SYNERGISTIC EFFECT OF MICROBUBBLE EMULSION

AND SONIC OR ULTRASONIC AGITATION

ON ENDODONTIC BIOFILM IN VITRO

by

Andrew Halford

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Dentistry

University of Toronto

Copyright by Andrew Halford (2012)

i
 SYNERGISTIC EFFECT OF MICROBUBBLE EMULSION AND
SONIC OR ULTRASONIC AGITATION ON ENDODONTIC
BIOFILM IN VITRO
Andrew Halford, BSc, DDS

Master of Science 2012

Graduate Department of Dentistry, Discipline of Endodontics

University of Toronto

ABSTRACT

This study examined the effects of a microbubble emulsion (ME) combined with sonic or

ultrasonic agitation, on irrigation dynamics and reduction of biofilm bacteria within root canal

models. First, high-speed imaging was used to characterize the bubble dynamics generated in

ME by sonic or ultrasonic agitation within canals of polymer tooth models. Second, 5.25%

NaOCl or ME was sonically or ultrasonically agitated in canals of extracted teeth with 7-day-old

Enterococcus faecalis biofilms. Dentinal shavings from canal walls were sampled at 1 mm and 3

mm from the apical terminus, and colony-forming units (CFU) enumerated. Mean log CFU/ml

values were analyzed with ANOVA and post-hoc tests. High-speed imaging demonstrated

strongly oscillating and vaporizing bubbles generated within ME during ultrasonic, but not sonic

agitation. Post-treatment biofilm CFU were significantly lower in the ultrasonic agitation

(P=0.000) of ME group than in the ultrasonic agitation (P=0.009) and sonic agitation (P=0.006)

of NaOCl groups. The synergistic effect of ME combined with ultrasonic agitation enhanced

bubble dynamics and reduced E. faecalis biofilm bacteria beyond the level achieved by sonic or

ultrasonic agitation of NaOCl.

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 ACKNOWLEDGEMENTS

First, I would like to thank my principal supervisor Dr. Anil Kishen for the opportunity to work
with him on this project. Dr. Kishen was always available and without his support and
supervision I would not have been able to accomplish this task. He is the best teacher I have ever
had and I feel privileged to have had him as my principle supervisor.

Second, I would like to thank Dr. Shimon Friedman for his guidance during my project and the
opportunity to be part of the Graduate Endodontics program at the University of Toronto. His
door was always open and I must specifically thank him for his meticulous eye for detail and
countless hours of corrections.

I would also like to thank Dr. Bettina Basrani for her work on my project and being a member of
my scientific advisory committee. Similarily, I would like to thank Dr. Amir Azarpazhooh for
his statistical support and Dr. Claus-Dieter Ohl for his help and guidance while I was in
Singapore.

Dr. Annie Shrestha and Zohra Aslamzada must also be thanked for their countless hours of help
during the execution of this project.

Finally, I would like to thank my mother, father, uncle, and brothers back home for their support
and always providing a laugh when one was needed.

This study was supported by the Canadian Academy of Endodontics Endowment Fund and the
American Association of Endodontists Foundation.

Andrew Halford, May 2012

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 TABLE OF CONTENTS

ABSTRACT ii

ACKNOWLEDGEMENTS iii

I. GENERAL INTRODUCTION 1

1. Role of bacteria in endodontic disease 3

1.1 Bacterial biofilm 3

1.2 Persistence of bacteria after endodontic treatment 4

1.3 Enterococcus faecalis 4

2. Sodium hypochlorite 5

2.1 Properties and mechanism of action 6

3. Microbubbles 9

4. Agitation of irrigants 9

4.1 Sonic activation (EndoActivator) 10

4.1.1 EndoActivator - Irrigant penetration 11

4.1.2 EndoActivator - Bacterial reduction 12

4.2 Ultrasonic irrigation 13

4.2.1 Passive ultrasonic irrigation - Irrigant penetration 14

4.2.2 Passive ultrasonic irrigation - Bacterial reduction 15

5. Mechanism of action for sonic or ultrasonic activation 19

5.1 Acoustic streaming and acoustic microstreaming 19

5.2 Cavitation 23

6. Conclusions from literature review 25

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II. OBJECTIVES AND HYPOTHESIS 27

1. Objectives 27

2. Hypothesis 27

III. ARTICLE 28

IV. DISCUSSION 47

1. Methodology 47

2. Results 49

3. Future directions 51

V. CONCLUSIONS 52

VI. REFERENCES 53

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I. GENERIAL INTRODUCTION

Root canal irrigation is a crucial component of endodontic treatment (1, 2). Its physical

objectives are to facilitate flow of the irrigant throughout the entire root canal system, so as to

flush out all debris. Its chemical objectives are to inactivate intracanal bacterial biofilms and

endotoxins, and to dissolve tissue remnants and smear layer present on the canal walls. Effective

delivery of the irrigant is required to distribute and frequently replenish the optimal

concentration and amount of irrigant throughout the root canal system. Sodium hypochlorite

(NaOCl) is the principal endodontic irrigant used, due to its excellent antibacterial property and

its ability to dissolve organic tissue (3). In spite of these favourable properties, conventional

irrigation delivery of NaOCl does not consistently achieve complete bacterial elimination from

root canal systems (4, 5).

The main limiting factors in conventional irrigation are the complexity of the root canal

anatomy, the ultra-structure of the dentin and the characteristics of the bacterial biofilms (3, 6,

7). Continuous attempts to surmount these limitations have recently led to the renewed interest in

the application of sonic and ultrasonic agitation of the irrigants within the root canal system (8).

Although several studies (9, 10) have reported that agitation enhanced the efficacy of NaOCl

irrigation, other studies (11, 12) have not corroborated these findings.The inconsistent outcomes

achieved with sonic or ultrasonic agitation of NaOCl might be attributed to the restricted

oscillation of the vibrating instruments within the confines of the root canal (13).

When sonic or ultrasonic agitation is applied to a liquid, it results in the formation of cavitational

bubbles that may exist in non-inertial and inertial forms. Non-inertial cavitation bubbles undergo

linear pulsation after being exposed to a low amplitude vibration (13). Inertial cavitation bubbles
1
undergo high-energy pulsations and their collapse generates powerful shock waves capable of

disrupting biofilms and directly killing bacteria (14). The desirable inertial cavitation effect may

not materialize within root canals, as the current ultrasonic systems generate predominantly

small non-cavitating bubbles that act mainly to increase acoustic microstreaming and produce

shear stresses along the root canal wall (15). The limited bubble dynamics may not be sufficient

to detach surface adherent biofilm bacteria or disrupt the biofilm structures within the root canal

(16). To overcome this limitation, it is reasonable to assume that the combination of intensified

bubble dynamics and increased chemical reactivity of the irrigant, would enhance the antibiofilm

efficacy of irrigation within root canals. One strategy that may intensify both the cavitation

bubbles and the chemical reactivity of the irrigant is by using microbubbles in root canal

irrigation.

Microbubbles, commonly employed as carriers for targeted drug delivery, are gas-filled bubbles

in the micron range produced in an emulsion. Inherently unstable, microbubbles require a

stabilizing shell of surfactant, lipid, protein, polymer or a combination of the above. Most of the

microbubble unit volume consists of the gas core (17). When exposed to ultrasonic waves, the

microbubble gas core expands during the rarefaction phase of the pressure wave and contracts

during the compression phase generating inertial bubbles (17). These bubbles coalesce with

those generated by the ultrasonic agitation, decreasing the threshold for bubble production and

increasing bubble dynamics (18). Furthermore, including an oxygen carrier and an oxidizer in

the microbubble emulsion will generate reactive oxygen species (19). The addition of sonic or

ultrasonic agitation to microbubbles could potentially enhance bubble dynamics producing an

antibacterial effect.

2
1. Role of bacteria in endodontic disease

Microorganisms cause endodontic infections and have been shown to be necessary for the

development of apical periodontitis (20-23). Typically, two to ten different bacterial species have

been found when the root canal system has been sampled (24-26). This environment initially

tends to favour Gram negative obligate anaerobes (24, 26-37) and the number of bacterial cells

present in the necrotic system has been reported to be from 102 to 108 per sample (4, 31, 38-45).

1.1 Bacterial biofilm

A biofilm can be defined as community of microorganism embedded in an extracellular matrix

and in most cases is attached to a surface (46, 47). Although Nair (46) did not use the term

biofilm, he was the first to describe the presence of surface adherent biofilm structures in the root

canal system. He examined the root canals of 31 teeth with large carious lesions that were

extracted with the periapical tissue intact. Light microscopy and transmission electron

microscopy was used for this purpose. It was suggested that the intracanal microorganisms

existed in loose collections of cocci, rods, filaments and spirochetes (46). Since that time

several authors have shown that the biofilm ultrastructure within the root canal varies between

cases (47-50) and our understanding of the conditions present during endodontic biofilm

formation are still limited (51).

Bacteria in a biofilm have advantages, which allow them to resist disinfection strategies, making

them more difficult to eliminate (52). The extracellular matrix provides the microorganisms with

a physical barrier against harmful environmental situations (53). Within the biofilm structure,

nutrients can be concentrated (54), producing niches for specific bacterial species requirements

3
(55). Bacteria present in different areas of a biofilm structure are in different growth phases,

which render them less susceptible to harsh environments (56). Biofilm bacteria undergo

physiological changes that can give added protection (57-59). Some of these physical changes

are a result of bacteria cells communicating with each other through quorum sensing. Quorum

sensing can allow bacteria in close proximity to coordinate their function (60). The exchange of

genetic material between different types of bacteria can also occur, allowing antibacterial

resistance to be transferred between bacterial strains and species co- existing in a biofilm (61,

62).

1.2 Persistence of bacteria after endodontic treatment

A number of studies have illustrated that in spite of elaborate cleaning and shaping efforts, we

are currently unable to completely eliminate bacteria from the root canal system (4, 40, 45, 47,

63, 64). Two main reasons for the persistence of endodontic bacteria are the biofilm

characteristics (57), and the anatomic complexities (65, 66) of the root canal system presents.

