An in vitro investigation into the antimicrobial activity of some endodontic medicaments and their bases using agar diffusion, biofilm testing and

medicament inactivation by dentine.

Basil Athanassiadis

BDSc – University of Queensland

A thesis presented for the degree of

Master of Science in Dentistry (by Research)

2007

School of Dentistry,

Faculty of Medicine and Dentistry,

University of Western Australia

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Acknowledgements I would like to express my sincere thanks and appreciation to those who have helped

me complete this thesis:

• Professor Paul V. Abbott for sparking my interest in root canal medicaments

by his lecture series in the late 1990’s. For his supervision, guidance, advice

and editing throughout this project.

• Professor Laurence J. Walsh for his enthusiasm, ideas, generosity of time,

support and editing throughout this project.

It has been an absolute pleasure and honour to have the two people above as my

supervisors.

Thanks also must go to the Royal Brisbane Hospital (RBH), in particular

• Associate Professor Narelle George for her assistance with the testing, advice,

encouragement and support over a three-year period in a diligent and

professional fashion.

• Professor Joan Faogali for encouraging alternate research at RBH, and the

support and advice given to me in establishing the microbiological protocols

for this project.

Finally, I wish to thank my wife Julie and my two boys Alex and Michael who were

supportive of me doing this research.

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DECLARATION I, Basil Athanassiadis, declare that the work presented in this collection of

manuscripts is, to the best of my knowledge and belief, original, except as otherwise

acknowledged. These manuscripts have not been submitted, either in whole or in

part, by me for any degree at this or any other University.

Basil Athanassiadis BDSc - University of Queensland

Witness – Professor Paul V. Abbott Head, School of Dentistry - University of Western Australia

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ABSTRACT

There is no question that micro-organisms are at the centre of the cause of pulpal

and periradicular pathoses. Antimicrobial agents have been used in endodontic

therapy for more than a century. It is well known that after instrumentation and

irrigation some type of further disinfection is required to enhance the chances for

treatment success; hence various types of medicaments have been used over the

last century.

Currently calcium hydroxide is the most commonly used medicament but others

include antibiotic/steroid combinations of which Ledermix™ paste is the most

popular. Although these ingredients are the active components of the medicaments,

their vehicles play an important role in determining the ionic dissociation of the active

components as well as the handling properties of the product.

This study used three different testing models to compare, by in vitro means, the

efficacy of a number of medicaments in terms of their antimicrobial spectrum,

medicament inactivation and ability to penetrate Enterococcus faecalis biofilms. Their

bases were also tested separately to establish what effect these components had.

The medicaments used in each test model were Ledermix™ paste, Pulpdent™

paste, Ultracal™ paste, and a 50:50 mixture of Ledermix and Pulpdent pastes, while

the bases tested included methyl cellulose and water, polyethylene glycol (PEG), and

PEG combined with zinc oxide, calcium chloride and the remainder of ingredients

which form the base for Ledermix paste.

Well diffusion assays were used to test the antimicrobial action of the above

commercial products and their bases. The zones of inhibition were recorded and

compared to assess the relative antimicrobial actions. Each medicament and base

was also applied to biofilms of E. faecalis generated on cellulose nitrate membrane

filters. Each of these biofilms were transferred to a saline solution which was seeded

onto agar plates and then the colony forming units were counted to determine the

number of bacterial cells destroyed.

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The final experiment investigated the antimicrobial effect of some current

medicaments and their bases on E. faecalis in the presence of dentine, and

evaluated the extent of inactivation of the antibacterial activity by dentine.

The results of the three studies showed that none of the bases had any major

antimicrobial effects against the bacteria tested. The combination of Ledermix paste

and Pulpdent paste produced the best antibacterial results in the agar diffusion

assay. Pulpdent and the Ledermix/Pulpdent combination produced the highest

percentage “kills” in E. faecalis biofilms, and this effect was also repeated whilst in

the presence of dentine.

Taken as a whole, the results of this series of studies suggest that of the

medicaments tested (Ledermix, Pulpdent, Ledermix/Pulpdent 50/50) the calcium

hydroxide containing medicaments have the greatest antimicrobial properties.

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TABLE OF CONTENTS

Page

TITLE PAGE 1

ACKNOWLEDGEMENTS 2

DECLARATION 3

ABSTRACT 4

TABLE OF CONTENTS 6

LIST OF TABLES and FIGURES 9 CHAPTER ONE: INTRODUCTION 10 CHAPTER TWO: LITERATURE REVIEW 12

Microbial Involvement 15 Microbial Invasion of Dentine 23 Medicament 28 Calcium Hydroxide 28 Antibiotics 32 Non-Phenolic Biocides 38 Phenolic Agents 43 Iodine Compounds 45 Medicament Vehicles 47 Biofilms 49 Antibiotic Resistance and Virulence of E. Faecalis 54 Conclusions 61 References 62

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INVESTIGATIVE PROTOCOL (Manuscripts prepared for submission to the International Endodontic Journal) CHAPTER THREE: AGAR DIFFUSION STUDY 86 Abstract Materials and Methods Results Discussion References Appendix CHAPTER FOUR: E. FAECALIS BIOFILM TESTING 109 Abstract Materials and Methods Results Discussion References Appendix CHAPTER FIVE: MEDICAMENT INACTIVATION STUDY 137 Abstract Materials and Methods Results Discussion References Appendix

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CHAPTER SIX: SUMMARY AND CONCLUSIONS 174 Summary/Conclusions Further Investigations Recommended 175

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LISTS OF TABLES and FIGURES Tables Page

2.1 Antibiotic Resistance in Enterococci 60 3.1 Mean (standard deviation) of zones of inhibition (mm) 104

and pH for each organism and preparation listed,

after 48 hours.

3.2 Mean dose responses for PEG 400/3350 and the 105

zone of inhibition (in mm) after 48 hours 4.1 Bacterial survival index of viable E. faecalis cells 123

for the various medicaments and their bases.

5.1 Bacterial survival index of E. faecalis against 153

medicaments/bases with pre-incubation (1hr)

and in the presence/absence of dentine.

(Dentine ground first then autoclaved)

5.2 Bacterial survival of E. faecalis against 154

medicaments/bases with pre-incubation (1 hr)

and in the presence/absence of dentine.

(Dentine autoclaved first then ground)

5.3 Mean pH measurements of preparations 155

Figures 4.1 Bacterial survival index of viable E. faecalis 124

cells for the various medicaments and their bases.

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CHAPTER ONE INTRODUCTION Bacteria play a central role in the development and progression of pulp and

periapical disease, as shown by Kakehashi et al. (1). Hence, endodontic treatment is

initially based on instrumentation, irrigation and medication of the root canal system

with the primary goal being to eliminate bacteria prior to placing a root canal filling.

Disinfection of the root canal system takes time, even with the strongest of

antimicrobial substances (2). Hence, the use of intracanal medicaments between

appointments is essential to reduce the number of viable bacteria within the root

canal system. Substantial periods of time are needed to allow medicaments to

penetrate to those regions where bacteria are found, such as the dentinal tubules,

any fins, transverse anastomoses, lateral canals and accessory canals. Moreover,

bacteria may be found not only in planktonic forms but as adherent well-organized

biofilms which are inherently more resistant to antimicrobial agents and may require

extended contact times for effective antimicrobial activity.

Since bacteria play a primary role in the initiation and progression of endodontic

diseases, their persistence within the root canal system is linked fundamentally to

poor clinical outcomes. Factors which may contribute to treatment failure include the

selection and dominance of a few microbial species, and the inhibitory effect of

dentine on certain root canal medicaments, particularly calcium hydroxide. Since E.

faecalis is reported to be the dominant microbe in infected canals in previously root-

filled teeth, it is a key species to consider in terms of its virulence and antibiotic

resistance. E. faecalis will also be one of the main microbes used in the three assay

systems that will be presented in this thesis.

The use of in vitro tests should provide insight into the relative benefits of the

currently available medicaments used in clinical practice, and should assist not only

in the decision making processes employed by clinicians but also in the further

evolution of root canal medicaments.

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References

1. Kakehashi S, Stanley HR, Fitzgerald RJ, Bethesda BS. The effects of surgical

exposures of dental pulps in germ-free and conventional laboratory rats. Oral

Surg Oral Med Oral Pathol 1965;20:340-9.

2. Orstavik D. Root canal disinfection: a review of concepts and recent

developments. Aust Endod J 2003;29:70-4.

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CHAPTER TWO LITERATURE REVIEW

The use of calcium hydroxide, antibiotics, biocides and phenol compounds as antimicrobial agents in endodontics

INTRODUCTION

The outcome of endodontic treatment is largely dependent on the effectiveness of

the control or elimination of micro-organisms from the root canal system. This can be

achieved with mechanical cleaning processes and through the use of antimicrobial

agents. Typically the antimicrobial agents can be used during two different stages of

endodontic treatment - firstly, as a chemically active irrigating solution during the

mechanical instrumentation phase of root canal treatment and, secondly, as an

antiseptic medication within the root canal system between appointments (1). This

review will focus on the use of inter-appointment antiseptic medications (commonly

referred to as “medicaments”).

The three most common situations requiring endodontic treatment are as follows (1-

4):

• the uninfected pulp (irreversible pulpitis, iatrogenic pulp exposures, trauma,

elective pulp therapy for mechanical or restorative dentistry reasons),

• infected root canal systems with or without apical periodontitis (and without

any previous endodontic treatment) where bacterial distribution is variable

within the main canal, dentine and lateral canals, and

• infected canals in previously root-filled teeth that have apical periodontitis,

including cases where the location of bacteria may reflect the quality of the

root filling.

Bacteria play a central role in the development and progression of pulp and

periapical diseases as shown by Kakehashi et al. (5), Möller et al. (6), Dahlén et al.

(7), and Fabricius et al. (8) These authors have also shown that root canal infections

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cause periapical inflammatory responses which manifest as periapical radiolucencies

on radiographs (6, 8). Several studies have shown that the microflora in teeth with

root canal fillings that have become infected again and have apical periodontitis (i.e.

they have a periapical radiolucency) differ from initial pulp necrosis with infection

(9,10). Molecular methods have demonstrated that bacteria can be detected in

virtually all cases of root-filled teeth with periapical radiolucencies (11).

Bacteria can exist within the root canal itself, or within other related regions such as

dentinal tubules, accessory canals, canal ramifications, apical deltas, fins, and

transverse anastomoses (12). Bacteria may also colonise cemental defects and

extruded root canal filling materials. Apart from the canal itself, all of these other

areas are inaccessible to endodontic mechanical instrumentation procedures and the

irrigating solutions used during endodontic treatment. The antimicrobial agents used

as inter-appointment medicaments must therefore be able to penetrate through the

hard and soft tissues in the presence of microbes to reach a sufficiently high

concentration in order to eliminate the disease-causing bacteria in a predictable

manner (13-15).

In order to predictably eliminate as many bacteria as possible from the entire root

canal system, a combination of mechanical instrumentation and irrigating solutions is

used to remove or dissolve organic and inorganic debris, to destroy bacteria, to

remove the smear layer and to increase or maintain dentine permeability (12). It is

essential to maintain (or increase) the permeability of dentine at its pre-operative

level as this will allow the active ingredients of any inter-appointment medicaments to

diffuse through the dentine and disinfect the inaccessible areas of the root canal

system such as the dentinal tubules, fins, transverse anastomoses, etc. This is

important since studies have shown that mechanical instrumentation with

antibacterial irrigation will only render 50%-70% of infected canals free of micro-

organisms, depending on which irrigants are used, while the remaining canals still

contain viable bacteria (6,16,17). Since there is no predictable way in one treatment

session to ensure complete elimination of root canal bacteria, an effective

antimicrobial agent in the root canal is required for a predetermined time period to

predictably eradicate or destroy any remaining bacteria (18,19).

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Medicaments are used as an aid to improve the predictability and prognosis of

endodontic treatment (12). They can also be used to reduce inflammation and hence

improve patient comfort following treatment. They play a very important adjunctive

antibacterial role in endodontic therapy. In stating this, it must be recognized that

other procedures performed during endodontic treatment help to prevent further

bacterial penetration both during and after endodontic treatment, namely removal of

the cause(s) of the diseases (such as any existing restorations, cracks and caries),

thorough mechanical instrumentation of the canals, irrigation, and the provision of a

restoration that prevents any further ingress of bacteria into the tooth.

In summary, medicaments are used in endodontic therapy in order to (12, 20):

• eliminate or destroy any remaining viable bacteria in the tooth and in the root

canal system that have not been destroyed by the chemo-mechanical

preparation processes (i.e. instrumentation and irrigation),

• reduce peri-radicular inflammation and hence reduce pain,

• help eliminate apical exudate if it is present,

• prevent or arrest inflammatory root resorption if it is present, and

• prevent re-infection of the root canal system by acting as both a chemical and

a physical barrier if the temporary restoration breaks down (13).

Inter-appointment intra-canal medication has been unequivocally shown to contribute

to a favourable outcome in treating endodontic infections in infected cases (21-27).

The need for intracanal medication is greater in those cases where bacteria are

resistant to routine treatment and where the therapy cannot be successfully

completed due to the presence of pain or continuing exudate (28). The inter-

appointment period is an ideal opportunity to influence peri-radicular tissue reactions

prior to the final filling of the root canal system.

Some endodontic conditions are ideally treated over several appointments which may

be extended over a long period of time. This allows various medicaments to be used

depending on the status of the pulp, the periapical tissues, the hard dental tissues

(such as cementum) and the condition of the apical foramen (i.e. “open” or fully

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developed and unaffected by resorption) (12). The minimum inter-appointment time

interval should be no less than 14 days, as it takes at least 10-14 days for

inflammation to subside or heal (29) but longer periods are generally more desirable

as most medicaments take 3-4 weeks to reach their maximum concentration within

the peripheral dentine (30). In addition, if signs or symptoms are not subsiding, then a

longer period of medication time may be necessary.

Antimicrobial intracanal medicaments have the potential to prevent post-operative

pain caused by persistent intra-radicular micro-organisms or by secondary microbial

invaders, provided they are not highly cytotoxic and are not extruded in significant

amounts into the periradicular tissues. However, intracanal medicaments are highly

unlikely to be effective in preventing “flare-ups” caused by extruded micro-organisms

during the chemomechanical processes (31) since the medicaments will not be

present in the periapical tissues at an effective therapeutic concentration.

Although a technically “good” root canal filling may entomb many micro-organisms

remaining in the root canal system, root canal fillings do not provide a complete “seal”

of root canals. Hence, if defects in the root canal filling exist that communicate with

the periapical tissues, tissue fluid may enter the voids and support bacterial growth.

Subsequently, bacterially-derived inflammatory mediators such as endotoxins will be

produced and will then be released into the periapical tissues, thereby initiating or

maintaining an inflammatory reaction in these tissues. This is the primary reason for

the use of antimicrobial agents in endodontic therapy (1).

MICROBIAL INVOLVEMENT

The oral cavity contains one of the most concentrated accumulations of micro-

organisms in the human body with more than 700 different species having been

detected so far. Almost all bacteria recovered from the root canal systems of infected

teeth belong to the oral microflora (30). However, it is estimated that some 50% of

species in the oral microflora can not be cultured or have not yet been cultivated.

Consequently, concepts of endodontic infections and the associated microflora may

need to be re-defined over time as more species become identified through more

refined research techniques (11, 32).

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Bacteria can be found in both planktonic form and in biofilm communities. The

configuration will affect the efficacy of antimicrobial agents with biofilms being much

more resistant because of their diffusion barriers and altered bacterial cell

metabolism and replication rates (33). It has been shown that some of the root canal

walls often remain untouched during chemomechanical preparation of the canals

regardless of the instrumentation technique and instruments used (34). Endodontic

treatment failure that can be attributed to micro-organisms remaining in the canal

may occur if the micro-organisms possess pathogenicity, reach sufficient numbers,

and gain access to the periradicular tissues to induce or maintain periradicular

inflammation (35). Bacteria remaining in the untouched areas within the root canal

system are recognized as the main cause of persistent infections after root canal

therapy (35, 36) and they can induce inflammatory responses in the periradicular

tissues (11).

Studies using bacterial cultivation methods have shown that infected necrotic pulps

and pulpless, infected teeth (i.e. teeth without any previous endodontic treatment,

also termed a primary endodontic infection) present with a polymicrobial flora

characterized by a wide variety of combinations of bacteria. They have an average of

between four and seven species per canal and there is a predominance of strict

anaerobic bacteria with approximately equal proportions of Gram-negative and

Gram-positive organisms (16, 37). In contrast, previously root-filled teeth with apical

periodontitis are characterized by one or two species per canal, and the microflora is

dominated by facultative anaerobes and Gram-positive bacteria (9, 10, 37), such as

E. faecalis.

If molecular methods are used to analyse the root canal microflora, the reported

microbial flora is more complex than that revealed by cultivation methods alone (11,

32). This reflects the fastidious growth requirements of some species such as strict

anaerobes which make their recovery and growth in culture very unlikely. It also

needs to be recognised that micro-organisms which resist the action of endodontic

treatment can exist in low numbers in the root canal or can be located in areas which

are difficult to access when using paper points for sampling.

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The polymerase chain reaction (PCR) method for identifying bacteria is arguably the

most sensitive method for microbial species or strain identification. It can provide

results that are more accurate and reliable in terms of the prevalence of various

microbes in root-filled teeth and it is less prone to interference - e.g., remnants of

medicaments can be gathered on the same paper point as the bacteria, which may

inhibit their subsequent growth (35, 38). According to Kaufman et al. (39), molecular

studies cannot be directly compared because of technical factors which influence the

PCR assay, such as using different oligonucleotide primers and different reaction

conditions (which may be sub-optimal).

The higher sensitivity of the quantitative PCR method, in part, could be attributed to

the fact that it potentially targets free floating DNA as well as the DNA from non-

viable, culturable viable cells, and viable but non-culturable (VBNC) cells. This

technique identifies the presence of specific DNA sequences in the samples and

hence the number of intact and viable micro-organisms is not known. Moreover the

remaining micro-organisms are destroyed. These are not culturable and it is not

possible to study the phenotype characteristics and pathogenicity associated with

individual strains recovered from root canals. The clinical significance of the

remaining non-viable cells or DNA fragments in root canals is not known. Therefore a

combination of both molecular and culture techniques will be required to provide a

comprehensive understanding of the role of E. faecalis in the root canal infection

process (40).

In studies using molecular methods to examine root-filled teeth, the mean number of

species in “adequately treated” cases was three (range 1-5) while cases designated

as “poorly treated” yielded a mean of five species (range 2-11) (11, 35). In another

study of root-filled teeth (41), a mean number of 5.2 species was present when the

tooth had a sound coronal restoration, but a mean of 8.6 species was present in

canals of teeth with defective coronal restorations. Based on studies using molecular

techniques, a reasonable estimate at present is that infected root canals contain

between 10 to 30 bacterial species and occasionally up to 50 species which

coincides well with the number of bacterial species found in dental plaque (11). The

microbiota of infected root canals also appears to be very similar to the flora of

periodontal pockets in teeth with active periodontal disease (11, 32). Thus it is

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conceivable that subgingival dental plaque is the origin of the bacterial species found

in the root canal system (42).

Once the pulp becomes necrotic, only a restricted subset of species can colonize it

(43). Using cultivation methods, the bacterial genera most frequent isolated from

infected root canals are Peptostreptococcus, Fusobacterium, Prevotella,

Porphyromonas, Eubacterium, Actinomyces, Bacteroides and facultative

Streptococcus spp (37,43,44,45). When molecular methods (i.e. PCR, checkerboard)

are used, the most prevalent species are T. denticola (68%), P. endodontalis (61%),

T. forsythia (58%), P. alactolysis (56%), D. pneumosintes (55%), F. alocis (46%), and

P. gingivalis (45%). It is interesting to note that T. forsythia and T. denticola have not

been identified using cultivation methods, which reflects their fastidious requirements

for growth (11).

Samples from root-filled teeth have frequently shown Gram-positive facultative

organisms including Enterococci, Streptococci and Lactobacilli (9,10, 46, 47). These

findings support the concept that endodontic treatment procedures apply a selective

pressure so that more resilient organisms survive whereas the more susceptible

Gram-negative anaerobes are readily eliminated (46). E. faecalis is occasionally

found in primary (previously untreated) root canal infections with its prevalence

varying from 3.2%-13.3% (37, 43, 48). It is, however, much more frequently

associated with asymptomatic primary endodontic infections than with symptomatic

ones (49). When E. faecalis is present in low numbers initially, it can usually be

eliminated but once established in the root canal system in higher numbers it is a

difficult organism to eradicate (10).

Culture-based studies have shown that the microbial flora in previously root filled

teeth with periapical lesions are predominately Gram-positive micro-organisms, with

approximately equal proportions of facultative and obligate anaerobes (10, 47, 50).

The most frequently isolated bacterial species are E. faecalis, Streptococcus,

Peptostreptococcus, and Actinomyces, with E. faecalis being the most frequent out of

this group. In the studies of Pinheiro et al. (47, 50), some 15-20% of the canals had

no bacteria that could be recovered. Of the total microbial species, 80-83% were

Gram-positive, 58% were facultative anaerobes, and 42% were obligate anaerobes.

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Teeth with poor quality root-canal fillings were more likely to have a flora similar to

that found in untreated canals (i.e. polymicrobial) than teeth with apparently well filled

canals (10, 50).

In culture-based studies, the presence of E. faecalis has been reported by Molander

et al. (9), Sundqvist et al. (10), Pinheiro et al. (47, 50), and Peciuliene et al. (51) in

78%, 38%, 45.8%, 52.94% and 64% (respectively) of previously root-filled canals

with positive cultures. It is noteworthy that in these studies E. faecalis was the

dominate or the only isolate in 19 of 21 canals (51), 18 of 27 canals (50) and 6 of 19

canals (37). Four molecular studies have found that E. faecalis is the most commonly

found species in root-filled teeth with an associated apical periodontitis (11, 35, 38,

49) ranging from 64-77% while two recent studies (39,52) have reported a lower

incidence varying from 12.1% up to 22%. After considering all the data from culture-

based and molecular studies, it is clear that E. faecalis may be encountered in root-

filled teeth with periradicular lesions where there are recoverable micro-organisms in

the root canal with the prevalence ranging from 12% to 70% (35).

