 5 seconds on the mesial and 5 seconds on the distal)16. ten minutes after brackets bonding the corresponding arch was placed for the split type group and the chemically cured group, (while for the light cure group it is placed immediately)12,13, and the first wire was 0.14 NiTi (svenska orthocut) to be replaced later by the corresponding sequence of arches. Bracket bond failure was recorded only for the first-time failures, and the observation period was 9months16. Bond-failure rates during the nine-month period were estimated for each type of adhesive and Failure rates were analyzed using 2 test statistics at ! = .05 level of significance.

RESULTS
The numbers and rates of brackets failure for the three groups are shown in table (1). The chi squared test showed no significant difference in brackets failure rate between the groups (p= 0.100).Table (2) shows the distribution of brackets failure rates among the teeth for all the groups. The result shows that the majority of failed brackets were for the premolars.

Table 1: Bond failure rates


Group Brackets(n) Failures(n) Failure rate(%) 249 18 7.2 control 237 9 3.7 Light cured 240 15 6.2 No mix The chi-squared test found no significant differences (p=0.14) in brackets failure between the groups

Table 2: Failure rate distribution among teeth


Group Incisors &canines premolars Control (n) 2 16 Rate (%) 11.1 88.8 No mix (%) 2 13 Rate (%) 13.3 86.6 Light cured (n) 1 8 Rate (%) 11.1 88.8

DISCUSSION
The advantages of the split mouth design is; time saving, fewer subjects required and the uniform reaction toward the material and technique regarding the affinity of the teeth to the adhesive, but the patient might use one side more than the other which makes it liable to errors, in addition to that, the comparison between split mouth and full mouth design provides an opportunity to test the material in a wider range of possibilities. The result of this study shows non significant difference between groups in bracket failure rates which is compatible to the result of in vivo study made by Armas etal 17,and coincide with the result
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of the invitro study of chamda and Stein 18 , while Saeytijd et al 19 found in a clinical study that the brackets bonded by a chemically cured composite failed significantly less than the light cured composite which disagree with our result, but the type of comparison made in this study strengthen the findings of armas et al 17 and chamda and Stein 18. The non significance in bracket failure rates between groups indicates that the ease of use of the light cured composite for brackets fixation will not be a problem regarding bond longevity in contrast with the traditionally used chemically cured one.

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The result of bracket failure rates for previously done studies were 6.6% (20), 6.34% (21) and 6.08 (22), which are close to the rate obtained in this study (7.2%) ,indicating that the use of light cured and chemically cured composites for bonding produces results close to this average, while the rates for the, light cured , chemically cured, composites separately were; 3.7% ,6.2% respectively which differs from that of Koupis et al 16 who found it 5.00% for the light cured with LED light source and 3.33% for the light cured with halogen light source, and Armas et al 17 who found it 11.3% for the light cured and 12% for the chemical cured . The distribution of the bracket failure rate in this study indicates that the higher rate was for the posterior teeth regarding both types of adhesives which differ from that of Saeytijd et al 19 who found it more posteriorly for the light cured and distributed more equally over the dental arches for the chemical cured.

REFERENCES
1. Leloup G, DHoore W, Bouter D, Degrange M, Vreven J. Meta analytical review of factors involved in dentin adherence. J Dent Res 2000;80:1605-14 2. Bishara SE, Laffoon JF, VonWald L, Warren J. The effect of repeated bonding on the shear bond strength of different orthodontic adhesives. Am J Orthod Dentofacial Orthop 2002b; 121(5): 521-5. 3. Sunna S, Rock WP. An ex-vivo investigation into the bond strength of orthodontic brackets and adhesive systems. Br J Orthod 1999; 26(1): 47-50. 4. Proffit WR, Fields HW, Sarver DM. Contemporary orthodontics. 4th ed. Mosby, Inc., anaffiliate of Elisive Inc., 2007. 5. Newman GV, Synder WH, Wilson CE Jr. Acrylic adhesives for bonding attachments to tooth surfaces. Angle Orthod 1968; 38(1): 12-8. 6. Greenlaw R, Way DC, Galil KA. An in vitro evaluation of a visible light-cured resin as analternative to conventional resin bonding systems. Am J Orthod Dentofacial Orthop 1989; 96(3): 214-20. 7. Albers HF. Tooth-colored restoratives: principles and techniques. 9th ed. BC Decker Inc 20 Hughson St. South, Hamilton, London 2002. 8. Fredericks HE. Mutagenic potentials of orthodontic materials. Am J Orthod 1981; 80:316-24. 9. Thompson IR, Miller EG, Bowles WH. Leaching of un polymerized materials from orthodontic bonding resin. J Dent Res 1982; 61: 989-92. 10. Miller EG, Thomson CR, Zimmerman E, Bowles WH. Invivo studies on the carcinogenic potential of an orthodontic bonding resin. Am J Orthod 1984; 86: 342-6. 11. Douglas WH, Craig RG, Chen CJ. A new composite restorative based on a hydrophobic matrix. J Dent Res 1979; 58 (10): 1981-6. 12. Sonis AL. Comparison of a light-cured adhesive with an autopolymerization bonding system. J Clin Orthod 1988; 22(11): 730-2. 13. Hamula DW. Technique clinic: Direct bonding with light-cured adhesives. J Clin Orthod 1991; 25: 437-8.

14. Read MJF. The bonding of orthodontic attachment using a visible light cure adhesive. Br J Orthod 1984; 11: 16-20. 15. Helvatjoglou-Antoniadi M, Papadigianis Y, Kolinioton-Kubia E, Kubias S. Surface hardness of light cured composite resins. J Prosthet Dent 1991; 65: 215-20. 16. Koupis NS, Eliades T, Athanasiou AE. Clinical evaluation of bracket bonding using two different polymerization sources. Angle Orthod 2008; 78(5): 922-5. 17. Armas Galindo HR, Sadwosky PL, Vlachos CH, Jacobson A, Wallance D. An in vitro comparison between a visible light- cured bonding system and a chemically cured bonding system. Am J Orthod Dentofac Orthop 1998; 113(3): 271-5. 18. Chamada RA, Stein E. Time related bond strengths of light cured and chemically cured bonding systems: An in vitro study. Am J Orthod Dentofac Orthop1996; 110: 378-82. 19. Saeytijd CD, Carels CEL, Lesaffre E. An evaluation of light curing composite for bracket placement. Eur J Orthod 1994; 16(6):541-5. 20. Sunna S, Rock WP. Clinical performance of orthodontic brackets and adhesive systems: a randomized clinical trial. Br J Orthod 1998; 25:283-7. 21. Linklater RA, Gordon PH. Bond failure patterns in vivo. Am J Orthod Dentofac Orthop 2003; 123:534-9. 22. Compay MD, Plasencia E, Vincente, Bravo LA, Cibrian R .Effect of saliva contamination on bracket failure with a self-etching primer: A prospective controlled clinical trial. Am J Orthod Dentofac Orthop 2010; 137:679-83.

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Correlation between caries related bacteria in plaque and saliva in different age group children
Zainab J. Jafar, B.D.S., M.Sc. (1) Yasameen A.A. Al-Bayati, B.D.S., M.Sc. (1) Ghada I. Taha, M.Sc, Ph.D. (2)

ABSTRACT
Background: Dental plaque contains bacteria that are both acidogenic and acidoduric. Different types of streptococci were identified in saliva. Although many bacterial subspecies have been shown to be associated with caries, streptococcus mutans is still believed to be the most important bacterium in the initiation of this disease while lactobacillus is correlated with the active caries episode. This study was conducted in order to estimate the correlation between caries related bacteria in plaque and saliva in different age group children. Materials and methods: Fifty three children aged 3-10 years old were chosen for this study. Recording of dental caries was carried out by the dmfs index for primary teeth and DMFS index for the permanent teeth according to the criteria suggested by the WHO. One ml of unstimulated (resting) whole saliva was collected from the children using spitting method then diluted and applied on the surface of agar media specific for streptococcus mutans and lactobacilli growth. Dental plaque sample was taken from the buccal surface of the maxillary second primary molar by a clean toothpick and store in Epindorf tube which contain 1 ml. normal saline then inoculated in the same growth media that were used with the salivary samples. Colonies of the bacteria were counted with the aid of dissection microscope (15 X) on the basis of their characteristic morphology. Results: Strong positive significant correlation was found between dmfs and ds components of the primary teeth, Positive results was found when correlating dmfs or ds with streptococcus mutans in dental plague while a negative correlation was found with lactobacilli. Negative correlation was found when correlating dmfs with streptococcus mutans in saliva while the relation is positive with lactobacilli. In dental plaque and in saliva there was a strong positive highly significant correlation between the DMF and the DS. Correlation coefficient between DMFS with the bacterial counts of the caries related microorganisms (streptococci and lactobacilli) in the dental plaque and in saliva revealed weak, negative not significant correlations. Conclusion: The relation is not significant between the caries related microorganisms with each other in different media; either the dental plaque or saliva. Key words: Correlation, streptococcus mutans, lactobacilli, saliva, plaque, dmfs, DMFS. (J Bagh Coll Dentistry 2012;24(3):140-144).

INTRODUCTION
Dental caries is a microbial disease of the calcified tissues of the teeth characterized by demineralization of the inorganic portion and destruction of the organic portion of the tooth (1). Several interrelated factors such as: the tooth and saliva (host), microorganisms and cariogenic substrate (2).These factors interact in a certain period of time, causing an imbalance in the demineralization and remineralization between tooth surface and the adjacent plaque (biofilm)( 3). Plaque is a complex mixture of dense microbial elements enmeshed within a gel like matrix of bacterial polysaccharide, salivary proteins and cellular components of the oral mucosa (4). Dental plaque contains bacteria that are both acidogenic and acidoduric. Although many bacterial subspecies have been shown to be associated with caries, streptococcus mutans is still believed to be the most important bacterium in the initiation of this disease while lactobacillus is correlated with the active caries episode (2, 5).
(1) (2) Assistant lecturer, Department of Pediatric and Preventive Dentistry, College of Dentistry, University of Baghdad. Lecturer, Department of Basic Sciences, College of Dentistry, University of Baghdad.

Saliva has several critical roles in the caries process; it is excreted at different rates and with different constituents depending on the presence or absence of stimulatory factors and it helps to balance the caries process and has critical role in remineralization as it provides a stabilized supersaturated solution of calcium and phosphate ions as well as fluoride ions from extrinsic sources (2).Different types of streptococci were identified in saliva (6). Different studies have shown a correlation between counts of streptococcus mutans in the oral cavity and both the prevalence and incidence of caries (7, 8, 9, 10).However, in other studies, no correlation has been found between the quantity of streptococcus mutans and the incidence of caries (11, 12). On the other hand, some researchers have suggested the importance of streptococci other than streptococcus mutans in the generation of dental caries (13).Usually there are only few lactobacilli in saliva, their number increases if mutans streptococci start to colonize in the oral cavity, since they produce a favorable acid environment for lactobacilli, so the pH-value decreases (14). Lactobacilli preferably settle in niches with a low pH-value and in the vicinity of plaque accumulation (15). Koroluk et al and

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Ziotopolus et al were found significant correlations between the plaque and salivary levels of mutans streptococci and caries experience in the primary teeth(16, 17).Concerning permanent teeth E. Bramilla et al found a statistically positive relationship between the levels of salivary streptococcus mutans and lactobacilli and both were significantly correlated to caries(18).

