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Irrigants and Intracanal Medicaments
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Date: 03/06/2011 17:09
Irrigants and Intracanal Medicaments
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Irrigants and Intracanal Medicaments - MARKUS HAAPASALO, WEI QIAN

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Infection Control in the Human Body


Successful elimination of opportunistic infections in most parts of the human body usually requires only involvement of normal host defense mechanisms. Occasionally, a systemic antibiotic therapy or manipulative treatment (such as drainage of pus) is applied in addition in order to resolve the infection. Elimination of endodontic infection, however, follows a different pathway. Host measures that are sufficient to eliminate the infective microorganisms in other sites are unable to completely eliminate endodontic infections. Because of the special anatomical challenges, control of an endodontic infection must be built on a joint effort by a number of host and treatment factors.1-3 Success in all parts of treatment will be needed for elimination of infection and healing of periapical pathosis. The sequence of events and procedures in the control of endodontic infections are host defense system, systemic antibiotic therapy (rarely used with specific indications only), instrumentation and irrigation ("cleaning and shaping"Smile, intracanal medicaments used between appointments, permanent root filling, and coronal restoration.1-3


Host defense is responsible for the prevention of spreading canal infections to the bone and to other parts of the body. The body defense is usually successful in stabilizing the lesion size and preventing its expansion after the initial growth period. However, because of the lack of circulation, host defense mechanisms cannot effectively reach the microbes residing inside the tooth in the necrotic root canal system. Mechanical instrumentation removes a portion of the microbes from the main root canal space, but its main purpose is to enhance irrigation and the placement of medication and the root filling. Irrigation supports mechanical instrumentation, by reducing friction and removing dead and living microbes from the root canal. In addition, many irrigating solutions have antimicrobial activity that effectively kill residual microbes in the canal. Intracanal antimicrobial medicaments, on the other hand, are used in multi-appointment endodontic treatments to complete the work started by instrumentation and irrigation and, optimally, to render the root canal system bacteria-free.


The classical study by Sjogren and his colleagues4 and by others indicated that a bacteria-free canal at the time of filling is a prerequisite for high success rate and that calcium hydroxide [Ca(OH)2] as an intracanal medicament will predictably help reach this goal.5-7 A number of other studies, however, have challenged these results, and at present, there is no clear consensus regarding the use of intracanal medicaments, or the microbiological and other advantages from their use.8-10


In this chapter, the focus is on the role of irrigation and intracanal medication in killing and reducing the number of bacteria in the root canal system. It is important, however, to understand that irrigation and local antibacterial dressings in the root canal are part of a concerted effort to control endodontic infections. Alone they cannot guarantee success if there are problems in quality of some other parts of the treatment.


Complete Elimination of Microbes in the Root Canal System


Pulpitis is caused by microbial antigens entering the pulp from a carious lesion or a leaking filling through dentinal tubules. As long as the pulp remains vital, the number of bacteria in the pulp is considered minimal and of no clinical significance. However, with proceeding infection, necrosis and apical periodontitis, the entire root canal system becomes invaded by bacteria. It has been shown beyond doubt that microorganisms are the etiological factor of apical periodontitis (Figure 1).11-13 It has been suggested that, in a small


[Figure 1. Bacteria are the etiological factor of apical periodontitis. Sixweek-old biofilm on the main root canal wall created by a mixed culture of oral bacteria under anaerobic conditions. Notice that several cells have died (hollow cells) because of limited availability of nutrients.]


number of cases, non-microbial factors are responsible for the persistence of the lesion after treatment.14,15 Nevertheless, there is general consensus that, in an optimal situation, the goal of endodontic treatment is to remove and kill all microorganisms in the root canal and to neutralize any antigens that may be left in the canal after killing the microbes. Reaching this goal is expected to guarantee healing of periapical lesions. It has become obvious that complete destruction of root canal microbes is a particularly difficult challenge. On the other hand, in a high number of cases, high quality treatment is followed by complete healing.16-18 It has also been shown that a majority of cases harboring living bacteria at the time of filling healed completely.4 It is clear, therefore, that although destroying 100% of the infective flora is the optimal goal, complete clinical and radiographic healing can also occur when the microbiological goal of treatment has not been fully realized. The situation may be compared to marginal periodontitis and gingivitis, where it is not possible to gain a totally bacteria-free environment in the gingival crevice area. Periodontal treatment of good quality, however, results in healing of the periodontal disease.