The dominance of E. faecalis is highly relevant to questions regarding the use of

inter-appointment medicaments. Several studies have shown that obligate anaerobes

are more easily eliminated by endodontic procedures than facultative micro-

organisms as the latter are more likely to be resistant to antimicrobial agents,

mechanical endodontic procedures and antibiotics (6, 21). The supposedly higher

resistance of Gram-positive bacteria may be related to their much more robust cell

wall structure, the metabolic products they secrete and their resistance to

medications (46).

The presence of the Gram-positive facultative organism E. faecalis may also be

relevant to the emergence of Gram-negative strict anaerobes. Several studies (6, 44,

53) have shown that the presence of facultative anaerobic bacteria is important for

the persistence of obligate anaerobes since, by reducing the oxidation-reduction

potential, the facultative anaerobic bacteria create an environment that is suitable for

the growth and survival of strict anaerobes. In order words, a synergistic effect may

be operating between these two groups of organisms.

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Facultative anaerobes are less susceptible to antimicrobial agents and therefore they

can be expected to persist more frequently in the root canal system following

inadequate treatment procedures (46, 53). Facultative anaerobes, especially some

Gram-positive species, can survive in a quiescent phase with low metabolic activity

for a period of time until nutritional conditions improve which then permit bacterial

growth (9,10). An example of such a change in nutritional conditions would be when

breakdown of the coronal restoration occurs– either during or after root canal

treatment.

Bacteria vary in their pH tolerance, and most species grow well within a pH range of

6-9. However, Enterococci are able to tolerate low and high pH values (ranging from

4.8-11) while fungi can tolerate pH ranges of 5-9 (13, 54). Bacterial tolerance to pH

changes may be due to several factors such as activation of specific proton pumps,

one or more specific enzymatic systems and/or internal buffering systems which help

to maintain their internal pH at a constant and approximately neutral level (13). This

phenomenon has been demonstrated by Molander et al. (9) who found that the use

of intracanal medicaments directed specifically towards the anaerobic microflora

provided a suitable environment for growth of Enterococcus species.

Microbiological investigations have shown that yeasts may be present in the

microflora of teeth with apical periodontitis (2, 9, 55). Yeasts have been found in

cases of persistent root canal infections (with apical periodontitis) in as few as 5% of

cases and up to as many as 18% of cases (2, 51). In a study by Waltimo et al. (2),

13% of cases had yeasts present in pure cultures. This suggests that the ecological

conditions in the necrotic root canal may allow yeasts to grow in pure culture without

the presence of bacteria. Moreover the isolation of pure cultures of yeasts from root

canal infections may indicate that they can contribute to the pathogenesis of apical

periodontitis. In the study by Waltimo et al. (2), yeasts were present together with

streptococcal species in 66% of the cases (2). The predominant yeasts were

Candida, with C. albicans being the most frequent and the most virulent (2). In

another study C. albicans was not isolated in pure culture but was found in 18% of

teeth with positive bacterial cultures (51). In 50% of the cases, it was present with E.

faecalis and in the remaining 50% it was present with other bacteria. C. albicans and

other Candida species have been shown to be even more resistant to calcium

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hydroxide in vitro than E. faecalis (56). C. albicans appears able to utilize

opportunities created by the removal of other microbes, and can grow in the low–

nutrient environment of the endodontically-treated canal (10).

Extra-radicular infections can be fostered by intra-radicular infections where some

oral bacteria have the potential to develop strategies to survive in inflamed

periradicular tissues and form biofilms. Alternatively, they may develop independent

of intra-radicular infections due to the presence of draining sinuses, periapical cysts,

foreign body reactions, scar tissue and cholesterol crystals situated outside the root

canal system (9,11, 57). Few oral micro-organisms have the ability to overcome host

defence mechanisms and thereby induce an extra-radicular infection but it is

currently recognized that some Actinomyces species and Propionibacterium

propionicum may be implicated in extra-radicular infections (27, 43, 57, 58).

Even though Actinomyces organisms were reported in 10-15% of cases of infected

root canals with periradicular disease, only 2.5% of these cases developed

periradicular actinomycosis (43, 59). The number of extra-radicular micro-organisms

present is small and not a common occurrence in untreated teeth, as shown by

Siquiera and Lopes (60) who demonstrated that only one out of 27 root tips from

untreated teeth with periradicular lesions had micro-organisms on the external root

surface. Actinomyces strains have fimbrial structures which enable them to adhere to

the root canal wall or to dentinal debris forced out through the apical foramen during

endodontic therapy. Such adhesive structures may also allow them to cling to other

bacteria or host cells as they advance into the periradicular tissues.

Actinomyces species are known to have the ability to thrive in tissues for long periods

of time without causing undue discomfort to the host. The low pathogenicity of these

micro-organisms and the consequent minimal host response may be contributing

factors in the perpetuation of chronic periradicular inflammation (43). Importantly,

extra-radicular bacteria can not be destroyed by conventional root canal irrigants or

antimicrobial dressings. If micro-organisms on the external surface of the root persist,

then the inflammatory periapical processes continue and healing does not occur.

Therefore, at present, apical surgery is the treatment of choice to resolve these

refractory cases (61).

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The conversion of an asymptomatic periradicular lesion to a symptomatic one

suggests that there has been a shift in the structure of the microbial flora which is

probably due to the arrival of a new pathogenic species or a variation in the number

and combinations of existing species (62). Clinical findings and symptoms such as

pain, history of previous pain, tenderness to percussion and swelling have been

associated with Gram-negative species, especially Prevotella, Porphyromonas spp

and fusobacteria (37, 63). The black-pigmented species (P. gingivalis, P.

endodontalis, P. intermedia and P. nigrescens) are found at higher frequency in teeth

with necrotic pulps or pulpless canals than in teeth with a previous root canal filling

that has apical periodontitis (63). Pulpless, infected teeth with radiographically visible

periapical radiolucencies show a predominance of anaerobic Gram-negative micro-

organisms and these produce by-products that are injurious to the periapical tissues,

such as proteases. They also have lipopolysaccharides (LPS) within their cell walls

which are released during duplication and on bacterial cell death. These can

stimulate production of bradykinin, a potent pain mediator (37), and trigger production

of inflammatory cytokines. LPS can also adhere irreversibly to mineralised tissue.

Gram-positive bacteria do not contain or release LPS. However their cell walls

contain muramyl dipeptide (MDP), which has a similar action on monocytes as LPS.

Although weaker than LPS, MDP may induce bone resorption (64). Because of the

effects of LPS and MDP, the aim of root canal treatment for infected teeth with apical

periodontitis is not only to eliminate micro-organisms and their substrates but also to

inactivate the toxic effects of LPS and other bacterial by-products so the periapical

tissues can heal (65) and bone resorption is inhibited.

In a recent study by Sakamoto et al. (66) the microbiota of asymptomatic and

symptomatic endodontic infections were compared. Molecular analysis revealed that

about one-half of the organisms detected had not been cultivated previously and the

number of bacteria associated with symptomatic samples was significantly greater

than those with no symptoms. Black-pigmented anaerobic bacteria have been

associated with symptomatic endodontic infections but recent molecular data failed to

show such an association. Prevotella was one of the most prevalent bacterial species

in this study but only P. intermedia was suspected to be associated with symptoms

Page 23

with Porphyromonas endodontalis detected in only two cases. There were several

species in this study that were detected exclusively in symptomatic cases - these

were Dialister pneumosintes, F. nucleatum, P. intermedia, and Prevotella species.

The prevalence of some species of black-pigmented bacteria (BPB) in infected root

canals have been underestimated by culturing techniques due to their fastidious

growth requirements (67). Hence multiplex PCR was used in a study (67) to

determine the presence of five BPB in infected root canals compared with

conventional culture procedures. The bacteria included Porphyromonas

endodontalis, P. gingivalis, Prevotella intermedia, P. nigrescens, and P. tannerae.

PCR showed at least one of the five BPB in 65% of the cases. P. intermedia was

found in 45%, P. endodontalis in 35%, P. gingivalis in 22.5%, P. nigrescens in 17.5%,

and P. tannerae in 5% of the samples. In contrast, culture techniques detected one of

the five BPB in 15% of the samples with P. intermedia found in 10% and P. gingivalis

in 5% of samples.

In a study by Sedgley et al. (68), E. faecalis was detected in more tongue samples

(42%) than gingival sulcus (14%), oral rinse (10%), and root canal samples (10%)

and there was a higher prevalence of E. faecalis in patients with unhealthy compared

to healthy periodontium (73% and 20% respectively). Hence further study is needed

to determine whether a relationship exists between the prevalence of E. faecalis in

healthy and unhealthy periodontium and whether oral E. faecalis harboured in the

tongue plaque biofilm is the main source of infection and pathogenicity in root canals,

perhaps due to the non-use or poor use of rubber dam during endodontic treatment.

The long-standing popular notion of entombment and perishing of intra-radicular

microbes following treatment still awaits scientific validity (69, 70). The presence of

micro-organisms inside a root canal may not necessarily lead to the failure of

treatment but their absence will certainly favour healing (48).

MICROBIAL INVASION OF DENTINE

Microbial invasion of the root canal system is time related and bacterial species

dependent. Hence, early endodontic treatment of a tooth should minimize the

Page 24

number of micro-organisms lodged in the dentinal tubules (71). The great majority of

micro-organisms in apical periodontitis are located in the main part of the root canal

system. As already discussed, bacteria do not usually proceed through the apical

foramen, although some species may also be found in the periapical tissues (19).

Micro-organisms in dentinal tubules may constitute a reservoir from which root canal

and surrounding tissue infection and reinfection may occur. Bacteria located inside

dentinal tubules are protected from the action of host defence cells (such as

neutrophils), systemic antibiotics and chemo-mechanical preparation. Therefore,

endodontic medicaments must be able to penetrate into dentinal tubules and kill

bacteria within them (13).

When bacteria invade the dentinal tubules, not all tubules will have been invaded to

the same extent (72). On the contrary, both in vitro and in vivo observations show

that bacterial penetration into dentinal tubules occurs as a random process, with

bacteria colonies seen as sporadic, dense accumulations of cells (rather than as a

continuous film), extending out from the main canal lumen towards the periphery

(22). The reported frequency of dentinal tubule invasion in necrotic teeth varies

between 50% and 90% (22).

Intact cementum is crucial for the limitation of the bacterial invasion of radicular

dentinal tubules from the root canal end of infected canals. Bacterial penetration is

enhanced when the overlying cementum has been removed (e.g. by periodontal

debridement) or resorbed as occurs with apical inflammatory conditions and

traumatic injuries that damage the periradicular tissues (e.g. inflammatory resorption)

(22, 72-74). If cementum is absent from the root surface, bacteria are able to colonize

the tubules from the periodontal aspect. However given the smaller tubule diameter

at the periphery, the ability to penetrate and the speed of penetration from the

periodontal side is less than from the root canal end (42,73). In addition, sclerosis of

the dentinal tubules is more advanced in the apical region than in the mid-root and

coronal dentine. Hence, chemo-mechanical preparation of root canals with minimal

apical enlargement and coronal flaring can effectively reduce the bulk of bacteria.

When this is combined with an effective inter-appointment root canal medication, the

number of bacteria surviving in the radicular dentine should be minimised.

Page 25

Dentinal tubule fluid contains albumin, IgG and fibrinogen which are involved in the

host defence reaction by inhibiting bacterial invasion of the radicular dentinal tubules

and by reducing the permeability of the dentine (74). However, in infected cases only

a few species are able to invade the coronal dentine and infect the root canal system

and subsequently invade radicular dentinal tubules. Tubule invasion and the

development of the inert tubular bacterial flora follow that seen in the formation of

plaque biofilms - that is, initial attachment and colonization by primary streptococcal

colonizers which then allow colonization by other “late” colonizers (74).

Bacterial penetration into radicular dentine is only evident in the presence of pulp

necrosis. The predentine is readily infected but the calcified dentine is less readily

infected. Bacterial species that penetrate the dentine are dominated by Gram-positive

rods (68%) and cocci (27%). The predominant genera are Lactobacillus (30%),

Streptococcus (13%), and Propionibacterium (9%) (3). The presence of Gram-

negative bacteria in root canal dentine has been confirmed indirectly by the detection

of high concentrations of lipopolysaccharide (LPS) in the inner layers up to 300 μm in

depth (3).

Invasion of the radicular dentinal tubules is a multifactorial event for which only a

limited number of oral bacterial species have the necessary properties (74). Bacteria

that penetrate dentine to a shallow depth may be removed by the mechanical

preparation of the root canal. In this regard, it is of note that 50 μm is the common

difference in diameters between two sequential stainless steel ISO root canal files. In

addition, bacteria may be destroyed by the proteolytic action of irrigant solutions such

as sodium hypochlorite. The pattern of invasion would favour a root canal preparation

with minimal apical preparation due to the higher amount of sclerotic apical dentine in

adult humans which prevents the bacterial penetration of tubules and may help to

explain the high success rate of endodontic therapy. The ideal canal preparation

should also flare coronally so as to effectively debride the heavily-infected mid-root

and cervical third dentine (72, 75).

Many hand and rotary instrumentation techniques tend to produce round

preparations (especially in oval canals) leaving uninstrumented and hence possibly

infected dentine in these areas. Approximately 50% of canal walls remains

Page 26

uninstrumented during preparation hence necrotic tissue remnants may provide a

source of nutrition for the surviving bacteria. (76, 77). No current technique is able to

remove the entire inner layer of infected dentine from a root canal, thus bacteria are

likely to remain in dentinal tubules after instrumentation – which in turn may result in

calcium hydroxide and other disinfectants that require direct contact with pathogens,

being ineffective (69).

In one study (71), it was reported that E. faecalis did not invade the dentine tubules

until after two weeks of incubation when cementum was intact. By three weeks, the

organisms had penetrated more than half way through the dentine in the cervical

third of the root, up to half way through the dentine in the middle third, and only a

third of the distance through the dentine in the apical third of the root. E. faecalis has

been shown to penetrate between 50-300 μm in human dentine (78, 79) although in

bovine dentine it has been shown to penetrate the entire width of circumpulpal

dentine after only 2 days of incubation (24).

With regard to the penetration by other species, in a study by Love (72),

Streptococcus gordoni gave a heavy infestation in the cervical and mid-root areas to

a depth of 200 μm but there were only limited numbers in the apical dentine to a

depth of 60 μm. Torabinejad et al. (80) showed in an in vitro study that bacteria could

infiltrate the root canal system between the dentine wall and the root filling material

after only three days when the coronal surface was not sealed. The bacteria reached

the apical area after 24 days. In a study by Perez et al. (81), the mean penetration of

S. sanguis varied little between 10 to 28 days with a mean penetration depth of 458

μm. No bacterial penetration was observed on the cementum side even after 28

days. In bovine dentine, S. sanguis required up to 14 days to penetrate the entire

length of the tubules (24). This finding was validated by other studies by Perez et al.

(82) and Berkiten et al. (83) who found that S. Sanguis was found 600 μm and 382

μm respectively inside the tubules. They also reported that Prevotella intermedia had

very little, if any, penetration into dentinal tubules and that Actinomyces naeslundi

covered the walls of the root canal but no penetration occurred. In another study by

Siqueira et al. (75), P. gingivalis, P. endodontalis, F. nucleatum, A. israelii and E.

faecalis heavily colonized the root canal walls but few organisms penetrated the

Page 27

tubules deeply. In particular P. gingivalis and P.endodontalis showed very little

penetration.

Dentine consists of a predentine layer (10-50 μm) that lines the innermost (i.e. canal)

portion. This layer consists principally of collagen and non-collagenous components.

It diminishes with age and is removed during chemomechanical preparation of the

root canal. The non-collagenous front mineralises into mature dentine which is made

up, on a weight basis, of 70% inorganic material (hydroxyapatite), 20% organic

material (of which 30% is collagen type 1, III and V) and 10 % water (84). Haapasalo

et al. (85) showed that dentine powder had an inhibitory effect on all endodontic

medicaments tested and that the effect was concentration dependent as well as

dependent upon the length of time the medicament was pre-incubated with dentine

powder. The effect of saturated calcium hydroxide solution on E. faecalis was totally

abolished by the presence of dentine powder which indicates effective buffering of

the alkalinity of calcium hydroxide by dentine. The effect of 0.05% chlorhexidine

(CHX) on E. faecalis was reduced but not totally eliminated by the presence of

dentine. In contrast, at the higher concentration of 0.5% CHX, the presence of

dentine did not impair its action against E. faecalis.

Portenier et al. (14, 86) studied the effects on medicaments of dentine powder,

hydroxyapatite (a major component of dentine), and bovine serum albumin

(representing inflammatory exudate), and found, as did Haapasalo et al. (85), that

calcium hydroxide had lost all of its antibacterial activity against E. faecalis after 24

hours in the presence of dentine, hydroxyapatite and bovine serum albumin. The

mixture of potassium iodide/iodine (0.2/0.4%) with dentine resulted in total inhibition

of E. faecalis growth after 24 hours. CHX acetate (0.05%) killed all E. faecalis

organisms in the presence of dentine, but bovine serum albumin was the strongest

inhibitor of CHX with 10% of E. faecalis cells still being viable after 24 hr incubation. Heat-killed cells of both E. faecalis and C. albicans were potent inhibitors of 0.02%

CHX and 0.1/0.2% iodine/potassium iodide (14). From these results, it can be

concluded that in order to achieve thorough and predictable disinfection of dentine, a

medicament must penetrate into the dentine at high enough concentrations to kill the

invading bacteria and it must also resist inactivation by the dentine and by the

microbial cells that occupy the tubules (14).

Page 28

MEDICAMENTS

Five groups of antimicrobial substances have been used commonly as root canal

medicaments:

a) Calcium hydroxide

b) Antibiotics

c) Non-phenolic biocides

d) Phenolic biocides, and

e) Iodine compounds.

a) Calcium Hydroxide

Calcium hydroxide - Ca(OH)2 - has been used extensively in dentistry since the

1920’s. Today, it is still the most commonly used medicament in endodontics

throughout the world (87). Calcium hydroxide has low solubility in water, an inherently

high pH (approximately 12.5-12.8), and is insoluble in alcohol. Its low water solubility

is a useful characteristic because a long period is necessary before it becomes

soluble in tissue fluids when in direct contact with vital tissues (88). Calcium

hydroxide paste kills bacteria by direct contact and hence it should occupy the apical

regions in a sufficient quantity to permit its biological effect to be exerted in close

proximity to the appropriate tissues (140). The antimicrobial activity of calcium

hydroxide is due to the release and diffusion of hydroxyl ions (OH-) leading to a high

alkaline environment which is not conducive to the survival of micro-organisms. The

rate of diffusion of hydroxyl ions is slow due to the inherent buffering capacity of the

dentine (13, 87, 89). The faster the diffusion and the higher the ultimate tissue pH

achieved, then the more efficient and effective the medicament would be (90).

Availability of calcium ions at the site of action appears to be useful for exerting

therapeutic effects which are mediated through ion channels. The role of calcium ions

in cell stimulation, migration, proliferation and mineralization is well established (89).

The lethal effect of calcium hydroxide is due to several mechanisms (13, 87):

a) chemically by:

Page 29

• damaging the cytoplasmic membrane, since hydroxyl ions affect the

structure of this membrane

• suppressing enzyme activity and disrupting cellular metabolism

• inhibiting DNA replication by splitting DNA

and

b) physically by:

• acting as a physical barrier that fills the space within the canal and

prevents the ingress of bacteria into the root canal system, and

• killing the remaining micro-organisms by withholding substrate for

growth and limiting space for multiplication.

Calcium hydroxide also inactivates LPS, and in so doing it can assist in periapical

tissue repair (28). The biological properties of calcium hydroxide include:

• biocompatibility (due to its low solubility in water and limited diffusion), the

ability to encourage periapical hard tissue healing around teeth with infected

canals,

• inhibition of root resorption and stimulation of periapical healing after trauma.

These properties are due to its antimicrobial activity, its ability to inactivate

LPS, its ability to promote hard tissue formation, and its long lasting action (91,

92).

The limited effectiveness of the short term use of calcium hydroxide in disinfecting

dentinal tubules is due to several factors (13), namely:

• inhibition by dentine, particularly in terms of the ability of hydroxyl ions to reach

the apical third and have an antibacterial effect,

• the low solubility and diffusibility of calcium hydroxide may make it difficult to

gain a rapid increase in pH to reach the level necessary to eliminate bacteria

within the dentinal tubules and anatomical variations,

• the varying alkaline potential of different formulations,

Page 30

• cells located at periphery of the dentinal tubules can protect those located

deeper inside the tubules,

• necrotic tissue in ramifications, isthmuses and irregularities may protect

bacteria from the action of calcium hydroxide,

• the ability of E. faecalis to colonise in the dentinal tubules and thus evade the

hydroxyl ions, and

• calcium hydroxide promotes the adhesion of bacteria to collagen (the main

organic component of dentine) which increases the extent of tubule invasion

and thereby resistance to further disinfection (93).

Other disadvantages of calcium hydroxide are the difficulties associated with

removing it from the root canal walls and its effect on decreasing the setting times of

zinc oxide (ZnO) based root canal cements. Some cements have brittle consistencies

when set and are granular in structure after contact with calcium hydroxide (94).

Since calcium hydroxide kills bacteria through the effects of hydroxyl ions, its efficacy

depends largely on the availability of these ions in solution which in turn is dependant

on the vehicle in which the calcium hydroxide is carried (90). Vehicles have different

water solubility and ideally they must not change the pH of the calcium hydroxide

significantly (13). The pH distribution has been shown to decrease from the canal

dentine walls outwards towards the cementum. The pH is lower in the apical third of

the root than in the cervical third (30) which results in a lack of sufficient alkalinity

(pH) to kill or inhibit E. faecalis. The hydroxyl ions rarely diffuse away from the bulk of

the material and when combined with the buffering effect of dentine and the

inaccessibility of the medicament to all areas in the root canal system, this results in

calcium hydroxide having a limited antibacterial effect. It remains to be seen whether

other root canal medicaments are more potent (69).