Complexities such as lateral canals, isthmuses and apical ramifications are often observed to still

harbour bacterial biofilms (47-49). Although the effect on healing caused by these residual

bacteria is debated it is generally agreed that complete elimination would be ideal to achieve

treatment goals (67).

1.3 Enterococcus faecalis

Enterococcus faecalis is a facultative gram positive coccus (68, 69). This species has been shown

to be a very hardy microorganism being able to survive temperatures up to 60 oC and growing at

temperature ranges from 10 to 45 oC (70). They are known to be resistant to substances such as

4
detergents, heavy metals, ethanol, bile salts and azide (68, 70). Survival at high pH (9.6),

desiccation and high salt environments have also been observed (68, 70). The ability to survive

these environmental extremes comes from a variety of characteristics it possesses. Wide spread

genetic polymorphism has been exhibited (71), along with the ability to express serine protease,

gelatinase and collagen binding protein which help in adhering to dentin substrate (72).

Prolonged periods of nutrient deprivation can be endured and once nutrients become available,

the cells can recover (73). Furthermore, these bacteria have been shown to form biofilms

making them 1000 times more resistant to antimicrobials (74). Resistance to calcium hydroxide

when present in dentinal tubules (75, 76) has also been reported. This species has been

extensively studied in endodontics as it has been found to occur in 16% to 77% of root filled

teeth (77-88). The high occurrence of this microorganism and its hardiness (73) make it an ideal

and relevant microorganism for studies on endodontic disinfection.

2. Sodium hypochlorite

Koch and Pasteur in the 19th century carried out laboratory studies that brought about the

acceptance of hypochlorite as a disinfecting agent (89). Dakin and Carrel (90) later irrigated

infected wounds with buffered 0.5% sodium hypochlorite solution, as a result of Dakins

investigation of the effect of different solutions on infected necrotic tissue. Hypochlorite has

been shown to be virucidal and sporicidal along with having a broad nonspecific ability to kill

bacteria (91). It also displays the ability to dissolve necrotic tissue more than vital tissue (92).

Since the 1920s the above characteristics have made aqueous sodium hypochlorite a main

endodontic irrigant (89, 93, 94).

5
2.1 Properties and mechanism of action

Sodium hypochlorite was reported to exist in a dynamic equilibrium (95). This equilibrium is

shown in the following equation:

NaOCl + H2O NaOH + HOCl Na+ + OH- + H+ +OCl-

The effect of sodium hypochlorite on organic tissue is illustrated in the following chemical

reaction (95-98) .

Chemical reaction 1. Chloramination reaction

Amino acid Hypochlorous Chloramine Water

acid

H O Cl O

R-C-O-C + HOCl R-C-O-C + H2 O

NH2 OH NH2 OH

Chemical reaction 1 illustrates hypochlorous acid acting as a solvent when in contact with

organic tissue. This process results in chlorine being released which combines with protein

amino groups to form chloramines. Amino acid degradation and hydrolysis is caused by both

hypochlorous acid and hypochlorite ions. The chloramines produced by the chloramination

reaction interfere with cell metabolism. The antimicrobial action comes from SH groups

(sulphydryl group) of bacterial enzymes being irreversible oxidated by chlorine.

6
Chemical reaction 2. Amino acid neutralization reaction.

Amino acid Sodium Salt Water

hydroxide

H O H O

R-C-O-C + NaOH R-C-O-C + H2 O

NH2 OH NH2 ONa

Chemical reaction 2 illustrates the production of water and salt from the neutralization of amino

acids. The pH is decreased due to the exit of hydroxyl ions.

Chemical reaction 3. Saponification reaction

Fatty acid Sodium Soap Glycerol

hydroxide

O O

R-C-O-R + NaOH R - C - O - Na + R - OH

Chemical reaction 3 illustrates that sodium hypochlorite degrades fatty acids by acting as an

organic and fat solvent. The degradation process results in the production of glycerol (alchol)

and fatty acid salts (soap) and reduces surface tension (99).

The physico-chemical characteristics of sodium hypochlorite have been examined by a number

of researchers (96, 99, 100). The effect of sodium hypochlorite to dissolve pulp tissue was

observed by Grossman and Meiman (101). Their findings were that the tissue would dissolve in

20 minutes to 2 hours (101). Previous studies on the dissolution of bovine pulp tissue (96-98)

7
highlighted that the rate of dissolution was proportional to the sodium hypochlorite concentration

and was faster without a surfactant. Further, the variation of surface tension was proportional to

the sodium hypochlorite concentration and was greater with no surfactant. Sodium hypochlorite

without a surfactant experienced a decrease in surface tension (96). Increasing the temperature

was found to result in rapid tissue dissolution (97). Furthermore, the greater the initial

concentration of the original solution the smaller the decrease in pH (98). The mechanism of

antimicrobial affect of sodium hypochlorite antimicrobial was discussed by examining its

reaction with organic tissue and its physico-chemical properties. Sodium hypochlorite has a high

pH over 11, this is one of the reasons for its negative effect on bacterial cells (99).

Enzyme activity of anaerobic bacteria at different pH levels was examined by Estrela (100). It

was suggested that the enzymatic sites at the cytoplasmic membrane is important to carry out

necessary cellular functions. The authors used calcium hydroxide and believed the hydroxyl ions

released were responsible for its action on the cytoplasmic membrane. The proteins on the

membrane are denatured as a result of the pH gradient created by the high hydroxyl ion

concentration. The high pH also changes the integrity of the cytoplasmic membrane. The change

in integrity is attributed to several mechanisms: combination of degradation of phospholipids or

unsaturated fatty acids, or from chemical injury to organic components (102). The hydroxyl ions

produced from sodium hypochlorite should behave in a similar manner to those produced by

calcium hydroxide (99). The production of chloramines interferes with cellular metabolism.

Hydrogen is replaced chlorine during the oxidation process, and causes irreversible enzyme

inhibition. The bacterial enzyme cystein is inactivated when the sulphydryl groups are

irreversibly oxidized (99).

8
3. Microbubbles

Microbubbles are gas-filled bubbles in the micron range produced in an emulsion. They are

commonly employed as carriers for targeted drug delivery. The microbubble emulsion (ME) is

inherently unstable, and requires a stabilizing shell of either surfactant, lipid, protein, polymer or

a combination of the above. Most of the ME unit volume consists of the gas core (17). When

exposed to ultrasonic waves, the microbubble gas core expands during the rarefaction phase of

the pressure wave and contracts during the compression phase generating inertial bubbles (17).

These bubbles coalesce with those generated by the ultrasonic agitation, decreasing the threshold

for bubble production and increasing bubble dynamics (18). George and Kishen (103) reported a

ME consisting of three ingredients: perfluoro-decahydronaphthalene an oxygen carrier, H2O2 an

oxidizing agent, and the non-ionic detergent triton-X100 in a ratio of 75.0:24.5:0.5. This formula

was reported to yield significantly higher reactive oxygen species and subsequently the best

antibacterial benefit of the several combinations tested in the study (103). When combined these

components form a gas filled bubble surrounded by the surfactant, which separated it from the

oxygen carrier and oxidizing agent. Furthermore, including an oxygen carrier and an oxidizer in

the microbubble emulsion will generate reactive oxygen species (19), while the properties of

microbubbles, in combination with sonic or ultrasonic agitation could potentially enhance bubble

dynamics, which would further improve its antibacterial effect within the root canal system.

4. Agitation of irrigants

The physical and chemical objectives of irrigation can be enhanced through agitation of the

irrigant by distributing the irrigant and creating shearing and streaming forces throughout the

root canal system. This review will focus on sonic and ultrasonic agitation. Agitation of an

9
irrigant is carried out by placing an instrument into the root canal and moving it in a rotating,

oscillating, or reciprocating motion (2). The agitation can be carried out with several methods

including manual reciprocation, a mechanically driven rotary instrument, and sonic or ultrasonic

devices (2).

4.1 Sonic agitation (EndoActivator)

Tronstad (104) was the first to utilize sonic instruments in endodontics. Sonic agitation occurs at

frequencies of 1 to 6 kHz and produces lower shear stresses compared to ultrasonic agitation

(105). Walmsley (106) evaluated the oscillatory patterns of sonically driven files in a Micro-

Mega 3000 handpiece. The authors found that movement of the file involves 1 node near the file

attachment and 1 antinode at the file tip. When the file movement was unimpeded an elliptical

pattern was observed. However when impeded the investigators found the file movement to a

longitudinal movement and the sideways movement was lost. The authors concluded that this

longitudinal movement would be effective for canal preparation (106). The EndoActivator

(Advanced Endodontics, Santa Barbara, CA) is marketed as a sonic device that is battery

operated and functions at cycles 2,000, 6,000 and 10,000 cycles per minute. The tips are made of

a medical grade polymer, with a snap-on or snap-off design and are available in sizes 0.15

mm/0.02 taper, 0.25 mm/0.04 taper, and 0.35 mm/0.04 taper (107). Jiang (107) made

observations of the EndoActivator tip in air and water. The oscillatory pattern was found to be

1.1+/- 0.1 mm and 0.6 +/- 0.1 at the attachment point and 3.1 +/- 0.1 mm and 1.2 +/- 0.1 mm at

the free end respectively in air and water. They also found that the frequencies of the sonic

device were different than those listed in the brochure. They observed that in mode 1, 160 +/- 5

10
Hz instead of 33 Hz, mode 2, 175 +/- 5 Hz instead of 100 Hz, and mode 3, was 190 +/- 5 Hz

insted of 166 Hz (107).

4.1.1 EndoActivator - irrigant penetration

The effect of the EndoActivator on irrigant penetration has been evaluated by several

investigators. de Gregorio (108) compared the effect of EndoActivator, needle irrigation, and

ultrasonic activation on irrigant penetration in simulated lateral canals in cleared extracted teeth.

