Review Article

Journal of Investigative Periodontology

Gingival Crevicular Fluid (GCF): An oral biomarker in the diagnosis and quantification of periodontal diseases
Punit Vaibhav Patel
Faculty of Dental Sciences, Institute of Medical Sciences, Banaras Hindu University (B.H.U.), Varanasi,-05, U.P., India Abstract Periodontitis is a disease characterized by loss of connective tissue attachment and bone around the teeth in conjunction with the formation of periodontal pockets due to the apical migration of the junctional epithelium. Early diagnosis and treatment of progressive periodontitis is important because of the irreversible nature of this disease The long-term aim is that treatment and prevention of periodontal disease will be founded on diagnostic tests based on aetiopathogenic factors rather than just clinical experience. Clinical measurements used in diagnosis of periodontal diseases are often of limited usefulness in that they are indications of previous periodontal disease rather than the present disease activity. Biochemical mediators in oral fluids like saliva and gingival crevicular fluid (GCF) are highly beneficial in the determination of current periodontal status. These substances known as biomarkers help in determination of inflammatory mediator levels, as they are good indicators of inflammatory activity. This review highlights recent advances in the use of salivary and gingival crevicular fluid (GCF) biomarkerbased disease diagnostics that focus on the identification of active periodontal disease. Introduction The presence of sulcular or gingival crevicular fluid has been known since the 19th century but its composition and possible role in oral defense mechanisms were elucidated by the pioneering work of Waerhuag , Brill and Krasse in the 1950s.1 The pioneer research of Waerhaug in the early 1950s was focused on the anatomy of the sulcus and its transformation into a gingival pocket during the course of periodontitis .In the late 1950s and early 1960s a series of ground breaking studies by Brill et al. laid the foundation for understanding the physiology of GCF formation and its composition.The studies of Le et al. contributed to this understanding and started to explore them use of GCF as an indicator of periodontal diseases Egelberg continued to analyze GCF and focused his studies on the dentogingival blood vessels and their permeability as they relate to GCF flow The GCF studies boomed in the 1970s. The rationale for

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understanding dentogingival structure and physiology was created by the outstanding electron microscopic studies of Schroeder and Listgarten . 2 Presence and functions of proteins, especially enzymes in GCF were first explored by Sueda, Bang and Cimasoni . Ohlsson, Golub and Uitto discovered that collagenase and elastase in GCF are derived primarily from human cells, most notably neutrophils, and that their activity is correlated with gingival inflammation and gingival pocket depth . In 1974 the first edition of the monograph The Crevicular Fluid by Cimasoni was published. This comprehensive revie gave a big boost to GCF studies and towards the end of the first millenium the research on GCF increased dramatically. 3-9 How is GCF formed? Brill et al 1959 systemically administered fluorescein in dogs GCF collected using filter paper strips Fluorescein appeared in the GCF collected Sample collected from other oral epithelia had not allowed the passage of the fluorochrome it was concluded that differences in permeability must exist between these oral epithelia and the epithelium lining the gingival pocket. Egelberg in 1966, in a first experiment obtained an increased permeability of the blood vessels of healthy gingivae by the use of three different methods: topical application of histamine, gentle massage of the gingiva by means of a ball-ended amalgam plugger and scraping of the gingiva crevice by means of a blunted dental explorer. Immediately after the application of histamine large amounts of gingival fluid were obtained and marked vascular labeling could be observed at the same time. Massage and scraping were also immediately followed by appearance of large amounts of gingival fluid. Formation of GCF is Physiological or Pathological process? Subsequent experiments showed that the flow of gingival fluid increased markedly following stimulation of the gingivae by Tooth Brushing By Chewing After Intravenous Injection Of Histamine Or The Development Of Inflammation . This led to the conclusion that some irritation, whether chemical or mechanical, was necessary to induce the production of GCF and that it should therefore be considered as a pathological phenomenon.

Is GCF a transudate of interstitial fluid? An alternative theory arose from the work of Alfano and Pashley (1974-76) which suggested that the initial fluid produced could simply represent interstitial fluid which appears in the crevice as a result of an osmotic gradient. This initial, pre-inflammatory fluid was considered to be a transudate, and, on stimulation, this changed to become an inflammatory exudate. A mechanistic analysis of gingival fluid production which analyzed the physiology of the gingival crevice area with respect to the factors described Originally by Starling was made by Pashley (1976). The model proposed by Pashley , predicted that GCF Production is governed by the passage of fluid from capillaries into the tissues (capillary filtrate) and the removal of this fluid by the lymphatic system (lymphatic uptake). When the rate of capillary filtrate exceeds that of lymphatic uptake, fluid will accumulate as edema and/or leave the area as GCF The mathematical model proposed by Pashley