Nerwich et al. (30) demonstrated that when calcium hydroxide dressings were placed

into the root canals of experimental teeth, hydroxyl ions diffused through dentine

faster in the cervical third of the root than in the apical third. They suggested that the

smaller number and size of dentinal tubules in the apical third may be an important

Page 31

factor in the diffusion of hydroxyl ions. They also showed that hydroxyl ions diffused

in a matter of hours into the inner root dentine (i.e. adjacent to the root canal) but

required 1-7 days to reach the outer root dentine (i.e. near the cementum) and 3-4

weeks to reach peak pH levels and to stabilise at these levels (30). It took nearly 7

days for the pH to rise to 9.0, a level at which many bacteria do not grow (30).

Esberard et al. (95) showed similar results. The pH in cavities on the root surface

rapidly increased from control values (pH 7.8) to greater than pH 9.0 within 3 days,

followed by a small decline to pH 9.0 over the next 18 days before finally rising and

remaining near pH 10.0 for 120 days without paste replacement.

This alkaline environment may interfere with the resorptive activity of dentinoclasts,

which require an acid environment to achieve mineral dissolution. Aqueous calcium

hydroxide and calcium hydroxide with camphorated monochlorophenol released

hydroxyl ions more rapidly than Pulpdent paste particularly in the apical region

wherein the pH in canals filled with Pulpdent remained almost a full pH unit below

those attained with the other two preparations (95).

Nerwich et al. (30) showed that the concentration of hydroxyl ions reached its peak

and stabilized in 2-3 weeks, while Gomes et al. (96) showed the concentration of

calcium ions peaked and stabilized at 2-3 weeks after packing the canals with

calcium hydroxide. Fresh filling with calcium hydroxide paste causes appreciably

more diffusion than the addition of saline solution to the paste already in the canal

(96). When calcium hydroxide comes into contact with carbon dioxide or carbonate

ions (bacterial metabolism), calcium carbonate is formed. This material has a very

low solubility, a pH of 8.0 and has neither biological nor antibacterial properties (97).

Kwon et al. (97) showed that 10% of the calcium hydroxide was converted to calcium

carbonate in the apical region within 2 days and the remainder was unchanged after

6 weeks. Little calcium carbonate was detected in samples from the middle portion of

the root canal even after 6 weeks.

The use of calcium hydroxide has been associated with the continual presence of E.

faecalis, which confirms its relative inefficiency as an antimicrobial agent against this

organism (18, 28, 73, 78, 79, 98, 99,100). A saturated calcium hydroxide solution has

been shown to be unable to kill E. faecalis in the presence of dentine, hydroxyapatite

Page 32

and bovine serum albumin (86). Hence, calcium hydroxide cannot be considered to

be a universal intracanal medicament (80) for all cases of infected root canal systems

with apical periodontitis.

The use of calcium hydroxide as the disinfectant of choice in endodontic re-treatment

of infected root-filled teeth with apical periodontitis has also been questioned (9, 98)

because of the above findings. In a study by Sundqvist et al. (10), calcium hydroxide

could eliminate E. faecalis when it was present in low numbers (as in infected teeth

without previous root fillings) but not in teeth with previous root fillings where E.

faecalis is found in higher numbers. In a study by De Souza et al. (98), the use of

calcium hydroxide resulted in a reduction in most of the species initially detected but

the authors reported a modest increase in the number of A. actinomycetemcomitans,

E. corrodens and E. nodatum organisms. It has been noted already that the presence

of Actinomyces species is also related to endodontic infections that are resistant to

therapy (98). Waltimo et al. (56) showed in an in vitro study that Candida species are

more resistant to calcium hydroxide than E. faecalis. All Candida species were highly

resistant to saturated aqueous calcium hydroxide. The majority of yeast strains

survived incubation for between one and six hours while E. faecalis was killed within

10-20 minutes (56).

b) Antibiotics

Commercial preparations in this group contain either one or a combination of

antibiotics and sometimes involve other compounds such as corticosteroids.

Antibiotics can be used as an adjunct to endodontic treatment in a number of ways -

locally (i.e. intracanal), systemically and prophylactically (101). The focus for this

review will be the local use of antibiotics in the form of intracanal medicaments. As

discussed above, bacteria may be present within areas of the root canal system that

are inaccessible to irrigants and to the mechanical cleaning processes within the

canal. They may be found in accessory canals, lateral canals, dentinal tubules,

transverse anastomoses, fins and other irregularities. Hence, an antibiotic contained

within an intracanal medicament must be able to diffuse into these areas to reduce

Page 33

the number of viable bacteria which will help promote the periapical healing response

(101).

In 1951, a polyantibiotic paste (PBSC) was devised as an intracanal medicament by

Grossman (102). PBSC consisted of penicillin to target Gram- positive organisms,

bacitracin for penicillin-resistant strains, streptomycin for Gram-negative organisms,

and caprylate sodium to target yeasts – these compounds all being suspended in a

silicone vehicle. Although clinical evaluation suggested that the paste conferred a

therapeutic effect, the composition was ineffective against the anaerobic species

which are now appreciated as the dominant organisms responsible for endodontic

diseases. In 1975, the Food and Drug Administration (FDA) banned PBSC for

endodontic use primarily because of the risks of sensitisation and allergic reactions

attributed to penicillin (103).

The two most common antibiotic-containing commercial paste preparations currently

available are Ledermix™ paste and Septomixine Forte™ paste. Both of these also

contain corticosteroids.

Septomixine Forte contains two antibiotics – neomycin and polymixin B sulphate.

Neither of these can be considered as suitable for use against the commonly

reported endodontic bacteria because of their inappropriate spectra of activity (104).

Neomycin is bactericidal against Gram-negative bacilli but ineffective against

Bacteroides and related species as well as fungi. Polymyxin B sulphate is ineffective

against Gram-positive bacteria, as shown by Tang et al. (87) who demonstrated that

a routine one-week application of Septomixine Forte was not effective in inhibiting

residual intracanal bacterial growth between appointments. In addition, the anti-

inflammatory (corticosteroid) agent used in Septomixine Forte is dexamethasone (at

a concentration of 0.05%) and, although this is effective, triamcinolone is considered

to be more potent and has less systemic side effects (101). However, Marshall (104)

has stated that dexamethasone is five times more potent than triamcinolone.

In 1948, the first synthetic tetracycline, chlortetracycline, was developed and

marketed by Lederle Laboratories (105). Subsequently, they developed the agent

demethylchlortetracycline HCl, which became the antibiotic component of Ledermix

Page 34

paste, in which it is used at a concentration of 3%. Ledermix paste was developed by

Triadan and Schroeder, and released for sale in Europe in 1962 by Lederle

Pharmaceuticals (106,107). Their primary interest in the development of Ledermix

paste was based on the use of corticosteroids to control pain and inflammation while

the antimicrobial properties at the time were catered for by a formalin-based paste

called Asphalin (introduced in 1921). The sole reason for Triadan and Schroeder

adding an antibiotic to their formula was to compensate for what was perceived to be

a reduction in the host immune response induced by the corticosteroid component.

They used chloramphenicol in their first trials but when Lederle became the

manufacturer, the antibiotic was changed to demethylchlortetracycline HCl (also

known as demeclocycline HCl) (107). Today, Ledermix paste remains a combination

of a tetracycline antibiotic, demeclocycline HCl (at a concentration of 3.2% w/w), and

a corticosteroid, triamcinolone acetonide (concentration 1%), in a polyethylene glycol

base.

The two therapeutic components of Ledermix paste (i.e triamcinolone and

demeclocycline) are capable of diffusing through dentinal tubules and cementum to

reach the periodontal and periapical tissues (12). Abbott et al. (108) showed that the

dentinal tubules were the major supply route of the active components to the peri-

radicular tissues while the apical foramen was not as significant as a supply route.

The concentration of demeclocycline within Ledermix paste itself (i.e. within the root

canal) is high enough to be effective against many species of bacteria but within the

peripheral parts of the dentine and in the periradicular tissues, the concentration

achieved through diffusion is insufficient to inactivate bacteria, especially over time

(101). Immediately adjacent to the root canal wall, inhibitory levels of demeclocycline

are achieved for all reported bacteria within the first day of application but this level

drops to about one tenth of the initial level after one week in both the mid-root and

apical third levels. Further away from the root canal towards the cementum, the

concentration of demeclocycline after one day is not high enough to inhibit growth of

twelve of the thirteen strains of commonly reported bacteria (101).

Tetracyclines are bacteriostatic, and it is well known that yeasts are resistant to

tetracyclines (12,106). Tetracyclines exhibit a level of substantivity due to their ability

to form complexes with bivalent and trivalent cations. It is for this same reason that

Page 35

they are deposited in teeth and bones during calcification (109). A study by Abbott et

al. (108) demonstrated that tetracyclines form a strong reversible bond with hard

tissues and that they exhibit slow release over an extended period of time. In a study

by Kim et al. (110), teeth with Ledermix paste in the root canals became dark brown

only upon exposure to sunlight and it was recommended that the paste should be

limited to the root canal and kept 2-3 mm below the cemento-enamel junction to

avoid discolouration of the tooth crown.

The combination of antibiotics with a corticosteroid paste as in Ledermix paste has

been used to arrest external inflammatory root resorption, and this effect has been

documented histologically in an in vivo study (111). Because it does not have

damaging effects upon the periodontal ligament tissues, Ledermix is an effective

medication for the treatment of inflammatory root resorption in traumatised teeth (12).

The immediate or early use of Ca(OH)2 following replantation has been shown to

exacerbate replacement resorption due to its high pH and toxicity, and therefore its

use in such situations should be questioned (111,112). Ledermix is the preferred

medicament immediately after replantation as it reduces both inflammatory and

replacement resorption. Not all authorities agree with this view, but this effect has

been shown in dogs by Bryson et al. (112). In a tooth experiencing 60 minutes of dry

time immediate Ledermix paste treatment allowed 59% favourable healing compared

with 14 % for calcium hydroxide. Ledermix paste also resulted in significantly greater

preservation of root mass hence maintaining the function of a replanted tooth longer

(112).

The combination of Ledermix paste with calcium hydroxide was advocated by

Schroeder initially for the treatment of necrotic teeth with incomplete root formation

(107). A 50:50 mixture of Ledermix paste and calcium hydroxide has also been

advocated as an intracanal dressing in cases of infected root canals, pulp necrosis

and infection with incomplete root formation (as an initial dressing prior to using

calcium hydroxide alone for apexification), perforations, inflammatory root resorption,

inflammatory periapical bone resorption and for the treatment of large periapical

radiolucent lesions (12). It has been shown that the 50:50 mixture resulted in slower

diffusion of the active components of Ledermix paste which makes the medicament

last longer in the canal. This in turn helps to maintain the sterility of the canal for

Page 36

longer and also maintains a higher concentration of all components (113) within the

canal.

The 50:50 mixture of Ledermix paste and calcium hydroxide pastes does not alter the

pH to any noticeable extent, and therefore it is expected that the mixture will act in a

similar manner to when calcium hydroxide is used alone (114). Taylor et al. (114)

also showed that for two micro-organisms Lactobaciluus casei and Streptococcus

mutans the 50:50 mixture was marginally more effective than either paste used

alone. However, Seow (115) showed that for Streptococcus sanguis and

Staphylococcus aureus the addition of only 25 % by volume of Calyxl™ (a calcium

hydroxide in saline paste) to Ledermix converted the zone of complete inhibition

originally seen in Ledermix to one of only partial inhibition. This study showed that

some endodontic medicaments should not be used in combinations and that when

two endodontic medicaments with strong antimicrobial activity are combined there

may be no additive or synergistic effects.

Due to the complexity of root canal infections, it is unlikely that any single antibiotic

could result in effective and predictable sterilization of all canals. More likely, a

combination would be needed to address the diverse flora encountered. A

combination of antibiotics would also decrease the likelihood of the development of

resistant bacterial strains (116). Hoshino et al. (117) determined that a combination of

ciprofloxacin, metronidazole, and minocycline, each at a concentration of 25 μg per

mL (0.0025%) of paste, was able to sterilize infected root dentine in vitro. Sato et al.

(118) found that this combination at 50 μg of each antibiotic per mL (0.005%) was

sufficient to sterilize infected root dentine in situ. However, it is questionable whether

this concentration would be adequate in vivo, particularly in immature teeth which

present many challenges to the disinfection of the root canals. The open apex allows

the potential for periapically-derived fluid to have a washing-out effect on the

antibiotic paste.

As already discussed, it was demonstrated by Portenier et al. (14) that dentine itself

can have an inhibitory effect on the bactericidal activity of intra-canal medicaments.

Therefore, Windley et al. used metronidazole, ciprofloxacin and minocycline in a thick

paste at a concentration of 20 mg of each drug per milliliter (2%) to counteract these

Page 37

potential effects. Of the 30 samples that cultured bacteria before treatment, 90%

remained positive following irrigation with 10 mL of sodium hypochlorite, but this

dropped to 30% following the application of the triple antibiotic paste for two weeks

(116).

In a study by Chu et al. (119), Ledermix paste, Septomixine Forte, and Calasept (a

calcium hydroxide in saline paste) were spiraled into canals and left for seven days.

Bacteriological samples were taken before and after two-visit endodontic treatment.

The mean CFU was reduced to 0.39% after seven days and the percentages of

canals that remained with positive growth were 48%, 31%, 31% respectively for each

medicament. There were no significant differences in the number of canals with

positive growth or mean CFU after instrumentation, irrigation and medication with

either Ledermix, Septomixine Forte, or Calasept. It was postulated that the results

may be due to the synergistic balance between strict anaerobes, facultative

anaerobes and other aerobes in endodontic infections being disrupted by antibiotics

not specifically targeting the predominant flora. Although the antimicrobial effects of

calcium hydroxide are more long lasting than antibiotics, this difference could not be

seen within the seven day test period.

The incidence of post–operative pain following endodontic treatment varies from 16%

- 48.5% and this can last for several hours up to several days (120). The

effectiveness of corticosteroid preparations in decreasing periapical inflammation

secondary to chemo-mechanical instrumentation of the root canals was

demonstrated by Smith et al. (121) who also showed histologically that fibroblastic

and osteoblastic activity was not eradicated by the use of 2.5% hydrocortisone.

Ledermix paste has also been shown to be useful in reducing the incidence of pain

following chemomechanical preparation of root canals (106, 122). Dentine acts as a

slow release mechanism for triamcinolone to the periodontal tissues which is the

probable basis of its therapeutic effect (108). In one study (122), it was shown that

Ledermix paste gave greater post-operative pain relief compared to teeth medicated

with calcium hydroxide. This has been confirmed by other studies by Rimmer et al.

(123) and Matthews et al. (124). The intracanal use of antibiotic-corticosteroid

combinations to effectively reduce post-treatment pain has been reported by several

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authors (106, 123, 120, 125). As an example, Negm (120) reported that more than 85

% of cases had complete relief of pain after a one hour interval and more than 93%

were free of pain within 24 hours of treatment.

c) Non-Phenolic Biocides

Biocides comprise a large group of diverse chemical agents that are capable of

inactivating a variety of micro-organisms. Biocides have a long history of safety and

therefore are used in a large variety of applications (e.g. as antiseptics and

disinfectants in public hospitals, oral rinses, water purification and as preservatives)

(126). Some of the commonly-used biocides are alcohols (e.g. ethanol), aldehydes

(e.g. formaldehyde, glutaraldehyde), biguanides (e.g. chlorhexidine), quaternary

ammonium compounds (QACs), zinc compounds, iodine compounds, and the

phenolic compounds including phenylethers (e.g. triclosan). Some biocides, (e.g.

CHX salts and QACs) are used predominately as antiseptics, disinfectants and

preservatives whereas others (e.g. glutaraldehyde) are used predominately for

disinfection, of endoscopes and water cooling towers in large air conditioning

systems (33).

While antibiotics affect a specific target site in micro-organisms resulting in

bacteriostatic and bacteriocidal effects at therapeutic concentrations, biocides have a

broader spectrum of activity as they work on multiple target sites. Hence, bacterial

resistance to biocides is unlikely to develop (126, 127). In general, biocides bind to

target molecules within the cell wall which becomes disrupted and this allows the

agent to then penetrate into the cell and interact with the cytoplasmic constituents

(128). The modes of action of biocides include membrane damage and leakage,

protein denaturing, binding of thiol groups, initiation of autolysis and congealing of

cytoplasmic contents at higher concentrations (126). Susceptibility to biocides is a

function of the permeability of the biocide through the cell wall; gram-positive bacteria

are more permeable and susceptible to biocides, whereas mycobacteria and gram-

negative bacteria, which have a more complex cell wall, are less permeable and

susceptible (128).

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When used at their recommended concentrations, biocides have multiple targets

within the microbial cell which confers bactericidal activity and prevents the

emergence of resistant bacteria (126, 128). While mutational resistance to antibiotics

is well known, target site mutations are rare with biocides and it is unlikely that

mutation to high level resistance occurs with biocides (33). Laboratory studies have

shown evidence of intrinsic or acquired mechanisms of non-susceptibility to biocides

but clinical failure has not yet emerged (128). Despite this, sub-optimal biocide

concentrations may result in the emergence of non-susceptible organisms which

could be resistant to antibiotics (128).

Epidemiological surveys show no increase in antibiotic resistance in clinical isolates

of oral bacteria from subjects using anti-plaque biocide formulations. Similarly,

analysis of the skin microflora from dental professionals routinely using biocides for

hand disinfection in dental clinics has indicated no alterations in the composition of

the microbial flora and no resistance to biocides (126).

Biocide activity is affected by several factors - notably concentration, period of

contact, pH, temperature, and the presence of organic matter (33). Concentration is a

factor of prime importance - QACs, CHX and glutaraldehyde retain much of their

activity when diluted but phenolics and alcohols rapidly lose activity on dilution (33).

Glutaraldehyde and cationic biocides (CHX, QACs) are most active at alkaline pH,

whereas hypochlorites and phenolics are most potent at acid pH. Interaction with

organic matter (such as blood, serum, pus) and non-ionic surfactants can adversely

affect the efficacy of many biocides (33). The activity of biocides can be enhanced by

the use of chemical agents (e.g. EDTA, polylysine) which increase the permeability of

bacterial cell membranes (33).

CHLORHEXIDINE

Chlorhexidine (CHX) has a reasonably wide range of activity against aerobic and

anaerobic organisms as well as Candida species. It is more effective at alkaline pH

than at acid pH, and its action is inhibited by the presence of soaps and organic

matter (129, 130). CHX is sporostatic (prevents the outgrowth but not the germination

of bacterial spores) but not sporicidal towards bacterial spores (130). CHX is a

Page 40

positively charged hydrophobic and lipophillic molecule that interacts with

phospholipids and lipopolysaccharides on the cell membrane of bacteria and then

enters the cell through some type of active or passive transport mechanism (131). Its

efficacy is based on the interaction between the positive charge of the molecule and

negatively charged phosphate groups on the bacterial cell wall. This increases the

permeability of the cell wall which allows the chlorhexidine molecule to penetrate into

the bacteria with intracellular toxic effects. CHX at low concentrations will result in a

bacteriostatic effect but at higher concentrations it is bactericidal due to precipitation

and/or coagulation of the cytoplasm which is probably caused by protein cross-linking

(28). The beneficial effect of CHX is due to its antibacterial, substantive properties

and its ability to inhibit adherence of certain bacteria (132). As attachment of bacteria

to oral surfaces represents a critical step in the pathogenic process, it is interesting

that CHX has been shown to inhibit adherence of P. gingivalis to host cells (132).

CHX has greater activity against Gram-positive than Gram-negative organisms. The

least susceptible of Gram-negative microorganisms include strains of Proteus,

followed by Pseudomonas, Enterobacter, Actinobacter and Kleibsiella (133). There is

no evidence that CHX resistance in Gram-negative bacteria is plasmid-borne or

transferable (130). This may explain why long term application of CHX in dogs led to

a domination in plaque samples of Gram-negative rods. The efficacy of CHX against

Gram-positive bacteria may thus cause an over-estimation of the clinical usefulness

of this agent (134).

CHX in gel formulations results in low toxicity to the periapical tissues. The viscosity

keeps the active agent in contact with the root canal walls and dentinal tubules (28).

According to Barthel et al. (135), CHX gel, when used to medicate root canals, does

not interfere with the sealing properties of root filling materials. However, unlike

sodium hypochlorite, CHX does not dissolve organic tissue or inactivate bacterial

LPS (65, 136).

CHX has a unique feature in that dentine medicated with it acquires antimicrobial

substantivity (100, 137-142). The positively-charged molecules of CHX can adsorb

onto dentine and prevent microbial colonization on the dentine surface for some time

beyond the actual medication period (142). This can inhibit re-infection of the canal

Page 41

subsequent to treatment (139). CHX is retained in root canal dentine in levels

sufficient to exert antimicrobial effects for at least 12 weeks (143). In order to achieve

this substantivity, prolonged interaction is required to allow saturation of the dentine

with the CHX. Thus, it should be applied as an intracanal medicament between

appointments for at least seven days rather than being used as an irrigant (139).

Antimicrobial substantivity depends on the number of CHX molecules available to

interact with the dentine. Therefore, medicating the canal with a more concentrated

CHX preparation should result in increased resistance to microbial colonization (142).

In large bovine root canals, 0.2% CHX imparted substantivity whereas in smaller

human root canals a higher concentration of CHX was required to achieve a

comparable effect (142).

When CHX is used as an irrigating solution, it has a relatively short exposure time in

the root canal (a few minutes at most) and this does not allow the medication to apply

its full antibacterial action. As a result, a large number of bacteria may persist within

the dentinal tubules and remain viable (144). The limited antibacterial effect of CHX

irrigation is a result of the lack of time of application. The dentine must reach

saturation point before there is any persistence of the antibacterial activity. During the

first hour of exposure, the dentine is absorbing the medication and therefore it is not

yet active (144). In a study by Lin et al. (144), a slow release device containing 5%

CHX was placed in root canals for seven days to allow sufficient time for penetration

into the dentinal tubules and to reach its maximum antibacterial effect. This resulted

in no bacteria being detected in bovine dentine up to 500 μm into the dentinal tubules

(144).