Agitation was carried out for one min at a distance of 2 mm from the apex. The amount of

colored dye entering the lateral canals was compared. It was observed that there was no

significant difference between the EndoActivator and ultrasonic activation; however, both were

more effective than conventional irrigation (109). Paragliola (110) evaluated irrigant penetration

into dental tubules of extracted teeth with EndoActivator, manual agitation with gutta percha or a

K file, two ultrasonic devices and without agitation. After treatment specimens were sectioned at

1, 3, and 5 mm from the apex (1 mm thick slabs) and viewed under fluorescence microscopy.

Greater irrigant penetration was found with the ultrasonic devices compared to the other groups

including the EndoActivator. The EndoActivatior did however provide a benefit over several

groups (110). In another study, de Gregario (111) observed irrigant penetration in simulated

lateral canals in cleared extracted teeth with a surgical operating microscope. The experimental

groups were the EndoActivator, F file, Ultrasonic and EndoVac. Agitation was done for 30 sec at

working length. The investigation found that the EndoVac was significantly more effective at

delivering irrigant to working length and the ultrasonic activation produced the best penetration

into lateral canals (111).

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4.1.2 EndoActivator - bacterial reduction

The effect of the EndoActivator on bacterial reduction was examined in several studies.

Townsend (112) evaluated the ability of different irrigation methods to reduce 7 day old E.

faecalis biofilm grown in simulated root canals in plastic blocks. A crystal violet assay was used

to quantify the biomass in this study. Sterile water was used as an irrigant and experimental

groups were EndoActivator needle irrigation, EndoVac, ultrasonic, sonic and F- file agitation.

Agitation was carried out at 2 mm short of the working length for a period of 30 sec. The authors

found that the ultrasonic was significantly better than needle irrigation and the EndoVac system.

There was no significant difference in residual bacteria between the EndoActivator, ultrasonic

device, F- file, and sonic device (112). Brito (113) also evaluated the reduction of a 7 day E.

faecalis biofilm grown in extracted teeth using colony forming units. These researchers

compared the EndoActivator, needle irrigation, and EndoVac. Agitation was carried out for 90

sec at a distance of 2 mm from the working length and found all resulted in significant bacterial

but none was any better than the other (113). Shen (114) grew multispecies biofilms for 21 days

on collagen- coated hydroxyapatite disks. EndoActivator or ultrasonic agitation was carried out

at a distance of 5 mm from the disks for 1 or 3 min. Using confocal laser scanning microscopy

the authors found that the EndoActivtor at both time periods had the highest levels of

bactericidal activity (114). Pasqualini (115) compared the EndoActivator and standard needle

irrigation in an extracted tooth model which was inoculated for 2 hours with an E. faecalis

medium. Agitation was carried out 2 mm short of the working length for 15 or 30 sec and colony

forming units were used to assess the outcome. The 30 sec agitation had the lowest bacterial

counts. While 15 seconds of agitation was similar to conventional irrigation of 5 % NaOCl and

leaving the irrigant for 15 or 30 seconds (115). Tardivo (12) compared EndoActivator agitation,

12
syringe irrigation, and ultrasonic agitation of 5.25 % NaOCl in an extracted tooth model.

Agitation was carried out for 3 min and colony forming units were used as an outcome measure.

This investigation found no difference between the three groups (12). One of the more

interesting in vivo investigations with the EndoActivator was carried by Huffaker (116). The

investigators compared agitation for a total 1 min with the EndoActivator at the end of the

treatment session and syringe irrigation. Treatment was carried out on any tooth with apical

periodontitis. 5 % NaOCl was used and bacterial samples were taken with paper points at the end

of the first treatment session. They found no significant difference between the two tested groups

(116).

4.2 Ultrasonic irrigation

Richman (117) was the first to introduce ultrasonic devices in Endodontics. In these devices,

files oscillate at frequencies of 25 to 30 kHz and operate in a transverse vibration. As such they

have a pattern of motion consisting of nodes and anti-nodes along their length (118-120). Two

methods of ultrasonic irrigation have been described. The first involves ultrasonic

instrumentation (UI) along with irrigation, while the second method is referred to as passive

ultrasonic irrigation (PUI) and does not involve instrumentation (120). This review will focus on

PUI which was first described by Weller (121). PUI has advantages over UI as contact with

canal walls will dampen acoustic streaming (122) and will potentially gouge the canal walls

(123). However, this is an active process even though the term passive is used since the activated

file is meant to be noncutting. When PUI is carried out, acoustic energy is transmitted from an

oscillating wire or file to a liquid medium (120). According to Krell (124) the basic PUI

13
procedure is that after the root canal system was shaped to the master apical file, and with

irrigant in the canal system a ultrasonic file or wire is placed to the apical area and activated.

4.2.1Passive ultrasonic irrigation - irrigant penetration

The effect of PUI on irrigant penetration has been evaluated by several investigators. de

Gregorio (108) compared the effect of ultrasonic activation, needle irrigation, and EndoActivator

on irrigant penetration in simulated lateral canals in cleared extracted teeth. Agitation was carried

out for one min at a distance of 2 mm from the apex. The amount of colored dye entering the

lateral canals was compared. They found that there was no significant difference between the

EndoActivator and ultrasonic activation, however, both were more effective than conventional

irrigation (109). de Gregario (111) observed irrigant penetration in simulated lateral canals in

cleared extracted teeth with a surgical operating microscope. The experimental groups were

ultrasonic activation, F file agitation, EndoActivator, and EndoVac. In all the tested groups,

agitation was done for 30 sec at working length. It was found that the EndoVac was significantly

more effective at delivering irrigant to working length and the ultrasonic activation produced the

best penetration into lateral canals (125).

Paragliola (126) evaluated irrigant penetration into dental tubules of extracted teeth with two

ultrasonic devices, manual agitation with gutta percha or a K-type file, two sonic devices

including the EndoActivator and without agitation. After treatment teeth were at 1, 3, and 5 mm

from the apex and viewed under fluorescence microscopy. Greater irrigant penetration was found

with the ultrasonic devices compared to the other groups including the EndoActivator (110).

Ferreira (127) evaluated methylene blue penetration in prepared root canals immersed in

14
methylene blue dye for 24 h under six different conditions: pre-agitation for 10 min using an

endodontic ultrasound; pre-agitation for 10 min using a cleaning ultrasound; 25-mmHg vacuum

for 10 min followed by passive immersion; 30 and 650-mmHg vacuum for 24 h and passive

immersion. No significant difference was found between any groups (127).

4.2.2 Passive ultrasonic irrigation - bacterial reduction

Bacterial reduction was examined and observed to be improved in several studies with PUI.

Martin (128) evaluated the effect of 5 min of ultrasound agitation with and without a bactericidal

medium (5% NaOCl or 1% potentiated acid pentanedial) on a medium containing several

bacterial species. Experiments were carried out in an extracted tooth model inoculated for 24 hrs

and it was found that ultrasound in a neutral medium, decreased the bacterial count but that

effect was greater when a bactericidal medium was used (128). Huque (129) compared

ultrasonically agitation of various concentrations of NaOCl and 15 % EDTA for 1 min and

syringe delivery of 12 % NaOCl. The investigators smeared a mixture of dentin debris and dental

plaque on the prepared canal surface of extracted teeth. SEM was used to measure the outcome

measure. They found that ultrasonic activation of the higher concentrations of NaOCl (12 % and

5.5 %) eradicated the bacteria from the artificial smear layer. The lower concentrations of NaOCl

0.5 % and 2.5 %, EDTA, and 12 % NaOCl syringe irrigation could not eradicate the bacteria.

Another aspect of this study was that some teeth had holes (1 mm in diameter and a depth of 3

mm) drilled parallel to the canal wall. Bacterial suspensions were subsequently placed in the

holes. The irrigation was then done as in the first part in the canals. The authors in this case

found the ultrasonic activation of 12 % NaOCl to be the only modality effective at eliminating

15
bacteria from the channels and the syringe irrigation of 12 % NaOCl to be the second most

effective (129).

Weber (130) evaluated the effect of 5.25 % NaOCl or 2 % chlorhexidine with or without PUI for

1 min. After agitation canals were filled with PBS, after 6 hrs the PBS was transferred to on agar

containing Streptoccus sanguinis and zones of inhibition were examined over several time

periods. With respect to both irrigants PUI resulted in increased residual antimicrobial activity

(130). Spoleti (131) ultrasonically agitated sterile saline for 10 sec or conventional saline

irrigation in extracted teeth with prepared canals inoculated with bacteria for 72 hours. After

irrigation all samples were positive for bacterial growth, however, levels were significantly lower

for the ultrasonically activated group (131). Townsend (112) evaluated the reduction of a 7 day

old E. faecalis biofilm grown in simulated root canals in plastic blocks with crystal violet assay.

In this investigation sterile water was used as an irrigant and along with needle irrigation the

experimental groups were ultrasonic, EndoVac, sonic, F- file and EndoActivator. Agitation was

carried out at 2 mm short of the working length for a period of 30 sec. The authors found that the

ultrasonic was significantly better than needle irrigation and the EndoVac. There was no

significant difference between the ultrasonic device and the F- file, sonic device and

EndoActivator (112).

Harrison (132) developed 4 week old E. faecalis biofilms and tested two experimental groups in

which the teeth were instrumented and either had 1 min of ultrasonic irrigation or calcium

hydroxide placement for 1 week. SEM was used and histological analysis was carried out at the

apical, middle, and coronal third. Both experimental groups were effective at removing bacteria

16
but neither removed bacteria from all roots (132). Alves (133) used 30 day old E. faecalis

biofilms grown in extracted teeth with oval shaped canals. Ultrasonic agitation or the use of a

Hedstrum file was tested. Ultrasonic agitation was carried out for 1 min at a distance 1 mm from

the apex. Teeth were then instrumented and 2.5 % NaOCl was ultrasonically activated followed

by 0.2 % chlorhexidine in the other experimental group each canal received additional

instrumentation to the buccal and lingual. PUI did not significantly increase bacterial reduction

in this case, however, the combination of chlorhexidine and ultrasonic agitation was found to

produce significant bacterial reduction (133).