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(a) Absence of inflammation: low vascular permeability and low permeability of the basement membrane results in low GCF flow and high % uptake by lymph vessels. (b) Macromolecules of plaque result in an osmotic gradient, increased vascular permeability and basement membrane changes, resulting in increased passage of fluid into the tissues and increased GCF production. Factors modulating the respective filtration and uptake processes include the Filtration coefficients of the lymphatic and Capillary endothelium as well as The osmotic pressure within the different compartments. Thus, even in health, if the osmotic pressure of the sulcular compartment exceeds that of the tissue fluid, Possibly because of accumulation of plaque-derived molecules, there will be a net increase in the flow of GCF. GCF is an exudate of varying composition found in the Sulcus /periodontal pocket between the tooth and marginal gingiva. GCF contains components of serum, inflammatory cells, connective tissue, epithelium, and microbial flora inhabiting the gingival margin or the sulcus/pocket . In the healthy sulcus the amount of GCF is very small. However, its Constituents participate in the normal maintenance of function of the junctional epithelium throughout its lateral and vertical dimensions, including the most coronal DAT cells. Permeability Of Junctional And Oral Sulcular Epithelia Substances that have been shown to penetrate the sulcular epithelium include Albumin, Endotoxin, Thymidine, Histamine, Phenytoin, Horseradish peroxidase. The main pathway for the transport of substances across the junctional and sulcular epithelia seems to be the intercellular spaces which according to Schroeder and Munzel Pedrazzoli (1970) form 18% of the total volume of the junctional epithelium and 12% that of the oral sulcular epithelium. According to Squier (1973) the degree of permeability of the oral mucosa does not seem to depend upon its degree of Keratinization. The mechanisms of penetration

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through an intact epithelium were reviewed by Squier and Johnson. Three routes have been described: 1. Passage Form CT Into The Sulcus: 2. Passage From The Sulcus Into The CT 3. Passage Of Substances Through Pathological or Experimentally Modified Gingival Sulcus Methods of collection Several techniques have been employed for the Collection of GCF and the technique chosen will Depend upon the objectives of the study as each Technique has advantages and disadvantages. Gingival washing methods In this technique the gingival crevice is perfused with an isotonic solution, such as Hanks balanced salt solution, usually of fixed volume. The fluid collected then represents a dilution of crevicular Fluid and contains both cells and soluble constituents such as plasma proteins. Two different techniques have been used. The simplest involved the instillation and re-aspiration of 10ml of Hanks balanced salt solution at the interdental papilla . This process was repeated 12 times to allow thorough mixing of the transport solution and GCF. This technique could therefore be applied either to individual interdental units or to multiple units which were then pooled. A more complicated method involved the construction of a customized acrylic stent which isolated the gingival tissues from the rest of the mouth. The tissues were then irrigated for 15min, with A saline solution, using a peristaltic pump, and the diluted GCF was removed. Advantages The washing technique is particularly valuable for harvesting cells from the gingival crevice region. Disadvantages 1. 2. 3. 4. 5. 6. 7. The production of customized acrylic stents is complicated and technically demanding. It is therefore a technique of limited application and has been restricted to the study of GCF obtained from only a few individuals. It has also usually only been applied to the maxillary arch, presumably because of the difficulties of producing a technically satisfactory appliance for the mandibular arch. It is also a disadvantage that GCF from individual sites cannot be analyzed. In contrast, the simpler technique may be applied to individual sites or groups of sites which may be categorized as healthy or inflamed. The major disadvantage of this technique is that all fluid may not be recovered during the aspiration and re-aspiration procedure. Thus accurate quantification of GCF volume or Composition is not possible as the precise dilution factor cannot be determined.

Capillary tubing or micropipettes Following the isolation and drying of a site, capillary tubes of known internal diameter ar inserted into the entrance of the gingival Crevice. GCF from the crevice migrates into the tube by capillary action and because the internal diameter is known the volume of fluid collected can

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be accurately determined by measuring the distance which the GCF has migrated Advantages This technique appears to be ideal as it provides an undiluted sample of native GCF whose volume can be accurately assessed. Disadvantages However, it is difficult to collect an adequate volume of GCF in a short period, unless the site are inflamed and contain large volumes of GCF. To collect a reasonable volume of fluid may, in some instances, mean that collection times from an individual site may exceed 30min and, even then, adequate samples from healthy crevices may be impossible to obtain. It is difficult to conceive that holding a capillary tube at the entrance to a gingival crevice for such lengthy periods ensures an atraumatic collection. A further complication of this technique is the difficulty of removing the complete sample from the tubing. This has either been forced out with a jet of air or, by passing a larger fixed volume of a diluting solution through the capillary or, more usually, by centrifugation of the tube. Absorbent filter paper strips There are considerable variations in the application of the filter paper strip method of collection The advantages of the technique are that it is quick and easy to use can be applied to individual sites and, possibly, is the least traumatic when correctly used. Methods of collection

(a) Extracrevicular method; (b) intracrevicular method superficial [Le & Holm-Pederson], (c) intracrevicular method deep[Brill ].