Figure 1. Bacteria are the etiological factor of apical periodontitis. Sixweek-old biofilm on the main root canal wall created by a mixed culture of oral bacteria under anaerobic conditions. Notice that several cells have died (hollow cells) because of limited availability of nutrients.0



The Goal of Endodontic Treatment


The goal, with the great majority of teeth requiring root canal treatment, is either the prevention or treatment of apical periodontitis.19 In other words, the goal is prevention or elimination of a microbial infection in the root canal system. In some special situations, such as resorptions and endodontic complications, there may be a variety of intermediate goals of a more "technical" nature. Even then, the final success is dependent on successful infection control. There is a widely accepted view that cleaning and shaping of the root canal system is the most important step toward a sterile canal free from a microbial presence.


Instrumentation of the Root Canal


The goal of hand and rotary instrumentation and irrigation is to remove all necrotic and vital organic tissue, as well as some hard tissue including dentin chips created by instrumentation, from the root canal system and give the canal system a shape that facilitates optimal irrigation, debridement and placement of local medicaments, and permanent root filling. From a biological point of view, the goal of instrumentation and irrigation is to remove and eradicate the microorganisms residing in the necrotic root canal system. Furthermore, the goal is to neutralize any residual antigenic material remaining in the canal after instrumentation and irrigation.


The Effect of Instrumentation on the Root Canal Microbes


Instrumentation has a key role in the cascade of treatment procedures to eradicate microbes in the root canal system. Instrumentation removes a great number of microbes from the accessible parts of the main root canal by direct mechanical cleaning action. Moreover, instrumentation shapes the root canal in such a way that effective irrigation becomes possible. In other words, instrumentation is a way of mechanically removing microbes from the root canal. In addition, it supports and facilitates mechanical removal and chemical eradication of the infection by irrigation during and following instrumentation.


The classical studies from Umea, Sweden have greatly influenced our understanding of the effects of instrumentation and irrigation of the intracanal microflora.20 In a series of studies on teeth with apical periodontitis, the authors demonstrated that thorough mechanical instrumentation with hand stainless steel instruments, together with irrigation with either physiological saline, ethylenediamine-tetra-acetic acid (EDTA), or EDTA and sodium hypochlorite (NaOCl), they were unable to predictably produce sterile root canals. Fifteen root canals were instrumented at five sequential appointments and sampled at the beginning and end of each appointment. The access cavity was sealed with a bacteria-tight temporary filling, but the canals were left empty between the appointments. This procedure resulted in a 100- to 1000-fold reduction in bacterial numbers, but it was difficult to obtain completely bacteria-free root canals. Corresponding results were also reported by Orstavik et al.21 and Cvek et al.22 in teeth with closed and immature apices. The antibacterial effect of mechanical cleansing with sterile saline was reported to be very low and limited to the teeth with fully developed roots. NaOCl increased the antibacterial effect as compared with saline irrigation. Interestingly, no statistical difference was found in the antibacterial effect between 0.5% and 5.0% NaOCl solutions.22


The effects of instrumentation and irrigation were investigated in an excellent series of studies by Dalton et al.23 They measured reduction in microbial counts in 48 patients, on teeth instrumented with 0.04 taper nickel-titanium (NiTi) rotary instruments or with stainless steel K-files using the step back technique with saline for irrigation. Bacteriological samples were obtained before, during, and after instrumentation. All teeth with apical periodontitis yielded positive growth at the beginning, whereas control teeth with vital pulp and irreversible pulpitis were sterile. A reduction in bacterial counts was detected with progressive enlargement of the root canals with both techniques. However, only 28% of the teeth became bacteria-free following instrumentation.


Similar observations were reported by Siqueira et al.24 when saline was used in irrigation. Interestingly, this study showed that increasing the size of apical preparation from #30 to #40 resulted in a significant reduction in microbial counts. Ex vivo studies by Pataky et al.,25 using 40 human first maxillary premolars extracted for orthodontic reasons, also verified the difficulty of obtained sterility of the infected canal space by instrumentation and saline irrigation. Although a considerable reduction in bacterial counts was detected after instrumentation, none of the teeth became bacteria-free. It should be mentioned, however, that the size of the master apical file in some of the studies was quite small, #25, and that may increase the possibility of positive cultures.25 It can be concluded that instrumentation and irrigation with saline alone cannot predictably eliminate all bacteria from infected root canals. Therefore, it is not surprising that the focus of activity in root canal disinfection is placed on the development and use of irrigating solutions and other intracanal disinfecting agents with strong antibacterial activity. In addition, there is growing interest in the combined effect of ultrasonic energy and irrigating solutions as well as other new ways of mechanical irrigation.