When used as an intracanal medicament, CHX was more effective than calcium

hydroxide in eliminating E. faecalis from inside dentinal tubules (100). In a study by

Almyroudi et al. (145) all of the chlorhexidine formulations used, including a CHX

/Ca(OH)2 50:50 mix, were efficient in eliminating E. faecalis from the dentinal tubules

with a 1% CHX gel working slightly better than the other preparations. These findings

were corroborated by Gomes et al. (28) in bovine dentine and Schafer et al. (78) in

human dentine where 2% CHX gel had greater activity against E. faecalis, followed

by CHX + Ca(OH)2, and then Ca(OH)2 used alone.

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In a study using agar diffusion (146), Haenni et al. could not demonstrate any

additive antibacterial effect by mixing Ca(OH)2 powder with CHX (0.5%) but they

showed that Ca(OH)2 did not lose its antibacterial properties in such a mixture

whereas the CHX did have a reduction of its antibacterial action. This may be due to

the deprotonation of CHX at a pH greater than 10 which reduces its solubility and

alters its interaction with bacterial surfaces as a result of the altered charge of the

molecule. In an in vitro study using human teeth Ercan et al. (147) showed 2% CHX

gel was the most effective agent against E. faecalis inside dentinal tubules followed

by Ca(OH)2 / 2% CHX mix whilst Ca(OH)2 was totally ineffective even after 30 days.

The 2% CHX gel was significantly more effective than Ca(OH)2 / 2% CHX against

C.albicans at 7 days however there was no significant difference at 15 and 30 days.

Ca(OH)2 was completely ineffective against C. albicans. In an in vivo study using

primary teeth (148) a 1% CHX gluconate gel with or without Ca(OH)2 was more

effective against E. faecalis than Ca(OH)2 within a 48 hour period.

In another study by Schafer et al. (149), 2% CHX gluconate was significantly more

effective against E. faecalis than a Ca(OH)2 paste (Calxyl) used alone, or a mixture

of the two. This was also confirmed by Lin et al. (91) although in a study by Evans et

al. (150) using bovine dentine, 2% CHX with Ca(OH)2 was shown to be more

effective than Ca(OH)2 in water. In an animal study, Lindskog et al. (151) reported

that teeth dressed with CHX for four weeks had reduced inflammatory reactions in

the periodontium (both apically and marginally) and reduced root resorption. Waltimo

et al. (152) used a filter paper method and reported that 0.5% CHX acetate was more

effective at killing C. albicans than saturated Ca(OH)2 while Ca(OH)2 combined with

CHX was more effective than Ca(OH)2 used alone The high pH of Ca(OH)2 was

unaffected when it was combined with CHX in this study.

CHX is also used as an endodontic irrigant. Its efficacy was shown by Parson et al.

(153), who reported that when bovine dentine was exposed for periods of 20 and 40

minutes to CHX concentrations of 0.2% and 1.0 %, the dentine acquired antibacterial

properties which completely inhibited the growth of E. faecalis for one week. Dametto

et al. (154) used 2% CHX gluconate gel and 2% CHX gluconate liquid as irrigants

between endodontic file changes. They also used 3mL of the liquid as a final flush of

the root canals. They reported substantivity for a period of seven days and the

Page 43

maintenance of a low E. faecalis CFU count. Oncag et al. (155) showed that the

antibacterial effect of 2% CHX gluconate when left in the canal for 5 minutes at the

end of the chemomechanical instrumentation process was substantive for a period

ranging from 5 minutes to 48 hours.

CHX has been used extensively in commercially available oral rinses and subgingival

irrigants at a concentration of 2% without any apparent adverse effects being

reported (156). In vivo and in vitro studies conducted by Wennberg (157) showed

CHX was no more toxic or irritant to tissues than other endodontic agents. Filho et al.

(136) found that 2% CHX gluconate produced the same inflammatory response in

mice as phosphate buffered saline while Tanomaru et al. (65) found that 2.0 % CHX

digluconate solution did not induce a significant inflammatory response nor damage

tissue, again indicating its biocompatibility. Hence, 0.1% to 2% chlorhexidine

solutions are classified as toxicologically safe and even at higher concentrations,

CHX has very low toxicity (72, 88, 158).

d) Phenolic Agents

These medicaments may be applied either on a cotton wool pellet placed in the pulp

chamber or on a paper point placed in the root canal with the rationale being that the

antimicrobial effect is delivered through vaporisation of the medicament (19).

Endodontic medicaments such as camphorated monochlorophenol (CMCP) depend

on the diffusion of their vapours to spread the material throughout the root canal

system and bring it into contact with micro-organisms remaining in the canal following

chemo-mechanical instrumentation and irrigation (159). The antimicrobial action in

the apical portion of the root and within the dentinal tubules is therefore dependent

on the volatility of the medicament. Hence it must convert to the vapour phase and

penetrate the entire root canal system to come into direct contact with the micro-

organisms. Depending on the volatility of the agent used, the amount which can be

loaded onto a cotton pellet or paper point is small and some of the medicament will

be lost through evaporation into the atmosphere before the access cavity is closed. If

this loss were great, the duration of effectiveness of the remaining material within the

canal will be limited. In studies by Menezes et al. (160) and Messer and Chen (161),

Page 44

there was 90% loss of CMCP from cotton pellets inserted into pulp chambers within

1-2 days of insertion. Vapours may be effective but the delivery to the apical part of

the root canal is unpredictable. Hence an endodontic antiseptic that is not in direct

contact with the root canal walls in the very apical end of the pulp space must be

considered as being unreliable at best (19).

The antibacterial action of phenolic materials may not persist for prolonged periods.

Hence some bacteria may survive and have the opportunity to multiply and persist in

the root canal system (161, 162).

CMCP can diffuse beyond the apical foramen and since it is not firmly bound to

periapical proteins, it is rapidly removed by the blood circulation with 80% of it being

excreted by the kidney. The kidney and liver contain less than 1% of the total CMCP

that is delivered which suggests that the CMCP primarily passes through the tissues

and into the blood, whilst not being concentrated or metabolised to a large extent

(152).

In a study by Byström et al. (23), calcium hydroxide had a higher antibacterial effect

in the root canal than camphorated phenol (CP) or CMCP. When infected root canals

were treated with CP or CMCP, bacteria persisted in 33% of the canals. When

compared with mechanical cleansing and irrigation, the antibacterial effect of CP and

CMCP was not impressive (23). When paramonochlorophenol (PMCP) is combined

with Calen {a Ca(OH)2 paste}, the amount of PMCP released is of sufficiently low

concentration not to harm the periapical tissues but sufficient to exert bactericidal

effects. A loss of approximately 50% of the PMCP occurred within 48 hours but there

was no further significant loss after 14 days (163).

In a study by Ayhan et al. (164), Cresophene (a mix of 30% paramonochloro-phenol,

5% thymol, 0.1% dexamethasone) (Septodont, Saint-Maur, France) had one of the

highest levels of antimicrobial activity using agar diffusion when compared with five

other irrigants. However, the authors cautioned against using Cresophene due to its

known cytotoxic and possible carcinogenic, mutagenic and teratogenic properties.

Page 45

The Calen and CMCP paste combination was reported as the most effective

intracanal medicament compared to a variety of medicaments including 2% CHX

solution, Calen paste and 2.5% sodium hypochlorite (NaOCl) for the elimination of E.

faecalis and C. albicans using sampling with sterile paper points after 15 days in

human teeth (160).

CMCP is the most toxic and irritating phenolic antiseptic agent followed by Cresatin,

formocresol, and camphorated phenol (CP) but they all have reasonable

antimicrobial properties. These findings concerning biocompatibility were

corroborated by Collet et al. (165). The toxic effects of CP ended when the dilution

exceeded 1:70 while CMCP required 1:2,000 times dilution to eliminate its toxicity to

cultured cells. CMCP has low solubility in water and a slow diffusion rate in agar –

hence, when assessed in vitro it appears to have only a limited antimicrobial effect. In

contrast, serial dilution experiments indicate that CMCP is a highly effective antiseptic

agent (166).

e) Iodine Compounds

Iodine is rapidly bactericidal, fungicidal, tuberculocidal, virucidal, and sporicidal.

Aqueous iodine solutions are unstable, with molecular iodine (I2) being mostly

responsible for the antimicrobial activity. Iodophors (iodine carriers) are complexes of

iodine and a solubilizing agent or carrier, which acts as a reservoir of the active free

iodine (4). Torneck in 1976 advocated the use of povidone-iodine solution as an

endodontic irrigant. This was based on its rapid antiseptic action against a broad

range of micro-organisms, low toxicity, hypoallergenicity, and greatly decreased

tendency to stain dentine than other iodine containing antiseptics (167). One study

(167) measuring the cytotoxicity of endodontic irrigants on cultured human gingival

fibroblasts for 15 minutes showed that 2% iodine potassium iodide (IPI) and 10%

calcium hydroxide were the least toxic.

The antimicrobial action of iodine is rapid, even at low concentrations, but the exact

mode of action is not fully known. It is thought that iodine attacks key groups such as

proteins, nucleotides, and fatty acids, resulting in cell death (4). In endodontics, IPI is

commonly used, with potassium iodide needed to dissolve iodine in water and form

Page 46

the tri-iodide ion (4). Formocresol, CMCP, and Cresatin are known to be highly toxic

and tissue irritating medicaments whilst 2% IPI has the lowest toxic and irritating

effect of the endodontic medicaments evaluated in some studies (166). In 2% IPI

treated root canals of human teeth, a period of 1-2 hours was required to prevent

growth of E. faecium in dentinal tubules whilst the calcium hydroxide specimens still

had positive cultures after 24 hours of calcium hydroxide therapy (79).

Molander et al. (168) failed to demonstrate enhanced antibacterial effect that

included medication with 5% IPI for a period of 3-7 days followed by calcium

hydroxide dressing for 2 months in human teeth where the smear layer was not

removed. When IPI was used, 44% of the 50 cases showed positive growth whilst the

addition of calcium hydroxide resulted in only 80% negative cultures. Peciuliene et al.

(51) studied the effect of calcium hydroxide medication for 10-14 days on 20 root-

filled teeth with apical periodontitis. Bacteria were isolated in 16 of the 20 teeth prior

to instrumentation, but only in 5 teeth after instrumentation and irrigation (smear layer

removed). The third sample taken after five minutes of irrigation with 2% IPI in 4%

potassium iodide (2% IPI 4%) resulted in growth in only one canal. The antifungal

effects of different medicaments and their combinations were compared (152) and

the results showed that 2% IPI 4% killed all of the C. albicans cells within 30 seconds

and the 10 fold dilution showed complete killing within 5 minutes. The 2% IPI 4%-

saturated calcium hydroxide combination was significantly less effective than 2% IPI

4%.

As with other medicaments and irrigants the presence of dentine and its components

are responsible for different inhibitory patterns on the activity of the iodine solutions.

Haapasalo et al. (85) demonstrated that dentine powder effectively abolished the

effect of 0.2% IPI 0.4% whilst dentine powder had a very limited capacity to inactivate

2% IPI 4% where it took only 5 minutes to kill E. faecalis. Portenier et al. (14) showed

that hydroxyapatite, the major component of dentine, caused little or no inhibition

whilst the collagen matrix in dentine effectively inhibited 0.1% IPI 0.2 %.

Abdullah et al. (169) showed that a 10% povidone iodine solution resulted in 100%

bacterial reduction after 30 minutes in an E. faecalis planktonic suspension and 30

minutes in an E. faecalis biofilm model whilst calcium hydroxide was unable to

Page 47

reduce to 100% bacterial reduction even after 60 minutes. In a model using bovine

teeth infected with E. faecalis, Baker et al. (170) showed that every iodine containing

agent tested (2% IPI, 2% IPI plus Tween 20, 10% povidone iodine, 10% povidone

iodine plus detergent) performed significantly better than 10% calcium hydroxide. The

most effective irrigant was 2% IPI with nearly total elimination of E. faecalis within 15

minutes whilst 10% calcium hydroxide failed to eliminate E. faecalis from any of the

samples. There was also no residual effect of the iodine-containing medicaments.

Siren et al. (171) used E. faecalis infected bovine root blocks to test the effectiveness

of 1 day and 7 day incubation of medicaments. The 2 % IKI medicament showed

results with no growth up to 700μm and 950μm at 1 and 7 days. However after 7

days 2% IKI saturated with calcium hydroxide had the same effect as 0.5%

chlorhexidine acetate and 2% IKI. Cwilka et al. (172) used human single rooted teeth

infected with E. faecalis and showed that Ca(OH)2/iodoform/silicone oil was the most

effective combination followed by 2% IKI/ Ca(OH)2 and then Ca(OH)2.

MEDICAMENT VEHICLES

The medicament vehicle plays a very important role in the overall disinfection

process because it determines the velocity of ionic dissociation causing the paste to

be solubilized and resorbed at various rates by the periapical tissues and from within

the root canal. The lower the viscosity, the higher will be the ionic dissociation. The

high molecular weight of commonly-used vehicles minimizes the dispersion of

calcium hydroxide into the tissue and maintains the paste in the desired area for

longer periods of time (88).

There are three main groups of paste vehicles (45,88):

• water-soluble substances - such as: water, saline, dental anaesthetics,

Ringers solution, methylcellulose, carboxymethylcellulose, anionic detergent

solutions (including sodium lauryl sulphate and sodium lauryl

Page 48

diethyleneglycol). Water-soluble proprietary brands include Calxyl, Pulpdent,

Calasept, Hypocal, and DT temporary dressing.

• viscous vehicles - such as: glycerine, polyethyleneglycol (PEG) and propylene

glycol. Some examples of proprietary brands are Calen and Ledermix paste.

• oily vehicles - such as: olive oil, silicone oil, camphor (the essential oil of

camphorated parachlorophenol), metacresylacetate, eugenol and some fatty

acids (including oleic, linoleic and isostearic acids). Examples of oil-based

proprietary brands include Endoapex and Vitapex.

Calcium hydroxide should be combined with a liquid carrier because the delivery of

dry calcium hydroxide powder alone is difficult or impossible in narrow, curved

canals. Sterile water or saline are the most commonly used carriers and they are also

effective in delivering hydroxyl ions. Aqueous solutions promote rapid ion liberation

and should be used in clinical situations involving intense exudate or tooth

replantation. Although anaesthetics have an acid pH range (between 4-5), they

provide an adequate mixing agent because calcium hydroxide is a very strong base

which is only minimally affected by acid.

Viscous vehicles are also water soluble substances that release calcium and

hydroxyl ions more slowly for extended periods (45). A viscous vehicle may remain

within root canals for 2-4 months and hence, the number of appointments required to

change the dressing will be reduced (88). Oily vehicles have restricted applications -

they are generally only used in situations that require very slow ionic dissociation

such as perforation defects (45) since they are difficult to remove and leave an oily

film on the canal walls.

In a study by Camoes et al. (173), the pH in an aqueous medium was tested outside

the roots of human teeth when various vehicles (aqueous or viscous) were used with

calcium hydroxide. They reported that vehicles with glycerine and PEG 400 showed a

trend to acidification during the first eight days after filling (pH 6.85 to 6.4 - PEG 400)

but then the pH returned to similar levels to other groups after 42 days (pH 7.1 - PEG

400). Even though anaesthetic solution is a weak acid, there were no significant

differences in the release of calcium and hydroxyl ions when compared with saline

Page 49

solution. The effects of glycerine and propylene glycol mixing vehicles on the pH of

calcium hydroxide preparations was investigated using conductivity testing by Safavi

et al. (174). A range of 10-30% for a glycerine/water mixture and 10-40% for a

propylene glycol/water mixture resulted in the greatest amount of conductivity. They

reported that the higher concentrations of these vehicles may decrease the

effectiveness of calcium hydroxide as a root canal dressing.

As well as the type of vehicle used, the thickness of the calcium hydroxide paste can

influence the antimicrobial activity. This was seen in a study by Behnen et al. (175) in

which thick mixtures of calcium hydroxide showed a significant reduction of

antibacterial activity against E. faecalis in dentine tubules compared to a thin mix and

Pulpdent paste.

PEG is one of the most commonly used vehicles in root canal medicaments and it

possesses an ideal array of properties including very low toxicity, excellent solubility

in aqueous solutions and extremely low immunogenicity and antigenicity (176).

Concentrated PEG 400 solutions have significant antibacterial activity against various

pathogenic bacteria including Klebsiella pneumoniae, Pseudomonas aeruginosa,

Eschericha coli, and Staphylococcus aureus (177).

BIOFILMS

Micro-organisms do not exist as isolated single cells suspended in an aqueous

environment (i.e. the planktonic model) and most in vivo populations of bacteria grow

as adherent biofilms (105). Biofilms are composed of micro-colonies of bacterial cells

that are non-randomly distributed in a matrix of polysaccharides, proteins, salts and

cell material in an aqueous solution. Since biofilms are the preferred method of

growth on a surface for most species of bacteria, it is likely that bacteria are present

in biofilms on the dentinal wall or on the external surface of the root tip (69).

Infection of the root canal system ultimately leads to liquefaction necrosis of the pulp

where the bacteria form biofilms on the root canal walls with the tissue remnants in

the canal (32). Planktonic bacteria that are either leaving or joining the biofilm

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surround it and this constant detachment of cells serves as a steady source for

chronic infection (178, 179).

Fluid flow is considered to be a principal determinant of biofilm structure. It provides

nutrient exchange, influences density and strength, and affects the dispersal of cells

from the biofilm. When a tooth undergoes pulp necrosis and subsequently develops

apical periodontitis, an exudate may cycle in and out of the canal. This fluid

exchange provides proteins, glycoproteins, and other nutrients to the bacteria

growing as a biofilm in the root canal and exerts shear force on the bacterial biofilm

(180).

Oral biofilms are complex and may comprise thirty or more bacterial species. Biofilm

formation in the root canal varies depending on the cause of the pulp breakdown.

Root canals may not always be fluid-filled and the canals of teeth with necrotic pulps

often appear dry on entering them, at least in the coronal portion. Hence the question

remains as to whether bacterial condensations in a biofilm structure can develop or

be retained in sites of the root canal system other than near the apex where host-

derived proteins and bacterially-produced adhesive substances may provide the

proper prerequisites (181).

Nair et al. (70) found that even after instrumentation, irrigation and obturation in a

one-visit treatment, microbes existed as biofilms in untouched locations in the main

canal, isthmuses and accessory canals in 14 of the 16 root-treated teeth examined.

Bacterial biofilms are reported to be the most common cause of persistent

inflammation and apical periodontitis is considered to be the result of an intra-

radicular biofilm-induced chronic disease (69, 70).

Mechanisms of resistance of bacterial biofilms to antimicrobial agents include (127,

182, 183):

a) the polysaccharide matrix retards diffusion of the antibiotic. Extracellular

enzymes such as ß-lactamase may become trapped and concentrated in the

matrix thereby inactivating ß-lactam antibiotics (180),

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b) communication with one another (known as “quorum sensing”) which can

influence the structure of the biofilm by encouraging growth of species

beneficial to the biofilm (181, 182). Transfer of genetic information (antibiotic

resistance and virulence traits) to other bacteria within the biofilm may provide

an effective defence against host protective mechanisms and antimicrobial

agents (32, 178) producing a highly resistant phenotypic state,

c) chemical changes to the environment in the biofilm where the lack of oxygen

inhibits some antibiotics and accumulated acidic waste leads to a difference in

pH which has an antagonizing effect on the antibiotic.

d) depletion of nutrients or accumulation of waste products can result in bacteria

entering a non-growing state which protects bacteria from the antibiotics (183),

as well as the dose and frequency of exposure to the antimicrobial agent

(156),

e) protecting themselves by being located within the interior part of a biofilm -

hence medicaments will only act on the micro-organisms in the peripheral

portion of the biofilm (98). Bacterial cells residing within a biofilm grow more

slowly than planktonic cells and as a result antimicrobial agents act more

slowly (180),

f) subpopulations of bacteria in a biofilm form a phenotypic state (altered gene

expression) where they are highly protected - this form of cell differentiation is

similar to spore formation (183).

g) biofilm bacteria existing in a low metabolic state, a slower growth rate and

production of exopolysaccharides (184).

Biofilms and microbial aggregates are a common mechanism for the survival of

bacteria in nature (185). Co-aggregation is highly specific and is considered a

virulence factor which is believed to play a role in the development of dental plaque

and other biofilms (185). In a study by Khemaleelakul et al. (185) co-aggregation was

very prominent between streptococci-prevotellae, streptococci-staphylococci, and

streptococci-corynebacteria. Prevotellae and streptococci were the genera that most

often co-aggregated with multiple bacterial species. Fusobacteria isolated from

endodontic abscesses demonstrated the ability to co-aggregate with members of

Page 52

every genus tested and it also had the ability to auto-aggregate. Prevotella spp. is

considered to play a key role in the pathogenesis of endodontic infections due to its

ability to co-aggregate with other species, especially streptococci (regarded as early

colonizers). It seems likely that multi-generic co-aggregation may occur in infected

root canals and periapical tissues in the same manner as dental plaque biofilms. The

aggregation of bacteria in biofilms is likely to result in these bacteria being more

resistant to antibiotics and other antimicrobials as well as being protected from the

host defences.

Wilson (186) has pointed out that the above refers to mono-species biofilms. At

present, there are no studies to show what happens in multi-species biofilms with low

oxygen content, low oxidation-reduction potential and high concentrations of

metabolic end-products which may lead to increased growth rates for anaerobic

bacteria. It would be useful to know their antimicrobial susceptibilities in relation to

their planktonic counterparts. The use of the membrane filter-based model using

single species biofilms offers a simple and more accurate assessment for

determining the susceptibility of oral bacterial biofilms to antimicrobial agents (186).

It is estimated that bacteria grown in a biofilm have a 1000-1500 times greater

resistance to antibiotics than planktonically-grown bacteria (32). For example, the

biofilm inhibitory concentrations for chlorhexidine and amine fluoride are 300 and 75

times greater, respectively, when Streptococcus sobrinus is grown in biofilm

compared with the minimum bactericidal concentration for planktonic cells (181). With

regard to host defences, bacteria in biofilms are less easily phagocytosed and less

susceptible to complement than their planktonic counterparts (105). Bacterial viability

may vary throughout dental plaque biofilm with the most viable bacteria being located

in the central part of the plaque. Lining the voids and channels are layers of viable

bacteria packed in layers of dead material (187).