The Intracrevicular method depends on the strip being inserted into the gingival crevice, whereas in the exttacrevicular method the strips are overlaid on the gingival crevice region in an attempt to minimize trauma. The intracrevicular method is the method used most frequently and can be further subdivided depending upon whether the strip is inserted just at the entrance of the crevice or periodontal pocket or whether the strip is inserted to the base of the pocket or until minimum resistance is felt Methods of estimating the volume collected In many early studies the only purpose of the investigation was to determine the amount of GCF

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produced at a given site. The amount of GCF collected on a strip was assessed by the distance the fluid had migrated up the strip. This was often taken as a simple linear measurement, but a more accurate value was achieved by assessing the area of filter paper wetted by the GCF sample.Further accuracy was achieved by staining the strips with ninhydrin to produce a purple color in the area where GCF had accumulated. A similar result was shown with 2g fluorescein given systemically to each patient 2 hours prior to the collection of GCF, following which the strips were examined under ultraviolet light (Weinstein 1961). They found that fluorescein labeling was 100 times more sensitive than ninhydrin for staining protein. The introduction of an electronic measuring device, The Periotron , has allowed accurate determination of The GCF volume and subsequent laboratory Investigation of the sample composition. The instrument measures the effect on the electrical current flow of the wetted paper strips. It has two metal jaws which act as the plates of an electrical condenser. If a Dry strip is placed between the jaws, the capacitance is translated via the electrical circuitry and registers zero on the digital readout. A wet strip will increase the capacitance in proportion to the volume of fluid and this can be measured as an increased value in the readout. The technique is rapid and has no discernible effect upon the GCF sample. Three models of PeriotronA have been produced (the 600, 6000 and now the 8000) and each one has been shown to be an efficient means of measuring the volume of fluid collected on filter paper strips. An alternative approach, involving the weighing of strips before and after sample collection, has been adopted by some workers . This has been successful but requires a very sensitive balance to estimate the very small amounts of fluid which may be collected from a healthy crevice. Problems with GCF collection The major sources of contamination of GCF samples would be blood, saliva, or plaque. Frank blood contamination is usually dealt with by discarding the sample and removing the data from analysis. The presence of dental plaque on filter paper strips used for collecting GCF has been shown to have a marked effect on the volume recorded. Experiments where dental plaque was applied directly to filter paper strips showed that a large plaque mass contained a considerable amount of fluid which would influence volume determinations, which would be expressed as volume of GCF. Careful isolation should be performed in an effort to minimize the potential for saliva contamination. Alpha-amylase was used in an assay to confirm, or refute, the presence of the contamination of GCF samples with saliva (Griffiths 1992). Application of the Assay to saliva samples collected using a good isolation and drying technique confirmed that the likelihood of a significant contribution from saliva was small Sampling time The early literature from the PeriotronA suggested that filter paper strips should be left in place for 5 seconds. Alternative approaches to sampling techniques have been developed which included either leaving the strip in place for a longer period or the use of a sequence of repeated strips, with Possible recovery periods in between. Other approaches have also included collecting until a Minimum volume has been recovered. This latter approach has sometimes resulted in collection times of 2030min.The problem with prolonged collection times isthat the nature of the GCF sample collected is likelyto change with the protein concentration of the initialGCF collected comparable to interstitial fluid,whereas prolonged sampling at the site resulted inprotein concentrations approaching those of serum . Volume determination

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Evaporation is considered to be a significant problem in accurate volume determination of GCF samples. This is particularly the case as the total volumes collected are usually Less than 1ml and more often than not are less than 0.5 ml. In practical terms it may be necessary to determine a Particular volume (for example 0.2ml) below which it is not possible to analyze the sample. Recovery from strips. Having collected the GCF sample and determined its volume the samples are usually then required for some investigation of the composition of GCF. To achieve this it is necessary to recover the GCF from the filter paper strips and initial work indicated that protein recovery was close to 100% using a centrifugal elution technique (Cimasoni 1988). A variety of other methods of elution have been employed, but it is essential in all instances to determine the percentage recovery from the original samples. Data reporting Constituents found within GCF samples have eitherbeen reported as absolute amount (mg), concentrations(mg/ml) or either of these two measurements with reference to pocket depth or duration ofsample collection. Gingival crevice fluid flow Gingival crevice fluid (GCF) flow is an important Determinant in the ecology of the periodontal pocketor sulcus. It creates a flushing action and an isolationeffect. In addition, it probably determines the growth level of subgingival microorganisms and is a potentialmarker for periodontal disease activity. The first important characteristic associated With GCF flow is its flushing action. Substances put into the periodontal pocket are rapidly washed out. The second important characteristic associatedwith GCF flow is the isolation effect. Substancesfrom the outside do not easily penetrate the Periodontal pocket. The concentration of immunoglobulin G (IgG) found in GCF from periodontal pockets is approximately 100 times that found in saliva . Clearly, this could not be the case if saliva gained ready access to the periodontal pocket. One may reasonably conclude That the net outward GCF flow inhibits retrograde salivaryflow and that salivary contents do not generally enter the periodontal pocket. This creates a relative isolation of the periodontal pocket from the rest of the oral cavity. The nutrient effect of GCF flow has been suggestedby associations between bacterial numbers or composition and GCF flow in several studies .Although it is widely suggested that accumulation of bacteria leads to increased GCF flow, it has not beenso widely appreciated that increased GCF flow maysupport larger bacterial plaque masses.GCF flow (or flow rate) is the process of fluid