In vivo antibacterial efficacy of ultrasonic activation was examined by Carver (9) . They

compared hand and rotary instrumentation with hand and rotary instrumentation followed by 1

minute of ultrasonic activation of irrigant to a pre measured depth just before the needle tip

bound. Procedures were carried out in the mesial roots of necrotic mandibular molars and

samples were taken with sterile paper points. This investigation showed a significant decrease in

CFUs when ultrasound was used, and it was found that when ultrasound was used there was a 7

times greater chance to obtain a negative culture (9). Two other in vivo investigations were

carried out by Burleson (134) and Gutarts (135) from the same group as the above study. The

experimental setup was the same except instead of paper point sampling this investigation

involved extraction of the sample tooth and histological observations were made of canals and

isthmuses for the amount of biofilm and necrotic debris present. Both investigations found the

ultrasonic activation to result in significantly cleaner canals and isthmuses. A more consistent

level of cleanliness was also found when ultrasonic was used (134, 135).

17
Bacterial reduction was examined in several studies and observed not to be improved with PUI.

Ahmad (136) in simulated glass root canals with 24 hour S. mitis biofilms grown in them,

investigated the effect of 2.5 % NaOCl and ultrasound or ultrasound alone (sterile saline).

Agitation was carried out at a low or medium power setting for 2 or 5 min at a distance of 1 mm

from the apical terminus. In this case the ultrasound alone was found to increase CFU counts,

while the ultrasound and NaOCl resulted in no viable bacteria (137). Siqueira (5) evaluated root

canals prepared and inoculated E. faecalis for 24 hours. Canals were then exposed to 4 % NaOCl

agitated with an ultrasonic unit, a hand file, or a combination of 4 % NaOCl and 3 % H2O2. After

treatment sampling was done with paper points and turbidity was the measure for viable bacteria.

The experimenters found no significant difference between any of the agitation techniques (5).

Shen (114) grew multispecies biofilms for 21 days on collagen- coated hydroxyapatite disks.

Ultrasonic or EndoActivator agitation was carried out at a distance of 5 mm from the disks for 1

or 3 min. Using confocal laser scanning microscopy the authors found the EndoActivtor at both

time periods had the highest levels of bactericidal activity(114).

Bhuva (11) compared passive ultrasonic agitation and syringe irrigation of 1 % NaOCl on 72

hour E. faecalis biofilms. In this case there was no significant difference between the two

experimental groups (11). Tardivo (12) compared ultrasonic agitation, syringe irrigation and

EndoActivator agitation of 5.25 % NaOCl in an extracted tooth model. Agitation was carried out

for 3 min and colony forming units were used as an outcome measure. This investigation found

no difference between the three groups (12). Peters (138) compared ultrasonic agitation,

conventional irrigation and erbium:YAG laser in root canals with 6 % NaOCl which had an oral

biofim grown in situ for 1 week and were incubated for a further 2 weeks. Laser activation for 30

18
sec was carried out with the tip placed in the chamber while ultrasonic agitation for 30 sec was

done at 1 mm from the apex. Laser activation produced superior disinfection and less biofilm

mass in the apical area (138). Grundling (139) used E. faecalis biofilms grown in bovine teeth to

test the efficacy of irrigation with saline, 2% NaOCl followed by 17% EDTA, with and without

ultrasonic agitation. This study demonstrated no bacterial growth in the NaOCl groups with and

without agitation (139). Case (140) used 14 day old E. faecalis biofilms grown in an extracted

tooth model to test the efficacy of irrigation with 1 % NaOCl, ozone enriched air, and sterile

saline, the latter two groups were ultrasonically agitated for 2 min. CFU analysis showed an

improvement in bacterial reduction when ultrasonic agitation was carried out. However, syringe

irrigation with the 1% NaOCl showed the most significant reduction (140).

5. Mechanism of action for sonic or ultrasonic activation

5.1 Acoustic streaming and acoustic microstreaming

Acoustic streaming is defined as the rapid movement of particles of fluid in a vortex-like motion

about a vibrating object (118). In the root canal this fluid movement may be caused by a

vibrating file, however, it can be associated with small gas bubbles set into oscillation by the

fluctuating pressure field generated by the file. With this in mind stable cavitation can be

included when describing acoustic streaming (105). Acoustic microstreaming is the streaming

pattern that occurs during ultrasonic irrigation in a root canal system and has been defined by

Leighton (141) as the streaming that occurs near small obstacles within a sound field, near small

sound sources, vibrating membranes or wires, which arise from the frictional forces between a

boundary and medium carrying vibrations of circular frequency.

19
Ahmad (105) evaluated acoustic streaming by viewing polystyrene spheres dispersed in a

solution of methylene blue. A Cavi-Endo unit was used with endosonic files #15, 20, and 25 and

diamonds #25, 35, and 45 were then partially submerged and oscillated at settings of 1,2, or 3.

As soon as the power was on time- dependent acoustic streaming was seen along the file. The

authors observed fluid movement along the file surface from the apical up toward the coronal.

Eddying motions were mainly near the apical half of the file, with the more rapid eddying being

near the tip and slow movement in the coronal (105). In a follow up study investigating acoustic

streaming Ahmad (122) observed the Cavi-Endo unit was used with endosonic files #15, 20, and

25 at power settings of 1.0, 1.5, 2.0, or 2.5. The authors observed each file oscillating in air to

determine displacement amplitudes and also in the irrigant to view flow patterns. The

information obtained from the first part of this study was then applied to an extracted tooth

model in which teeth had a final step of ultrasonic instrumentation or passively ultrasonic

irrigation which was discussed earlier. A number of observations were made on the flow

patterns. With all files once the activated there was a streaming field created that had two

components. The primary field was rapidly moving eddies in which there is oscillation of the

fluid elements about a mean position. Superimposing the secondary field consists of relatively

slow time dependent flow. The majority of eddies were seen toward the tip and eddies were

found to be in clusters of four along the file. The smaller files #15 and 20 produced a maximum

of four clusters of eddies, while the #25 file produced a smaller number of eddies but in a similar

distribution pattern. It was also observed that in the primary field neighboring eddies had flowed

in the opposite direction. In the secondary field fluid had a movement was laminar from the

apical to coronal. At the tip of the file there was some apical movement of fluid. Increasing the

20
power did not increase the number of eddy currents, however, both the eddy size and velocity of

streaming appeared to increase (105).

Lumley (142) evaluated the ultrasonic and sonic streaming patterns around a number 20 K-type

file by agitating the file in 2 mm of water over top of plaster in a petri dish. The file was placed

parallel and at right angles to the surface and agitated for 20 sec. The effects of restraint were

also tested by placing the file against the dish. Measurements were taken from the plaster and

acoustic microstreaming patterns were noted that coincided with the nodal and anti-nodal pattern

of the sonic and ultrasonic agitation. Streaming was found to occur mainly in front or behind the

ultrasonic file, and was dependent on power setting and whether or not the file was constrained.

The streaming was found to be evenly distributed around the sonic file, produced a large

disturbance when freely oscillating and was unaffected by constraint. The majority of activity

was found to occur around the file tip and decrease toward the hub with both agitation types

(142).

Ahmad (143) evaluated the streaming pattern around straight and precurved files agitated in the

ultrasonic device Piezon-Master 400 unit (Electro-Medical Systems, Le Sentier, Switzerland).

Agitation in the free field was done in a container filled with a solution containing methylene

blue and a white PVC powder dispersed on the surface. In the second phase agitation was

observed in straight, tapered and curved channels prepared in resin blocks. The authors reported

that acoustic streaming was generated in both the free field and channel scenarios, with light wall

contact reducing streaming and severe wall contact resulting in no streaming. Smaller files that

were precurved resulted in the highest streaming velocities. Two rows of vortices along the side

21
of the file were seen during agitation in the free field. In the channel the position of the vortices

were above the surface of the file. The authors concluded that streaming was possible even in

small channels and that these streaming patterns should be utilized during root canal treatment

(143).

In another study Ahmad (144) evaluated the Piezon-Master 400 ultrasonic file that was driven by

a piezoelectric transducer. The experimental set up was similar to his 1987 (105) study and the

results were compared to those results. The oscillating file was found to produce a standing wave

with nodes and antinodes, with the largest deflection at the tip. When compared to the Cavi-Endo

file the displacement amplitudes were higher. The authors suggested that the power transmission

to the file of this device may be more efficient, contributing to the larger amplitudes seen (144).

Roy (13) evaluated the Piezon-Master 400 at manufactures settings in glass capillary tubes with

an internal diameter of 1.09 mm. Experiments were carried out with straight glass tubes or tubes

bent to a 30 o curvature. Contact with the canal wall was either as minimal as possible or severe

to simulate a clinical situation. Different file designs and sizes were evaluated with visual

observations and sonoluminescence being used as an outcome measure for transient cavitation in

water, carbonated water and 2.5 % NaOCl and made observations on streaming patterns. The

cavitation results will be discussed later, with respect to streaming patterns the authors found that

along the sides of the file streaming could be seen but no obvious signs of cavitation. With

respect to the tip the majority of activity was determined to be small scale microstreaming and

some transient cavitation. When files motion was restricted the authors still saw stable bubbles

being produced and steady streaming patterns (145). In another study, Jiang (146) evaluated the

22
flow pattern around an oscillating file with high speed imaging. Agitation was produced with the

Suprasson PMax Newtron system at a ultrasonic setting of blue 4. The file tip restricted by

placing it through a hole into a water tank, and hollow glass spheres were placed in the water to

trace motion. They observed flow consisted of 2 jets in the same direction of the oscillating file.

An unsteady flow was located within a distance of 1 diameter of the file was also present (146).

5.2 Cavitation

Cavitation can be defined in a number of different ways, generally the definition includes the

growth and subsequent collapse of a small gas-filled bubbles pre-existing in a fluid (15, 147).

Leighton (141) defined acoustic cavitation as the creation of new bubbles or the expansion,

contraction and or distortion of pre-existing bubbles in a liquid, this being coupled with acoustic

energy. Roy (13) divided cavitation to stable and transient. Stable cavitation was defined as the

linear pulsation of bubbles in a low amplitude ultrasound field. Transient cavitation involves

bubbles undergoing highly energetic pulsations. When the energies are high enough the bubbles

can collapse producing shock waves high pressures and temperatures (13).