During the irrigation of root canals, the outer layer of the biofilm will be directly

affected by the high concentration of the irrigating solution but the extracellular matrix

of the biofilm may prevent the solution penetrating into the deeper layers at full

strength (188). Endodontic instrumentation helps to disrupt and expose the full

thickness of the biofilm to the irrigant solution. However, since not all of the root canal

Page 53

walls are contacted by instruments, some micro-organisms may still remain in the

canal and within the tubules (189).

Thrower et al. (190) showed that chlorhexidine was by far the superior agent in the

membrane-based biofilm model and the planktonic model of A.

actinomycetemcomitans, followed by cetylpyridinium chloride with triclosan being the

least active. In a study by Lima et al. (191), medications containing 2% chlorhexidine

gluconate were able to completely eliminate most of the biofilms while medications

containing clindamycin, with or without metronidazole, did not significantly affect the

biofilms. Biofilms of oral bacteria have also been found to be more resistant to

amoxycillin, doxycycline and metronidazole (182).

In polymicrobial (bacteria and fungi) biofilms, in particular the bacterial species, were

found to be more resistant to biocide treatment than the single species biofilms (192).

Hence Clegg et al. (193) used polymicrobial biofilms to asses the antimicrobial

effectiveness of various irrigants including 2% CHX. The root apices were incubated

for 7 days to allow biofilm formation and immersed in test solutions for 15 minutes.

No cultivable organisms were found after dentine sections were placed in 2% CHX

for 15 minutes. However, when the samples were viewed under SEM, the biofilm was

virtually intact. This result may be due to the potential fixation of cells by CHX and if

CHX is to be used as an irrigant, additional agents (e.g. sodium hypochlorite) may be

needed to physically disrupt the biofilm.

E. faecalis has been shown to form biofilms in the root canals of human teeth, with

or without intracanal medicaments, after only two days with depths of 21 μm for 86-

day biofilms and 28-30 μm for 160-day biofilms. Root canals inoculated with E.

faecalis for 86 days resulted in the bacteria becoming embedded in branching

filamentous material which represented an extracellular polysaccharide produced by

the bacteria. Biofilms maintained for 160 days had a highly organized structure

consisting of mushroom-shaped clumps of bacteria with vacant areas which were

thought to contain water channels for the delivery of nutrients and to remove waste

from the biofilm bacteria (178).

Page 54

Alterations in the micro-environment of root canals (as occurs during endodontic

treatment) could stimulate calcification of E. faecalis biofilms, as shown in a study by

George et al. (179). They also reported that biofilms which formed under rich nutrient

conditions had increased calcium levels within the biofilm whereas poor nutrient

conditions did not produce this effect. This implies a possible role of the mineralized

matrix as a shelter for viable bacteria which helps to protect them against

antimicrobial agents (179).

Biofilms produced in the laboratory are often less adherent and less virulent than

those found in nature and this should be taken into account when trying to achieve in

vivo-in vitro correlations (190). More studies are needed to explore the conditions that

may affect the efficacy of endodontic antimicrobial agents in vivo so their clinical

effects can be better predicted. Indeed, conclusions reached on studies of

monocultures on standard laboratory surfaces must be interpreted with great caution

as such models do not reflect the clinical scenario and may give misleading data,

especially in terms of the effects of antimicrobials on bacteria within biofilms (181).

ANTIBIOTIC RESISTANCE AND THE VIRULENCE OF E. FAECALIS

Anaerobes and Gram-negative rods which are found frequently in untreated infected

root canals are often associated with acute exacerbations of chronic apical

periodontitis. Since the Gram-negative cell wall is more fragile, these bacteria are

typically more sensitive to biocides and easier to eliminate than Gram-positive and/or

facultative bacteria (46, 194). Bacteria have been able to develop or acquire

resistance to every class of antibiotics in use and, as a result of this, every antibiotic

has a limited period of use before resistance emerges (195).

Enterococcus species are now second only to E. coli as the most common

nosocomial pathogen isolated overall. They are the leading cause of surgical site

infections and the third leading cause of urinary tract infections (196). Enterococcus

faecalis (formerly designated Streptococcus faecalis) accounts for 85-90% of

Page 55

infections caused by enterococci and Enterococcus faecium accounts for 5-10% (54,

197, 198).

E. faecalis is a normal intestinal organism and may inhabit the oral cavity and

gingival sulcus. In its intestinal environment, it is considered a commensal organism

that contributes to carbohydrate, amino acid and vitamin metabolism. However, a

subset of this species appears to be pathogenic because it has acquired a number of

genes which confer infectivity and virulence, plus resistance to multiple antibiotics

(39).

The low prevalence of E. faecalis reported in primary endodontic infections (i.e. no

previous endodontic treatment) may be a result of other species inhibiting its growth.

Thus, the possibility exists that during chemomechanical preparation, the most

prevalent anaerobic bacterial species (which are Gram-negative) are more likely to

be eliminated than the facultative Gram-positive E. faecalis, which is resistant to

many intra-canal antimicrobial procedures and medications.

Despite its ability to adapt extremely well to the surrounding environment, E. faecalis

does not appear to exert major pathogenic actions within the root canal system when

it is present as a mono-infection (39). It has been shown that the addition of E.

faecalis to a four-strain combination increased the survival and pathogenicity of the

other bacteria in the root canal system (53). Increased pathogenicity of E. faecalis

has also been found in animal models (199). Currently, it is unclear whether E.

faecalis is the major pathogen associated with endodontic treatment failures or

merely a survivor that takes advantage of its ability to endure adverse conditions

within obturated root canals (39).

Limitations of studies into the pathogenic role of E. faecalis in endodontic failures

include (200):

a) in some cases a periradicular lesion can be present but remains

radiographically undetectable until a certain amount of bone is resorbed,

b) for a disease to develop, a certain amount of bacterial load is required. Hence

it is possible that the number of E. faecalis bacteria is lower in root-filled teeth

with no lesions,

Page 56

c) a more virulent clone of E. faecalis is responsible for teeth with periradicular

disease,

d) host resistance differs from subject to subject and may result in different

patterns of microbiological infection. Enterococci have both:

a) intrinsic resistance (that is, a usual species characteristic in all or most of the

strains where the gene for intrinsic resistance resides on the chromosome),

and

b) acquired resistance (that is, resistance due to either a mutation in the existing

DNA or acquisition of new DNA)

to many antibiotics (191, 198). Enterococci are inherently more resistant to

antimicrobial drugs than other clinically-important Gram-positive bacteria (201).

Enterococci inhabit the gastrointestinal tract and environments contaminated by

human waste and therefore they may be exposed to antibiotics that pass through the

gastrointestinal tract. The intrinsic resistance of enterococci to many antimicrobials

might have resulted from their need to survive and persist in highly competitive and

potentially detrimental ecosystems (54).

Enterococci are intrinsically resistant to many antimicrobial agents, including

cephalosporins, clindamycin, penicillinase-resistant penicillins and the antibiotic of

last resort, vancomycin (47, 54, 201). E. faecalis has intrinsic resistance to

clindamycin, cephalosporins and aminoglycosides (198, 202). In addition to these

intrinsic resistances, enterococci have acquired genetic determinants for resistance

to many classes of antimicrobials, including tetracycline, doxycycline, erythromycin,

chloramphenicol, ciprofloxacin and vancomycin (198, 201, 202, 203).

E. faecalis has many remarkable and distinct features which make it an exceptional

survivor (9). It can:

a) live and persist in the poor nutrient environment of endodontically-treated

teeth (9, 10, 46),

Page 57

b) survive in the presence of several medications, sodium hypochlorite (24),

clindamycin (204) and the most popular medication, calcium hydroxide (28),

c) form biofilms in medicated canals (178),

d) invade and metabolise fluids within the dentinal tubules and adhere to

collagen in the presence of human serum (76),

e) convert into a viable but non-cultivable state (VBNC) (205),

f) endure prolonged periods of starvation and utilize tissue fluid (human serum)

that flows from the periodontal ligament and bathes the alveolar bone to

recover (206, 207),

g) establish mono-infections in medicated root canals (21, 204, 208),

h) acquire gene-encoding antibiotic resistance combined with natural resistance

to various antimicrobials agents, and

i) survive in extreme environments with low pH, high salinity, and high

temperatures.

Some of the virulence factors of E. faecalis are (183):

a) Cytolysin - this is a toxin expressed by E. faecalis that causes direct tissue

damage and inflammation. The precise mechanisms for this remain

speculative but it is thought that it plays a role in the evasion of the host

immune response.

b) Gel E gene - E. faecalis secretes two proteases, namely gelatinase (liquefied

gelatin) and a serine protease which together are responsible for tissue

damage by hydrolizing gelatin, collagen, casein, porcine proteins and small

peptides. Serine proteases aid E. faecalis in binding to dentine and they

posses the ability to remain stable and active over a high pH range from 6-12

(209).

c) Aggregation substance - this is a surface binding protein that promotes

survival within neutrophils and enhances adherence and uptake into

eukaryotic target cells thereby increasing the density of bacteria to the level

necessary to achieve quorum status. Cytolysin and aggregation substance

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work synergistically to enhance virulence factors, resulting in tissue damage

and potentially deeper penetration (54).

d) ESP (enterococcal surface protein) - a strong association between ESP and

biofilm formation in E. faecalis has been reported. It is also a good marker for

the identification of strains that are highly adherent to abiotic surfaces (197).

The ESP gene (210) and the cytolysin operon occur close to each other within

a specific region of the E. faecalis chromosome and this generates an

environment where a quorum of bacteria can accumulate (e.g. in biofilms)

(54).

e) ACE (adhesion of collagen E. faecalis) - ACE is a collagen binding protein of

E. faecalis. Microbial infections are initiated by bacterial adherence to host

cells and/or extracellular matrix proteins, collagen (Types I, II, and IV), and

laminin (54). Since collagen accounts for nearly 90% of the organic part of

dentine and most of this collagen is type I, it is not surprising that ACE

mediates binding to dentine (93, 209). Love et al. (76) showed inhibition of E.

faecalis invasion of dentinal tubules by acid soluble collagen and this was

confirmed by Kowalski et al. (207) where the ACE deficient mutant strain of E.

faecalis significantly inhibited attachment.

In a study by Sedgley et al. (199), all isolates had ACE and the gel E gene, 61% had

ESP gene, 93% expressed gelatinase activity in primary endodontic infections and

25% in re-treatment cases. Due to the proximity of bacteria in biofilms, E. faecalis can

participate in plasmid-mediated horizontal transfer of virulence determinants. It also

has the ability to acquire plasmid-encoded resistance genes from other bacteria

which results in intra-species propagation of resistance (199). Resistance is a major

issue with this bacterial species (Table 2.1). When the opportunity arises, E. faecalis

could be released from dentinal tubules into the root canal space and act as a source

of bacteria for re-infection of the canal (199).

E. faecalis can gain entry into the root canal system during endodontic treatment,

between appointments or even after the endodontic treatment has been completed.

Hence, current methods used to eliminate E. faecalis include (211):

Page 59

a) using aseptic techniques consisting of a pre-treatment CHX rinse, placement

of rubber dam, disinfection of the tooth and the rubber dam with CHX or

NaOCl and disinfection of the gutta-percha with NaOCl,

b) minimum instrumentation of the apical third of the canal to a size 30 file with

sufficient taper of the canal towards the coronal orifice in order to allow further

penetration of the irrigating solutions (212),

c) irrigation with 1% NaOCl, 17% EDTAC and 2% CHX solution,

d) intracanal medicaments consisting of 2% CHX gel or 2% CHX gel plus

calcium hydroxide, and

e) the use of AH 26, AH Plus or Grossman’s sealer with a well placed and

adapted coronal restoration.

Clindamycin offers no additional antimicrobial advantage over conventional root canal

medicaments such as calcium hydroxide and therefore it has not been recommended

for routine use in endodontic therapy (204). Dahlén et al. (208) reported that 26

isolates of E. faecalis and 3 isolates of E. faecium were found in 17 of 29 teeth that

had calcium hydroxide dressings and 4 of 29 teeth that had iodine tincture as root

canal dressings. All strains were resistant to metronidazole and most strains were

resistant to clindamycin.

In another study by Sedgley & Clewell (213), all E. faecalis isolates from oral rinse

samples except one were resistant to clindamycin and aminoglycosides. The LSA

gene is responsible for intrinsic resistance to lincosamides (such as clindamycin) and

streptogramins (214). Tetracycline resistance is present in at least 60 to 80% of

enterococcal isolates, even though these antibiotics are not routinely used to treat

enterococcal infections (54, 198). Tetracycline resistance has also been associated

with oral isolates of Prevotella intermedia and Prevotella nigrescens. Tsai et al. (215)

found 21% of the P.intermedia and 34% of the P. nigrescens isolates either showed

intermediate susceptibility or were resistant to tetracycline, with resistance carried by

the tet Q gene.

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Table 2.1 Antibiotic Resistance in Enterococci, based on several studies (54, 195, 196)

Antibiotic

Mechanisms of Resistance

Type of Resistance

β-Lactams

(e.g. penicillins, carbapenems, cephlosporins)

a) Overproduction of low-affinity penicillin binding proteins and/or decreased affinity for binding ß-Lactams

(NB: Intrinsic resistance to almost all cephalosporins)

b) β-Lactamase production

Intrinsic

Acquired

Tetracyclines

a) Ribosomal protection systems (Tet L,Tet M, Tet O genes)

b) Efflux pump

Acquired

Acquired

Lincosamides

(e.g. clindamycin)

a) Low level

b) High level (MLSb phenotype-methylation in 23S ribosomal RNA)

Intrinsic

Acquired

Macrolides

(e.g. eryhromycin, azithromycin, clarithromycin)

a) MLSb phenotype

b) Efflux pump

Acquired

Acquired

Streptogramin B

(e.g. quinupristin)

Streptogramin A

(e.g. dalfopristin)

a) MLSb phenotype

NB: Virtually all E. faecalis isolates are resistant

Acquired

Intrinsic

Aminoglycosides

(e.g. gentamycin, streptomycin, kanamycin)

a) Aminoglycoside modifying enzyme

(NB: Not effective as monotherapy needs addition of penicillin)

b) Low level (limiting transport of drug across cell membrane)

Acquired

Intrinsic

Glycopeptides

(e.g. vamcomycin, teicoplanin)

a) Van A phenotype

b) Van B phenotype(suseptible to teicoplanin)

Acquired

Acquired

Fluroquinolones

(e.g. moxifloxacin)

a) DNA gyrase(topoisomerase II,IV)

b) Efflux pump

Acquired

Acquired

Chloramphenicol

a) Chloramphenicol acetyltransferase enzyme

b) Efflux pump

(NB: 50% of enterococci are resistant to chloramphenicol)

Acquired

Acquired

Trimethoprim and

Sulfamethoxazole a) Chromosomal mutations in gene encodes

dihydrofolate reductase Acquired

Page 61

Bacterial resistance to tetracycline usually results in cross resistance to other

tetracyclines and this was observed by Byström et al. (59) where the strains resistant

to tetracycline were also resistant to doxycycline. In his thesis, Gulabivala (216) found

that Enterococcus, Staphylococcus and Lactobacillus species contributed the

majority of strains that were resistant and they also had the highest degree of multiple

antibiotic resistance. This was also noted by Noda et al. (217). Enterococcus strains

showed low susceptibility for azithromycin and erythromycin while clindamycin and

the cephalosporins were not clinically effective for Enterococcus spp (47).

Sublethal concentrations of antibiotics (e.g. tetracycline and chloramphenicol) can act

as inducers of multi-drug resistance (127). It is possible that during root canal

therapy, only sublethal doses may be in contact with the infecting organisms,

particularly in the narrow and more inaccessible parts of the canals (216).

CONCLUSIONS

• Calcium hydroxide may not be an ideal medicament for all cases with infected

root canal systems with or without apical periodontitis and in previously root-filled

teeth.

• Antibiotics may not be ideal as the active component for intracanal medicaments

• Corticosteroid antibiotic pastes are best suited in situations were pain control is

required.

• Combination of Calcium hydroxide and Ledermix paste is not synergistic.

• Current medicaments used in association with instrumentation and irrigation are

unlikely to predictably achieve a bacteria-free root canal system.

• Chlorhexidine may have promise as an intracanal medicament in teeth with

infected canals - both when there has not been any previous root canal filling, and

when there has been a previous root filling.

• More testing, involving intracanal medicaments and single/multiple species

biofilms combined with the effects of dentine, is required.

Page 62

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CHAPTER THREE Agar Diffusion Study

(Manuscript prepared for the International Dental Journal)

Title: An in vitro study of the anti-microbial activity of some endodontic medicaments and their bases using agar diffusion

Authors: Basil Athanassiadis

Postgraduate Student School of Dentistry University of Western Australia

Paul V. Abbott School of Dentistry University of Western Australia

Laurence J. Walsh School of Dentistry University of Queensland

Address for correspondence: Prof. Paul V. Abbott

School of Dentistry

University of Western Australia

17 Monash Avenue

NEDLANDS WA 6009 Australia

Phone: + 61 8 9346 7665

Fax: +61 8 9346 7666

E-mail: paul.v.abbott@uwa.edu.au

Short running title: Anti-microbial action of medicaments Keywords: Endodontics, medicaments, anti-microbial action

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Abstract

Aim: To determine the anti-microbial activities of various endodontic medicaments

and their bases against selected organisms using agar diffusion.

Materials and Methods: Well diffusion assays were used to test the anti-microbial

action of some commonly used endodontic medicaments (Ledermix paste, Pulpdent

paste, Ultracal paste, and a 50:50 mix of Ledermix and Pulpdent pastes) against

three bacteria (E. faecalis, P. micros, P. intermedia) and one yeast (C. albicans) and

the bases which included polyethylene glycol (PEG), PEG plus zinc oxide and

calcium chloride, and methyl cellulose. The zones of inhibition were recorded to

assess the relative antimicrobial actions. This assay system was repeated to

determine the effect of alkalinity on the antimicrobial activity of demeclocycline

hydrochloride. The pH of each preparation and component was also measured.

Results: The PEG base without any other active ingredients inhibited the growth of

P. intermedia but not the Gram-positive organisms (E. faecalis and P. micros).

Overall, the most resistant organism was P. micros. All of the commercial products

showed similar effects against E. faecalis. Pulpdent and Ultracal pastes had the

highest pH (12.0 and 12.1 respectively). The addition of calcium hydroxide to a

PEG/demeclocycline mix reduced its antimicrobial activity.

Conclusions: All the commercial products showed some anti-microbial activity in the

well diffusion assay, but those with PEG bases (i.e. Ledermix paste and the 50:50

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Ledermix/Pulpdent mixture) showed the greatest zones of inhibition in the agar

diffusion model. The addition of calcium hydroxide to Ledermix did not appear to be

synergistic in terms of activity against any of the organisms tested.

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Introduction

Micro-organisms play a central role in the development and progression of

pulpal and periapical diseases (Kakehashi et al. 1965). Anti-microbial medicaments

placed in the canals between appointments have been shown to significantly reduce

the number of organisms, and thereby increase the number of canals that are

rendered free of bacteria (Haapasalo et al. 2005). Rapid disinfection appears to be

impossible to achieve with medicaments alone, as the killing of micro-organisms

takes time even with powerful antimicrobial agents (Orstavik et al. 2003).

The major factor associated with unfavourable outcomes of endodontic

treatment is persistence of micro-organisms within the root canal system (Siqueira

2001). When this occurs, the microbial flora usually consists of only one species or a

small number of bacterial species. Gram-positive facultative anaerobic species

including enterococci, streptococci, lactobacilli and yeast-like organisms have been

reported in such cases, and it is noteworthy that these organisms have shown

resistance to the commonly-used medicaments such as calcium hydroxide [Ca(OH)2]

(Siqueira 2001, Chávez De Paz et al. 2003).

The aim of this study was to determine the relative anti-microbial effectiveness

of some commercially available medicaments and their bases against several

bacterial species commonly found in root canals, using a standardized assay system

to measure inhibition of bacterial growth.

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Materials and Methods

The root canal medicaments evaluated were a commercially-available

corticosteroid-antibiotic compound, Ledermix paste (Lederle Pharmaceuticals,

Wolfsratshausen, Germany), two commercially-available Ca(OH)2 pastes, Pulpdent

paste (Pulpdent Corporation of America, Watertown, MA, USA) and Ultracal paste

(Ultradent Products, South Jordan, UT, USA), plus a 50:50 mixture of Ledermix and

Pulpdent pastes. Because polyethylene glycol (400/3350) (also known as PEG)

(Huntsman, Victoria, Australia) is used as part of the base of Ledermix paste, the

anti-microbial action of this vehicle was tested with water and in combination with

zinc oxide, calcium chloride, triethanolamine and sodium sulphite, which together

make up the remainder of the base of Ledermix paste (Abbott 1999). The bases used

in the Pulpdent and Ultracal pastes consist mainly of water and methylcellulose,

hence a mixture of methylcellulose (Methocel A4M premium, Swift and Company,

Rosehill, NSW, Australia) and water was tested. The calcium hydroxide used to

replicate the commercial pastes was from Mallinckrodt Baker Inc (Phillipsburg, New

Jersey, USA).

The organisms tested were Enterococcus faecalis ATCC (American Type

Culture Collection) 29212, Peptostreptococcus micros ATCC 33270, Prevotella

intermedia wild strain, and Candida albicans ATCC14053. These organisms were

cultured on Brain Heart Infusion agar (BHI) (BBL 211065 Becton Dickinson, Sparks,

MD, USA) with added yeast extract, haematin, Vitamin K and 5% whole horse blood.

They were incubated at 35°C for 48 hours prior to testing the medicaments and their

base components. Purity of strains was checked by subculture of inocula, onto horse

blood agar for E. faecalis and C. albicans, and Brain Heart Infusion agar for

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Peptostreptococcus and Prevotella. Purity checks were performed at the time of

inoculation of test samples.