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moving into and out of the gingival crevice or pocket .It is a small stream, usually only a few microliters per hour. GCF in a gingival sulcus or periodontal pocket is like a spring-fed pond. Fluid enters the pond from an unseen source and leaves by flowingout at the margin. Fluid flow is a rate measure. It is the volume that crosses a defined boundary over a given time, mathematically symbolized as dV/dt, the first derivative of volume with respect to time.In the gingival environment at equilibrium, the influx (fidVi/dt) should equal the efflux (fodVo/dt) so that measurement of either could be considered the GCF flow. As a practical matter, however, only the influx is measured. The gingival sulcus or pocket also has a resting volume (Vr) through Which the GCF flows. The washout ratio (fi/Vr) is the number of times the pocket volume is replaced in unit time.The resting volume (Vr) is the result of forming apool of fluid in the crevice or pocket. The incrementin volume as a result of GCF flow (fiDt) results becausethe GCF flow cannot be turned off while takingthe measurement.There are at least two approaches to measuring GCF flow using conventional methods for measurementof GCF volume.The concept is that by pumping a marker substance into a Periodontal pocket at a constant rate, an equilibrium concentrationwill be established which is the result of the fluid flow rate and the pump delivery rate. An intrapocket drug delivery system is a small pump that can deliver a marker substance into the periodontal pocket. Although many devices of this type have been developed , only one, the Tetracycline fiber, establishes and maintains the constantconcentration required for this method.Application of this method requires that a measuredlength of tetracycline fiber be placed into a periodontalpocket and the equilibrium concentration be measured at a later time. To date, no published study has been conducted to evaluate GCF flow in this manner. The measurement of tetracycline concentration requires two measurements, the GCF volume of the sample and the amount of Tetracycline in the sample.

COMPOSITION OF GCF: The GCF consists of: 1. Cellular elements 2. Electrolytes

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3. Organic compounds 4. Metabolic and bacterial products 5. Enzyme and enzyme inhibitors

Cellular Elements: 1. The cellular elements found in the gingival fluid include bacteria, desquamated epithelial cells, and leukocytes (PMNs, lymphocytes and monocytes, erythrocytes) which migrate through the sulcular epithelium. The major cellular components of the gingival crevicular fluid The anatomical origin of the cells of the gingival crevice fluid (GCF) With the accumulation of bacterialplaque in the vicinity of the gingival sulcus, the flow of GCF increases. Cells that originate from plaque and Their metabolic by-products can induce the egress of inflammatorycells from the gingival plexus of blood vessels, located in the connective tissue, just below the Junctional epithelium. Electrolytes: Potassium, sodium calcium magnesium and fluoride have been studied in gingival fluid. Most studies have shown a positive correlation of calcium and sodium concentrations and the sodium to potassium ratio with inflammation. According to Krasse and Egelberg (1962) the sodium and potassium ratio of fluid from inflamed pockets was more than two times that of fluid from healthy sulci. According to Kaslick et al (1970) a significantly higher sodium: potassium ratio was also found in the moderately inflamed as compared to the nearly normal gingivae. It has also been reported that in case of deep pockets sodium values tend to be lower, while the values for potassium tended to increase. In more severe cases of periodontitis, the higher number of degenerating epithelial, CT and blood cells contribute to increase the potassium concentration of exudate by liberating their intracellular content.

(iii) Organic Compounds: Carbohydrates, proteins and lipids have been investigated. Glucose hexosamine and hexuronic acid are two of the compounds found in gingival fluid. Glucose concentration in gingival fluid is 3-4 times greater than that in serum. This is interpreted not only as a result of metabolic activity of adjacent tissues, but also as a function of the local microbial flora.

The total protein content of gingival fluid is much less than that of serum. No significant correlations have been found between the concentration of proteins in the Journal of Investigative Periodontology 3 (6) Special Issue: 45-63, 2010

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gingival fluid and the severity of gingivitis, pocket depth and extent of bone loss. Proteins namely , , 2 and 1 globulins, transferrin, albumin, immunoglobulins such as IgG, IgM and IgA, complement components such as C1, C4, C3, C5, have been reported to be present in GCF.

Proteins Include: - fibrinogen, ceruloplasmin, - lipoprotein, transferrin, 1 antitrypsin and 2 macroglobulin.

(iv) Metabolic And Bacterial Products: Metabolic and bacterial products identified in gingival fluid include lactic acid, urea, hydroxy proline, endotoxins, prostaglandins, cytotoxic substances, hydrogen sulphide and antibacterial factors.

(v) Enzyme And Enzyme Inhibitors: Various enzymes known to be present in gingival fluid include: Acid phosphatase, alkaline phosphatase, pyrophosphatase, - Glucoronidase, lysozyme, Hyaluronidase, Proteolytic enzymes such as Mammalian proteinases which includes Cathepsin D,Elastase, Cathepsin G, Plasminogen activators, Collagenase and bacterial proteinases (i.e. endo and exopeptidases), and lastly lactic dehydrogenase serum proteinase inhibitors such as 2 macroglobulin, 1 antitrypsin, 1 antichymotrypsin have also been known to be present in GCF.