Root Canal Disinfection by Chemical Means


THE CHALLENGES AND PITFALLS IN STUDYING THE ANTIMICROBIAL EFFECTIVENESS OF ENDODONTIC IRRIGATING SOLUTIONS AND INTRACANAL MEDICAMENTS

In Vitro Models

The testing of the antimicrobial activity of various chemical compounds may appear to be straightforward and simple procedure. In theory, this may be true—the microbes of interest are exposed to the antimicrobial agent to be examined. At certain time intervals, microbiological samples are collected and cultured on suitable media. The results are then expressed as the length of time required to kill all microbes. In reality, regarding endodontic disinfecting agents, it is, however, quite different. Research focusing on the antimicrobial effectiveness of irrigating solutions and temporarily used intracanal medicaments has in many occasions adopted techniques originally designed for some other context, for example, antibiotic susceptibility using agar plates. Testing of systemically used antibiotics is based on tens of years of international standardization.26-30 The chemical composition of both the antibiotic discs and the culture media used in testing are defined in detail in order to secure predictable diffusion of the active ingredient of the antibiotic and the absence of chemical reactions between the agar plate ingredients and the antibiotic. Zones of inhibition of growth around the antibiotic disc have been compared through clinical studies to the serum and tissue concentrations that are possible to reach with safe dosages of low toxicity, as well as the clinical effectiveness of the antibiotics. In addition, reference strains from culture collections with known antibiotic susceptibilities are used as internal standards for quality testing of each new batch of plates. Most aerobic and facultative bacteria can be reliably tested using the disc diffusion method. However, despite years of extensive research, there is still no generally accepted standard for susceptibility testing anaerobic bacteria using the disc diffusion method.31


The use of agar diffusion method in endodontics, despite good intentions, is not based on any kind of standardization of the media or the tested materials. Chemical interactions between the media and the disinfecting agents are largely unknown. Furthermore, there are no true comparative studies helping to draw conclusions from the zones of inhibition to the performance of the disinfectants in vivo. The antimicrobial effect of some endodontic medicaments is based on the pH effect; therefore, the buffering capacity of the agar plate is in key position in determining the diameter of the growth inhibition zone. Another example is EDTA, that causes a zone of inhibition on an agar plate, but fails to reduce the number of viable microbes even after 24-hour incubation in a test tube. The growth inhibition on agar plates is based on removal of important ingredients from the media by EDTA, that makes the medium unsuitable for growth of many species. Such bacteria, even if alive, cannot multiply on the plate which has been nutritionally altered by EDTA. In conclusion, the information obtained from agar diffusion studies is at best of limited value and should not be used to compare and select disinfecting agents for clinical use.


Another matter of importance in susceptibility testing is understanding the difference between bacteriostatic and bactericidal activities. Bacteriostatic means prevention of growth of the microbial cells. However, they are not killed, unlike when the medicament has bactericidal activity. In endodontic studies, this difference is not always clearly addressed, and the results are often reported as "antibacterial activity." The agar diffusion test, when properly used, is an example of measuring bacteriostatic activity. In endodontics, a bactericidal effect of a disinfecting agent is more important than a bacteriostatic effect. In the necrotic root canal system, a temporary prevention of bacterial growth (as long as the disinfecting agent remains in the canal) is of limited value, because the microbes may still grow in numbers afterwards causing a new challenge to the host defense.


Testing endodontic disinfecting agents in vitro can be done in test tubes using a mixture of the microbes (suspended in sterile water) and the medicament. The presence of a culture medium in the mixture is commonly seen in endodontic literature. However, culture medium or other organic material is a confounding factor: the disinfecting agents vary in their susceptibility to the inactivating effect of the various organic substances.32 Experiments done under such conditions do not give a reliable picture of the characteristics and activity of the medicament/disinfectant against the tested microbe.