MATERIALS AND METHODS


Fifty three children aged 3-10 years old were chosen for this study, all of them were with carious lesion, attended the department of Pediatric and Preventive Dentistry, Baghdad teaching hospital for Dentistry, and neighboring primary schools and kindergartens. Recording of dental caries was carried out by the dmfs index for primary teeth and DMFS index for the permanent teeth according to the criteria suggested by the WHO in 1987(19), with no history of any systemic disease. Salivary samples were collected in the morning between (10-11) a.m. at least one hour after breakfast, and then the children were asked to rinse out their mouths with water. The first mouthful of saliva was discarded, and then one ml unstimulated (resting) whole saliva was collected into small labeled plastic polyethylene tubes using spitting method for collection. The following points were followed for collection whole saliva according to Fejerskov and Thylstrup (20). 1. The patient should not eat or drink (except water) one hour before saliva collection. 2. A pre sampling period of one minute is recommended. 3. A fixed collection time (10-15 min. for unstimulated saliva) should be used. 4. The patient should sit in a relaxed position in an ordinary chair. 5. Samples containing blood should be discarded if chemical analyses of saliva are planned. After collection of the saliva, it was diluted with normal saline in the bacteriology laboratory of Baghdad university college and then by using micropipette the saliva was applied on the surface of Mitis salivaris agar and Rogosa agar medium which were used for mutans streptococci and lactobacilli growth. The plates were incubated in an anaerobic atmosphere for 48 hours at 37C. CFU with morphology characteristic of s.mutans and lactobacilli were counted and expressed as numbers of CFU per milliliter of saliva (21).

Dental plaque sample was taken from the buccal surface of the maxillary second primary molar which should be sound, otherwise the sample was taken from the maxillary first primary molar, by a clean toothpick and store in Epindorf tube which contain 1 ml. normal saline to prevent dryness while reaching the laboratory to be disperse for 30 seconds by vortex mix(22), then tenfold dilutions was made by normal saline to have a full colony counting for the caries related microorganisms (streptococcus mutans and lactobacilli), then inoculated in Mitis-Salivarius-Bacitracin agar which is the special selective media for the streptococcus mutans, and Rogosa agar media which is the special selective media for the lactobacilli. Colonies of the bacteria were counted with the aid of dissection microscope (15 X) on the basis of their characteristic morphology (21). Statistical analysis was done by using correlation test by the aid of the SPSS application version 13. Correlation is significant at 0.01 levels (2-tailed).

RESULTS
Descriptive statistics revealed that the caries experience for primary teeth was 31.517.56 with range 10(minimum) -88(maximum), while for the permanent dentition was 1.1251.39 with a range 0.0-4.0 (Table 1), age range was 3-10 years demonstrated in table 2. Correlation coefficient revealed that there was a weak negative not significant correlation between the caries related microorganisms (streptococcus mutans and lactobacillus) in the dental plaque. Also, the same result was found between the streptococcus mutans bacterial count in different media (plaque and saliva). On the other hand, when we correlated the lactobacillus bacterial counts in dental plaque and saliva, we found a weak positive not significant correlation. The same result was found when we correlate the caries related microorganisms (streptococcus mutans and lactobacillus) with each other in saliva (Table 3). Data analysis of the present study showed that there was a strong positive significant correlation between dmfs and ds components of the primary teeth. The results revealed that there was a weak positive not significant correlation between the caries experience of the primary teeth (dmfs or ds) and the streptococcus mutans bacterial counts in the dental plaque, while there was a weak negative not significant correlation between the caries experience in the primary teeth (dmfs or ds) with the lactobacillus bacterial counts in the dental plaque (Table 4).

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Different pictures were found concerning the relation of caries experience for the primary teeth with the bacterial count (streptococcus mutans and lactobacilli),the relation was a weak negative not significant with the streptococcus bacterial counts, and a weak positive non significant with the lactobacillus bacterial counts( Table 5). Concerning permanent teeth, data analysis of the present study showed that there was a strong positive significant correlation between DMFS and DS components. Correlation coefficient between caries experience in the permanent teeth with the bacterial counts of the caries related microorganisms (streptococci and lactobacilli) in the dental plaque revealed weak, negative not significant correlations (table 6). The same results were found when correlating the caries experience in the permanent teeth with bacterial counts of the caries related microorganisms (streptococci and lactobacilli) in saliva (Table 7).

DISCUSSION
There is a concept that the streptococcus mutans are responsible for the initiation of dental caries and lactobacilli are responsible for its progression (2, 5) , the same result was found in the present study as in dental plaque, negative correlation was found between streptococcus mutans and lactobacilli. Negative, weak, not significant correlation was found when correlating the streptococcus mutans bacterial count in dental plaque and saliva which agrees with Orstavik et al who stated that when bacteria were suspended in whole saliva, the adherence of streptococcus mutans was inhibited (23) . Opposite results was found when correlating lactobacilli bacterial count in dental plaque and saliva, the outcome was positive but weak not significant, this is in agreement with Nancy and Dorignac(24), while it was found that the correlation is positive between streptococcus mutans and lactobacilli in saliva, and this is in accordance with Brambilla et al(18).This may be due to the caries activity in dental carious patients(2,5). Strong positive significant correlation between dmfs and ds and the same result is obtained concerning the permanent teeth(DMFS,DS), may give a clue that the most apparent component of dmfs of the patients is ds and not the filling or missing surfaces, which indicates that these patients are with urgent need for motivation and visit their dentists for treatment. Positive results when correlating dmfs or ds with streptococcus mutans in dental plague, this may be explained by that the accumulation of plaque
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on teeth surface is the main causative factor of dental caries. While a negative correlation was found with lactobacilli, which could be attributed to the fact that the lactobacilli are responsible for the progression and not the initiation of dental caries. Negative correlation was found when correlating caries experience in the primary teeth with streptococcus mutans in saliva and this disagree with Ge et al (25); it may be due to different age group and different sample size. Positive correlation was found between caries experience in primary teeth with lactobacilli in saliva which can be explained by the fact that lactobacilli appear during the first years of a childs life, and are present in high numbers in saliva (26). Correlation coefficient between caries experience in the permanent teeth with the bacterial counts of the caries related microorganisms (streptococci and lactobacilli) in the dental plaque and in saliva revealed weak, negative not significant correlations. These results could be due to that this study involves different age group children which contain mixed dentition.

REFRENCES
Damle SG. Text book of pediatric dentistry. 3rd edition. Arya publishing house; 2009: p33. 2. Cameron AC, Widmer RP. Handbook of pediatric dentistry, 3rd edition. Elsevier limited; 2008:p39. 3. Harris R, Nicoll AD, Adair PM, Pine CM. Risk factors for dental caries in young children: a systematic review of the literature. Community Dent Health 2004; 21(Suppl):S71-85. 4. Rao A. Principles and practice of pedodontics.2nd edition. Jaypee brothers medical publishers ltd; 2008: p164. 5. Aguilera Galavis LA, Premoli G ,Gonzales A, Rodreguez RA. Caries risk in children determined by numbers of mutans streptococci and lactobacillus.J Clin Pediatr Dent 2005; 29(4):329-33. 6. Philip K, Wuen YT, Muniay S, Yaakob H. Identification of Major Cultivable Aerobic Bacteria in the Oral Cavity of Mandlaysian Subjects. Americ J Biochemist and Biotechno 2008; 4 (4): 367-70. 7. Loesche W. Role of Streptococcus mutansin human dental decay. Microbiology Review1986; 50: 353380. 8. Lang N, Hotz P, Gusberti F,Joss A. Longitudinal clinical and microbiological study on the relationship between infection with Streptococcus mutans and the development of caries in human. Oral Microbiology Immunology 1987; 2: 39-47. 9. Beighton D, Manji F, Baelum V, Fejerskov O, Johnson N, Wilton J. Associations between salivary levels of Streptococcus mutans, Streptococcus sobrinus, lactobacilli, and caries experience in Kenyan adolescents. J Dent Res 1989; 68: 1242-1246 (IVSL). 10. Crielaard W, Zaura E , Annemarie AS, Huse SM , Montijn RC, Keijser BJF. Exploring the oral 1.

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11.

12.

13.

14.

15.

16.

17.

18.

microbiota of children at various developmental stages of their dentition in the relation to their oral health.BMC medical Genomic 2011,4:-22 doi:10.1186/1755-8794-4-22.(IVSL) Marsh P, Featherstone A, MckeeA,Hallsworth A, Robinson C, Weatherell J, Newman H, Pitter A. A microbiological study of early caries of approximal surfaces in school children. J Dent Res1989; 68: 1151-4. Macpherson L, Macfarlane T, Geddes D, Stephen K. Assessment of the cariogenic potential of Streptococcus mutans strains and its relationship in vivo caries experience. Oral Microbiology Immunology 1992; 7: 142-7. Van Houte J, Sansone C, Joshipura K, Kent R. Mutans streptococci and non-mutans streptococci acidogenic at low pH, and in vitro acidogenic potential of dental plaque in two different areas of the human dentition. J Dent Res 1991; 70: 1503-7. Newbrun E. Preventing dental caries: breaking the chain of transmission. J Am Dent Assoc 1992; 123: 55-9. Beighton D, Brailsford S. Lactobacilli and actinomyces; their role in the caries process in: L.stosser (Hrsg.) Kariesdynamic und Kariesrisilko; Quintessenz verlags-Gmbh, Berlin 1998. Koroluk LD, Hoover JN, Komiyama K. The effect of caries scoring systems on the association between dental caries and Streptococcus mutans. ASDC J Dent Child 1995; 62(3):187-91. Zoitopoulos L, Brailsford SR, Gelbier S, Ludford RW, Marchant SH, Beighton D . Dental caries and caries associated micro organisms in the saliva and plaque of 3- and 4-year-old Afro-Caribbean and Caucasian children in south London. Arch Oral Biol 1996 Nov; 41(11):1011-8. Brambilla E, Twetman S, Felloni A, Cagetti M, Canegallo L, Garcia-Godoy F, Strohmenger L.

19. 20.

21.

22.

23.

24.

25.

26.