The primary reason for this intense interest is the realization that the most widely used method for assessing disease progression is not precise enough to detect small amounts of periodontal damage. The general approach used in most investigations has been to first determine in cross sectional studies if a specified component of GCF is either, present in Periodontitis and absent in health or gingivitis. Strongly related to the severity of periodontitis, the potential markers have been grouped into three general categories:

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Inflammatory mediators and products Host derived enzymes Tissue breakdown products (i) Inflammatory Mediators And Products: (a) Prostaglandin E2 (PGE2 ): PGE2 is a product of the cyclooxygenase pathway. Elevated levels of PGE2 in GCF were found in patients with periodontitis compared to patients with gingivitis. PGE2 levels were three times higher in patients with juvenile periodontitis compared to adult periodontitis. (b) Cytokines: Cytokines are potent local mediators of inflammation that are produced by variety of cells. Cytokines that are present in GCF and have been investigated as potential diagnostic makers for periodontal disease include: - interleukin - 1, 1, interleukin 6, interleukin 8 and tumor necrosis factor (TNF -).Both IL - 1 and IL - 1 have pro-inflammatory effects and depending on a variety of factors can stimulate either bone resorption or formation.It has also been reported that in adult periodontitis patients, a higher percentage of sites are positive for IL 1 (87%) and IL - 1 (56%) IL-6 has also been associated with bone resorption. GCF from sites with progressing periodontitis contains elevated amounts of IL-6.IL-8 was formerly called monocyte-derived neutrophil chemotactic factor. GCF from sites with periodontitis contains significantly more total IL-8 than GCF from healthy sites. (c) Antibacterial Antibodies: Several reports indicate that certain antibacterial antibodies and antibody subclasses are significantly higher in GCF than in serum.This suggests that local production of specific antibody might occur in response to infecting bacteria in periodontal pockets. In one of the studies, GCF from unstable or progressing sites had significantly elevated levels of Ig G1 and Ig G4. One of the other study demonstrated that GCF from progressing sites had depressed levels of IgA thus suggesting the protective role of IgA. (d) Auto Antibodies: Some reports have demonstrated that sera from patients with periodontitis have elevated levels of antibody to type I collagen compared to control sera. Presence of anti-desmosomal antibodies in sera and GCF from patients with periodontitis has also been demonstrated. To date, the possible use of these autoantibodies as diagnostic markers has not been investigated. (e) Total Protein: Several reports suggest that, compared to periodontally healthy controls, GCF from sites withperiodontitis has significantly elevated levels of total protein.Some study has reported that GCF from inflamed sites in patients with periodontitis havesignificantly lower protein concentrations than GCF from inflamed sites in patients with gingivitis alone. (2) Host Derived Enzymes: (a) Aspartate Aminotransferase: It has been demonstrated that on a total amount per-site basis, the GCF levels of aspartate aminotransferase increase during the development of experimental gingivitis.In ligature induced experimental periodontitis studies in dogs, the GCF levels of AST increase during the

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development of attachment loss and bone resorption.The association between GCF AST levels and the risk of progression of periodontitis has led to the commercial development of a chairside test. Some uncertainity remains regarding the ability of the test to reliably distinguish between progressing sites and those that are inflamed but not progressing. (b) Alkaline Phosphatase: Its been confirmed that the levels of ALP in GCF are significantly higher than those in serum thus suggesting the local production of enzyme.Cross-sectional data indicate that GCF from sites with gingivitis or periodontitis have significantly higher concentrations of ALP than healthy sites. (c) -Glucuronidase: -Glucuronidase is a lysosomal enzyme but its presence in GCF is probably due to contribution from numerous host cells. GCF levels of -Glucuronidase were positively corelated to depth of periodontal pockets and bone loss. In experimental gingivitis, -glucuronidase and arylasulphatase activity in GCF was significantly higher in gingivitis and periodontitis than in healthy patients. Elastase: It has been demonstrated that the GCF levels of functional and antigenic elastase increase during the development of experimental gingivitis. It was also noted that as the experimental gingivitis developed, the GCF content of the two major inhibitors of neutrophil elastase (i.e. 2macroglobulin and 1 proteinase inhibitor) also increased. The value of GCF Neutrophil elastase has also been evaluated as a possible screening device to identify periodontitis sites as increased risk of developing additional attachment loss.