Ahmad (148) attempted to study cavitation by detecting the light released when cavitation

bubbles collapse. A Cavi-Endo unit was used with endosonic files #15, 20, and 25 were activated

along with #25, 35, and 45 diamond coated files in a container filled with 2.5% sodium

hypochlorite and allowed to oscillate freely. The power settings were from 1 to 3 which

corresponded to a displacement of 5.6 to 41.3 m. The experiment was repeated with a sickle

shaped ultrasonic scaler. All of the files in this investigation failed to produce any light when

23
oscillated. The scaler did produce some high intensity light at the power setting of 2. The authors

concluded transient cavitation did not play a role in cleaning (148).

Lumley (149) examined cavitation production with the Cavi-Endo system and # 20 K type files.

This author used the oxidation of potassium iodide to iodine in the presence of carbon

tetrachloride to determine the amount of cavitation occurring. The solution was either passed

over an activated drive, activated driver and file, activated file alone, the driver with no

activation, and finally an activated file in a glass tube of the solution. This investigation found

the cavitation decreased with an increase in flow rate to 7 ml/min at this point there was a rapid

decrease. After this point the amount of cavitation for the driver alone and the driver and file

remained stable. There was no cavitation detected when the solution was activated in the glass

tube or when the solution was only placed over the file. The authors concluded this to be due to

the displacement amplitude of the file to be too low to produce cavitation. These authors

concluded that cavitation observed would not be clinically relevant (149).

Roy (13) in the previously mentioned study found that as long as severe wall contact was

avoided transient cavitation was observed. With respect to file surface characteristics when

severe wall contact was made no transient cavitation was observed with the standard file. The

pitted file produced transient cavitation while the smooth file produced the most. Curved files in

sever contact with canal walls produced some transient cavitation but this was only minimal. All

three irrigants produced low amounts of transient cavitation when the file was in sever contact

with the canal wall. When power settings were evaluated it was observed that transient cavitation

was independent of the settings. Visually, streaming could be seen along the sides of the file but

24
no obvious signs of cavitation were noticed. With respect to the tip the majority of activity was

determined to be small scale microstreaming and some transient cavitation. Stable bubbles were

noted even when the file motion was restricted along with steady streaming patterns (145). Jiang

(107) in a two part study used high speed imaging to evaluate the EndoActivator for production

of cavitation. Glass tubes were used for a root canal model, however, no cavitation was observed

(107).

6. Conclusions from literature review

It is apparent from the current literature that both sonic and ultrasonic agitation have the potential

to benefit endodontic disinfection. However, the literature clearly demonstrates that this is not

always the case. The conflicting results are for a variety of reasons including the models tested,

how agitation was carried out, and the outcome assessment. For example models tested varied

from resin blocks (112), extracted teeth (150), to collagen-coated hydroxyapatite disks (114).

Biofilm growth also varied with number of species and incubation times varying form hours

(150) to weeks (114). In addition, the irrigants tested ranged from sterile saline (131),

chlorhexidine (114) to full strength NaOCl (130). Parameters used for agitation also varied

between studies. For example, agitation was done for periods ranging from 15 sec (115) to 5 min

(151) and at distances ranging from 2 mm from the working length (115) to just before the file

binds in the root canal (9). The settings of devices varied resulting in different energy levels

being transferred to the irrigant. Finally, the outcome measures ranged from visual observations

of turbidity (150) to CFU analysis (9). The differences among study design illustrate a

reasonable explanation for the disparity in results obtained after the sonic or ultrasonic agitation

experiments.

25
Even with the conflicting results, several conclusions can be made with respect to the mechanism

of action of these devices. The unique environment presented by the root canal system presents

several challenges, which limit the potential benefits of sonic and passive ultrasonic irrigation.

With respect to passive ultrasonic irrigation the effect is dampened when the file contacts the

canal wall. When the contact occurs at an antinode the dampening is more severe than when

contact occurs at a node (16, 143, 145, 152, 153). This can be further illustrated by the fact that

in curved canals pre curving files results in better acoustic microstreaming (143, 152). Sonic

agitation is less prone to the anatomical constraints produced within a root canal. However, when

loaded the elliptical oscillatory pattern is eliminated leaving a pure longitudinal motion (106).

The mechanism of action for sonic and ultrasonic devices is fairly well described along with the

possible factors which can limit there efficacy. The literature on bacterial reduction shows

potential benefits with agitation but it is far from conclusive. As discussed above this is likely

due to differences in the study design. Nonetheless, it would be very difficult to have a model

which addressed all the different variables associated with root canal irrigation. With these

challenges in mind it is important to couple models designed for evaluating the bacterial

reduction with a model to understand the observations.

26
II. OBJECTIVES AND HYPOTHESIS

1. Objectives

The first objective was to characterize the interaction of the microbubble emulsion and the

bubbles generated by sonic or ultrasonic agitation by high speed image analysis.

The second objective was to determine the antibacterial efficacy of the microbubble emulsion

combined with sonic or ultrasonic agitation by colony forming unit analysis.

Specifically the study aimed to:

Study the interaction of pre-introduced microbubbles and the bubbles generated by

ultrasonic or sonic activation.

Determine the ability of the bubble dynamics produced by combining sonic or ultrasonic

activation with the microbubble emulsion to kill biofilm bacterial.

2. Hypothesis

We hypothesized that compared to sonic or ultrasonic vibration of NaOCl alone:

Sonic or ultrasonic vibration of the microbubble emulsion would result in an increase in

stable and transient bubbles improving bubble dynamics (Physical effect)

Addition of an oxygen carrier (PFC) on H2O2 (oxidizer) would further increase its

chemical effect

27
III. ARTICLE

(Submitted for publication)

Synergistic effect of microbubble emulsion and sonic or ultrasonic agitation

on endodontic biofilm in vitro

A. Halford BSc, DDS, C-D. Ohl PhD*, A. Azarpazhooh DDS, MSc, PhD, B. Basrani DDS, PhD,

S. Friedman DMD, A. Kishen BDS, MDS, PhD**,

Discipline of Endodontics, University of Toronto, Toronto, Canada

* Nanyang Technological University, Singapore, Singapore

**Corresponding author

Dr. Anil Kishen

Associate Professor, Discipline of Endodontics

University of Toronto, Faculty of Dentistry

124 Edward Street, Toronto, ON, M5G 1G6

E mail: anil.kishen@utoronto.ca

28
Abstract

Introduction: Irrigation dynamics and antibacterial activity determine the efficacy of root canal

disinfection. Sonic or ultrasonic agitation of irrigants is expected to improve irrigation dynamics.

This study examined the effects of microbubble emulsion (ME) combined with sonic or

ultrasonic agitation, on irrigation dynamics and reduction of biofilm bacteria within root canal

models. Methods: Two experiments were conducted: First, high-speed imaging was used to

characterize the bubble dynamics generated in ME by sonic or ultrasonic agitation within canals

of polymer tooth models. Second, 5.25% NaOCl irrigation or ME was sonically or ultrasonically

agitated in canals of extracted teeth with 7-day-grown Enterococcus faecalis biofilms. Dentinal

shavings from canal walls were sampled at 1 mm and 3 mm from the apical terminus, and

colony-forming units (CFU) enumerated. Mean log CFU/ml values were analyzed with ANOVA

and post-hoc tests. Results: High-speed imaging demonstrated strongly oscillating and

vaporizing bubbles generated within ME during ultrasonic, but not sonic agitation. Compared to

CFU counts in controls, NaOCl-sonic and NaOCl-ultrasonic yielded significantly lower (P <

0.05) counts at both measurement levels. ME-sonic yielded significantly lower (P = 0.002)

counts at 3 mm, while ME-ultrasonic yielded highly significantly lower (P = 0.000) counts at

both measurement levels. At 3 mm, ME-ultrasonic yielded significantly lower (P=0.000) CFU

counts than ME-sonic, NaOCl-sonic and NaOCl-ultrasonic. Conclusions: Enhanced bubble

dynamics and reduced E. faecalis biofilm bacteria beyond the level achieved by sonic or

ultrasonic agitation of NaOCl, suggested a synergistic effect of ME combined with ultrasonic

agitation.

Key Words: Irrigation dynamics, cavitation, acoustic microstreaming, microbubble

emulsion, ultrasonic, sonic, sodium hypochlorite.

29
 Introduction

Root canal irrigation is a crucial component of endodontic treatment (1, 2). Its physical

objectives are to generate flow of the irrigant throughout the entire root canal system, so as to

flush out all debris. Its chemical objectives are to inactivate intracanal bacterial biofilms and

endotoxins, and to dissolve tissue remnants and smear layer present on canal walls. Effective

delivery of the irrigation solutions is required to distribute and frequently replenish the optimal

concentration and amount of irrigant throughout the root canal system. Sodium hypochlorite

(NaOCl) is the principal endodontic irrigant used, for its excellent antibacterial property and its

ability to dissolve organic tissue (3). In spite of these favourable properties, conventional

irrigation delivery of NaOCl does not consistently achieve complete bacterial elimination from

root canal systems (4, 5).

The main limiting factors in conventional irrigation are the complexity of the root canal

anatomy, the ultra-structure of the dentin and the characteristics of the bacterial biofilms (3, 6,

7). Continuous attempts to surmount these limitations have recently led to the renewed interest in

the application of sonic and ultrasonic agitation of the irrigants within the root canal system (8).

Although several studies (9, 10) have reported that agitation enhanced the efficacy of NaOCl

irrigation, other studies (11, 12) have not corroborated these findings. The inconsistent outcomes

achieved with sonic or ultrasonic agitation of NaOCl might be attributed to the restricted

oscillation of the vibrating instruments within the confines of the root canal (13).

When sonic or ultrasonic agitation is applied to a liquid like an endodontic irrigant, it results in

the formation of cavitational bubbles that may exist in non-inertial and inertial forms. Non-

inertial cavitation bubbles undergo linear pulsation after being exposed to a low amplitude
30
vibration (13). Inertial cavitation bubbles undergo high-energy pulsations and their collapse

generates powerful shock waves capable of disrupting biofilms and directly killing bacteria (14).