Salivary mutans streptococci and lactobacilli in 9and 13-year-old Italian schoolchildren and the relation to oral health. Clinical Oral Investigations 1999; 3(1). WHO. Oral health surveys: Basic methods. 3rd ed. Geneva, Switzerland 1987. Fejerskov O, Thylstrup A. The oral environment and introduction. Textbook of Clinical Cariology 2nd edit. Munksgaard. Copenhagen 1994; p: 13-17. Kishi M, Abe A, Kishi K, Ohara-Nemoto Y, Kimura S, Yonemitsu M. Relationship of quantitative salivary levels of s. mutans and s. sorbinus in mothers to caries status and colonization of mutans streptococci in plaque in their 2.5 year-old children. Community Dent Oral Epidemiol 2009; 37(3):241-9. Krishnakumar R, Singh S, Subba Reddy VV, Davangere. Comparison of level of mutans streptococci and lactobacilli in children with nursing bottle caries rampant caries, healthy children with 3-5 dmft/DMFT and healthy caries free children. J Indian Soc Pedo Prev Dent 2002; 20:1:1-5. Orstavik D, Kraus FW, Henshaw LC. In vitro attachment of streptococci to the tooth surface. Infect Immun 1974; 9(5):794-800. Nancy J, Dorignac G. Lactobacilli from the dentin and saliva in children. J Clin Pediatr Dent 1992; 16(2):107-11. Ge Y, Caufield PW, Fisch GS, Li Y. Streptococcus mutans and Streptococcus sanguinis Colonization Correlated with Caries Experience in Children Caries Res 2008; 42(6):444-8. Straetemans MM, van Loveren C, de Soet JJ, de Graaff J, ten Cate JM. Colonization with mutans streptococci and lactobacilli and the caries experience of children after the age of five. J Dent Res 1998; 77(10):18515.

Table 1: Caries experience for primary and permanent dentition.


Caries experience N Minimum Maximum Mean SD 53 10.00 88.00 31.5094 17.55718 dmfs* 24 .00 4.00 1.1250 1.39292 DMFS**
* decayed, missed, filled, surfaces in the primary teeth. ** decayed, missed, filled, surfaces in the permanent teeth.

Table 2: Distribution of the age groups


Age group N percent 3-6 35 66 7-10 18 34 53 100

Table 3: Correlation coefficient of the bacterial counts (streptococcus mutans and lactobacillus) in the dental plaque with the bacterial counts in the dental plaque and saliva.
Streptococcus mutans-plaque r P N -.010 .959 32 Lactobacillus-plaque Streptococcus mutans -saliva -.068 .711 32 .008 .964 32 Lactobacillus- plaque Lactobacillus- plaque .247 .174 32 Streptococcus mutans -saliva Lactobacillus- plaque

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Table 4: Correlation coefficient between the caries experience and the bacterial colony counts (streptococcus mutans and lactobacillus) in the dental plaque.
Caries experience Streptococcus mutans-plaque dmfs r P N .289 .109 32

ds*

-.107 .559 32 Lactobacillus- plaque Streptococcus mutans-plaque .320 .074 32 Lactobacillus- plaque
* decayed surfaces in the primary teeth.

-.195 .284 32

Table 5: Correlations between the caries experience in the primary teeth and the bacterial counts (streptococcus mutans and lactobacillus) in saliva.
Caries experience dmfs ds Saliva r P N Streptococcus mutans -.097 .597 32 .110 .551 32 Lactobacillus Streptococcus mutans -.083 .651 32 .200 .273 32 Lactobacillus

Table 6: Correlations between the caries experience in the permanent teeth and the bacterial counts (streptococci and lactobacilli) in the dental plaque:
Caries experience DMFS DS* plaque r P N Streptococcus mutans -.346 .174 17 -.198 .446 17 Lactobacillus Streptococcus mutans -.346 .174 17 -.198 .446 17 Lactobacillus

*decayed surfaces in the permanent teeth.

Table 7: Correlations between the caries experience in the permanent teeth and the bacterial counts (streptococci and lactobacilli) in saliva:
Caries experience DMFS DS Saliva r P N Streptococcus mutans -.081 .757 17 -.117 .654 17 Lactobacillus Streptococcus mutans -.081 .757 17 -.117 .654 17 Lactobacillus

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Quantitative analysis of IgG antinuclear antibody in chronic periodontitis patients


Batool H. Al-Ghurabei, B.Sc., M.Sc., Ph.D. (1) Hind Wael Al-Alousi, B.Sc., M.Sc. (2) Ahmed A. Al-Hassan, BVMS, M.Sc., Ph.D. (3)

ABSTRACT
Background: Periodontitis is a bacterial infection of tooth-supporting tissues; the immunopathologic mechanisms include inflammatory cells and chemical mediators, which persist inflammation and develop a local autoimmune. The presence of autoantibodies against extracellular matrix components, anti-neutrophil cytoplasmic antibodies (ANCA) and anti-DNA was detected. This study aimed to provide evidence of altered humoral immune response in chronic periodontitis, as well as to determine the presence of auto-antibodies in this disease. Subjects and Methods: Blood samples were collected from 35 patients with chronic periodontitis (20 with sever periodontitis and 15 with moderate periodontitis) and from 30 healthy age and sex matched individuals served as controls. Clinical periodontal parameters used in this study were plaque index, gingival index, probing pocket depth, clinical attachment level and bleeding on probing. The levels of serum IgG-antinuclear antibody were determined using enzyme-linked immunosorbent assays, whereas serum immunoglobulin (IgG, IgM and IgA) were estimated by single radial immune diffusion method. Results: Serum levels of IgG-antinuclear antibody and IgG were significantly higher in sever chronic periodontitis than in moderate chronic periodontitis and healthy controls (p<0.05). On the other hand, the serum levels of IgM and IgA showed no significant differences among three studied groups (p>0.05). Concerning the correlation between serum IgG-antinuclear antibody and clinical periodontal parameters, the level of this autoantibody did not show any correlation with clinical parameters of periodontitis (p>0.05). Conclusion: The production of antibodies against self structures could be involved in the pathogenic mechanism of chronic periodontitis. Key words: Chronic periodontitis; Anti-nuclear antibody; Serum immunoglobulin. (J Bagh Coll Dentistry 2012;24(3):145148).

INTRODUCTION
Periodontitis is a multifactorial infection characterized by a destructive inflammatory process affecting tooth supporting tissues and resulting in periodontal pocket formation and alveolar bone resorption, which might eventually lead to tooth loss (1). The pathogenesis of periodontal disease includes locally synthesized biological products, such as enzymes produced by fibroblasts or bone cells, bacterial-specific immunoglobulin secretion, and soluble inflammatory mediators, including cytokines and prostaglandins and cellular or tissue degradation products (2, 3, 4) Although many products have been associated with the presence of inflammation, few have been demonstrated to be associated with a progressive autoimmune disorder. Systemic alterations of the cellular and humoral immune responses to periodontal pathogens among chronic periodontitis patients have been evaluated, including immunosuppression, exaggerated inflammatory cell responses, impaired neutrophils, and antibody production (5).
(1)Assistant professor. Department of Basic Science, College of Dentistry, University of Baghdad. (2)Assistant lecturer. Department of Basic Science, College of Dentistry, University of Baghdad. (3) Assistant professor. Department of Microbiology, Medical College, Al-Nahrain University.

In periodontitis lesions, plasma cells are the most common cell type and represent about 50% of all cells, while B cells comprise about 18%. The proportion of B cells is larger than that of T cells (6). The involvement of host response to bacteria involved in periodontal disease can be detected by the serum immunoglobulins to particular bacteria and/or their antigens (7). Because of the relationship between antibodies and periodontal infection, the potential application of antibody titers for periodontal diagnostic purposes has received considerable research attention (8, 9). This study was designed to provide evidence of altered humoral immune response in chronic periodontitis, as well as to determine the presence of auto-antibodies in this disease.

SUBJECTS AND METHODS


The present study included 35 patients (10 females and 25 males) were from attendants seeking treatment in the private clinic, with an age mean of 39 6.2 years, and ranged between (3357years). CP patients were divided in to two groups based on their clinical attachment loss (CAL), [15 moderate CP with CAL>2mm and 20 sever CP with CAL 5mm], compared with 30 age and sex-matched apparently healthy individuals considered as a controls. Clinical attachment level is defined as the distance from the cement enamel junction (CEJ) to the location of the inserted
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probe tip. The measurements were made at four surfaces of each tooth. Serum level of IgG-ANA was estimated by using commercially available enzyme-linked immunosorbent assay (ELISA) kit and performed as recommended in leaflet with kits (AESKU. DIAGNOSTICS. Germany). Statistical analysis was assessed using P (Mann-Whitney-test), and (Kruskal-Wallis-test) (10).

RESULTS
In the present study the age of CP patients ranged between 33-57 years with a mean age of 396.2 years. There was males predominance among patients as shown in table (1). The differences in clinical periodontal parameters in patients and healthy controls are summarized in table (1). Serum levels of IgG-ANA antibody and IgG were significantly higher in sever chronic periodontitis (1.90 IU and 1194 mg/dl respectively) than in moderate chronic periodontitis (0.82 IU and 1179.2 mg/dl respectively) and healthy controls (0.4 IU 1182.5 and mg/dl respectively), (p<0.05). On the other hand, the serum levels of IgM and IgA showed no significant differences among three studied groups (p>0.05), as shown in table (2). Concerning the correlation between serum IgG-antinuclear antibody and clinical periodontal parameters, the level of this autoantibody did not show any correlation with clinical parameters of periodontitis (p>0.05), table (3).

DISCUSSION
Periodontal diseases are inflammatory diseases resulting in the destruction of tissues of the periodontium. Although bacteria must be present for periodontal disease to occur, a susceptible host is also required, which is determined by genetic, environmental, and acquired factors. One such factor, autoimmunity, may play a role in the tissue destruction (11, 12). The present study showed that the mean serum level of IgG-ANA was significantly higher in patients with severe CP as compared with moderate CP and healthy control groups. These findings are consistent with other studies reported by (13, 14). The first control study to explore the possible link between ANA and periodontal disease was conducted by Novo and Viera (13), and they found that number of ANA positive patients was statistically significant in the periodontitis group than in the healthy controls. On the other hand Galaviz and colleagues (14) reported that 57% of periodontitis patients were positives for ANA and ANCA. So they concluded that the nature of
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the immune response in periodontal disease is influenced by the microflora, however the production of antibodies against self structures, could play a crucial role in pathogenesis of CP, and these autoantibodies may be associated with necrotic or apoptotic phenomena. Although the mechanisms that trigger the development of ANCA are not completely understood, several hypotheses have been postulated, including immunospecific genes such as alleles of human leukocyte antigens and other genes that determine the level of host immune response. Recently, microbial super-antigens and mechanisms related to disturbed apoptosis or removal of apoptotic cells have been proposed for the induction of ANCA (15). The involvement of host response to bacteria involved in periodontal disease can be detected by the serum immunoglobulins to particular bacteria and/or their antigens. The current study shows that severe CP patients have significant elevation of the serum IgG level, but normal levels of IgM and IgA as compared to healthy control, this is in agreement with other reports (16-18) who mentioned that the elevation in IgG level as a result of host response to bacterial colonization. Similarly Quinn and associates noticed that IgG antibodies have been considered important in preventing periodontal destruction in patients with aggressive and chronic periodontitis (19). On the other hand Craig et al. reported that the serum IgG antibody may be reflective of the destructive periodontal disease, and its level can be considered a risk indicator for disease progression (20). In conclusion the production of antibodies against self structures could be involved in the pathogenic mechanism of CP.