Well diffusion assays were undertaken in 90 mm diameter/4 mm deep Petri

dishes using Mueller Hinton Agar (BBL 211438 Becton Dickinson, Sparks, MD, USA)

for Enterococcus, Brain Heart Infusion Agar (BBL 211065 Becton Dickinson, Sparks,

MD, USA) for Peptostreptococcus and Prevotella, and Mueller Hinton agar for

Candida albicans. A 2 mL suspension of each test isolate was placed in 0.85% sterile

saline, and the turbidity was adjusted to a 0.5 McFarland standard for E. faecalis and

a 1.0 McFarland standard for Peptostreptococcus, Prevotella and Candida. The agar

plates were flooded with the test suspension and a well (4 mm deep and 7 mm in

diameter) was cut into the centre of the agar. A sterile pipette was used to place 500

μL of the test medicament or component into each well. The plates were then

incubated at 35°C for 48 hours in 5% CO2 for Enterococcus and Prevotella

specimens were cultured in anaerobic conditions achieved using a gas generating kit

(bioMerieux, Mitsubishi Gas Chemical Company Inc, Japan). Each test was carried

out five times. After incubation for 48 hours, the diameters of the zones of inhibition

were measured in millimetres to the nearest 0.1 mm using electronic callipers.

The pH of each medicament and component was measured in triplicate (to the

nearest 0.01 pH unit) using a pH meter with temperature compensation (Aqua-pH,

TPS, Brisbane, Australia). The meter was calibrated to known standard solutions of

pH 4 and 6.88, and the materials to be tested were placed in direct contact with the

sensor, and left in places for at least 30 seconds or until a stable pH reading was

obtained.

Page 92

The same procedures described above were used to measure the pH and

zones of inhibition in three preparations made to evaluate the effect of alkalinity on

the antimicrobial properties of demeclocycline hydrochloride (Sigma 6140-5G, St

Louis, USA). The three preparations consisted of a) PEG 400/3350, water and 3.2%

demeclocycline HCl, b) PEG 400/3350, water, 3.2% demeclocycline HCl and 35%

Ca(OH)2 , and c) PEG 400/3350, water, 3.2% demeclocycline HCl plus the inactive

components of Ledermix paste base (thereby replicating Ledermix paste but without

the corticosteroid).

The results were tabulated and data sets pooled from replicates examined for

their distribution using the Kolmogorov-Smirnov test (GraphPad Instat version 3.0

software, Palo Alto, California). As some groups showed non-normal distributions, all

data was examined using the Kruskal-Wallis (non- parametric) test, followed by

Dunn’s multiple comparison test, with significance set at the 5% level.

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Results

The mean zones of inhibition and the pH values for each preparation are

presented in Table 1. The range of inhibition values observed between the

preparations varied over a large range, and not all preparations exhibited anti-

microbial properties. Considering all the commercial products tested and their bases,

the largest zones of inhibition in the well diffusion assay were seen with Ledermix

paste and with the 50:50 mix of Ledermix and Pulpdent pastes. The PEG and water

combination showed a small inhibitory effect on P. intermedia, however the addition

of zinc oxide, calcium chloride, triethanolamine and sodium sulphite to PEG resulted

in growth inhibition of C. albicans as well.

.

All commercial products showed similar effectiveness against E. faecalis, but

none were active against Peptostreptococcus micros. This latter species was

therefore the most resistant organism to all medicaments tested. Ultracal and

Pulpdent pastes showed the least effectiveness of the commercial products tested,

with no inhibition of Peptostreptococcus micros or Prevotella intermedia.

Methylcellulose was not effective against any of the organisms tested when used

alone.

Both Pulpdent and Ultracal pastes had a similar pH of 12.6, while the

PEG/water and PEG/Ledermix base inactive components gave notably lower pH

readings. The addition of 35% calcium hydroxide to methylcellulose and water

resulted in a pH which was similar to that seen with the commercial products.

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Table 2 lists the mean zones of inhibition of varying dilutions of PEG 400/3350

combinations to P. intermedia done in duplicate. There was no antimicrobial activity

of PEG 400/3350 and its various dilutions to E. faecalis, P. micros and C. albicans,

however a graduated decrease was noted in its activity to P. intermedia as the

dilution factor increased. The PEG/water/demeclocycline combination resulted in the

greatest zone of inhibition. The addition of calcium hydroxide to this mix resulted in a

significant reduction in the zones of inhibition seen, except in the case of C. albicans.

Results of the statistical analyses pewrfromed are presented as an Appendix

to this Chapter.

Discussion

The bacterial species Peptostreptocosus micros and Prevotella intermedia

were chosen to represent organisms typically isolated from untreated infected root

canal systems (Sundqvist 1994, Gomes et al. 2004) whilst E. faecalis and C. albicans

were selected to represent organisms commonly isolated from the root canals of

teeth that have been previously root-filled (Siqueira 2001). Both E. faecalis and C.

albicans are known to be more resistant to endodontic therapy, and have been linked

to unfavourable treatment outcomes, as shown by Chu et al. (2005).

The agar diffusion test method employed in this study is useful for evaluating

the anti-microbial activity of medicaments before other more advanced tests are

performed. The results obtained from this in vitro test must be interpreted with

caution as this assay does not demonstrate the full clinical potential of the material

Page 95

being tested. In 2007, the Clinical and Laboratory Standards Institute (CLSI)

published a warning regarding the interpretation of broth dilution and agar dilution

studies. It stated that cephalosporins, aminoglycosides, clindamycin and

trimethoprim-sulfamethoxazole may appear active in vitro against Enterococcus spp.,

but they are not effective clinically, and isolates should not be reported as

susceptible. However, this point is not directly relevant to the present study as the

agents examined were not in these classes.

Resistance of E. faecalis, Peptostreptococcus spp., Fusobacteria spp.,

Propionibacterium spp., and some other species to doxycycline tetracycline and

minocycline have been reported (Antibiotic Guidelines 2003). Moreover, resistance of

Enterococcus and Peptostreptococcus to clindamycin is common. Both tetracycline

and clindamycin are bacteriostatic antibiotics (Martindale 1996, Siqueira et al. 2003,

Murray et al. 2007), and this characteristic may explain the inactivity of these

medicaments and their bases seen in the agar diffusion model for E. faecalis and P.

micros. This further highlights the point that antibiotics may not be the ideal agent for

use in intracanal medicaments.

The size of the inhibition zone seen in agar diffusion studies will depend upon

the solubility and diffusibility of the test substance in agar. The vehicles on which

these pastes are based may dramatically influence their chemical characteristics

(such as solubility and diffusion). Materials in water-based vehicles (i.e. distilled water

or saline solution) will have a higher rate of ionic dissociation in agar than those in

viscous vehicles (e.g. PEG) and oil-based vehicles (e.g. camphorated

paramonochlorophenol). As Estrela et al. (2001) reported, the vehicle plays a

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supportive role by giving pastes their chemical characteristics (i.e. dissociation and

diffusion) as well as the appropriate consistency for placement within, and removal

from, the root canal system. While agar diffusion tests, although aqueous in nature,

do not exactly mirror the anti-microbial effect of the medicaments within dentinal

tubules, they do allow direct comparisons of the ability of various agents to diffuse

through agar and inhibit the test organisms. Poorly diffusing materials will give very

small zones of inhibition, even if they are potent antimicrobial agents (Portenier et al.

2003). In addition, agar diffusion tests only show inhibition of growth, which may not

be the same as bacterial death (Haapasalo et al. 2005).

Substances that act by means of extreme values of pH, such as acids and

bases, may have their activities altered by the buffering effect of the environment.

Since calcium hydroxide has low solubility in aqueous media, it will diffuse slowly,

and thus an extended time will be needed to alkalinise a broad zone of the agar

medium to the point where microbial growth is inhibited. However, in the clinical

situation, the buffering actions of protein and ionic (phosphate and bicarbonate)

components of blood, tissue fluids and particularly of dentine itself may cause similar

effects which may reduce the action of calcium hydroxide (Siqueira & De Uzeda

1997). Since calcium hydroxide is known to have poor efficacy against E. faecalis in

agar plates, Basrani et al. (2003) compared the action of calcium hydroxide against

E. faecalis with that against S. mutans, an aciduric species which is known to be

killed readily by the high pH of calcium hydroxide. The growth of S. mutans was

inhibited, a finding which confirmed the ability of calcium hydroxide to provide

sufficient hydroxyl ions to diffuse through agar. On this basis, the observed failure of

calcium hydroxide in the current study to inhibit the growth of E. faecalis under the

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same conditions is likely to be a true reflection of its limited anti-microbial properties

against this particular organism, rather than merely an effect caused by limited

diffusion through agar.

An important finding in the present study was the inhibitory effect of PEG

against Prevotella intermedia, a species which was inhibited only by PEG and by

preparations based on PEG. The lack of inhibition of this organism has been reported

with calcium hydroxide preparations based on water and glycerine (such as Hy-Cal,

Root-cal and Hypo-cal) (Morrier et al. 2002), rather than those with PEG bases.

However the effect of PEG per se on endodontic pathogens has not hitherto been

recognized. The inhibitory effect of PEG against Gram-negative species is clearly an

advantage when used as a base for the formulation of endodontic medicaments.

PEG has been shown to exert anti-microbial effects by lowering the water content of

Gram-negative bacterial cells, causing plasmolysis, clumping and morphological

changes (Chirife et al. 1983). PEG 400 meets many of the physio-chemical

requirements for an ideal topical disinfectant – it is neutral, highly soluble in water,

non-irritating to the skin, does not decompose, and its toxicity from acute oral

administration is extremely low (Chirife et al. 1983). Currently, PEG 400 is used to

form the base for pastes such as Ledermix and Calen (a calcium hydroxide paste)

(SS White, Rio de Janeiro, Brazil). Its biocompatibility is demonstrated by its wide

use in cosmetic and medical preparations.

There are no studies reported in the literature that have examined the anti-

microbial properties of the individual components that make up the base for Ledermix

paste and the various calcium hydroxide pastes. The results of the current study

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indicate that PEG provides some anti-microbial activity against P.intermedia, whilst

methyl cellulose has no anti-microbial properties. In the study by Chirife et al. (1983),

PEG 400 showed significant antibacterial activity against Gram-negative bacteria,

including Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli

while Gram-positive bacteria such as Staphylococcus aureus were far more resistant.

This effect has also been demonstrated in the current study.

Taylor et al. (1989) showed that the 50:50 mixture of Ledermix and calcium

hydroxide pastes was marginally more effective than either paste alone at killing two

cariogenic micro-organisms, Lactobacillus casei and Streptococcus mutans.

However, this finding was not corroborated by Seow (1990), who reported that the

50:50 combination showed reduced activity against two different organisms,

Streptococcus sanguis and Staphylococcus aureus. The results presented in Table1

show that, when compared to Pulpdent paste, the addition of Ledermix paste to

Pulpdent increased the activity against P. intermedia only. This increased activity is in

fact most likely to be due to the carryover effect of the PEG 400/3350 and

demeclocycline HCl within the Ledermix paste. When compared to Ledermix paste

used alone, the combination showed improved activity against C. albicans.

Demeclocycline hydrochloride, as with other tetracyclines, is a yellow,

odourless, crystalline powder that darkens in strong sunlight and in moisture. The

potency of all tetracyclines is reduced in solutions having a pH below 2, and they are

rapidly destroyed in solutions of alkali hydroxides. A 1% demeclocycline HCl solution

in water has a pH of 2 to 3. Other properties of demeclocycline HCl include an

extreme affinity for binding to calcium ions, the greatest of all the tetracyclines

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(Martindales, 1996). The effect of pH on demeclocycline was investigated in this

study, and the results presented in Table 1 show a significant reduction in inhibition

zones when 35% calcium hydroxide was added to the PEG/demeclocycline HCl mix.

Thus, the addition of calcium hydroxide negated the effect of demeclocycline HCl, by

raising its pH. The PEG/water/demeclocycline mixture with a low pH (3.58) gave the

largest inhibition zones against P. micros and E. faecalis, and most of this can be

attributed to the low pH of the mix.

The pH of PEG and water was very similar to the pH of PEG combined with

all the inactive components found in the Ledermix paste base. The methylcellulose

base was alkaline, and hence the addition of calcium hydroxide gave it an even

higher pH. The maximum amount of calcium hydroxide, without any radiopaquing

agent, that can be added to water/methylcellulose or PEG before the mix becomes

too dry is approximately 55%. Between 15-25% of most medicaments is a

radiopaque agent, and therefore 35-40% would be the maximum amount of calcium

hydroxide that could be incorporated into either PEG or methylcellulose. The pH of

the 50:50 mix of Ledermix and Pulpdent pastes was found to be lowered from the

12.64 of Pulpdent paste alone to12.00. This was similar to the results reported by

Taylor et al. (1989), who found that mixing these two pastes marginally lowered the

pH from 12.8 to 12.5. Anderson and Seow (1990) also noted a small decrease in pH

from 12.18 to 12.06.

The zone of growth inhibition decreased as the PEG 400/3350 combination

was diluted with water. The greatest zone of inhibition occurred within the 0-10%

range of water dilution. It was also within this range that the combination remained in

Page 100

a set state. In the current study, E. faecalis, P. micros and C. albicans were not

affected by the PEG 400/3350 combination.

In summary, this agar diffusion study has shown the importance of the

composition of the paste bases used for endodontic medicaments. PEG alone

exerted a limited level of antimicrobial action, whilst methyl cellulose had no

antimicrobial action. The addition of a tetracycline to the PEG base (as in Ledermix

paste) improved the anti-microbial properties. The 50:50 mixture of Ledermix and

Pulpdent pastes showed no additive effects against most of the tested organisms

when compared to Ledermix paste alone, and this was confirmed by testing

demeclocycline HCl combined with calcium hydroxide in PEG. When the commercial

pastes were compared, Ledermix paste and the 50:50 combination with Pulpdent

paste were more effective than Pulpdent and Ultracal pastes used alone.

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Table 1: Means and standard deviations (in parenthesis) of the zones of inhibition

(in mm) after 48 hours for each organism and preparation. The pH value of

each preparation is also listed. Means and standard deviations were

calculated from the data obtained from five trials of each organism and

preparation.

Preparation E. faecalis Peptostrep.

micros

Prevotella

intermedia

Candida

albicans pH

PEG + water only  0.0 ( 0) 0.0 (0) 12.56 (0.39) 0.0 (0) 8.83 (0.05)

PEG + Ledermix paste

inactive components

(i.e. ZnO, CaCl, etc)

0.0 ( 0) 0.0 (0) 17.36 (3.36) 13.54 (0.60) 8.50 (0.01)

Methyl Cellulose 2% + water 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 9.64 (0.15)

Ledermix paste 9.58 (0.42) 0.0 (0) 37.84 (2.38) 12.4 (1.52) 7.63 (0.05)

Pulpdent paste 10.38 (0.59) 0.0 (0) 0.0 (0) 20.78 (1.13) 12.64 (0.03)

Ultracal paste 10.90 (1.19) 0.0 (0) 0.0 (0) 20.66 (1.92) 12.53 (0.09)

50:50 mix of Ledermix

+ Pulpdent pastes 10.8 (0.89) 0.0 (0) 35.76 (6.25) 19.45 (0.55) 12.00 (0.06)

PEG + water + demeclocycline HCl 3.2%

21.56 (0.46) 22.10 (1.41) 49.90 (1.50) 12.76 (1.61) 3.58 (0.03)

PEG + water + demeclocycline HCl 3.2% + inactives

12.20 (0.57) 11.94 (0.95) 60.8 (1.30) 12.66 (0.51) 7.52 (0.02)

PEG + water + demeclocycline HCl 3.2% + Ca(OH)2 35%

0.0 (0) 0.0 (0) 21.90 (1.19) 20.11(2.48) 11.47 (0.05)

Methylcellulose 2 % + water + Ca(OH)2 35% 0.0 (0) 0.0 (0) 0.0 (0) 0.0 (0) 12.43 (0.01)

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Table 2: Mean dose responses for PEG 400/3350 and the zone of inhibition

(in mm) after 48 hours when tested against Prevotella intermedia.

PEG 400/3350 Dilutions with water (%)

Prevotella

intermedia

0 14.5

5 14.3

10 13.7

20 12.1

40 0

60 0

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Appendix Table 1: Descriptive Data.

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Table 2: Kruskal-Wallis Test (Nonparametric ANOVA).

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Table 3: Dunn’s Multiple Comparisons Test. Comparing between groups.

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CHAPTER FOUR E. faecalis Biofilm Study

(Manuscript prepared for the International Endodontic Journal)

Title: An in vitro study of the anti-microbial activity of some endodontic medicaments and their bases to Enterococcus faecalis biofilms

Authors: Basil Athanassiadis

Postgraduate Student School of Dentistry University of Western Australia

Paul V. Abbott School of Dentistry University of Western Australia

Laurence J. Walsh School of Dentistry University of Queensland

Address for correspondence: Prof. Paul V. Abbott

School of Dentistry

University of Western Australia

17 Monash Avenue

NEDLANDS WA 6009 Australia

Phone: + 61 8 9346 7665

Fax: +61 8 9346 7666

E-mail: paul.v.abbott@uwa.edu.au

Short running title: Antimicrobial action on E. faecalis biofilms

Keywords: Endodontics, medicaments, E. faecalis biofilms

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Abstract

Aim: The aim of this study was to determine the anti-microbial activities of various

endodontic medicaments and their bases in eliminating Enterococcus faecalis

biofilms.

Materials and Methods: E. faecalis biofilms were induced on cellulose nitrate

membrane filters and the number of colony forming units (CFU) were determined at

designated time intervals of 1 day, 3 days, and 5 days. The medicaments tested

were: Ledermix paste, Pulpdent paste, a 50:50 mix of Ledermix and Pulpdent pastes

and a replica of Ledermix paste. Bases included polyethylene glycol (PEG), PEG with

zinc oxide, calcium chloride and the other components (inactives) that make up the

Ledermix paste base, and methyl cellulose with water. Serial dilutions were prepared,

and CFU were determined at each dilution. The bacterial survival index (BSI) of E.

faecalis was measured against each medicament and their respective bases.

Results: All of the bases and the Ledermix paste showed a very small reduction of

the BSI index for E. faecalis, whilst the reduction of the remaining two medicaments

varied. Pulpdent produced the greatest reduction, followed by the 50:50combination

of Ledermix and Pulpdent pastes.

Conclusion: None of the bases or Ledermix paste had any significant activity

against E. faecalis biofilms using the cellulose nitrate disc model. Pulpdent paste

produced the greatest reduction of E. faecalis growth, whilst the 50:50 mixture of

Ledermix and Pulpdent pastes was the second most effective of the medicaments

tested

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Introduction

Micro-organisms in the oral cavity generally do not exist as isolated single cells

in aqueous environments (planktonic forms) but rather exist as populations growing

at interfaces in moist environments as adherent biofilms. The most important features

of a biofilm are that it forms at interfaces, and that the bacteria within it display a

marked decrease in susceptibility to antibacterial agents and to host defence systems

compared to their planktonic counterparts (Mascaretti 2003).

Oral biofilms are complex and may comprise thirty or more bacterial species.

The adhesion of micro-organisms to a surface triggers an altered expression in a

large number of genes, and thus their phenotypes are changed. It is possible that

attachment gives rise to new phenotypes in dental biofilms, however other factors

such as the polysaccharide matrix, quorum sensing, genetic transfer of information,

growth phase of the bacteria, and location within the biofilm may also affect the

efficacy of medicaments against biofilms (Svensater & Bergenholtz 2004, Mascaretti

2003).

E. faecalis is occasionally found in untreated root canal infections, but it is

more often associated with previously root-filled teeth that have apical periodontitis

(Siqueira & Rôças 2005). Its prevalence in untreated root canal infections varies from

3.2%-13.3%, according to studies by Lana et al. (2001), Gomes et al. (2004), and

Siqueira et al. (2002). E. faecalis was significantly more often associated with

asymptomatic cases of primary endodontic infections than with symptomatic cases

(Rôças et al.2004). In cases of infected canals in previously root-filled teeth, E.

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faecalis was detected in up to 77% of the cases examined by Siqueira & Rôças

(2005).

E. faecalis is relatively easily destroyed in planktonic forms in vitro, but it

appears to become more resistant when it is in an infected root canal system. This

may be due to the activation of virulence factors, biofilm formation, or invasion of

dentinal tubules. It displays resistance to a wide range of antimicrobial agents

(Molander et al. 1998, Love 2001). Enterococci have both intrinsic and acquired

resistance to many antibiotics, and they are inherently more resistant to antimicrobial

drugs than other clinically important Gram-positive bacteria (Shepard & Gilmore,

2002). E. faecalis has intrinsic resistance to clindamycin, cephalosporins and

aminoglycosides (Gilmore, 2002). E. faecalis has also shown resistance to one of the

most widely used medicaments in endodontics, namely calcium hydroxide, especially

when tested in the presence of dentine (Haapasalo & Orstavik 1987).

The aim of this study was to evaluate the effectiveness of a series of

commercially available medicaments and their bases on E. faecalis within a biofilm

model.

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Materials and Methods

The root canal medicaments evaluated were a commercially-available

corticosteroid-antibiotic compound, Ledermix paste (Lederle Pharmaceuticals,

Wolfsratshausen, Germany), a commercially-available Ca(OH)2 paste, Pulpdent

paste (Pulpdent Corporation of America, Watertown, MA, USA) , and a 50:50 mix of

Ledermix and Pulpdent pastes. Polyethylene glycol (400/3350) (also known as PEG)

(Huntsman, Victoria, Australia) is used as part of the base of Ledermix paste, and

therefore the anti-microbial action of this vehicle was tested with water and in

combination with zinc oxide, calcium chloride, triethanolamine and sodium sulphite

which make up the remainder of the base of Ledermix paste (Abbott 1999). To

replicate Ledermix paste, a combination of PEG, demeclocycline HCl (Sigma 6140-

5G, St Louis, USA), and the inactive components of Ledermix paste was used. The

base used in Pulpdent consists mainly of water and methyl cellulose; hence a

combination of water and methyl cellulose (Methocel A4M premium, Swift and

Company, Rosehill, NSW, Australia) was used.