The desirable inertial cavitation effect may not materialize within root canals, as the current

ultrasonic systems generate predominantly small non-cavitating bubbles that act mainly to

increase acoustic microstreaming and produce shear stresses along the root canal wall (15). The

limited bubble dynamics may not be sufficient to detach surface adherent bacteria or disrupt the

biofilm structures within the root canal (16). To overcome this limitation, it is reasonable to

assume that the combination of intensified bubble dynamics and increased chemical reactivity of

the irrigant, would enhance the antibiofilm efficacy of irrigation within root canals. One strategy

that may intensify both the cavitation bubbles and the chemical reactivity of the irrigant is use of

microbubbles in root canal irrigation.

Microbubbles, commonly employed as carriers for targeted drug delivery, are gas-filled bubbles

in the micron range produced in an emulsion. Inherently unstable, microbubbles require a

stabilizing shell of either surfactant, lipid, protein, polymer or a combination of the above. Most

of the microbubble unit volume consists of the gas core (17). When exposed to ultrasonic waves,

the microbubble gas core expands during the rarefaction phase of the pressure wave and

contracts during the compression phase generating inertial bubbles (17). These bubbles coalesce

with those generated by the ultrasonic agitation, decreasing the threshold for bubble production

and increasing bubble dynamics (18). Furthermore, including an oxygen carrier and an oxidizer

in the microbubble emulsion will generate reactive oxygen species (19). Considering the

properties of microbubbles, their combination with sonic or ultrasonic agitation could potentially

enhance bubble dynamics also having an antibacterial effect.

31
This in vitro study explored the combination of microbubbles with sonic or ultrasonic agitation

within root canal models, with two objectives: (1) to characterize the bubble dynamics generated

within a simulated root canal, and (2) to assess the effect on biofilm bacteria grown in canals of

extracted teeth.

Materials and Methods

Microbubbles-containing emulsion

All chemicals (analytical grades) and bacteriological media used in this study were purchased

from Sigma-Aldrich Inc. (St. Louis, MO) unless stated otherwise. A microbubbles-containing

emulsion (ME) was prepared using a combination of oxygen carrier (perfluoro-

decahydronaphthalene [PFC]), oxidizer (30% H2O2) and non-ionic detergent surfactant (triton-

X100, Bio-Rad Laboratories, Hercules, CA). The ratio of PFC:H2O2:triton-X100 in the prepared

ME was 75.0:24.5:0.5 (19).

Bubble dynamics characterization

Five polymer central incisor models (Advanced Endodontics, Santa Barbara, CA) were

decoronated. The mesial and distal aspects of each tooth model were ground flat with a slow-

speed diamond disc (Brasseler USA, Savannah, GA) and the surfaces polished with a diamond

polishing bur and pumice slurry, to enhance visualization of the root canals. Canals were shaped

with ProTaper Universal instruments (Dentsply Maillefer, Balleigues, Switzerland) to size F4

(400 m tip size) at the apical terminus, while maintaining apical patency with a #10 K-type file

(Dentsply Maillefer). Intermittent 5.25% NaOCl irrigation and final flush of 5 ml was applied,

followed by 1 ml of 17 % EDTA and sterile water to remove debris. The apical terminus and
32
lateral canals were occluded with rope wax (Moyco Technologies, inc, York, PA, and the tooth

models stabilized on a petri dish with All Purpose Stickyputty (Yiwu Changqing Toys Co., Ltd.,

Zhejiang, China). The stabilized tooth model was placed on the viewing table of an Olympus

IX71 inverted microscope (Olympus Inc., Center Valley, PA).

The EndoActivator (Dentsply Tulsa Dental Specialities, Tulsa, OK) or ultrasonic handpiece (P5

Newtron, Satelec, Bordeaux, France) was secured in a custom-made jig allowing X- and Y-axis

adjustments. This setup ensured accurate and reproducible positioning of the agitation tip inside

the root canal. The irrigant was delivered with a 27 G needle (ProRinse, Dentsply Tulsa Dental

Specialities), with the needle tip placed 2 mm short of the apical terminus. Agitation was applied

for 20 sec, with the medium sized EndoActivator tip or #10 K-type ultrasonic file, placed at a

distance of 1.5 or 4 mm from the apical terminus.

The pattern of bubble dynamics was recorded for 1 sec at the beginning and end of the 20 sec

agitation period. A charged-couple-device (CCD) camera (Fastcam SA 1.1 Photron, Motion

Engineering Company Inc., San Diego, CA) was used to acquire digital recordings at 125,000

frames/sec (20). Each recorded frame was analyzed separately for presence of oscillating

bubbles, their location, size and direction of movement within the root canal space. Four groups

were tested, 15 times each (Table 1).

Assessment of antibacterial activity

The Research Ethics Board of the University of Toronto approved the use of extracted human

teeth for this study. Forty single-rooted, freshly extracted human teeth were stored in phosphate

buffered saline solution (PBS) until use. To standardize the length of canals, teeth were

33
decoronated 12 mm from the root end and apical patency established with #10 K-type files.

Canals were shaped with ProTaper Universal instruments (Dentsply Maillefer) to size F4 at the

apical terminus, and apically enlarged with an ISO size #50 K-type file (Dentsply Maillefer) to

ensure consistent exchange of bacterial culture medium during biofilm development. Intermittent

5.25% NaOCl irrigation was applied with a 27 G ProRinse needle (Dentsply Tulsa Dental

Specialities) placed 1 mm short of the apical terminus, followed by 1 ml of 17 % EDTA and

sterile water to remove debris.

Shallow grooves were prepared with a diamond disc (Brasseler USA, Savannah, GA) along the

mesial and distal root surfaces to facilitate splitting of the teeth before sampling. These grooves

extended approximately 1.5 mm deep, and did not perforate the canal space. Roots were

externally coated with two layers of nail varnish and apical patency was again verified with a

size #50 K-type file. Subsequently, roots were placed in a falcon tube with BHI media and

autoclaved at 1200 C for 30 min. Canals were conditioned with 1% bovine serum albumin to aid

in initial bacterial colonization of the canal surface, and each root placed in an eppendorf tube

with 1 ml of sterile BHI.

The inoculum was prepared from a single colony of Enterococcus faecalis (ATCC 29212)

incubated in BHI broth for 24 hrs. The optical density of the culture was adjusted to 1 at 600 nm

(corresponding to 108 cells/mL) (21). Under aseptic conditions, the E. faecalis culture was

directly injected into the canal lumen, with the needle positioned at the apical terminus and

slowly withdrawn coronally up to the canal orifice. A sterile size #10 K-type file (Denstply

Maillefer) was then gently inserted into the canal to the apical terminus and retrieved to

uniformly coat the canal wall with the inoculum. The inoculated roots were each placed in a

34
sterile eppendorf tube containing 1 ml of fresh BHI broth and incubated in an orbital incubator

(120 rpm) at 37 C under aerobic conditions for 7 d, to grow a 7-day old E. faecalis biofilm

within the root canal (22). Fresh culture medium was gently injected into canals every other day,

to remove dead cells and to ensure proper growth of the bacterial biofilm. To verify the purity of

the grown biofilms, samples obtained from the canals of two randomly selected roots were plated

on chromocult E. faecalis agar. In addition, two additional inoculated roots were longitudinally

split and the canal walls observed under scanning electron microscope (JEOL, Tokyo) to verify

that the biofilm had the typical morphologic characteristics.

Six experimental one untreated control groups (n = 10) were established (Table 2). All

procedures and sampling were done under strict aseptic conditions within a biosafety cabinet.

Apices were occluded with wax and sealed with nail varnish to ensure a closed root canal system

for irrigation. In all experiments, the EndoActivator (Dentsply Tulsa Dental Specialities) was

used to generate sonic agitation with a medium size tip at full energy setting. The P5 Newtron

unit (Satelec) was used to generate ultrasonic agitation with a 21 mm endodontic K-type file at a

power setting of 10. Both the sonic and ultrasonic vibrating tips were inserted to 2 mm from the

apical terminus. Irrigation was delivered with ProRinse (Dentsply Tulsa Dental specialties)

irrigating needles at 2 mm from the apical terminus. A total of 3 ml of sterile water was

delivered, followed by 3 ml of the test solution and 1-minute agitation period. Finally, 3 ml of

either sterile water or sodium thiosulfate was used to deactivate NaOCl and ME. A total

irrigation volume of 9 ml/canal was used.

After completion of the experimental procedures, roots were split and canals exposed. Sterile #2

round burs mounted in an electric motor handpiece were used to collect root dentin samples from

35
canal walls at a depth of 0.5 mm. Four samples/canal were collected, two from each the buccal

and lingual walls, at a distance of 1 mm and 3 mm from the apical terminus. The dentinal

shavings were transferred to 1 ml of BHI in an eppendorf tube, and incubated in an orbital

incubator (120 rpm) at 37 C under aerobic conditions for 6 hrs to enrich the bacterial cells

before plating. The enriched samples were serially diluted and plated in triplicates on BHI agar

plates. Plates were then incubated for 12 hrs and the colony forming units (CFU) enumerated.

Data was analyzed with one-way analysis of variance (ANOVA) and post-hoc Tukey tests. All

tests were carried out with SPSS version 19.0 (SPSS, Chicago, IL). Significance was set at 0.05.

Results

Bubble dynamics

Sonic agitation of different irrigants did not produce distinct bubble dynamics at either 1.5 mm

or 4 mm from the apical terminus. In contrast, the ultrasonic agitation of sterile water and ME

generated bubbles on the lateral aspect of the file tip. The bubbles generated in ME were larger

than those generated in water (Fig. 1). These larger oscillating bubbles were also observed

laterally and away from the file tip, throughout the field of view. Strongly oscillating and

vaporizing bubbles (Fig. 1 B-D) that coalesced (Fig. 1 E-G) were observed at the end of the 20

sec agitation period (Fig. 1 H). These strongly oscillating bubbles occurred conspicuously less at

the file tip than on the lateral aspect of the file. Greater bubble dynamics was observed with

agitation at 4 mm than at 1.5 mm from the apical terminus.