REFERENCES
1. Cazalis J, Tanabe S, Gagnon G, Sorsa T and Grenier D. Tetracyclines and Chemically Modified Tetracycline-3 (CMT-3) Modulate Cytokine Secretion by Lipopolysaccharide-Stimulated Whole Blood. Inflammation. 2009; 32: 130-137. 2. Deo V, Bhongade M. Pathogenesis of periodontitis: role of cytokines in host response. Dent Today 2010; 29(9): 60-2. 3. Holmlund A, Hnstrm L, Lerner UH. Bone resorbing activity and cytokine levels in gingival crevicular fluid befoire and after treatment of periodontal disease. J Clin Periodontol 2004; 31: 475482. 4. Heasman PA, Collins JG, Offenbacher S. Changes in crevicular fluid levels in IL-1!, LTB4, PGE2, TXB4 and TNF" in experimental gingivitis in humans. J Periodontal Res 1993; 28: 241247. 5. Palmer RM, Wilson RF, Hasan AS, Scott DA. Mechanisms of action of environmental factors Tobacco smoking. J Clin Periodontol 2005; 32(6):180195.

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6. Berglundh T and Donati M. Aspects of adaptive host response in periodontitis. J Clinical Periodontol 2005; 32(6): 87107. 7. Ebersole JL. Systemic humoral immune responses in periodontal disease. Crit Rev Ora Biol Med 1990; 1: 283-331. 8. Pietrzak ER, Polak B, Walsh LJ, Savage NW, Seymour GJ. Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis. Oral Microbiol Immunol 1998; 13: 65-72. 9. Pussinen PJ, Vilkuna-Rautiainen T, Alfthan G, Mattila K, Asikainen S. Multiserotype enzyme-linked immunosorbent assay as a diagnostic aid for periodontitis in largescale studies. J Clin Microbiol 2002; 40: 512-518. 10. Sorlie DE. Medical biostatistics and epidemiology: Examination and board review. 1st ed. Norwalk Connecticut: Appleton and Lange; 1995. pp. 47-88. 11. Koutouzis T, Haber D, Shaddox L, Aukhil I, Wallet SM. Autoreactivity of serum immunoglobulin to periodontal tissue components: a pilot study. J Periodontol 2009; 80(4): 625-33. 12. Schenkein HA. Host responses in maintaining periodontal health and determining periodontal disease. Periodontol 2000; 40: 7793. 13. Novo E, Viera N. Antineutrophil cytoplasmic antibodies:A missing link in the pathogenesis of periodontal disease? J periodontal Res 1996; 31: 3658. 14. Galaviz LA, Bernal MP, Medina MA, Rodrguez SS, Luna ML. Nuclear and cytoplasmic antibodies in severe periodontytis. J Immunology 2010; 184(48):16. 15. Patil BS, Patil S, Gururaj TR. Probable autoimmune causal relationship between periodontitis andHashimatosis thyroditis: A systemic review. Nigerian J Clinical Practic 2011; 14(3): 253-61. 16. Califano JV, Chou D, Lewis JP, Rogers JD, Best AM, Schenkein HA. Antibody reactive with Porphyromonas gingivalis hemagglutinin in chronic and generalized aggressive periodontitis. J. Periodontal Res 2004; 39: 263-268. 17. Engstrom PE, George M, Larsson P, Lally ET, Taichman NS, Norhagen G. Oral and systemic immunoglobulin G-subclass antibodies to Actinobacillus actinomycetemcomitans leukotoxin. Oral Microbiol Immunol 1999; 14:104-108. 18. Kobayashi T, Kaneko S, Tahara T, Hayakawa M, Abiko Y, Yoshie H. Antibody responses to Porphyromonas gingivalis hemagglutinin A and outer membrane protein in chronic periodontitis. J Periodontolm 2006; 77: 364-369. 19. Quinn SM, Zhang JB, Gunsolley JC, Schenkein JG, Schenkein HA , Tew JG. Influence of smoking and race on immunoglobulin G subclass concentrations in early-onset periodontitis patients. Infect Immun 1996; 64: 2500-2505. 20. Craig RG, Boylan R, Yip J, et al. Serum IgG antibody response to periodontal pathogens in minority populations: Relationship to periodontal disease status and progression. J Periodontal Res 2002; 37: 132-146.

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Table 1: Demographic and Clinical l Parameters in Patients and healthy Control groups
Patients (n=35) Demographic Parameters Age Range Age Mean SD Male Female Clinical Parameters PI GI PD AL BOP 33-57 396.2 25 10 1.30 1.22 2.05 1.69 28.62 Healthy Control (n=30) 28-53 36.65.567 18 12 0.61 0.53 1.12 0 4.76 P -Value

0.74

P<0.001 P<0.001 P<0.001 P<0.001 P<0.001

Table 2: Mean serum level of IgG ANA, IgG, IgM and IgA in sever chronic peridontitis, Moderate chronic peridontitis and controls.
Sever CP Serum IgG-ANA Mean SD Number P (T-test) Sever X Healthy = p<0.05 Sever X Moderate=NS Moderate X Healthy =NS Serum IgG Mean SD Number P (Mann-Whitney) Sever X Healthy = p<0.05 Sever X Moderate= NS Moderate X Healthy = NS Serum IgM Mean SD Number Serum IgA Mean SD Number 1.90 0.96 20 Moderate CP 0.82 0.55 15 Healthy control 0.4 0.488 30 P (ANOVA)

p<0.05

1194 39.08 20

1179.2 36.36 15

1182.5 19.45 30

p<0.05

117.4 7.43 20 220.2 9.19 20

118.6 9.49 15 217.5 13.44 15

111.3 5.41 30 208.4 10.84 30

NS

NS

Table 3: Correlation between serum level IgG-ANA and clinical periodontal parameters in chronic peridontitis cases.
Clinical Parameters IgG-ANA PI Correlation P (Mann- Whitney) 0.279 0.233 GI C. P (Mann- Whitney) 0.266 0.259 AL C. P (Mann- Whitney) 0.155 0.515 PD C. P (Mann- Whitney) 0.257 0.274 BOP C. P (Mann- Whitney) 0.190 0.421

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Comparison between severe haemophilic A and healthy children in Streptococcus mutans, oral Lactobacilli and Candida albicans counts
Zainab A. AlDhaher, B.Sc., M.Sc. (1) Maha F. Almelan, V.M.B.Ch.B., M.Sc. (1) Luma M. Alhadi, B.Sc., M.Sc. (1)

ABSTRACT
Background: Haemophilia is a common hereditary hemorrhagic disorder, however little is known about the oral microflora of hemophilic patients. The purpose of this research is to compare between the viable count of Streptococcus mutans, oral Lactobacilli and Candida albicans from saliva of children suffering from haemophilia A aged (6-12) years and the viable count of the same microorganisms isolated from saliva of healthy subject (healthy supject group) aged (6-12) years. Materials and methods: - Saliva samples were collected from 30 children with severe Haemophilia A (patients group and 30 healthy children (healthy subject group). Microbial counts of Streptococcus mutans, oral Lactobacilli and Candida albicans were recorded for each group by using colony counter and expressed as colony forming unit multiplied by the dilution factor per millimeter saliva (CFU/ml). Results: - The present study observed that the viable count of Streptococcus mutans and oral Lactobacilli in patient group was higher than the count of the healthy subject group while no significant differences were observed between the viable count of Candida albicans in patients group and healthy subject group. Conclusion: - education, prevention and effort among parents and dental professionals can aid in improving the oral health of Haemophilia children. Key words: Haemophilia,Streptococcus mutans, Lactobacilli, Candida albicans. (J Bagh Coll Dentistry 2012;24(3):149-153).

INTRODUCTION
Haemophilia is group of inherited bleeding disorders termed congenital disorder characterized by a lifelong defect in the clotting mechanism. This disease is caused by hereditary deficiency of factor VIII or IX. And it shows an X.linked recessive pattern affecting predominantly males (1) . It can further be classified according to the clotting factors affected. Haemophilia (A) or classic haemophilia is a deficiency of factor (F) VIII that occurs in 85% of patients and has an estimated frequency of 1 in 10000 males in Caucasian population most of haemophiliacs cases were first diagnosed following an episode of severe oral bleeding (2). Thus the dentist may be the first to diagnose patient with haemophilia(3). Dental caries (holes in teeth) occur when the hard tissues of the tooth and dentine are softened by demineralization caused by bacteria on foods especially sugars, producing an acid that demineralizes the hard tooth (4). Streptococcus mutans is a gram positive cocci facultative anaerobic bacterium commonly found in the human oral cavity and it is a significant contributor to tooth decay (5).
(1) Lecturer. Department of Basic sciences. College of Dentistry. University of Baghdad.

These streptococci can attach to the proteins covering the tooth enamel, where they then convert sucrose into extra cellular polysaccharides (mutan, dextran, levan) (6). These sticky substances, in which the original bacterial layers along with secondary bacterial colonizers are embedded, form dental plaque. The final metabolites of the numerous plaque bacteria are organic acids that breach the enamel, allowing the different caries bacteria to begin destroying the dentin (7). Oral Lactobacilli (LB) are the most aciduric of the plaque bacteria, but these organisms only predominate by the time the carious lesion has extended into the dentin, this aciduricity best explains the involvement of Lactobacilli in human decay(8). A few fungi have developed a commensal relationship with humans and are part of the indigenous microbial flora (e.g., various species of Candida, especially Candida albicans). The first exposure to fungi that most humans experience occurs during birth, when they encounter the yeast Candida albicans (C albicans) while passing through the vaginal canal. C albicans accidentally penetrate barriers such as intact mucous membrane linings, or when immunologic defects or other debilitating conditions exist in the host, these conditions favorable for fungal infections (9).