(e) Cathepsins: Cathepsins B, H and L are cysteine proteinases that play an important role in intracellular protein degradation. Significant decreases in the GCF levels of Cathepsin B/L activity have been noted after scaling and root planing. (f) Trypsin like and Other Enzymes: Besides the trypsin-like enzymes other host derived GCF enzymes with potential diagnostic importance include: immunoglobulin degrading enzymes, glycosidases, dipeptidyl peptidases and myeloperoxidase. On a concentration and total activity per site basis, the GCF levels dipeptidyl peptidase II and IV were significantly elevated at the progressing sites (g)Collagenases / Gelatinases / Neutral proteinases / Stromelysins: GCF from sites with adult or juvenile forms of periodontitis exhibit significantly elevated collagenolytic activities compared to GCF from healthy or gingivitis sites.In ligature-induced periodontitis in the beagle dog, GCF collagenase activity increased to maximum values within weeks after ligature placement, active collagenase was elevated during active periodontitis and active collagenase was strongly corelated with attachment loss. Latent collagenase and collagenase inhibitors were prominent during gingivitis. (3) Tissue Breakdown Products:

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(a) Glycosaminoglycans (GAGs): The GAGs in GCF that have been most examined as possible diagnostic markers for periodontal diseases are: Chondroitin 4 sulfate, chondroitin 6 sulfate and hyaluronic acid. The appearance of C-4-S in GCF has been suggested as a marker for bone resorption associated with periodontal disease or orthodontic tooth movement. But no studies have been conducted to determine its role in the progression of periodontitis. (b) Hydroxyproline: It is a prominent aminoacid of collagen and its appearance in GCF has been preliminary investigated as a marker for the destruction of periodontal connective tissue. Data from one cross-sectional study in humans indicate that GCF hydroxyproline levels cannot distinguish between sites with gingivitis or periodontitis. Because of this it is not an attractive candidate as a potential marker for the progression of periodontitis. (c) Fibronectins: These are a large group of heterogenous glycoproteins present in blood and connective tissues. Data from most studies indicate that GCF fibronectin is not a promising diagnostic marker. (d) Connective Tissue Proteins: Increased GCF levels of the aminoterminal propeptide of type I collagen have been reported at periodontitis sites. On a concentration basis, the amount of osteocalcin in GCF does not appear to be different at sites with gingivitis or periodontitis.Osteonectin another non-collagenous protein of bone and a variety of other tissues, has been reported to be elevated in GCF at sites with severe periodontitis. Neither Osteocalcin nor Osteonectin levels in GCF have been systematically evaluated as diagnostic markers for periodontitis.Biochemical markers of the progression of periodontitis The composition of the gingival crevice fluid (GCF) is the result of the interplay between the bacterial biofilm adherent to the tooth surfaces and the cells of the periodontal tissues. The collection of GCF Is a minimally invasive procedure and the analysis of specific constituents in the GCF provides a quantitative biochemicalindicator for the evaluation of the localcellular metabolism that reflects a persons periodontal health status. Many studies have looked at the association between total amount or Concentration levels of different constituents of GCF and periodontal health status. Since host response is a critical determinant in periodontal disease pathogenesis, the measure of inflammatory mediator levels in the GCF has been used to evaluate risk: Risk for a tooth, or more precisely a site, to lose clinical attachment and alveolar bone, or risk for an individual to develop periodontal disease. Pathogenesis of periodontitis; Periodontitis is a chronic inflammatory response to the subgingival bacteria, producing irreversible periodontal Tissue destruction and tooth loss. The progression of periodontitis is chronic, with cyclic periods of exacerbation and remission, and may remain unnoticed with Minimal symptoms in the early stages. Periodontitis is Diagnosed clinically by loss of attachment between the tooth and the supporting tissues (clinical attachment loss), by deepening of the pocket between The root of the tooth and the supporting tissues (pocket depth), and/or by radiographic evidence of bone loss.

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Lipopolysaccharide is a key microbial stimulus that will trigger the host response at periodontal disease Sites. Locally, Lipopolysaccharide triggers monocytes to release inflammatory mediators (prostaglandin E2, thromboxane B, interleukins -1, -6 and -8, tumor necrosis factor, And collagenase) that increase local destruction of the connective tissues structural elements. Therefore, levels of monocytic inflammatory mediators (including prostaglandin E2, interleukin-1, and tumor necrosis factor) in GCF may well represent the ideal markers of disease activity at a site level. Elevated GCF levels of neutrophil Markers (including neutrophil elastase, b-glucuronidase, and leukotriene B4) may reflect acute episodes of localized tissue destruction.Taken together, these monocytic and neutrophilimediator levels in the GCF may also give an indication of the quality of the host response, andof the level of risk for the individual to develop periodontal disease.Many studies have reported that GCF Prostaglandin E2 levels are significantly elevated in patients suffering from severe forms of diseases (juvenile periodontitis and refractory periodontitis) compared to healthy controls or patients suffering from a mild form of the disease (gingivitis or chronic adult periodontitis). It seems that GCF prostaglandin E2 levels are significantly elevated in gingivitis patients compared to controls (seymour 2001). This would indicate that the determination of GCF prostaglandin E2 levels is a good indicator of inflammatory activity.Patients with mild forms of adult periodontitis did not Have much higher levels than gingivitis patients, althoughGCF prostaglandin E2 levels seemed to increase with increasing disease severity. Studies by others haveconfirmed that GCF prostaglandin E2 levels are elevatedin periodontitis patients compared to controls and gingivitis patients (preshaw 1999,Tsai 1998). In addition, various periodontal treatmenttherapiesinduced a decrease in GCF prostaglandin E2 levels GCF interleukin-1 levels are also significantly Elevated in all forms of periodontitis compared to Health or gingivitis. In the Salvi et al. study, GCF Interleukin-1 levels were 16.85.3 for healthy controls 115.852.6 for gingivitis patients, 263.273.7 for adult periodontitis patients , 836.8284.2 for juvenile periodontitis patients, and 457.9157.9 for refractory periodontitis patients , expressed in mg/ml. Overall individual response to bacterial challenge One way to investigate the idea that GCF Inflammatory mediator levels reflect the overall ability of an individual to produce inflammatory mediators is to evaluate the individuals ability to produce inflammatory mediators. This can be accomplished by measuring the levels of inflammatory mediators released from isolated peripheral blood monocytes. Clearly, monocytes isolated from periodontitispatients produce higher levels of prostaglandin E2 than healthy controls, and monocytes isolated from patients suffering from severe forms ofperiodontal diseases produced even higher levels ofprostaglandin E2 than monocytes isolated from Patients suffering from milder forms of the disease.Interestingly, levels of prostaglandin E2 released By isolated peripheral blood monocytes were highlycorrelated to the levels of prostaglandin E2 Measured in the GCF .