36
Antibiofilm efficacy

Figs. 2 and 3 present the mean bacterial log CFU/ml values corresponding to the control and

experimental groups, for sonic and ultrasonic agitation, respectively. Compared to the control

group, sonic agitation of NaOCl yielded a significantly (P < 0.04) lower CFU count both at 1

mm and 3 mm from the apical terminus (Fig. 2). Sonic agitation of ME yielded a significantly (P

= 0.002) lower CFU count only at the 3 mm level (Fig. 2). Ultrasonic agitation of NaOCl also

yielded significantly lower CFU counts (P < 0.05), both at 1 mm and 3 mm levels, while

ultrasonic agitation of ME yielded a highly significantly (P = 0.000) lower CFU count, both at 1

mm and 3 mm levels (Fig. 3).

At 1 mm from the apical terminus, the CFU counts did not differ significantly for ultrasonic

agitation of ME and both sonic and ultrasonic agitation of NaOCl (P > 0.2). At the 3 mm level

(Fig. 4), the CFU count at the 3 mm level was significantly lower for ultrasonic agitation of ME

than for both sonic and ultrasonic agitation of NaOCl (P < 0.01).

Discussion

Characteristics of bubble dynamics and elimination of biofilm bacteria by sonically or

ultrasonically agitating ME were explored in vitro. For the former, the polymer tooth model was

developed; it proved to provide adequate visualization of the bubble dynamics generated within

the simulated root canal. For the latter, an extracted tooth model with a straight single canal and

a mono-species E. faecalis biofilm model were chosen in order to control different variables in

such analysis. A 7-day old biofilm was chosen based on previous experiments conducted in our

laboratory (23), that have confirmed the 7-day growth phase as optimal for developing

37
standardized E. faecalis biofilm models for testing the efficacy of antimicrobials (21, 22).

Developing a standardized biofilm in an in vitro root canal model allowed for the controlled

assessment and comparison of the tested antibiofilm strategies. Shen et al.(24) suggest that a

more mature biofilm, grown for 21 days, is less vulnerable to antimicrobials and, therefore,

better simulates the clinical scenario. However, even if the biofilm is grown for a longer period,

it still remains an in vitro scenario quite remote from the clinical one, while it may introduce

other biofilm-related factors that would jeopardize standardization, deemed paramount for the

current study. To approximate irrigation dynamics as close to a clinical application as possible,

canals were sealed apically to form a closed system (25).

ME was formulated in order to allow bubble production at a lower threshold of energy. The

inability of sonic agitation to generate bubble dynamics in combination with ME suggested a low

energy transfer to the irrigant by this device, below the threshold required. This finding

corroborates the report by Jiang et al. (26) of similar observations when using the EndoActivator

in a simulated glass root canal filled with water or NaOCl. Even without evident bubble

production, sonic agitation of ME effectively reduced viable bacteria at a level of 3 mm from the

apical terminus. This antibacterial activity could be due to the chemical effect of ME, whereby

the presence of PFC, an oxygen carrier, would increase the oxidizing potential of hydrogen

peroxide within ME (19). Nevertheless, sonic agitation of ME failed to effectively reduce viable

bacteria at the 1 mm level, suggesting a limited penetration of the irrigant to the proximity of the

apical terminus.

Ultrasonic agitation has been suggested to generate bubbles as long as the file can oscillate freely

within the root canal (13). In the present study, ultrasonic agitation in combination with ME

38
generated increased bubble dynamics characterized by larger, more strongly

oscillating/vaporizing bubbles compared to smaller bubbles produced in water. The intensified

bubble dynamics suggested that when ME was exposed to ultrasonic energy, the microbubbles

coalesced with other bubbles to form large bubbles. Consequently, the applied energy was

concentrated so that the threshold for the formation of bubbles was reduced (18).

Ultrasonic agitation combined with ME also effectively reduced viable bacteria in the root canals

at both 3 mm and 1 mm levels. At the 3 mm level, the antibacterial efficacy of agitated ME was

better than that of agitated NaOCl. This antibacterial activity might be due to the intensified

bubble dynamics as suggested above, as well as the oxidizing effect of ME. The intense bubble

dynamics could enhance apical penetration of ME while concomitantly generating a more

turbulent flow of irrigant along the root canal walls to the proximity of the apical terminus.

Furthermore, the presence of strongly oscillating and vaporizing bubbles might also affect direct

killing of bacteria, disruption of biofilms and increase in biofilm susceptibility to the chemical

effect of the ME.

In conclusion, within the limitations of this in vitro study its results suggested a synergistic effect

of ME combined with ultrasonic agitation, resulting in enhanced bubble dynamics and

antibiofilm efficacy. Because such synergistic strategy could offer potential advantages in root

canal disinfection, further research is warranted to investigate its effect on multispecies biofilms

in complex root canal configurations. The physical and chemical effects of ME on dentin and its

cytotoxicity also require investigation.

39
 Acknowledgements

The authors thank Zohra Aslamzada and Dr. Annie Shrestha for their valuable help in carrying

out experiments. The authors deny any conflict of interest related to this study.

40
References

1. Haapasalo M, Endal U, Zandi H, Coil JM. Eradication of endodontic infection by

instrumentation and irrigation solutions. Endodontic Topics. 2005;10(1):77-102.

2. Gulabivala K, Ng YL, Gilbertson M, Eames I. The fluid mechanics of root canal irrigation.
Physiol Meas. 2010 Dec;31(12):R49-84.

3. Leonardo MR, Tanomaru Filho M, Silva LA, Nelson Filho P, Bonifacio KC, Ito IY. In vivo
antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution. J Endod. 1999
Mar;25(3):167-71.

4. Bystrm A, Sundqvist G. The antibacterial action of sodium hypochlorite and EDTA in 60

cases of endodontic therapy. Int Endod J. 1985 Jan;18(1):35-40.

5. Siqueira JF, Machado AG, Silveira RM, Lopes HP, de Uzeda M. Evaluation of the
effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of
enterococcus faecalis from the root canal, in vitro. Int Endod J. 1997 Jul;30(4):279-82.

6. Love RM. The effect of tissue molecules on bacterial invasion of dentine. Oral Microbiol
Immunol. 2002 Feb;17(1):32-7.

7. Svensater G, Bergenholtz G. Biofilms in endodontic infections. Endodontic Topics.

2004;9:27-36.

8. Gu XH, Mao CY, Kern M. Effect of different irrigation on smear layer removal after post
space preparation. J Endod. 2009 Apr;35(4):583-6.

9. Carver K, Nusstein J, Reader A, Beck M. In vivo antibacterial efficacy of ultrasound after

hand and rotary instrumentation in human mandibular molars. J Endod. 2007 Sep;33(9):1038-43.

10. Jensen SA, Walker TL, Hutter JW, Nicoll BK. Comparison of the cleaning efficacy of
passive sonic activation and passive ultrasonic activation after hand instrumentation in molar
root canals. J Endod. 1999 11;25(11):735-8.

11. Bhuva B, Patel S, Wilson R, Niazi S, Beighton D, Mannocci F. The effectiveness of passive
ultrasonic irrigation on intraradicular enterococcus faecalis biofilms in extracted single-rooted
human teeth. Int Endod J. 2010 Mar;43(3):241-50.

12. Tardivo D, Pommel L, La Scola B, About I, Camps J. Antibacterial efficiency of passive

ultrasonic versus sonic irrigation. ultrasonic root canal irrigation. Odontostomatol Trop. 2010
Mar;33(129):29-35.

13. Roy RA, Ahmad M, Crum LA. Physical mechanisms governing the hydrodynamic response
of an oscillating ultrasonic file. Int Endod J. 1994 Jul;27(4):197-207.
41
14. Ohl SW, A Shrestha A, Khoo BC, A Kishen A. Chacterizing bubble dynamics created by
high intensity focused ultrasound for the delivery of antibacterial nanoparticles intodental hard
tissue. J Eng Med (in press).

15. Crum LA. Acoustic cavitation. Proceedings of the 1982 IEEE International Symposium on
Sonics and Ultrasonics, San Diego, CA. 1982:1-12.

16. Ahmad M, Pitt Ford TR, Crum LA, Walton AJ. Ultrasonic debridement of root canals:
Acoustic cavitation and its relevance. J Endod. 1988;14(10):486-93.

17. Sirsi S, Borden M. Microbubble compositions, properties and biomedical applications.

Bubble Sci Eng Technol. 2009 Nov;1(1-2):3-17.

18. Ferrara KW. Driving delivery vehicles with ultrasound. Adv Drug Deliv Rev. 2008 Jun
30;60(10):1097-102.

19. George S, Kishen A. Augmenting the antibiofilm efficacy of advanced noninvasive light
activated disinfection with emulsified oxidizer and oxygen carrier. J Endod. 2008 9;34(9):1119-
23.

20. Shrestha A, Fong SW, Khoo BC, Kishen A. Delivery of antibacterial nanoparticles into
dentinal tubules using high-intensity focused ultrasound. J Endod. 2009 Jul;35(7):1028-33.

21. Kishen A, Upadya M, Tegos GP, Hamblin MR. Efflux pump inhibitor potentiates
antimicrobial photodynamic inactivation of enterococcus faecalis biofilm. Photochem Photobiol.
2010 Nov-Dec;86(6):1343-9.

22. Shrestha A, Kishen A. Polycationic chitosan-conjugated photosensitizer for antibacterial

photodynamic therapy(dagger). Photochem Photobiol. 2011 Nov 2.

23. Shrestha A, Shi Z, Neoh KG, Kishen A. Nanoparticulates for antibiofilm treatment and effect
of aging on its antibacterial activity. J Endod. 2010 Jun;36(6):1030-5.

24. Shen Y, Stojicic S, Qian W, Olsen I, Haapasalo M. The synergistic antimicrobial effect by
mechanical agitation and two chlorhexidine preparations on biofilm bacteria. J Endod. 2010
Jan;36(1):100-4.