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MATERIALS AND METHODS

Sample collection:- 30 children with severe Haemophilia A, aged (6-12) years that attended Al-Mansur hospital for children in Baghdad medical city and matched with 30 healthy healthy supject children aged (6-12) years were included in this study. Stimulated saliva samples were collected under standard conditions (10) from patient and healthy supject group. Each individual was instructed to chew a piece of Arabic chewing gum (0.4-0.5g) for five minutes to stimulate salivary flow as much as possible then saliva was collected in sterilized screw capped bottles. Inoculation: - The collected saliva was homogenized by vortex mixer for two minutes. Ten-fold serial dilutions were prepared using sterile normal saline. Two dilutions were selected for each microbial type and inoculated on the following culture media: 1. Mitis-Salivarius Bacitracin Agar (MSB Agar), the selective media for Streptococcus mutans: 0.1ml was withdrawn from dilutions 10-2 and 10-3 using adjustable micropipette with disposable tips and then spread in duplicate by using sterile microbiological glass spreader on the plates of MSB agar, 2. The plates were then incubated anaerobically by using a gas pack supplied in an anaerobic jar for 48 hrs at 37C followed by aerobic incubation for 24hrs at 37C. 2. Rogosa Selective Lactobacilli Agar (RSL Agar), this is selective for cultivation of oral LB: The inoculum was withdrawn from 10-2 and 10-3 dilutions; 0.1ml from each dilution was inoculated by using pour plate method. The plates were incubated aerobically for 48 hrs at RESULTS 37C. 1. Identification of Streptococcus mutans, oral 3. Sabouraud Dextrose Agar (SD Agar), the Lactobacilli and Candida albicans was medium is selective for the cultivation and carried out by:isolation of C albicans: 0.1ml was withdrawn a) Colony morphology. from dilutions 10-2 and 10-3 using adjustable 1. On the selective MSB agar plates, S micropipette with disposable tips and then spread mutans colonies appeared light blue in duplicate by using sterile microbiological in color about 1-2mm in diameter as glassspreader on the plates of SD agar then the spherical or ovoid in shape with plates were incubated aerobically for 48 hrs at raised or convex surface. 37 C. 2. On the selective RSL agar plates, Identification: LB colonies appeared as spindle, a) Colony morphology: - the colony on MSB star like shaped, circular, ovoid or agar, RSL agar and SD agar were examined heart like in appearance, white in directly and under dissecting microscope color. (magnification 15). Basic Sciences 150

b) Morphology of the Microbial Cells: - a colony was picked up from MSB agar, RSL agar and SD agar plates separately under sterilized conditions and subjected to gram`s stain. c) Biochemical Tests: - Bacterial colonies of different morphology were picked up from MSB agar, RSL agar and SD agar separately under sterilized conditions using inoculating loop and then inoculated in 10 ml of sterilized (BHI-B) and incubated aerobically at 37C for 18 hrs. The following tests were conducted:1. Catalase Production test: - this test was conducted on both types of cells (S mutans and LB) separately. Hydrogen peroxide 3% (H2O2) had been used to detect the activity of catalase enzyme production. 2. Carbohydrate fermentation test for: S. mutans - CTA- mannitol media had been used to test the ability of S mutans to ferment the mannitol which was added in a concentration of 1% to the CTA- mannitol media. d) Identification system of API (analytical profile index) strep: - API 20 strep was a standardized system used in the identification of S. mutans. It is combining of 20 biochemical tests that offer wide spread capabilities. The strip consists of 20 microtubes containing dehydrated substrates for the demonstration of enzymatic activity or the fermentation of sugars. The enzymatic tests were inoculated with a dense suspension of organisms made from a pure culture Microbial counts of S. mutans, LB and C. albicans were recorded by colony counter taking in consideration the dilution factor and expressed as colony forming unit multiplied by the dilution factor per milliliter saliva (CFU/ml)

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3. Colonies of C. albicans appeared smooth, creamy in color with a yeast odor and typically medium sized 1.5-2mm diameter which later develop into high convex, off-white larger colonies after 2 days. b) Morphological test of bacterial cells. 1. S. mutans cells were gram positive, spherical or ovoid in shape, arranged in short or medium length non spore forming chains. 2. Microscopical appearance of LB includes the presence of gram`s positive, non-spore forming rods 3. Candida albicans under light microscope are rounded or oval yeast cells which stained Gram positive. c) Biochemical test: - All colonies of S. mutans were catalase negative and had the ability to ferment mannitol. A positive reaction indicated by the change in color of indicator from red to yellow by the formation of acid after incubation. All colonies of LB were catalase negative. d) Identification system of API strep: - The reaction read according to the reading table and the identification was obtained by referring to the analytical profile index. The fermentation of carbohydrates was detected by a shift on the PH indicator. 2) The viable count of microbial isolates. As shown in table 1 there are significant differences between healthy subject group and patients group for the viable count of S. mutans isolates

Table 2: Comparison in viable count of Lactobacilli 103 CFU/ml. in saliva of haemophilia group and healthy subject group.
Viable count mean SD P(ttest) Healthy subject group 24.2 1.83 11.76 2.98 2.14771 E-14 P<0.00000 Patient group

Result in table 3 revealed that there are no significant differences between patients group and healthy subject group for the viable count of Candida albicans.

Table 3: Comparison in viable count of Candida albicans 103 CFU/ml. in saliva of haemophilia group and healthy subject group
Viable count mean SD P(ttest) Patient group Healthy subject group 1.9 1 3.04 0 P = 0.11 Not significant

Table 1: Comparison in viable count of Streptococcus mutans 103 CFU/ml. in saliva of haemophilia group and healthy subject group
Viable count mean SD P(t-test) Patient Healthy group subject group 20.67 2.27 9.54 3.25 3.12488 E-14 P<0.0000

Figure 1: Comparison between the viable count of Streptococcus mutans, Oral Lactobacilli and Candida albicans of patients group and the viable count of Streptococcus mutans, Oral Lactobacilli and Candida albicans of healthy subject group.
Results analyzed by using SPSS 15 statistical package (SPSS LTD working UK) T test showed differences between the viable count of Streptococcus mutans, oral Lactobacilli and Candida albicans from saliva of patients group
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Table 2 demonstrate that statistically there are significant differences between patients group and healthy subject group for the viable count of Lactobacilli isolates
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and the viable count of the same microorganisms from saliva of healthy subject group .

DISCUSSION
Haemophilia is the most common sex linked bleeding disorder worldwide (11). In developing countries most the patients with haemophilia are in the pediatric age group as they seldom reach adulthood because of inadequate treatment. Haemophilia in these areas are not given the priority it deserves as there are high numbers of other serious health problems (12). The results of this recent study showed that the count of Streptococcus mutans and oral Lactobacilli was higher in patients group in comparison to healthy subject group while there is no significant differences between patient group and healthy subject group in Candida albicans and these results disagree the with results of other studies in developed countries like the study of Boyd D. &Kinirons M. (13) who found that the prevalence of caries was low in haemophilia patients. Moreover, Sonbol et al. (14) also disagree with our results because they found that a significantly greater group of children with severe haemophilia were caries free compared with the healthy subject and the mean number of colony forming units of Streptococcus mutans in healthy subject group was significantly greater than patients group. The possible explanation for the lower dental caries experience in these developed countries is to the existence of comprehensive haemophilic centers which provide children diagnosed as haemophilics, regular dental periodic checkups, a comprehensive preventive dental programme including topical application of fluoride by the delivery of fluoride to teeth topically or systemically in order to prevent dental caries(15) and fissure sealant application which is a method of preventing caries from developing in the pits and fissure of the teeth by sealing them off with a special varnish called fissure sealant and strict dietary and oral hygiene instructions from an early age(16). These differences reflect the higher tendency for conservative treatment and the greater concern about health education and preventive measures among both healthcare workers and haemophilic patients and their caregivers in these developed countries. Oral care of haemophiliacs is not of primary importance in developing countries, as they have tended to receive less oral health care, or of lower quality, than the general population, yet
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they may have oral problems that can affect their systemic health also The fear of the dentist to deal with these patients who might be bearing dangerous transmissible viruses as hepatitis and human immunodeficiency virus and the fear from complications like uncontrollable haemorrhage or life-threatening haematomas from simple procedures as anaesthesia and extraction have participated in the exaggeration of their dental problems(17). These also may be explained by the fact that haemophilics either refrain from the use of the tooth brush all together or use it inefficiently to avoid gingival bleeding and that they are more concerned with their medical health than their dental health (18). It is clear that education, intervention and prevention make positive changes in oral health of haemophiliacs. A team effort among parents/guardians, children, and dental professionals can aid in improving the oral health of those who may not have the necessary means to dental care.

REFERENCES
1. Blanchette VS and Sparling C, Turner C. Inherited bleeding disorders. Bailliere's ClinHaematol 1991; 4 (2): 291-332. 2. Behrman RE and Vaughan VS. eds Nelson's Textbook of Paediatrics, 13th edn. Philadelphia: WB Saunders Company 1987; 1065-1074. 3. Sonis A, Musselman RJ. Oral bleeding in classic haemophilia. Oral Surg Oral Med Oral Pathol 1982; 53: 363-369. 4. Brook MD, Carroll MD, Janet S and Stephen A. Medical Microbiology. Twenty fourth edition printed in the United State of America 2007; 31-32. 5. Harrrington B. Primary dental care of patient with haemophilia, (treatment hemophilia) 2004; 3:127-35. 6. Cleaton-Jones P, Hargreaves JA, Fatti LP, Chandler HD and Grossman ES. Dental caries diagnosis and calibration for clinical eld surveys. Caries Res 1989; 23(3): 195-204. 7. Franco E, Saunders CP, Roberts GJ and Suwanprasit A. Dental disease caries related microflora and salivary IgA of children with severe congenital cardiac disease an epidemiological and oral microbial survey. Paediatr Dent 1996; 18 (3): 228-63. 8. Ainamo J. An epidemiological index of developmental defects of dental enamel (DDE Index). Int Dent J 1982; 32: 159-215. 9. Gow N A R, Gadd G M. The Growing Fungus. London: Chapman and Hall 1994; 324-32. 10. Dasanyake AP, Rosemaa JM, Caufield PW. Distribution and determinants of Mutans Streptococci among African American children and association with selected variables. Pediatr Dent 1995; 17 (3): 192-6. 11. Girolami A, Luzzatto G, Varvarikis C, Pellati D, Sartori R and Girolami B. Main clinical manifestations of a bleeding diathesis: an often disregarded aspect of medical and surgical history taking. Haemophilia 2005; 11: 193202.

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12. Chuansumrit A. Treatment of haemophilia in the developing countries Haemophilia 2003; 9: 38790. 13. Boyd D and Kinirons M. Dental caries experience of children with haemophilia in Northern Ireland. BSPD and IAPD, Int J Paediatr Dent 1997; 7: 149-53. 14. Sonbol H, Pelargidou M, Lucas V S, Gelbier M J, Mason C and Roberts G J. Dental health indices and caries-related microflora in children with severe haemophilia. Blackwell Science Ltd. 2001; (7), 46874. 15. Mohire N,Yadav A and Gaikwad V. Good Oral Hygiene for Good Overall Hygiene :A Review Research. J Pharm and Tech 2009; 2:262-4.