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CLINICAL SIGNIFICANCE: (a) General Health And Gingival Fluid: (i) Circadian Periodicity: There is a gradual increase in gingival fluid amount from 6:00AM to 10:00PM and adecrease afterwardOn the other hand in the studies conducted by a group of investigators, there are no systematic differences between the flow of fluid measured at 9:00a.m and that of the fluid collected at 3p.m (ii) Gingival Fluid Flow And Sex Hormones: Clinical investigations have shown an exacerbation of gingivitis during pregnancy (loe 1965) during the menstrual cycle (Lemann 1948) and at puberty (Sutcliffe 1972). Female sex hormones increase the gingival fluid flow, probably because they enhance vascularpermeability.Pregnancy, ovulation and hormonal contraceptives all increase gingival fluid production. (iii) Gingival Fluid in Diabetic Patients: Ringelberg et al in 1977 described a higher flow rate of gingival fluid in a group of diabetic children, when compared to the flow rate measured in a group of children without diabetes. In healthy individuals Hara and Le found exudate glucose values upto 6 times those of serum. Kjellman (1970) reported glucose values much lower in gingival fluid when compared to serum, this being true for both healthy and diabetic patients. (b) Measurement of Gingival Inflammation: Brill was the first to suggest that measurements of the fluid recovered from gingival pockets by means of filter paper strips might be used for determining the degree of inflammation of the gingival. Presence or Absence of Fluid In healthy sulci: According to Egelberg when a deep intracrevicular technique of collection is used, a low but definite amount of gingival fluid can be collected from clinically healthy sulci. With this technique, the mechanical irritation supplied by the filter paper will elicit an increase of vascular permeability and a subsequent production of fluid.According to Loe and Holm Pedersen when the filter paper strip is not inserted deeply into the sulcus but placedat its very entrance, no or very little fluid can be collected from healthy sulci. (ii) Gingival Fluid As a Sign of Subclinical Inflammation: Le and Holm-Pedersen (1965) in a study found a gradual increase of the amount of fluid collected with their own technique during the period of no cleansing, and reported that flow from the gingival crevice was regularly demonstrated before clinical gingivitis was observable. The increase in amount of fluid preceding the earliest clinical evidence of inflammation during the development of gingivitis has also been confirmed by several other studies. (iii)Gingival Fluid flow as Related to Histological Inflammatory Changes: It is generally agreed that the histological signs of inflammation as recorded on biopsies of marginal gingiva, are not well co-related with the intensity of gingival fluid

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flow.Muhlemann and Mazor found a significant and positive co-relation between the exudate scores and both the clinical recordings of gingival inflammation and the roundcell infiltration.Sulcus fluid and the number of connective tissue cells or blood vessels were, however not co-related. (c) Drugs In Gingival Fluid: Drugs that are excreted through the gingival fluid may be used advantageously in periodontal therapy. Bader and Goldhaber were able to show that intravenouslyadministered tetracycline in dogs rapidly emerges within the sulcus. Ciancio et al in 1976 measured the concentration of tetracycline in blood and gingival fluid of 5 adult patients with advanced periodontitis, who were given 1g of tetracycline HCL daily for 2 weeks and 0.5g for 10 weeks. The concentration of the drug in gingivalfluid was only 1/10 of that found in serum. In a second study from the same laboratory the concentrations of the drug were found to be 5 times higher in samples of gingival fluid as compared to the concentrations in blood (d) Influence Of Mechanical Stimuli: Chewing and vigorous gingival brushing stimulate the oozing of gingival fluid. Even the minor stimuli represented by intrasulcular placement of paper strips increase the production of fluid. Smoking produces as immediate transient but marked increase in the gingival fluid flow. (e) Periodontal Therapy And Crevicular Fluid: There is an increase in gingival fluid production during the healing period after periodontal surgery. According to Arnold et al 1966 this increase was probably the result of the inflammatory reaction from gingival trauma and the loss of an intact epithelial barrier, especially considering the fact that fluid had been collected by deep intracrevicular technique. Suppipat et al in 1978 sampled gingival fluid 14, 21, 28 and 35 days after gingivectomy and found an increase in gingival fluid flow during the first 2 weeks after surgery followed by a gradual decrease. This decrease was same when using mechanical or chemical plaque control. RECENT CONCEPTS ON THE MECHANISM OF GINGIVAL FLUID PRODUCTION: (i) Effect of Histamine: Brill in 1959 and Egelberg in 1966 showed an increase in gingival fluid production after intravenous or topical administration of histamine respectively. It is known, however, that histamine can modify capillary filtration co-efficients, transmural capillary pressure and transmural osmotic pressure but, for the moment, the relative contribution of each of the three changes to the overall effect of increased rate of gingival fluid production by histamine is unknown. (ii) Preinflammatory Gingival Fluid: Most of the investigators agree that the gingival fluid flow increases several days prior to detectable clinical inflammation. (iii) Concentration Of Proteins In Gingival Fluid: Hattingh and Ho in 1980 confirmed that fluid collected from cases of inflamed gingivae