A single species biofilm was prepared using the test strain Enterococcus

faecalis ATCC 29212. This was subcultured onto Horse Blood Agar (HBA)

(bioMeriuex 04059, Brisbane, Australia) to check for purity and viability. Four to five

colonies of E. faecalis were cultured from the 24-hour HBA into Brain Heart Infusion

(BHI) (BBL 211059, Becton Dickinson, Sparks, Maryland, USA) and incubated at

35°C for 24 hours. The turbidity of the BHI culture was adjusted to a suspension

equivalent to a 0.5 McFarland standard. Three sterile 0.2 μm nitrocellulose

membrane filter disks (Whatman Ltd 7182-002, Maidstone, England) were placed

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onto a HBA plate. The testing employed three nitrocellulose filters per preparation in

order to obtain separate results for each of the time periods (1 day, 3 days, 5 days),

with a further three filters for the control biofilm. A 20 μl suspension of the broth was

placed onto the centre of each membrane, and the HBA plates incubated for 48

hours in air at 35°C. Purity of the test strain was checked at the time of inoculation of

the membranes by subculture onto horse blood agar. A 0.2 μm pore size was chosen

for the cellulose discs, since this would prevent bacteria penetrating to the

undersurface of the cellulose disc, but would allow sufficient nutrients to be absorbed

for the biofilm to grow. There was no biofilm growth detected on the underside of any

of the cellulose discs used in the study.

A 0.1 gram sample of the test material (weighed with a microbalance - Mettler

PC4400, Mettler Toledo, Mettler Instrument Company, Zurich, Switzerland) was

placed on the surface of a sterile 22 mm glass coverslip and this was then inverted

over one cellulose disk containing the biofilm so that the medicament was in direct

contact with the entire biofilm. Gentle pressure was applied to the coverslip so that

the medicament spread to cover the biofilm, but did not reach the edge of the filter.

The nutrient media was not removed from the cellulose disc/biofilm/medicament

combination as this would affect the survival of the E. faecalis biofilm, hence any

changes could not be totally attributed to the effects of the medicament.

Control discs were included which did not contain any medicament but were

covered with a sterile coverslip. Day 0 data was obtained prior to the addition of the

medicaments, but after the biofilms had been allowed to develop. The baseline (Day

0) count was determined by performing a quantitative count on biofilm membranes

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prior to addition of the coverslips. This value was then used as the baseline control

CFU count for subsequent determination of the bacterial survival indices (BSI).

At designated time intervals of 1 day, 3 days, and 5 days, the coverslip and

nitrocellulose biofilm were removed and placed into a sterile container with 10mL of

sterile distilled water. This was vortexed for one minute to disperse the bacteria, and

serial dilutions prepared to 10-6 in 2 mL Tryptone Soya Broth (TSB) (bioMerieux

04269, Brisbane, Australia) using 200 μl aliquots. A 100 μl sample of the inoculum

was placed on the centre of the HBA plate and spread across the surface for each

dilution (10-1 to 10-6 including the undiluted suspension). The plates were then

incubated for 24 hours at 35°C.

The colonies were counted for each dilution, and the number of CFU

determined using the following formula: Colonies x dilution x 100 = CFU per biofilm.

The Bacterial Survival Index (BSI) was determined by taking the CFU for each test

sample and dividing this by the CFU of the baseline control multiplied by 100, then

the log was taken (i.e. CFU test divided by CFU control X100- logg transformed). The

value thereby obtained was not the log of the counts but a log of the reduction in the

count of the test sample relative to the control biofilm tested after the same length of

incubation as the test. Hence the log reduction for untreated controls was consistently

2.0. The entire experiment was performed six times for each individual root canal

medicament and the respective base components.

The data from the individual experiments were collated and the distribution of

the data sets examined using the Kolmogorov-Smirnov test (GraphPad Instat

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software, version 3.0). Differences between groups were analysed using the Kruskal-

Wallis (non- parametric) test, with post-hoc tests using Dunn’s multiple comparison

test. Significance was set at the 5% level.

Results

Summary data for the bacterial survival indices for E. faecalis against the

various medicaments/bases over time is presented in Table 1. The results of the

statistical analyses are presented as an Appendix to this Chapter.

The overall trend for Pulpdent and the 50:50 combination of Ledermix and

Pulpdent pastes was for a reduction in viable bacterial cells over the 5 days. Pulpdent

showed a 1-2 log reduction at day 5. Ledermix paste and its replica showed no

significant reduction. The Pulpdent and Ledermix combination had a 1 log reduction.

None of the bases exerted a significant inhibitory effect. Thus, of the commercial

products tested, Pulpdent paste gave the greatest overall reduction of the BSI of

viable E. faecalis cells in this biofilm model.

Discussion

Bacterial biofilms are responsible for a wide range of dental diseases including

caries, periodontitis, root canal infections and osteomyelitis (Wilson 1996).

Knowledge of biofilm formation in root canals is limited, although it is clear that

biofilms may be present on the dentine walls of root canals and also on the external

Page 117

root surface of the apical portion of teeth with infected root canal systems (Svensater

& Bergenholtz, 2004). Biofilms produced in the laboratory setting are often less

adherent and less virulent than those found in nature, and this should be taken into

account when comparing in vitro-in vivo correlations (Thrower et al. 1997).

Appropriate agents can be used to prevent biofilm formation, disrupt existing biofilms,

prevent further biofilm growth or kill particular organisms in the biofilm. The latter two

roles are generally undertaken by antimicrobial agents.

The membrane filter-based model offers a simple means of assessing the

susceptibility of oral bacterial biofilms to antimicrobial agents (Wilson 1996). This

model does not account for anatomical variations seen in the root canal environment

but it does allow for a number of agents or variables to be tested for activity under

reproducible conditions. Hence this method can be useful as a rapid primary screen

to test the antimicrobial effect against biofilms. Using a biofilm model is more

representative of the clinical situation than traditional testing methods using cultures

growing on agar plates (Spratt et al. 2001).

E. faecalis is a normal intestinal organism which may inhabit the oral cavity

and gingival sulcus. However, a subset of this species appears to be particularly

pathogenic, having acquired a number of genes conferring infectivity and virulence,

including resistance to multiple antibiotics. It is unclear whether E. faecalis is the

major pathogen associated with endodontic treatment failures, or whether it is merely

a survivor that can take advantage of its ability to endure adverse conditions within

filled root canals (Kaufman et al. 2005, Möller et al. 2004).

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There are several mechanisms of resistance of bacterial biofilms to antibiotics.

These include the ability of the polysaccharide matrix to retard diffusion of antibiotics,

quorum sensing, transfer of antibiotic and virulence traits, inability to penetrate deep

into the biofilm, and the inability to affect bacteria which are slow growing and in a

lower metabolic state (Athanassiaids et al. 2007). The inability of antibiotics to

penetrate biofilms was shown by Lima et al. (2001), where 2% clindamycin allowed

the overgrowth of E. faecalis biofilms in the cellulose–nitrate membrane filter model,

whereas combinations of 2% clindamycin and 10% metronidazole have very small

reductions in viable bacterial cell counts. Takahashi et al. (2006) showed that

tetracycline HCl, minocycline HCl, doxycycline hyclate and ofloxacin had no effect on

three out of the four strains of P. intermedia tested in the same biofilm model. Distel

et al. (2002) reported that when single-rooted human teeth were left empty with no

medication, total colonization of the root canals by E. faecalis occurred after two days

incubation whilst the canals medicated with Pulpdent paste showed total colonization

after 77 days.

Abdullah et al. (2005) showed that 100% bacterial reduction of E. faecalis

biofilms on cellulose nitrate filters was achieved with 3% sodium hypochlorite

(NaOCl) for 2 minutes and 10% povidone iodine for 30 minutes. In comparison, a

saturated aqueous calcium hydroxide solution achieved a 7 x log10 reduction, and a

0.2% chlorhexidine gluconate solution resulted in a 5 x log10 reduction, after 60

minutes. These differences may be a result of the limited ability of these various

materials to penetrate into the exopolysaccharide matrix of biofilms.

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The biofilm model used in this study has produced different results to those

reported using the agar diffusion model, which showed a similar zone of inhibition for

E. faecalis with each of the same commercial products tested in this study

(Athanassiadis et al. 2007a).

An explanation for the greater effectiveness of Ca(OH)2 pastes in the biofilm

model may be due to the ability of hydroxyl ions to penetrating the biofilm, compared

to antibiotics with their larger and more complex molecular structures. Antibiotics will

also not be as effective as biocides due to the various mechanisms of resistance of

bacterial biofilms outlined above.

In summary, this study has shown that Pulpdent paste had the greatest activity

against mono-species E. faecalis biofilms using the cellulose-nitrate disc model. The

50:50 combination of Ledermix and Pulpdent pastes also significantly impaired

bacterial growth, whilst the bases, Ledermix paste and its replica did not cause

significant reductions in the number of viable bacterial cells.

Page 120

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Endodontics. The University of Western Australia, Perth. 61.

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Enterococcus faecalis phenotypes to root canal medications. Journal of Endodontics

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activity of some endodontic medicaments and their bases using agar diffusion.

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antibiotics, biocides and phenol compounds as antimicrobial agents in endodontics.

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Distel JW, Hatton JF, Gillespie MJ (2002) Biofilm formation in medicated root canals.

Journal of Endodontics 28, 689-693.

Gilmore MS (2002) The Enterococci: pathogenesis, molecular biology, and antibiotic

resistance. American Society of Microbiology Press (Text), Washington, USA. 114-

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TeixeiraFB, Souza-Filho FJ (2004) Microbiological examination of infected dental root

canals. Oral Microbiology and Immunology 19, 71-76.

Haapasalo M, Orstravik D (1987). In vitro infection and disinfection of dentinal

tubules. Journal of Dental Research 66, 1375-1379.

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endodontically treated teeth with and without periradicular lesions. Journal of

Endodontics 31, 851-856.

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Lana MA, Ribeiro-Sobrinho AP, Stehling R, Garcia GD, Silva BK, Hamdan JS, Nicoli

JR, Carvalho MA, Farias LdeM (2001) Micro-organisms isolated from root canals

presenting necrotic pulp and their drug susceptibility in vitro. Oral Microbiology and

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biofilms to some antimicrobial medications. Journal of Endodontics 27, 616-619.

Love MR (2001) Enterococcus faecalis - a mechanism for its role in endodontic

failure. International Endodontic Journal 34, 399-405.

Mascaretti OA (2003) Bacteria versus antibacterial agents: an integrated approach.

American Society of Microbiology Press (Text). Washington, USA. 40.

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apical periodontitis. International Endodontic Journal 31, 1-7.

Möller AJ, Fabricius L, Dahlén G, Sundqvist G, Happonen RP (2004) Apical

periodontitis development and bacterial response to endodontic treatment.

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with different forms of periradicular diseases. Journal of Endodontics 30, 315-320.

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Siqueira JF, Rôças IN, Souto R, de Uzeda M, Colombo AP (2002) Actinomyces

species, streptococci, and Enterococcus faecalis in primary root canal infections.

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Topics 9, 27-36.

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46, 425-429.

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Table 1: Bacterial survival index of viable E. faecalis cells for the various

medicaments and their bases. Data show medians and upper/lower

95% confidence limits from six trials. Baseline CFU at day zero in all cases

was 100 (log 2.00).

* indicates significant reduction (P<0.05) compared with the baseline.

Medicament Day 1 Day 3 Day 5

PEG + water 1.75 (1.57-1.91) 1.95 (1.62-2.02) 1.71 (1.61-1.81)

PEG + inactives 1.78 (1.69-1.86) 1.95 (1.75-2.14) 1.95 (1.63-2.12)

PEG + Demeclocycline + inactives 1.74 (1.49-1.88) 1.84 (1.75-1.91) 1.89 (1.84-1.94)

Methyl Cellulose + water 1.89 (1.82-1.98) 1.72 (1.45-2.07) 1.84 (1.71-2.12)

Pulpdent 1.39 (1.03-1.67) * 1.52 (1.34-1.75) 0.92 (0.52-1.17) *

Pulpdent + Ledermix 1.56 (1.43-1.66) * 1.26 (0.95-1.58) * 1.19 (0.67-1.69)

Ledermix 1.86 (1.73-2.01) 1.85 (1.66-2.16) 1.94 (1.77-2.15)

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Figure 1: Bacterial survival index of viable E. faecalis cells for the various

medicaments and their bases. Data show medians and upper/lower

95% confidence limits from six trials.

Page 125

Appendix Table1: Means of the BSI of viable E. faecalis for the medicaments and their bases.

Preparation Day 1 Day 3 Day 5

Ledermix 1.871 1.908 1.957

Pulpdent 1.348 1.542 0.8497

Ledermix/Pulpdent 1.546 1.262 1.182

PEG/water 1.742 1.82 1.711

PEG/base 1.775 1.946 1.875

Methyl Cellulose + water 1.897 1.761 1.915

PEG/demeclocycline + inactives 1.689 1.833 1.891

Preparation Day 1 Day 3 Day 5

Ledermix 1.871 1.908 1.957

Pulpdent 1.348 1.542 0.8497

Ledermix/Pulpdent 1.546 1.262 1.182

PEG/water 1.742 1.82 1.711

PEG/base 1.775 1.946 1.875

Methyl Cellulose + water 1.897 1.761 1.915

PEG/demeclocycline + inactives 1.689 1.833 1.891

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Table 2: Standard deviations of the BSI of viable E. faecalis for the medicaments

and their bases.

Preparation  Day 1 Day 3 Day 5

Ledermix 0.1366 0.2413 0.1808

Pulpdent 0.3486 0.2212 0.352

Ledermix/Pulpdent 0.1087 0.2997 0.4815

PEG/water 0.1826 0.2203 0.1056

PEG/base 0.09055 0.2063 0.2628

Methyl Cellulose + water 0.07645 0.2952 0.1948

PEG/demeclocycline + inactives 0.1856 0.07497 0.04461

Table 3: Medians of the BSI of viable E. faecalis for the medicaments and their bases.

Preparation Day 1 Day 3 Day 5

Ledermix 1.86 1.848 1.935

Pulpdent 1.398 1.523 0.9208

Ledermix/Pulpdent 1.562 1.261 1.194

PEG/water 1.745 1.949 1.711

PEG/base 1.778 1.949 1.954

Methyl Cellulose + water 1.893 1.716 1.837

PEG/demeclocycline + inactives 1.737 1.835 1.889

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Table 4: 95% lower confidence limits of BSI of viable E.faecalis for the medicaments and their bases.

Preparation Day 1 Day 3 Day 5

Ledermix 1.727 1.655 1.767

Pulpdent 1.025 1.338 0.5241

Ledermix/Pulpdent 1.432 0.9471 0.6763

PEG/water 1.573 1.616 1.614

PEG/base 1.691 1.755 1.632

Methyl Cellulose + water 1.817 1.451 1.711

PEG + demeclocycline + inactives 1.494 1.754 1.844

Table 5: 95% upper confidence limits of BSI of viable E.faecalis for the medicaments and their bases.

Preparation Day 1 Day 3 Day 5

Ledermix 2.014 2.161 2.146

Pulpdent 1.67 1.747 1.175

Ledermix/Pulpdent 1.66 1.576 1.687

PEG/water 1.911 2.024 1.809

PEG/base 1.859 2.137 2.118

Methyl Cellulose + water 1.977 2.071 2.12

PEG + demeclocycline + inactives 1.884 1.912 1.937

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Table 6: Day 1 - Descriptive Statistics

Page 129

Table 7: Day 1 - Kruskal- Wallis Test (Nonparametric ANOVA). Comparison

between all groups.

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Table 8: Day 1 - Dunn’s Multiple Comparison Test. Comparison between groups.

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Table 9: Day 3 - Descriptive Data.

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Table 10: Day 3 - Kruskal- Wallis Test (Nonparametric ANOVA).

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Table 11: Day 3 - Dunn’s Multiple Comparison Test. Comparison between groups.

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Table 12: Day 5 - Descriptive Data.

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Table 13: Day 5 - Kruskal- Wallis Test (Nonparametric ANOVA).

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Table 14: Day 5 - Dunn’s Multiple Comparison Test. Comparison between groups.

Page 137

CHAPTER FIVE Medicament Inactivation Study (Manuscript prepared for the International Dental Journal)

Title: An in vitro study of the inactivation of endodontic medicaments and their bases by dentine against E. faecalis Authors: Basil Athanassiadis

Postgraduate Student School of Dentistry University of Western Australia

Paul V. Abbott School of Dentistry University of Western Australia

Laurence J. Walsh School of Dentistry University of Queensland

Narelle George Medical Scientist, Division of Microbiology Royal Brisbane Hospital

Address for correspondence: Prof. Paul V. Abbott

School of Dentistry

University of Western Australia

17 Monash Avenue

NEDLANDS WA 6009 Australia

Phone: + 61 8 9346 7665

Fax: +61 8 9346 7666

E-mail: paul.v.abbott@uwa.edu.au Short running title: Medicament inactivation by dentine Keywords: Endodontics, medicaments, diffusion

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Abstract

Aim: To investigate the antimicrobial effect of endodontic medicaments and their

bases in the presence of dentine and to evaluate the extent of inactivation by the

dentine.

Materials and Methods: The medicaments tested were Ledermix paste, Pulpdent

paste, 50:50 combination of the Pulpdent and Ledermix, their bases and a saturated

Ca(OH)2 solution. The test organism was E. faecalis ATCC 29212 and the dentine

(25-50 μm diameter particles mixed with water) was used at a concentration of 28 mg

per 50 μl. Aliquots of the dentine/medicament suspension were incubated at 35°C for

1 hour before adding E. faecalis (pre-incubation group). Bacteria were also added

simultaneously with the dentine/medicament suspension (no pre-incubation group).

Serial dilutions were done at 1 hr, 1 day, and 3 days, and the colonies were counted.

The pH was measured for the saturated Ca(OH)2 solution and Pulpdent paste alone,

with dentine, and after 1 hr incubation with dentine.

Results: Except for PEG + inactives, the medicament bases were ineffective in

reducing the bacterial survival index of E. faecalis significantly, whilst the saturated

Ca(OH)2 solution resulted in total elimination of E. faecalis only in the absence of

dentine. The effect of Pulpdent and 50:50 mixture of Pulpdent/Ledermix pastes was

total elimination of E. faecalis, irrespective of whether dentine was present or absent,

or if there was a pre-incubation period or not. Ledermix was the least effective of the

commercial products tested.

Conclusion: Ca(OH)2-containing medicaments produced the greatest reduction of

E. faecalis growth in this model.

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Introduction

The aim of endodontic treatment of teeth with infected root canal systems is to

achieve healing of the periapical tissues. Ideally, there should not be any apical

periodontitis following endodontic treatment, not only as observed clinically and

radiographically, but also if the periapical tissues could be examined histologically.

Apical periodontitis associated with root-filled teeth has been reported to be present

in 44%-65% of cases in several different studies and some of the causes of this

inflammation included persistent bacteria remaining in inaccessible areas in the

apical third of the root canal, extra-radicular infections and poor quality root canal

treatment (Wu et al. 2006).

The ability of medicaments to diffuse through dentinal tubules to reach the peri-

radicular and periapical tissues at an adequate therapeutic concentration is essential

to eliminate microbes from both intra-radicular and extra-radicular sites. None of the

medicaments currently used in endodontics appear to have this property. Agar

diffusion and biofilm testing do not account for the buffering effects of dentine on the

medicaments placed in canals and hence Haapasalo et al. (2000) developed a model

to evaluate these properties. This model involved using crushed dentine (25-50 μm

diameter particles) and premixing the dentine with the medicament for 1 hour

followed by the addition of bacteria (E. faecalis). A saturated Ca(OH)2 solution was

used, which was achieved by dissolving Ca(OH)2 powder in distilled water at room

temperature until a solid phase occurred at the bottom. The liquid phase was then

used as the medicament in the tests.

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Calcium hydroxide [Ca(OH)2 ] has been used extensively as an inter-

appointment endodontic medicament. The antibacterial action of Ca(OH)2 is based

upon its ability to maintain an elevated pH due to the slow release of hydroxyl ions.

Its efficacy depends on the ability to maintain these hydroxyl ions in the dentinal

tubules which is related to the type of vehicle in which the Ca(OH)2 is delivered

(Robert et al. 2005). Nerwich et al. (1993) showed that dentine has the capacity to

buffer hydroxyl ions as these ions diffuse through the dentine and that a 2-3 week

period is required for peak levels to be reached in the outer root dentine near the

cementum.

Abbott et al. (1990) showed that the concentration of the antibiotic component of

Ledermix paste (demeclocycline) in the outer dentine would be unlikely to inactivate

most of the bacteria involved in a root canal infection. They advocated using a 50:50

mix of Ledermix paste and Pulpdent paste to overcome the limitations of the

individual pastes but, in another study (Abbott et al. 1989), significantly less

demeclocycline reached the periodontal tissues and therefore its antibacterial

potential would be no better than other pastes even though the addition of Ca(OH)2

was synergistic.

The aims of this study were to investigate the antimicrobial effect of some

current medicaments and their bases on E. faecalis in the presence/absence of

dentine.

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Materials and Methods

Twelve extracted human molar and premolar teeth had their crowns removed

using a high speed diamond bur. The working length of each canal was determined

visually by inserting a size 10 K-File (Dentsply/Maillefer, Ballaigues, Switzerland)

until it could be seen at the apical foramen and then subtracting 0.5mm. In order to

reduce the organic load within the root canals, the orifice and the cervical portion of

each canal was enlarged using sizes 2, 3 and 4 Gates-Glidden drills

(Dentsply/Maillefer, Ballaigues, Switzerland) followed by irrigation with 3 mL of 1%

sodium hypochlorite (NaOCl). The canals were then prepared with Profile files

(Dentsply/Maillefer, Ballaigues, Switzerland), lubricated with Glyde

(Dentsply/Maillefer, Ballaigues, Switzerland). Initially, a size 40 0.06 taper file was

used followed by a size 35 0.04 taper file inserted as close as possible to the working

length. The canals were irrigated with 3 mL of NaOCl and 1 mL 17%

ethylenediaminetetraacetic acid with cetrimide (EDTAC) between the use of each file.

After the canals were fully prepared, they were thoroughly rinsed several times with

distilled water to remove all traces of NaOCl and EDTAC.