16. Boyd D, Kinirons M. Dental experience of children with haemophilia in Northern Ireland. Int J Paediatr Dent 1997; 7: 14953. 17. Zanon E, Martinelli F and Bacci C. Proposal of a standard approach to dental extraction in haemophilia patients. Haemophilia 2000; 6: 336. 18. Kabil N, El Alfy M and Metwalli N. Evaluation of the oral health situation of a group of Egyptian haemophilic children and their re-evaluation following an oral hygiene and diet education programme. Journal compilation Blackwell Publishing Ltd. 2007; (13) 287-92.

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Effect of Nigella Sativa

Effect of Nigella Sativa L. extracts against Streptococcus mutans and Streptococcus mitis in Vitro
Najah A. Mohammed, Ph.D. (1)

ABSTRACT
Background: Seeds of Nigella sativa L., commonly known as black seed, have been used in traditional medicine. Streptococcus mutans and Streptococcus mitis are normal flora bacteria found in human oral cavity, which cause dental caries and bad breathe oder. This project considered as an explorer study for the inhibitory effect of Nigella Sativa L. seeds extract against S. mutans and S. mitis. Material and methods: Two different extracts of Nigella sativa L. has been evaluated in vitro against Streptococcus mutans & Streptococcus mitis, the antibacterial activity was determined by the agar well diffusion method. Results: The results showed the zone of inhibition was found 12.7mm and 10.4mm at a ethanol extract against Strept. mutans & Strept. mitis respectively,while the inhibition zone of ether extract was found 6.3mm and 5.1mm against Strept. mutans & Strept. mitis respectively. Conclusion: Methanol extract was more effective in comparison with the ether extracts. The highest inhibition zone was observed with ethanol fraction and it inhibited the growth of two careiogenic bacteria. Key words: Nigella sativa L., well diffusion, Antibacterial activity. (J Bagh Coll Dentistry 2012;24(3):154-157).

INTRODUCTION
The use of plants and plant products as medicines could be traced as far back as the beginning of human civilization. Medicinal plants are a source of great economic value all over the world. Nature has bestowed on us a very rich botanical wealth and diverse types of plants grow in different parts of the country (1). Herbal medicine is still the mainstay of 75-80% of the whole population and the major part of traditional therapy involves the use of plant extract and their active constituents. Following the advent of modern medicine, herbal medicine suffered a setback, but during last two or three decades, advances in phytochemistry and in identification of plant compounds, effective against certain diseases have renewed the interest in herbal medicines (2). In recent years, human pathogenic microorganisms have developed resistance in response to the indiscriminate use of commercial antimicrobial drugs commonly employed in the treatment of infectious diseases. Therefore, alternative antimicrobial strategies are urgently needed, and thus this situation has led to a reevaluation of the therapeutic use of ancient remedies, such as plants and plant-based products (3) . Nigella sativa L. is commonly known as which belongs to the botanical family of Ranunculaceae. N. sativa seeds have been used for nutritional and medicinal purposes in many Middle Eastern countries and other parts of the world (4, 5). N. sativa is considered a natural food additive and a condiment. Also, it had been used for medicinal purposes as a natural remedy in many ancient cultures, as those of Egypts Greece and Rome (6).
(1)Assistant professors. Technical medical institute/Baghdad

The N. sativa is the black seed medicine for every disease except death (7, 8). Recently, many biological activities of Nigella sativa L. seeds have been reported, including: antioxidant, anti-inflammatory, anticancer and antimicrobial. Nigella sativa L. seeds contain a large amount of fixed oils (9) and the main constituent of the seed extract is thymoquinone (10). Several pharmacological effects have been attributed to active principles of Nigella sativa L. which includes thymoquinone, thymohydroquinone, dithymoquinone, thymol, carvacrol, nigellicine, nigellimine-x-oxide, nigellidine and alpha-hedrin (11). The seeds or its oil is believed to have carminative, diuretic, lactagoge and vermifuge (12). N.sativa seeds have a wide spectrum of medicinal properties including antimicrobial, antihelminthic, antiinflammatory, analgesic, hypoglycemic, smooth muscles relaxant and immunostimulant activities (12,1 3). The anti-microbial effects of N. sativa seeds against different pathogenic microbes were investigated. The volatile oil of N. sativa seeds, prepared by steam distillation, was proved to be more effective against many strains of bacteria, including those known to be highly resistant to drugs (14). The diethyl ether extract was found to cause concentration dependent inhibition of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and a pathogenic yeast Candida albicans (15). The methanol and chloroform extracts have high inhibitory effects against S. aureus, P. aeruginosa and C. albicans (16) . The essential oil of the seeds have also dosedependent antibacterial effects on Gram-positive
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(S. aureus) and Gram-negative (E. coli) bacteria (17) . Among the various oral-micro-organisms, Sterptococcus mutans has been identified s a plaque-forming bacterium capable of producing dental caries in human (18). Many attempts have been made to eliminate S.mutans from the oral flora. The present study aimed to screen and evaluates antibacterial activity of two different extracts of Nigella sativa L. against Streptococcus mutans & Streptococcus mitis.

MATERIALS AND METHODS


Preparation of extracts: Nigella sativa seeds were obtained from the local seed supplier; the seeds were crushed manually in a mortar with a pestle. A volume of 100 ml of distilled water was added to 20 g of dry powder. It was vortexed continuously until there was no further change in color of the solution. This solution was centrifuged for 15 min. The supernatant (brownish-orange in color) was filtered through Whatman filter No.1using Buchner funnel and stored at 4 C in sterile tubes until use. Methanolic extraction: 10 grams of powdered plant was soaked separately in 50 ml of methanol and rotated on a rotary shaker at 150 rpm for 24 h. The extracts were filtered through Whatman filter paper no. 1 using Buchner funnel and the filtrates concentrated in vacuo at 40C using a rotary evaporator and then stored in refrigerator at 4C until used (17). Ether extract: 10 grams of seeds were placed to a soxhalet and extracted with diethylether at 35 0C. Ether was evoparated after extraction by a rotary evaporator connected to a vacuum pump (17). Antibacterial activity: The tested bacteria firstly isolated from saliva patients on mitis salivarius + bacitracin medium

and identification by API-20 Strep. System (bioMrieux, France). Secondly the antibacterial activity was determined using micro well dilution methods (19), and by using the Kirby-Bauer method (20) bacterial cultures were first grown in nutrient broth at 37C for 18-24 h incubated till turbidity became equivalent to McFarland 0.5 turbidity standard was obtained. The inocula of the respective bacteria were streaked on to the Muller Hinton agar (Oxoid) plates using a sterile swab in order to ensure a uniform thick lawn of growth following incubation. Wells of 6 mm in diameter were formed on to nutrient agar plates using a sterile cork borer. The dried plant extracts were dissolved in dimethylsulfoxide (DMSO) to give a concentration of 30 mg/ml. Wells were cut into the agar and filled with 75 l of the plant extracts and one well with (DMSO) as negative control. Inoculated plates were incubated at 37 C for 18- 24hrs. Finally, the plates were incubated at 37C for 18-24 h and the resulting diameters of zones of inhibition were measured. These studies were performed in triplicate for each plant extract. Statistical analysis: The results were calculated as mean diameter of inhibition zone in mm standard deviation (mean SD).

RESULT AND DISCUSSION


Spices are frequently used as an active ingredient in certain medicines and reported to possess a number of pharmacological effects to treat different human ailments (21). Two different extracts of Nigella sativa L. has been evaluated in vitro against two oral bacteria, which are known as causative agents of dental careis in humans. In the plates of Strep. mutans and Strep. mitis no inhibition zones seen around the methanol wells which were used as negative control. Among the different extracts, the methanolic extract showed most pronounced antibacterial activity. The inhibition zone around the discs containing ether and ethanol extract of N.sativa seeds shown in Table (1).

Table 1: The diameter zone of inhibition (mm) around discs containing the two N.sativa seeds extracts against Streptococcus mutans & Streptococcus mitis
Extract Ethanol Ether Negative control Zone of inhibition (mm) Strep. mutans Strep. mitis (mm) SD (mm) SD (12.7) 2.1 ( 10.4) 0.9 ( 6.3) 0.6 ( 5.1) 0.59 SD = Standard deviation.

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Methanol extract was more effective in comparison with the ether extracts. The highest inhibition zone was observed with ethanol

fraction and it inhibited the growth of two careiogenic bacteria figure (1&2).

3 Figure 1: Inhibition zone of Nigella sativa L. & against Strep. mutans. 1=negative control. 2= ether extract. 3=Methanol extract.

3 2

Figure 2: Inhibition zone of Nigella sativa L. & against Strep. mitis. 1=negative control. 2= ether extract. 3= Methanol extract.
It is interesting to note that G+ve bacterial isolates were sensitive to ether and ethanol extract of N.sativa seeds. Present study is in agreement with (14) who reported that N.sativa extracts produce antibacterial activity against a broad range of microbes and especially G+ve strains and multiple antibiotic resistant bacteria. This result also correlates with (22) who reported that Thymoquinone exhibited potent growth inhibitory effect against Gram-positive bacteria, During extraction process, solvents diffuse into the solid plant material and soluble compounds of similar polarity (23). The polarity of solvent affects quantity and composition of secondary metabolite of an extract. Traditional healers primarily use water for extract preparation from plant extracts but organic solvents have been found to give more consistent antimicrobial activity compared to water extracts (24). Most other bioactive
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compounds including phenols are generally soluble in polar solvents such as methanol. Many bioactive components are not water soluble and thus organic solvent extracts of plants have been found to be more effective (25). Methanol, ethanol and water are the most commonly used solvents for antimicrobial investigations. (26) Since most of the identified components from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained through ethanol or methanol extraction. The curative properties of medicinal plants are perhaps due to the resence of various secondary metabolites such as alkaloids, flavonoids, lycosides, phenols, saponins, sterols. One of these active ingredients is Thymoquinone (volatile oil of these seeds) and Melanine (fixed oil) (27). Some investigations performed to show the possibility of antimicrobial and antibacterial activities of

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these seeds using their extracts or oil (28). Generally, higher levels protein and carbohydrate content of the extract had better antimicrobial activities (29). Many proteins are involved in the microbial defense mechanism of plants. Puroindoline is the main component of a new family of proteins that has been suggested to exert an antimicrobial activity in plant seeds (30, 31). The study provides support to the antibacterial potential of N. sativa extracts as a potent antibacterial agent.

15.