25. Tay FR, Gu LS, Schoeffel GJ, Wimmer C, Susin L, Zhang K, et al. Effect of vapor lock on
root canal debridement by using a side-vented needle for positive-pressure irrigant delivery. J
Endod. 2010 Apr;36(4):745-50.

26. Jiang L, Verhaagen B, Versluis M, van der Sluis LWM. Evaluation of a sonic device
designed to activate irrigant in the root canal. J Endod. 2010 1;36(1):143-6.

42
 Figure legends

Table 1.Experimental groups for stage-1for bubble characterization experiments.

Table 2. Experimental groups for stage-2 to evaluate the antibiofilm efficacy in root canal
models.

Figure 1. Typical images obtained from the samples of ultrasonic agitation with ME at a distance
of 4 mm from apex. The pictures proceed in direction of broken arrows and are labeled A
through H. These pictures illustrate strongly oscillating and vaporizing bubbles (B-D) as shown
by the arrowheads. These bubbles expand (E), collapse (F), and coalesce (G) while moving
within the field of view.

Figure 2. Comparison of the CFU/ ml for sonic (EndoActivator) groups compared to the control
group. Significance was set at P < 0.05 and is indicated by *

Figure 3. Comparison of the CFU/ ml for ultrasonic groups compared to the control group.
Significance of P < 0.05 and is indicated by * and P = 0.000 is indicated by **

Figure 4. Comparison of the CFU/ml at 3 mm level of ultrasonic and sonic agitation of ME

compared to ultrasonic and sonic agitation of NaOCl. Significance was P < 0.01 and is indicated
by *

43
Table 1.

Irrigant Agitation
Sterile water Ultrasonic (Set 10)
Sterile water Sonic
ME/Sterile water Ultrasonic (Set 10)
ME/Sterile water Sonic

Table 2.

Irrigation protocol Activation

none None
Water/Water/Water Ultrasonic (set 10)
Water/Water/Water Sonic
Water/Sodium hypochlorite/Sodium thiosulfate Ultrasonic (set 10)
Water/Sodium hypochlorite/Sodium thiosulfate Sonic
Water/ ME/Sodium thiosulfate Ultrasonic (set 10)
Water/ ME/Sodium thiosulfate Sonic

44
 A B C D
File tip

H G F E

Figure 1.

Figure 2.

45
Figure 3.

Figure 4.

46
IV. DISCUSSION

1. Methodology

The current in vitro experiment was carried out for two purposes: (1) to characterize the bubble

dynamics of ME when sonically or ultrasonically agitated and (2) to examine the ability of the

sonically or ultrasonically agitated ME to eliminate biofilm bacteria in root canal. The ME was

formulated based on the hypothesis that agitation of ME would allow bubble production at a

lower threshold of energy increasing the physical effects of the irrigants. The sonically or

ultrasonically agitated ME might interact with the bubbles generated by the ultrasonic/sonic

agitation to enhance the bubble dynamics within the root canal. The improved chemical effect

was attributed to the combination of the oxygen carrier and oxidizer in the ME, which resulted in

increased production of reactive oxygen species.

The majority of simulated root canal models used to investigate the effects of sonic and

ultrasonic agitation have been made of glass (107, 145, 149). The polymer tooth model chosen

in this study has similar hardness to that of dentin. This material also allowed the preparation of

clinically realistic tooth/root canal models. Furthermore, once polished the polymer was a

suitable material for visualization of the bubble dynamics/irrigation dynamics within the root

canal space.

An extracted tooth model with a single straight canal was chosen to investigate the simplest

clinical situation, where sonic or ultrasonic agitation would be applied. Controlling for variables

such as curved canals (152) or narrow canals, which could limit the effects of these devices (13),

47
allowed for the preliminary investigation of the antibacterial efficacy of the bubble dynamics

produced by the agitation of ME.

A single species E. faecalis biofilm model was chosen to control different variables in such

analysis. A 7-day old biofilm was chosen in this study since previous experiments from our

laboratory have confirmed this growth phase to be optimum to develop standardized E. faecalis

biofilm models to test the efficacy of antimicrobials (154, 155). Developing a standardized in

vitro biofilm in a root canal model would allow the assessment and comparison of different

antibiofilm strategies. A longer period of biofilm growth might introduce other biofilm related

factors that could influence the efficacy of antimicrobials, which was beyond the scope of this

study. Furthermore, a closed system was used in this study to approximate irrigation dynamics as

close to a clinical situation as possible (156). Nevertheless, the possibility of extrusion of ME

due to the increased bubble dynamics and possibility of cytotoxic sequelae on the periapical

tissue must still be evaluated.

Antibacterial efficacy was assessed by counting colony forming units. This is a commonly

performed method to quantify viable bacteria post treatment. The 6 hour inoculation of samples

before plating (enrichment phase) ensured there were optimal numbers of activated bacteria for

quantification by plating. Samples were plated on triplicate plates and in triplicate per plate to

ensure counts were valid (157).

Ultrasonic agitation was done at a setting of ten, in stage 1 of our pilot experiments. This setting

produced increased bubble production when compared to the setting of five. The high setting for

the sonic device was chosen by default as there was no bubble production observed at any

48
settings. The 60 second agitation period was based on Sabins (158) findings that there was no

difference in debris removal in the apical region with 30 or 60 seconds of PUI. The longer

agitation period was chosen to simulate a clinical situation (9, 135) and allowed longer sonic

agitation (158). Finally, file placement 2 mm from the working length (108, 113) was used to

evaluate the ability of increased bubble dynamics produced by the agitation of ME to improve

the apical penetration of the irrigant.

2. Results

The lack of bubble production with the sonic agitation of both the sterile water and ME was

attributed to the low energy transferred to the irrigants with this device. Jiang (107) found similar

observations when the agitation was done with the EndoActivator in a simulated glass root canal,

filled with either water or NaOCl. Although the components of the ME were chosen to promote

bubble production at lower energies, it was still not possible with this sonic device. Even with no

bubble production, sonic agitation of ME produced a significant reduction in viable bacteria at

the 3 mm level when compared to the control, possibly due to the chemical effect of the ME. The

presence of PFC, an oxygen carrier, would increase the oxidizing potential of hydrogen peroxide

in the ME resulting in an increase in reactive oxygen species being produced (103).

Nevertheless, sonic agitation of ME at the 1 mm level did not significantly reduce in viable

bacteria when compared to the control. The limited bacterial killing may be because of the lack

of penetration of irrigant, and the lack of bubble activation in the apical 1 mm of the root canal

space. Thus the antibacterial effects seen with sonic agitation of NaOCl were attributed to the

antibacterial properties of the irrigant (95). The chemical effect of ME was further supported by

the comparable results for NaOCl and ME with sonic agitation at the 1 and 3 mm levels.

49
The production of bubbles with ultrasonic agitation has been shown as long as the file oscillated

freely (145). The ultrasonic agitation of ME showed increased bubble dynamics and produced

strongly oscillating/vaporizing bubbles, when compared to the small bubbles produced in water.

When microbubbles in a medium were exposed to ultrasonic agitation, they coalesced with other

bubbles to form larger bubbles. Consequently, the applied energy was concentrated so that the

threshold for the formation of bubbles was reduced (18). The ultrasonic agitation of ME

produced a significant reduction in viable bacteria at both 3 mm and 1 mm levels. This reduction

in viable biofilm bacteria may be due to the increased bubble dynamics as shown by the stage 1

experiment, which enhanced the chemical and physical effects of the ME. The increased bubble

dynamics may have allowed apical penetration of ME and concomitantly generated a more

turbulent flow of irrigant along the root canal walls at both levels. Furthermore, the presence of

violently oscillating and vaporizing bubbles may directly kill bacteria, disrupt biofilms and/or

make biofilm bacteria more susceptible to the chemical effect of the ME.

Antibacterial effects after ultrasonic agitation of NaOCl at both levels were again attributed to

the antibacterial properties of the irrigant (95). The antibacterial properties of the ME were

further supported by the comparable counts of viable bacteria after the agitation of the ME when

compared to NaOCl. At the 3 mm level the ultrasonic agitation of the ME did have a significant

reduction in viable bacteria compared to sonic or ultrasonic agitation of NaOCl. This was

attributed to the synergistic effect of the increased bubble dynamics and the reactive oxygen

species produced during agitation.

50
3. Future directions
Further research is required to examine the effect of microbubble dynamics in more complex

root canal models. The influence of complexities in the root canal anatomy (ex: isthmuses), root

canal curvature and other factors that induce constraints on the agitation devices, on the bubble

dynamics should provide insight into the advantages of using ME in such clinical scenarios.

The effects of the ME on more mature and robust multispecies biofilms is also essential;

however it is important to realize that even these models will only be an in vitro approximation

of what is found in a clinical situation. Hence more clinically realistic in vivo models have to be

tested finally.

The use of confocal laser scanning microscopy combined with fluorescent stains would allow for

qualitative observations of the effects on biofilm structure and bacteria viability. Investigation

into the effects on smear layer on the efficacy of ME and the alteration to the mechanical

properties of dentin post-ME treatment should also be evaluated to determine potential impacts

on resistance to fracture and restorability.

The cytotoxicity of ME and its potential to extrude beyond the root apex must be determined to

ensure that any detrimental effects to host tissues are minimized.

51
V. CONCLUSIONS

Within the limitations of this in vitro study, ultrasonic agitation of ME resulted in improved

bubble dynamics (irrigation dynamics) and chemical effect (antibacterial effect), which resulted

in its significant antibiofilm efficacy. Further research is required to investigate the effect of ME

on multispecies biofilms in more complex root canal models. The physico-chemical effect of ME

on dentin and their cytotoxicity must also be examined. The increased bubble dynamics and

antibiofilm efficacy produced with the agitation of the ME will have potential advantages in

endodontic disinfection.

52
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hand and rotary instrumentation in human mandibular molars. J Endod. 2007 Sep;33(9):1038-43.

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passive sonic activation and passive ultrasonic activation after hand instrumentation in molar
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