The roots were crushed with two machines into a fine powder, which produced

6.6 grams of dentine. The first machine used a chrome steel mill (Rocklabs, New

Zealand) sitting on a swing mill (Tema, Germany) to reduce the particle size to 50-

100 μm over a period of 2 minutes. The second machine was a micronizing mill

(McCrone Scientific, UK) which used agate beads with ethanol to further reduce the

particle size to approximately 25-50 μm for a period of 6 minutes. The powder was

dried overnight in a drying oven at 60°C (Townson and Mercer, Australia) and then

autoclaved. The effect of autoclaving dentine before or after being crushed was

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examined hence the above procedure was repeated but with the teeth being

autoclaved first.

The root canal medicaments evaluated were a commercially-available

corticosteroid-antibiotic compound, Ledermix paste (Lederle Pharmaceuticals,

Wolfsratshausen, Germany), a commercially-available Ca(OH)2 paste, Pulpdent

paste (Pulpdent Corporation of America, Watertown, MA, USA), and a 50:50 mix of

Ledermix and Pulpdent pastes. Polyethylene glycol (400/3350) (also known as PEG)

(Huntsman, Victoria, Australia) is used as part of the base of Ledermix paste and

therefore the anti-microbial action of this vehicle was tested with water and in

combination with zinc oxide, calcium chloride, EDTA, triethanolamine, sodium

bisulphite and Germaben IIE which make up the remainder of the Ledermix paste

base (Abbott 1999). The base used in Pulpdent paste consists mainly of water and

methyl cellulose. Hence, methyl cellulose (Methocel A4M premium, Swift and

Company, Rosehill, NSW, Australia) was tested alone. The medicaments and their

bases were warmed to room temperature prior to use and placed into a sterile petri

dish from which 50 μL was used for testing.

Several colonies from an overnight Horse Blood Agar (HBA) (bioMeriuex

04297, Brisbane, Australia) culture of Enterococcus faecalis ATCC 29212 was

emulsified in Tryptone Soy Broth and the turbidity adjusted to that of a 0.5 McFarland

standard (1.5 x 108 CFU/mL) using a nephelometer. Purity of test strain was checked

by subculture of inoculum onto HBA for E. faecalis. Purity checks were performed at

the time of inoculation of the tubes. This suspension was used as the test inoculum.

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Dentine powder (0.56 grams) was aseptically dispensed into a sterile tube and

1mL of sterile water was added to give a final concentration of 28 mg per 50 μL (0.56

g/mL). The solution was vortexed to ensure that the dentine powder had been evenly

mixed through the solution, even though all the powder did not dissolve.

The pH of the saturated Ca(OH)2 solution was measured in duplicate to the

nearest 0.01 pH unit using a pH meter (model Q1416, Dick Smith Electronics, North

Ryde). The meter was calibrated to a known standard solution of pH 7.0 and the

material tested was placed in direct contact with the sensor. The pH was then also

measured when this solution was combined with dentine (25 mg/50 μL) after 2

minutes, and again after 1 hour, keeping the mixture at room temperature. This

process was then repeated using Pulpdent, and Pulpdent plus dentine (25 mg/50 μL)

after 2 minutes and 1 hr incubation.

In order to measure the bacterial growth, two tubes were prepared for each

medicament, which contained 50 μL of the test material. To one tube, 50 μL of sterile

saline was added, and to the other 50 μL of dentine solution was added. There were

two negative controls (non-dentine and dentine) for each batch of tests and these

were prepared at the same time. The non-dentine control consisted of 100 μL saline

only to which 50 μL of bacteria was added after 1 hour .The dentine control

consisted of a second tube containing 50 μL sterile saline and 50 μL dentine

solution which were pre-incubated for 1 hour after which 50 μL of bacteria was

added. Tubes were vortexed to mix both suspensions. The same controls were used

for the no pre-incubation group but without a 1 hour pre-incubation period. The

mixture was vortexed again and 10 μL of the test mix was removed and pipetted into

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1 mL of sterile tryptone soy broth from which serial dilution prepared of the test mix

from 10-1 to 10-6 using tryptone soy broth. Then 100 μL of each dilution was cultured

onto a Horse blood agar plate. All plates were incubated at 35°C for 24 hours, after

which the colonies were counted. The count obtained represented the initial colony

count at zero time. The above test mixes were re-incubated at 35°C for 1 hour, 24

hours and 3 days. The number of colony forming units was determined by the

following formula: Colonies x dilution x 100 = CFU per film. The bacterial survival

index was determined by the CFU per medicament/base tested divided by the CFU

of the control (dentine or non- dentine control) x 100 and log transformed.

The sampling process and the quantitative counts outlined above were

repeated for each of the test mixes and for the dentine and organism controls. The

experiment employed five replicates per group. Bacterial survival indices were

calculated and expressed as the median with 95% confidence limits. Differences

between groups were assessed using the non-parametric Kruskall-Wallis test, with

post-hoc Dunn’s multiple comparison tests.

Results

The bacterial survival indices (BSI) of E. faecalis against the medicaments and

their bases with a pre-incubation period (1 hr) and in the presence/absence of

dentine are shown in Tables 1-3. Similar results were obtained when no pre-

incubation of the medicaments and bases occurred (not shown). The bases (PEG +

water, Methyl Cellulose + water) had no effect on survival of E. faecalis, whilst PEG

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with the other inactive components resulted in a steady and significant decrease in

BSI over the 3 days. Medicaments containing calcium hydroxide showed total

inhibition of the growth of E. faecalis within one hour. Ledermix paste when used

alone was effective, but required 3 days before a significant difference was observed.

Pulpdent paste and the 50:50 combination of Ledermix and Pulpdent were effective

within 1 hour. The pre-incubation period made no difference to the results, nor did the

presence or absence of dentine. Of note, in the two separate series of experiments,

there was no difference for dentine prepared by autoclaving then powdering (Table

2), versus the converse (Table 1).

Table 3 shows the pH values of the various materials and the effect of adding

dentine. These results clearly show that the initially high pH of the saturated Ca(OH)2

solution (12.25) was immediately reduced when dentine was added, with a further

reduction occurring over the following hour. In contrast, addition of dentine did not

affect the pH of Pulpdent paste.

Discussion

In order to completely disinfect dentine, endodontic medicaments must resist

inactivation by dentine, which occurs because of its various organic and inorganic

components, and by the microbes within the dentinal tubules. The space-filling ability

of Ca(OH)2 may be important in retarding root canal recontamination. Despite the

vehicle used to deliver it, Ca(OH)2 has the ability to act as a physical barrier,

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withholding substrate for growth as well as limiting space for bacterial multiplication

(Siqueira & Lopes 1999).

The vehicle components affect diffusion of the medicament through the

dentinal tubules. Water soluble vehicles such as methylcellulose (e.g. Pulpdent,

Ultracal) result in rapid ion release and they will require more frequent appointments

to replace the medicament compared to viscous vehicles (e.g. Calen, Ledermix

paste) (Fava & Saunders 1999). However, even the water soluble preparations would

not be expected to have as high a bio-availability of hydroxyl and calcium ions as a

saturated solution of calcium hydroxide. This point is relevant clinically since pure

saturated solutions of calcium hydroxide would be difficult to use as medicaments,

but may form over time around calcium hydroxide pastes when these interact with

aqueous fluids.

Chemical changes over time may reduce the effectiveness of calcium

hydroxide as an antimicrobial agent. For example, if dissolved CO2 is present in

water or saline, rapid carbonation of Ca(OH)2 may occur, resulting in largely insoluble

calcium carbonate (which is a weaker alkali) and water as reaction products. Within

the root canal system, CO2 may be generated by tissue decomposition (Simon et al.

1995). The problem of carbonation may be less with other (non-aqueous) vehicles

and in such cases the pH should stabilize eventually to a high value, once initial

reactions involving an excess of hydroxyl ions have terminated. In a study by

Camoes et al. (2003), the pH of Ca(OH)2 medicaments with PEG or glycerine

vehicles showed a decline in pH during the first 8 days after filling the canals but then

the pH rose again up to 42 days.

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The notion of instant clearing of microbial life is unfounded and there is no

shortcut to achieving effective disinfection. Killing of bacteria takes time, even with

the strongest of antimicrobial substances (Orstavik 2003). In primary apical

periodontitis, the great majority of microbes occupy the main root canal with some

invasion of dentinal tubules, and less of the cemental surface and periapical tissues.

Only some regions of the canal walls are cleaned by mechanical instrumentation and

irrigation, and the dentine in these areas would therefore be exposed to

medicaments at their full strength. In a perfectly clean region, the main concern

would be buffering of pH by dentine proteins as the hydroxyl ions interact with them.

In other areas within the root canal system, such as the apical third and dentinal

tubules, where removal of debris is often incomplete, the effective strength of the

medicament may be reduced even further because of its interactions with organic

debris, biofilms and inflammatory exudate (Haapasalo et al. 2007). Such buffering,

inactivation and dilution effects would be expected to occur with calcium hydroxide,

and this was seen in the present study in a dramatic way with the saturated calcium

hydroxide solution when dentine powder was added.

A saturated Ca(OH)2 solution has been shown to be unable to kill E. faecalis in

the presence of either dentine, hydroxyapatite and bovine serum albumin and this

inhibitory effect was maintained even when only one-tenth of the dentine was used

(Haapasalo et al. 2000, Portenier et al. 2000, 2001). Waltimo et al. (1999) also

showed that in vitro (filter paper and broth dilution) Candida species are more

resistant to a saturated Ca(OH)2 solution than E. faecalis. The use of the saturated

Ca(OH)2 solution in the above studies does not seem to reflect the concentration of

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Ca(OH)2 that would be present clinically especially in the main root canal, but it

would reflect deeper areas within the root canal system and dentinal tubules. The

pre-incubation of the medicament and dentine also does not appear to be

representative of what happens in a root canal where the bacteria have colonized the

tubules for varying periods of time before the medicament is added.

The results of the saturated Ca(OH)2 solution in pre-incubation with dentine

replicate those of the Haapasalo et al. (2000) model with a 3 log 10 reduction or

greater against E. faecalis without dentine and total inhibition of the saturated

Ca(OH)2 solution with dentine. The current study also included Pulpdent paste,

Ledermix paste, a 50:50 combination of these two pastes, and their bases. Pulpdent

and the 50:50 Pulpdent/Ledermix combination resulted in elimination of E. faecalis

beyond detectible limits (> 5 log 10 reduction) irrespective of the presence/absence

of dentine or the presence/absence of a pre-incubation period. This total elimination

of E. faecalis occurred within 1 hour, and continued over 3 days. One of the bases

(PEG + inactives) showed significant reduction in the BSI at 3 days whilst the other

two showed no reduction against E. faecalis, irrespective of the conditions examined.

This reduction may be due to the presence of zinc within the inactive components.

Pulpdent (pre-incubation 1 hr plus dentine) diluted 1:5 still achieved total

elimination of E. faecalis within 1 hour, but at a dilution of 1:100 in distilled water the

antibacterial effect was reduced (results not shown). Ledermix paste took 3 days

before a significant reduction in BSI of E. faecalis occurred. A possible explanation

for the different results with saturated Ca(OH)2 solution could be that the hydroxyl

ions are buffered by the dentine and hence are inhibited in their action, as proposed

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by Haapasalo et al. (2000). In the present study, the pH for a saturated Ca(OH)2

solution drops with the addition of dentine powder from 12.25 to 8.70, whilst in the

commercial product (Pulpdent paste) no such change was seen. This empirical

finding confirms the prediction of Haapasalo et al. (2000). The lack of significant pH

changes in Pulpdent after addition of dentine powder could be due to several causes

including the lower bio-availability of hydroxyl ions because of diffusion through a

more viscous material, the reservoir effect of undissolved calcium hydroxide

providing a gross excess of hydroxyl ions, reduced solubility or destabilization of the

dentine proteins which were added to the mixture (“salting out”), or other protein-

water-ion effects involving kosmotrope/chaotrope interactions.

If the conditions present in the main canal of a tooth are considered, the

results of this study combined with the biofilm study by Athanassiadis et al. (2007b)

indicate that Pulpdent paste or the 50:50 combination of Ledermix/Pulpdent pastes

are the most effective against biofilms and the effects of dentine. The biofilm study

also showed that antibiotic containing pastes such as Ledermix and the bases of the

medicaments tested have minimal effect on biofilms. In this study, Ledermix paste

took 3 days to overcome the buffering effects of dentine compared to Pulpdent paste

which took only 1 hour to totally eliminate E. faecalis in the presence of dentine. This

study has shown that Pulpdent paste can tolerate a certain amount of buffering by

dentine and still be effective against E. faecalis whilst saturated Ca(OH)2 solution is

ineffective.

In summary, this study has shown that Pulpdent paste and the 50:50

Pulpdent/Ledermix combination had the greatest activity against E. faecalis,

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irrespective of the presence or absence of dentine or the presence or absence of a

one hour pre-incubation period. Ledermix paste was the least effective of the

medicaments tested. These results are similar to the conclusions reached in the

biofilm study by Athanassiadis et al. (2007b).

The agar diffusion study should be used as an initial screening tool with the

biofilm and dentine inactivation studies being more clinically relevant. The agar

diffusion study Athanassiadis et al. (2007a) showed that the 50:50 combination of

Ledermix and Pulpdent pastes was not additive, PEG as a base may have some

limited antibacterial activity, and that the inactive components of Ledermix paste may

have some limited antibacterial and antifungal properties. The later two studies have

demonstrated that, in the presence of E. faecalis, calcium hydroxide in the form of

Pulpdent paste (and in conditions similar to that in the main part of the root canal) is

more effective than Ledermix paste. Overall the three studies suggest that antibiotics

(due to resistance, limited spectrum of antimicrobial activity, inability to penetrate

biofilms effectively and a delayed effect in the presence of dentine) may not be the

ideal active ingredient in intracanal medicaments and that calcium hydroxide is more

effective.

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References

Abbott PV (1999) Endodontics and Dental Traumatology - An Overview of Modern

Endodontics. The University of Western Australia, Perth. 61. Abbott PV, Hume WR, Heithersay GS (1989) The effect of combining Ledermix and

calcium hydroxide pastes on the diffusion of corticosteroid and tetracycline through

human tooth roots in vitro. Endodontics & Dental Traumatology 5, 188-192.

Abbott PV, Hume WR, Pearman JM (1990) Antibiotics and endodontics. Australian

Dental Journal 35,50-60.

Athanassiadis B, Abbott PV, Walsh LJ (2007a) An in vitro study of the anti-microbial

activity of some endodontic medicaments and their bases using agar diffusion.

International Endodontic Journal 40, in preparation.

Athanassiadis B, Abbott PV, Walsh LJ (2007b) An in vitro study of the anti-microbial

activity of some endodontic medicaments and their bases to Enterococcus faecalis

biofilms. International Endodontic Journal 40, in preparation.

Camoes ICG, Salles MR, Chevitarese O, Gomes GC (2003) Influence on pH of

vehicle containing glycerin used with calcium hydroxide. Dental Traumatology

19,132-138.

Fava LRG, Saunders WP (1999) Calcium hydroxide pastes: classification and clinical

indications. International Endodontic Journal 32,257-282.

Haapasalo M, Qian W, Portenier I, Waltimo T (2007) Effects of dentin on the

antimicrobial properties of endodontic medicaments. Journal of Endodontics 33, 917-

925.

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Haapasalo HK, Sirén EK, Waltimo TMT, Orstavik D, Haapasalo MPP (2000)

Inactivation of local root canal medicaments by dentine: an in vitro study.

International Endodontic Journal 33,126-131.

Nerwich A, Figdor D, Messer H (1993) pH changes in root dentin over a 4-week

period following root canal dressing with calcium hydroxide. Journal of Endodontics

19,302-306.

Orstavik D (2003) Root canal disinfection: a review of concepts and recent

developments. Australian Endodontic Journal 29,70-74.

Porteinier I, Haapasalo H, Rye A, Waltimo T, Orstavik D, Haapasalo M (2001)

Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum

albumin. International Endodontic Journal 34,184-188.

Robert GH, Liewehr FR, Buxton TB, McPherson JC (2005) Apical diffusion of calcium

hydroxide in an in vitro model. Journal of Endodontics 31,57-60.

Simon ST, Bhat KS, Francis R (1995) Effect of four vehicles on the pH of calcium

hydroxide and the release of calcium ion. Oral Surgery Oral Medicine Oral Pathology

80,459-464.

Siqueira JF Jr, Lopes HP (1999) Mechanisms of antimicrobial activity of calcium

hydroxide: a critical review. International Endodontic Journal 32,361-369.

Waltimo TMT, Sirén EK, Orstavik D, Haapasalo MP (1999). Susceptibility of oral

candida species to calcium hydroxide in vitro. International Endodontic Journal 32,94-

98.

Wu MK, Dummer PM, Wesselink PR (2006) Consequences of and strategies to deal

with residual post-treatment root canal infection. International Endodontic Journal

39,343-356.

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Table 1: Bacterial survival of E. faecalis against medicaments and bases with

pre-incubation (1hr) and in the presence/absence of dentine. Dentine

was first ground into a fine powder, and then autoclaved. Data show

medians and upper/lower 95% confidence limits from five experiments.

1 hour 1 hour 1 day 1 day 3 days 3 days Preparations

No Dentine Dentine No Dentine Dentine No Dentine Dentine

PEG + water 2.15

(1.97-2.28)

1.98

(1.86-2.03)

2.39

(2.03-2.64)

2.16

(1.86-2.32)

1.78

(1.65-1.98)

2.00

(1.88-2.05)

PEG + inactives 2.00

(1.94-2.22)

1.92

(1.81-1.99)

1.57

(1.29-1.91)

1.17

(0.8-1.43)

1.30

(0.7-1.55)

0.69

(0.31-1.16)

Methyl Cellulose + water

2.00

(1.99-2.02)

1.82

(1.72-1.96)

2.30

(2.08-2.48)

2.00

(1.86-2.17)

2.25

(2.02-2.53)

2.00

(1.71-2.36)

PEG + inactives

+ demeclocycline

2.00

(1.82-2.06)

1.82

(1.76-1.87)

1.47

(1.39-1.54)

1.17

(1.08-1.37)

0.00

(0.00-0.00)

0.00

(-0.07-0.00)

Ledermix 2.00

(1.87-2.28)

1.82

(1.76-1.92)

2.30

(2.03-2.47)

1.52

(1.37-1.75)

0.00

(-0.16-0.23)

0.00

(-0.04-0.15)

Pulpdent 0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

Ledermix/Pulpdent

50:50

0.00

(0.00-0.00)

1.65

(0.40-2.31)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

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Table 2: Bacterial survival of E. faecalis against medicaments and bases with

pre-incubation (1 hr) in the presence/absence dentine. Dentine was

autoclaved first and then ground into fine powder. Data show medians

and upper/lower 95% confidence limits from three experiments.

1 hour 1 hour 1 day 1 day 3 days 3 days Preparations

No Dentine Dentine No Dentine Dentine No dentine Dentine

PEG + inactives 1.52

(1.48-2.27)

1.69

(1.53-1.81)

1.96

(1.47-2.29)

1.65

(1.52-1.77)

0.69

(0.41-1.15)

1.25

(0.34-1.83)

Methyl Cellulose + water

2.09

(1.87-2.31)

2.00

(1.89-2.17)

2.39

(2.14-2.63)

2.00

(1.95-2.03)

2.18

(1.62-2.81)

2.08

(1.78-2.26)

Ledermix 1.63

(1.19-2.17)

1.39

(0.76-2.28)

2.17

(1.78-2.64)

1.69

(0.97-2.18)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

Pulpdent 0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

Ledermix/Pulpdent

50:50

0.00

(0.00-0.00)

1.65

(0.40-2.31)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

0.00

(0.00-0.00)

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Table 3: Mean pH measurements of preparations

Preparation pH

Ca(OH)2 solution - above the undissolved powder 12.25

Ca(OH)2 fluid + dentine powder, - after 2 min

9.25

Ca(OH)2 fluid + dentine powder, - after 1 hr

8.70

Pulpdent paste 12.40

Pulpdent paste + dentine powder, - after 2 min

12.30

Pulpdent paste + dentine powder, - after 1 hr

12.30

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Appendix Table 1: 1 Hour - Descriptive Data

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Table 2: 1 Hour - Kruskal-Wallis Test (Nonparametric ANOVA)

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Table 3: 1 Hour - Dunn’s Multiple Comparison Test. Comparing between groups

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Table 4: Day 1 - Descriptive Data

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Table 5: Day 1 - Kruskal- Wallis Test (Nonparametric ANOVA)

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Table 6: Day 1 - Dunn’s Multiple Comparison Test. Comparison between groups.

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Table 7: Day 3 - Descriptive Data

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Table 8: Day 3 - Kruskal- Wallis Test (Nonparametric ANOVA)

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Table 9: Day 3 - Dunn’s Multiple Comparison Test. Comparing between groups

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CHAPTER SIX SUMMARY AND CONCLUSIONS

• A series of in vitro models were established to assess the antimicrobial

efficacy of some current endodontic medicaments and their bases using test

methods involving agar diffusion, biofilm penetration and dentine inactvation.

• Ledermix paste and the combination of Ledermix paste and Pulpdent paste

produced the best results in the agar diffusion model.

• The PEG 400/3350 base has some antimicrobial activity against P.

intermedia in the agar diffusion model.

• Pulpdent provided the best activity against the E. faecalis biofilm whilst the

bases and Ledermix paste had no effect.

• Pulpdent and the 50:50 Ledermix/Pulpdent combination provided the best

activity against E. faecalis in the presence of dentine.

• The Ledermix/Pulpdent 50:50 combination is not synergistic when compared

to using Pulpdent alone.

• Where possible, the use of medicaments in experiments should be confined

to those used commercially.

• Overall, Pulpdent paste had the best antibacterial activity of the

medicaments tested against E. faecalis.

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Further investigations recommended The following are suggested for future investigations:

• The use of human tooth roots in an agar plate to evaluate the anti-microbial

effect and the amount of diffusion through dentine of the medicaments and

their bases.

• The use of scanning electron microscopy (SEM) to detect colonization of

root canals medicated with the above medicaments and their bases.

• Studies of the effectiveness of biocides (e.g. chlorhexidine) in the above

three in-vitro models.

• Testing of various medicaments and their bases using multi-species biofilms.