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REFERENCES
1. Ahmed L, Mohammed Z, Mohammed F. Screening of some Indian medicinal plants for their antimicrobial properties. J Ethnopharmacol 1998; 62: 183-193. 2. Arora D, Kaur J. Antimicrobial activity of spices. Int J Antimicrobial Agents 1999; 12: 257-262. 3. Gupta C, Amar P, Ramesh G, Uniyal C, Kumari A. Antimicrobial activity of some herbal oils against common food-borne pathogens. African J Microbiol Res 2008; 2: 258-261. 4. Al-Ghamdi MS. Anti-inflammatory, analgesic and anti-pyretic activity of Nigella sativa. J Ethnopharmacol 2001; 76: 4548. 5. El-Dakhakhny M, Mady NA, Halim MA. Nigella sativa L. oil protects against induced hepatotoxicity and improves serum lipid profile in rats. Arzneimittelforschung 2000; 50: 832-836. 6. Al-Haider A, Aqeel M, Hasan Z. Hypoglycemic effect of volatile oil of Nigella sativa seeds. Pharma Biol 1993; 31: 96-100. 7. Ghosheh OA, Houdi AA, Crooks PA. High performance liquid chromatographic analysis of the pharmacologically active quinines and related compounds in the oil of the black seed (Nigella sativa L.). J Pharm Biomed Anal 1999; 5: 757-762. 8. Takruri HR. Black cumin in prophet hadith and modern medicine. Presented in Symposium on Medicine in the Prophet Sunnah Islamic Hospital, Amman 2003. 9. Kokdil G, Yilmaz H. Analysis of the fixed oils of the genus Nigella L. (Ranunculaceae) in Turkey. Biochemical Systematics and Ecology 2005; 33: 12031209. 10. Aboul-Ela E.I. Cytogenetic studies on Nigella sativa seeds extract and thymoquinone on mouse cells infected with schistosomiasis using karyotyping. Mutation Research 2002; 516: 11-17. 11. Aljabre SHM, Randhawa MA, Akhtar N, Alakloby OM, Alqurashi AM, Aldossary A. Antidermatophyte activity of ether extract of Nigella sativa and its active principle, thymoquinone. J Ethnopharmacology 2005; 101: 116-119. 12. Hassieb AM. Melanin is the secret of Nigella Sativa. Al-Faisal Scientific J 2006; 4(3):110143. 13. Houghton PJ, Zarka R, Heras B, Hoult RS. Fixed oil of N. sativa and derived thymoquinone inhibit eicosanoid generation in leucoytes and membrane lipid peroxidation. Planta Med 1995; 61: 33-36. 14. Salman MT, Khan RA, Shukla I. Anti-microbial activity of Nigella sativa Linn. Seed oil against multi19. 20.

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drug resistant bacteria from clinical isolates. Nat Prod Rad 2008; 7: 10-14. Hanafy MS, Hatem ME. Studies on the anti-microbial activity of Nigella sativa seed (black cumin). J Ethnopharmacol 1991; 34: 275-278. Mashhadian NV, Rakhshandeh H. Antibacterial and anti-fungal effects of Nigella sativa extracts against S. aureus, P. aeruginosa and C. albicans. Pakistan J Med Sci 2005; 21: 47-52. Hosseinzadeh H, Bazzaz BSF, Haghi MM. Antibacterial activity of total extracts and essential oil of Nigella sativa L. seeds in Mice. Pharmacol 2007; 2: 429-435. Michalek SM, McGhee JR, Shiota T. Low sucrose levels promote extensive S. mutans-induced dental caries. Infect Immun 1977; 16:712-4. Hadacek F, Greger H. Testing of antifungal natural products: methodologies, comparability of results and assay choice. Phytochem Analys 2000; 11: 137-147. Bauer AW, Kirby M. Antibiotic Susceptibility testing by standard disc method. Am J Clin Patho 1966; 10 (45): 493-496. Bonjar GHS. Screening for antibacterial properties of some Iranian plants against two strains of E. coli. Asian J Plant Sci 2004; 3(3): 310-314. Kokoska L, Havlik J, Valterova I, Sovova H, Sajfrtova M, Jankovska I. Comparison of chemical composition and antibacterial activity of Nigella sativa seed essential oils obtained by different extraction methods. J Food Prot 2008; 71: 2475-2480. Green RJ. Antioxidant Activity of Peanut Plant Tissues. Masters Thesis. North Carolina State University. USA, 2004. Parekh J, Jadeja D, Chanda S. Efficacy of Aqueous and Methanol Extracts of Some Medicinal Plants for Potential Antibacterial Activity. Turk J Biol 2005; 29: 203-210. Parekh J, Karathia N, Chanda S. Screening of some traditionally used medicinal plants for potential antibacterial activity. Indian J Pharm Sci 2006; 68(6): 832-834. Bisignino G, Sanogo R, Marino A, Aquino R, Dangelo V, Germano MP, De Pasquale R, Pizza C. Antimicrobial activities of Mitracarpus scaber extract and isolated constituents. Lett Appl Microbiol 1999; 30: 105-108. Roy J, Shaklega D, Callery P, Thomas J. Chemical constituents and antimicrobial activity of a traditional herbal medicine containing garlic and black cumin. Afr J Tradit Complement Altern Med 2006; 3(2): 17. Ali BH, Blunden G. Pharmacological and toxicological properties of Nagila Sativa. Phytother Res 2003; 17: 299305. [IVSL]. Morsi NM. Antimicrobial effect of crude extracts of NS and multiple antibiotics-resistant bacteria. Acta Microbiol Pol 2000; 49: 6374. [IVSL]. Wafaa AH, Hassan A, Nefisa MAE. Biological and Anti-microbial Activities of Aqueous Extracts from Neem Tree (Azadirachta indica A. Juss., Meliaceae) J Appl Sci Res 2007; 3(10): 1050-1055. Dhatwalia VK, Sati OP, Tripathi MK, Kumar A. Isolation, Characterization and Antimicrobial Activity at Diverse Dilution of Wheat Puroindoline Protein. World J Ag Sci 2009; 5(3): 297-300.

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Evaluation of Entamoeba gingivalis and Trichomonas tenax in patients with periodontitis and gingivitis and its correlation with some risk factors
Sumaiah Ibrahim, B.Sc, M.Sc. (1) Rasha Abbas, B.Sc., M.Sc. (2)

ABSTRACT
Background: It was shown that two protozoans, Entamoeba gingivalis and Trichomonas tenax may be responsible for oral parasitic infection. This study was designed to detect these parasites in oral cavity of patients with periodontitis and gingivitis with some risk factors and compare it with healthy oral persons. Material and Method: A total of 60 patients with periodontitis and gingivitis and 25 healthy person (control group) enrolled in the present study. Two samples were collected from each patient, dental plaque and saliva samples. These samples were stained with Giemsa stain and examined under light microscope, some factors were taken such as PH saliva, age, sex, smoking habits, diabetes mellitus, blood pressure, heart disease and any history of antibiotic consumption during the last six months. Results: This result shown Entamoeba gingivalis and Trichomonas tenax in dental plaque samples was higher than in saliva samples in patients with periodontitis (70%, 60%), (33%,20%) respectively. While in gingivitis it was shown that these two parasites higher in saliva than the dental plaque (60%,46.6%),(46.6%,30.9%)respectively. these two parasites are present in healthy individuals less than in patients with periodontitis and gingivitis . The presence of these protozoans was related to the type of periodontal disease(p=0.0257),sex(p=0.043),age(p=0.0058),PH of saliva(p=0.043) and risk factors (p=0.0168). Conclusion: This study showed Entamoeba gingivalis more common in patients with periodontal disease whereas Trichomonas tenax is that considered a protozoan in oral cavity. There was relationship between the presence of these parasites and the type of periodontal disease and the risk factors. Key words: Entamoeba gingivalis, Trichomonas tenax, periodontitis, gingivitis, risk factors. (J Bagh Coll Dentistry 2012;24(3):158-162).

INTRODUCTION
The oral cavity is suitable for invasion of many microorganism, the protozoan Entamoeba gingivalis and Trichomonas tenax are recognized eukaryotic representatives. Although these forms are not generally associated with pathogenesis, their presence in the oral cavity is taken as a sign of poor dental hygiene (1,2,3). Since mouth infection symptoms derive from the interaction between pathogenic microbiota and the hosts defense mechanisms, it is extremely important to study the microorganisms that cause periodontitis and gingivitis in human(4). Entamoeba gingivalis and Trichomonas tenax were the first commensal foud in human oral cavity, they occure only as a trophozoit, and these are found in gingival tissues, particularly in suppurative, inflammatory processes, due to there are preference for anaerobic environments (5). Some authors believe that these commensal could be opportunistic, that these, capable of proliferating in a gingival environment modified by periodontal and gingivites disease (6).

The trophozoites of Entamoeba gingivalis and Trichomonas tenax are most probably transmitted from person to person by close contact,since they exhibit only sligth resistance to the enviroment. Kissing may be play a role in transmission, but direct passage by many ways such as mutual usages of cups, spoon, fork, and subjects contaminated by an infected person probably is a mode of transmission also (7). This study was designed to detected of these parasites in oral cavity of patients with periodontitis and gingivitis with some oral risk factors and compare it with healthy oral persons.

MATERIALS AND METHODS


Patients This study was conducted on 60 patients(39 males and 21 females)with gingivitis and periodontitis and 25 samples presented a healthy periodontium attending the Teaching Hospital of the College of Dentistry/Baghdad University during the period first of February to the end of April 2012. Samples collection and parasitological examination The samples of saliva and dental plaque were collected from all patients at the morning, in the first visit for each patient.Saliva samples were placed in sterile cup and diluted with normal
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(1) Lecturer, Department of Basic sciences, College of Dentistry, University of Baghdad (2) Assistant Lecturer .Department of Basis sciences, College of Dentistry, University of Baghdad.

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saline at room temperature (25-28C),immediately after dilution, these samples were stained with Giemsa stain and examined under a light microscope (40x,100x,200x,400x)(8) The dental plaque samples were collected by scraping the area with sterile swab rubbed around the surface of teeth from caries and around the gingival crevices, the swabs were dipped in sterile vials containing normal saline, after that the swab rolled on the slide, also stained with Giemsa stain and examined under light microscope (40x,100x,200x,400x). The parasites of Entamoeba gingivalis were identified by their shape depending on the expansion of the pseudopodia formation and presence of vacuoles while Trichomonas tenax was identified by its flagella and characteristic locomotion Questionnaire for these patients who entered in the present study, this includes: general health, age, sex, smoking habits, diabetes mellitus, Blood pressure, heart disease and any history of any type of antibiotic consumption during the last six months for each patients(most antibiotics used in this country). Determination of saliva PH Universal indicator strips (PH 0-14) were used to determine the saliva PH and a possible relationship between PH and presence of the study microorganisms. Statistical analyses The data were analyzed by the non parametric chi square test( 2 test).The significance level was set at (p< 0.05).