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contains the same concentration of total proteins as serum but indicates also that the concentration of proteins in the minute amounts of fluid collected in the absence of clinical inflammation is lower and similar to that of extracellular fluids. (iv) Passage of fluid and PMNL in the sulcus: The passage of fluid is governed, at least at the initiation of the exudation process by osmotic gradients, whereas that of cells is governed by chemotactic factors thus suggesting their independent phenomena. v) Inflammatory Changes Of The Basal Membrane: During inflammation the basal membrane becomes thinner and even to partially disappear. Such ultra structural lesions may decrease the coefficient of filtration of the junctional epithelium, thus allowing more fluid to enter the sulcus. (vi) Morphology Of The Junctional Epithelium: According to Pashley the loose organization of the junctional epithelium will also have an influence on its coefficient of filtration and may explain the relative ease with which large molecules and even cells can permeate this epithelial covering. (viii) Mechanical Stimuli And Gingival Fluid: Pressure sources such as mastication and tooth brushing may cause increase of gingival fluid production CONCLUSION: It is obvious that GCF provides a unique window for analysis of periodontal condition. Collection of GCF is a noninvasive and relatively simple Procedure.The potential of GCF in early detection Of periodontitis and healing of periodontal tissues Following therapy was already understood in the 1950s.However, we still do not have a practical and accurate periodontal indicator based on GCF. Several tests have been developed that areaimed at specifically and sensitively revealing the metabolic status of periodontal tissues. Unfortunately only a handful of GCF tests have made their way into clinical practice. Many longitudinal studies are required which will eventually pave the way for thedevelopment of practical GCF indicators that will aid in the accurate diagnosis and appropriate treatment ofperiodontal diseases

References
1. Patel PV, Kumar S, Kumar V, Vidya GD. Quantitative cytomorphometric analysis of exfoliated normal gingival cells. J Cytol 2011;28:66-72. 2. Gujjari GK, Gujjari AK, Patel PV, Shubhashini PV. Comparative evaluation of ultraviolet and microwave techniques for toothbrush decontamination. J Int Soc Prevent Communit Dent 2011;1:20-6 3. Patel PV, Kumar S, Vidya GD, Patel A, Holmes JC, Kumar V. Cytological Assessment of Healing Palatal Donor Site wound and Grafted Gingival Wound after Application of Ozonated Oil: An Eighteen Months Randomized Controlled Clinical Trial. Acta Cytol. 2012;56(3):277-84. 4. Patel PV, Kumar V, Kumar S,GD V, Patel A. Therapeutic effect of topical ozonated oil on the epithelial healing of palatal wound sites: a planimetrical and cytological study. Journal of Investigative and Clinical Dentistry 2011;2: 248258.

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5. Patel PV, Kumar GS, Patel A. Periodontal abscess: a review. Journal of Clinical and Diagnostic Research. 2011;5:404-409 6. Patel PV, Shruthi S, Kumar S. Clinical effect of miswak as an adjunct to tooth brushing on gingivitis. J Indian Soc Periodontol 2012;16:84-8. 7. SB Vishwanath, Kumar V, Gujjari S, Sashikumar P, Sashikumar Y, Patel PV, Correlation of periodontal status and bone mineral density in postmenopausal women-a digital radiographic and quantitative ultrasound study, Indian J Dent Res 2011; 22 (2), 271-276 8. Patel PV, Patel A, Kumar S, Holmes JC. Effect of subgingival application of topical ozonated olive oil in the treatment of chronic periodontitis: a randomized, controlled, double blind, clinical and microbiological study. Minerva Stomatol 2012;61:381-98. 9. S S, Patel PV, Kumar S. Management of a Persistent Periapical Lesion due to Apicomarginal Defect Associated with Root End Fracture in an Endodontically Treated Tooth: A Clinical Report. J Clin Diagn Res 2012;6:1593-6. 10. Patel PV, Gujjari S, Patel A, Acquired Immune Deficiency Syndrome (AIDS)in Periodontal Practice: A pactitioners handbook 1st ed. LAP LAMBERT Academic Publishing; HeinrichBocking-Str. 6-8, 661 21 Saarbrucken,Germany: 2012

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