RESULTS
Table (1) showed that the microscopical examination of the fresh samples revealed a total 35 dental plaque samples(58.3%) were positive for Entamoeba gingivalis, where 21cases (70%) were taken from patients with periodontitis,14 cases (46.6%) were taken from patients with gingivitis and it found in 4cases (16%) in control individuals,19 cases (31.6%)of dental plaque samples were positive for Trichomonas tenax, where 10 cases(33.3%)were taken from patients with periodontitis and 9 cases (30%)from patients with gingivitis and 5cases (20%) in control group.As for the saliva samples,36 (60%) were

positive for Entamoeba gigivalis,where18 cases (60%) were from patients with periodontitis, 18 (60%) were from patients with gingivitis while it found in 6 cases (24%) in control individuals. Trichomonas tenax was identified in 20 saliva samples (33.3%), where 6 cases (20%) were from patients with periodontitis, 14 cases (46.6%) were from patients with gingivitis and only in 3 cases (12%) in control group.There was a significant correlation between presence of these two parasites and type of periodontal disease ( 2 =4.73,p=0.0257). Table (2)showed higher Entamoeba gingivalis and Trichomonas tenax in males than in females,(38.4%,28.5%)(5.1%,0) respectively, so there was a significant correlation between the sex of the patients and the presence of these protozoan parasites ( 2 =3.860,p=0.043) Regarding the relationship between patients age and presence of these protozoan, Entamoeba gingivalis was more common in patients age 61 to 70 years(60%), whereas Trichomonas tenax was more common in patients age 31 to 40years(28.5%), Both commensals were found in higher rate in age 21 to 30 years(80%) (table 3),There was statistically significant correlation between the age of patients and presence of these protozoan ( 2 = 10.307, P=0.0058). The saliva PH of the participants ranged from 5 to 7.5,Entamoeba gingivalis was found in higher rate in PH 6-6.5 (44.4%)while Trichomonas tenax was found in higher rate in PH 7-7.5(5%),Both protozoan were found in higher rate in PH 55.5(100%) (table 4), There was statistically significant correlation between the saliva PH and presence of these parasites( 2 =3.563,P=0.043). 52 of 60 patients examined in this study presented risk factors,9 individuals were negative for these parasites and 43 were positive, Entamoeba gingivalis was found in higher rate in patients with hypertension in periodontitis and gingivitis (60%), while the parasite Trichomonas tenax was founed in higher rate in patients who taken antibiotics at last six months (7.6%),Both parasites were present in higher rate in patients with diabetic (56.2%) (table5),There was statistically significant correlation between the risk factors of patients and presence of these parasites( 2 =8.175,P=0.0168).

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Table 1: Detection rate of Entamoeba gingivalis and Trichomonas tenax in dental plaque and saliva samples according to the periodontal status of the patients.
Clinical status Periodontitis Gingivitis Total Control 30 30 60 25
2

No.of samples

Entamoeba gingivalis Dental plaque No % 21 70 14 46.6 35 58.3 4 16 saliva No % 18 60 18 60 36 60 6 24

Trichomoas tenax Dental plaque No % 10 33.3 9 30 19 31.6 5 20 saliva No % 6 20 14 46.6 20 33.3 3 12

=4.73 P=0.0257 P < 0.05 significant

Table 2: Detection rate of Entamoeba gingivalis and Trichomonas tenax in dental plaque and /or saliva samples according to the patients sex.
Sex Male Female Total No. of samples 39 21 60
2

Entamoeba gingivalis No % 15 38.4 6 21 28.5 35

Trichomonas tenax No % 2 5.1 0 2 0 3.3

both No 16 10 26 % 41 47.6 43.3

=3.860 P=0.043 P < 0.05 significant

Table 3: Detection rate of Entamoeba gingivalis and Trichomnas tenax in dental plaque and /or saliva samples according to the patients age. Entamoeba Trichomonas Both Age gingivalis tenax Total (years) No. % No. % No. % 11 3 27.2 0 0 3 27.2 >10 9 5 55.5 0 0 3 33.3 11-20 10 1 10 0 0 8 80 21-30 7 1 14.2 2 28.5 3 42.8 31-40 7 3 42.8 0 0 3 42.8 41-50 11 5 45.4 0 0 5 45.4 51-60 5 3 60 0 0 1 20 61-70 60 21 35 2 3.3 26 43.3 Total
2

=10.307 P=0.0058 P < 0.05 significant

Table 4: Detection rate of Entamaeba gingivalis and Trichomonas tenax in dental plaque and/or saliva samples according to the salivary PH.
PH 5-5.5 6-6.5 7-7.5 Total No. of samples 2 18 40 60
2

Entamoeba gingivalis No. % 0 0 8 44.4 13 32.5 21 35

Trichomonas. tenax No. % 0 0 0 0 2 5 2 3.3

Both No. 2 6 18 26 % 100 33.3 45 43.3

=3.563 P=0.043 P < 0.05 significant

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Table 5: Detection rate of Entamoeba gingivalis and Trichomonas.tenax in dental plaque and/or in saliva samples according to the risk factors of patients.
Risk factor Smoking Diabetic B.pressure Herat.Dis Antibiotic Total No. sample 14 16 5 4 13 52
2

Entamoeba gingivalis No. % 5 35.7 6 37.5 3 60 2 50 7 53.8 21 40.3

Trichomonas tenax No. % 1 7.1 0 0 0 0 0 0 1 7.6 2 3.8

Both No. 5 9 2 1 2 19 % 35.7 56.2 40 25 15.3 36.5

Negative sample 3 1 1 1 3 9

=8.175 P=0.0168 P < 0.05 significant

DISCUSSION
There are only few reports on the role of oral commensals in the pathogenesis of periodontitis and gingivitis despite the high incidence of certain protozoa, such as Entamoeba gingivalis and Trichomonas tenax. In the present study,the result revealed that the incidence of Entamoeba gingivalis was higher than the parasite Trichomonas tenax, very similar results were found by other studis (3,4,9,10.11),and the rate of oral parasites higher than in control individuals, this also in agreement with many studies(4,7,11).this study indicating that the incidence rate of Entamoeba gingivalis was (35%), this rate approximately for many studis (4,7,9,12). It has been suggested that these protozoans could affect the formation of contribute to the development and progression of periodontal disease. The other researchers state that this protozoans may be opportunistic, since they are capable of proliferating in the microenvironment of the mucobuccal fold affected by periodontal disease (6) . Therefore, if Entamoeba gingivalis helps the development and progression of periodontitis and gingivitis, these diseases increasingly facilitate the proliferation of these protozoa, this vicious circle could explain the increased incidence of these microorganisms in the dental plaque and saliva samples of patients with periodontitis and gingivitis. A suspension of Entamoeba gingivalis was spread on the gingival margins of rats immunosuppressed with (prednisolone acetate), leading to the development of the clinical signs and inflammatory process of periodontal disease much faster than that observed in immunocompetent rats(13) ,these data indicate that immunosuppression may play role in the pathogenesis of periodontitis and gingivitis induced by this commensal. The rate of incidence of Trichomonas tenax in this study was a little(3.3%),this approximately to other researche(14). Prevalence of patients with Trichomonas tenax world wide ranges from 4.0 to
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53% (15),but some reports found this parasite much less (2.17%)(3). the role of Trichomonas tenax in periodontitis and gingivitis is still controversial. Although a relationship between the increased occurrence of this protozoan and progression of this disease has been demonstrated recently (15), the precise mechanism of tissue damage is still unknown. However, further studies are necessary for the complete elucidation of this mechanism. Saliva PH ranged from 5.5-7.5, other research suggested that the peak incidence of commensals in salivary samples occurred between 6-6.5(4), while other study have not found a relationship between saliva PH and the presence of these protozoa(10). Regarding gender both parasites were more common in male, these data are in agreement with those of other studies(7,16)and the difference was statistically. Regarding age, several researchers reported that the presence of oral protozoan increases with age(16,17)as such in this study, where Entamoeba gingivalis was found in higher rate in age group (61-70), this finding support the idea that Entamoeba gingivalis may play an active role in the mouth disease, the both parasites were found in higher rate in age group(21-30),the parasite Trichomonas tenax was found in high rate in age group(31-40),this results are in general agreement with other studies (12,19). However, statistically significant correlation between age and presence of oral commensals was observed in this study. the complexity of the oral environment and the multifactorial nature of the caries lesion, with the periodontal disease , requires the cooperation of other disciplines such as microbiology, chemistry and dietetics. Other factors that increase the risk of periodontitis and gingivitis disease are diabetes, hypertension, smoking, heart disease and antibiotic consumption, in this study, there is statistically significance between these factors and the presence of these parasite, the reasons may be that the immunity which reduced in these patients,

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and the presence of these parasite are making facilitates. The present study recorded a relationship between the presence of Entamoeba gingivalis and Trichomonas tenax and these factors. In fact, risk factors for periodontitis and gingivitis were present in only a few participants, so any inference or deliberation on this subject should be cautious. Other studies did not find relationship between the presence oral parasites and these factors (4,10). The results of this study suggest that Entamoeba gingivalis occur more frequently in the patients with periodontal and gingivitis disease, whereas Trichomonas tenax sees the oral cavity as the natural habitat given its commonness in individual with healthy gums. However, further studies are necessary for determining the real nature of the relationship between these species and the periodontal disease.

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disease and healthy population in Sthiraz, Southern Iran. Indian J Dent Res 2010; 21(1):89-91. Onyido AE, Amadi ES, Olofin I, Onwusmma AA, Okoh I, Chikwendu CI. Prevalence of Entamoeba gingivali and Trichomonas tenax among dental attending Federal School of Dental Technology and Therapy Clinic, Enugu, Nigeria. Nature and Science 2011; 9 (9):59-62. Al-Saeed WM. Pathogenic effect of Entamoeba gingivalis on gingival tissue of rats. Al-Rafidain Dent J 2003; 3(1):70-73. Rodriguez DR, Garza-Ramot DM, Gonzalez DF, Mata DB. Entamoeba gingivalis in children attending the Dentistry School in Unal. Intern Assoc Dent Res 2010; 996. Athari A, Soghandi L, Haghighi A, Kazemi B. Prevalence of oral trichomoniasis` in patients with periodontitis and gingivitis using pcr and direct smear. Iranian J Publ Health 2007; 36 (3) :33-7. Vrablic J, Tomova S, Cattar G, Randova L, Suttova S. Morphology and diagnosis of Entamoeba gingivalis and Trichomonas tenax and their occurrence in children and adolescents. Bratisl Lek Listy 1991; 92(5):241-6. Jawad SQ. Frequency of Entamoeba gingivalis and Trichomonas tenax among patients with dental prosthesis-fixed or removable. J of College of Basic Education 2011; 68:97-100. Osorio EM, Ibanez AB, Ibenoz CM, Moro AM. Entamoeba gingivalis and Trichomonas tenax en cavided bucal de patients de la clinica intergral del adulto de la faculated de odontologia,Maracaibo, Venzula. Rev Soc Ven Microbiol 2009;29 (2):122-7.

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