"Carry-over effect" means that the medicament, in active form, follows along with the sample into the dilution series and even to the culture plate (or liquid culture), where surviving microbes are calculated [e.g., "percentage of colony forming units (CFU) surviving"]. A high enough concentration of the disinfectant, in such a situation, can cause a false-negative result: the microbes are not killed, but residual medicament in the culture media prevents their growth by a bacteriostatic effect. Thus, carry-over, if undetected, gives a too positive picture of the antibacterial effectiveness of the medicament. Endodontic irrigants and disinfecting agents, containing local antibiotics, are at particularly high risk of causing false-negative results. Effective inactivators are not available for many antibiotics. If they are used in high concentration, carry-over effect is possible even after several 10-fold dilutions.32


Various inactivating agents are used to prevent the effects of carry-over. Citric acid has been used in the root canal to neutralize Ca(OH)2, sodium thiosulfate neutralizes NaOCl,33 and a mixture of Tween 80 and alpha-lecithin inactivates chlorhexidine (CHX).34 Inactivation, however, is dependent on the concentration of the medicaments. When they are used in high concentration, inactivation may not be complete. A good example of this is CHX that cannot be effectively inactivated with Tween and alpha-lecithin if CHX concentrations of 1% or more are used (Figure 2 A). The importance of the careful design of the experiments and proper controls cannot be overestimated, in order to avoid the possibility of false negative results.


Ex Vivo and In Vivo Models

In vitro models give valuable information about the spectrum and antimicrobial potential of endodontic disinfecting agents. However, information from such experiments alone is not enough to predict their performance in a clinical situation in the root canal. The effectiveness of the medicament in vivo can be reduced by a variety of factors. These include problems in delivery, low


[Figure 2. A, Samples of Enterococcus faecalis from 10-fold dilution series after a brief contact with 2% chlorhexidine. Because of short contact time (2 seconds), no killing has taken place. Chlorhexidine (CHX) "carry-over" has prevented growth of the first two samples (arrows) on the plate (upper row, left) despite the presence of Tween-lecithin inactivator in the dilution series. Lower row: control sample with no CHX. If only the first two dilutions had been made, the result would have indicated complete killing of the microbes in just 2 seconds. B, The size of the initial inoculum and the length of the dilution series determine the depth of measurement of the colony forming units. In this example, killing effectiveness can by calculated to a level of about 99.99%. Upper row: medicament; lower row: control.]


overall volume, poor/incomplete penetration in the main root canal system, poor penetration into dentin, short contact time, or inactivation of the activity of the disinfecting agent by one or more of the chemical compounds present in the necrotic root canal. Therefore, a number of ex vivo and in vivo models have been developed in order to meet the challenge by the various confounding factors of the root canal and to improve the correlation between the test results and clinical performance. The ex vivo and in vivo models include the dentin block model,35,36 dentin powder model,37-39 and several modifications using roots/root canals from extracted teeth.40,41 More studies are also made in vivo during the treatment of endodontic infections.42,43


The dentin powder model, with its modifications, makes it possible to obtain information about the inhibition of the medicament activity by dentin and other compounds (biomolecules, microbial biomass, etc.) in various concentrations.37 It also allows standardization of the experimental conditions for large series of tests. Prolonged incubation time to create dentin infection is not required because the dentin is powdered. The downsides of the powder model include partial loss of the microanatomical structure of the tooth and the difficulty to use/create microbial biofilms. In short, the dentin powder model is an effort to simulate the chemical environment of the tooth.


The dentin block model has been widely used for testing endodontic medicaments.35 The benefits of the model include simulation of the chemical and microanatomical environment of the tooth and the root canal system. The root canal is usually standardized in size, making it easier to obtain comparable samples of dentin in different blocks. It also allows the use of microbial biofilms in the experiments. On the other hand, the dentin block model is quite laborious in use; it cannot be fully standardized as different blocks may vary in thickness and dentin microstructure. Handling of the block presents some challenges which may increase the risk for false-positive results (contaminations from outside the sampling area). Despite its limitations, the dentin block model has greatly contributed to our understanding of dentin disinfection, and it is still frequently used in endodontic research.


Newer ex vivo and in vivo models use "natural" root canals either in extracted teeth or directly in vivo. The obvious benefit of these experiments is the more complete simulation of the clinical situation; the results obtained with the various materials may best reflect their true activity clinically. However, in addition to the well-known difficulty to organize controlled clinical studies, and to gather patients for such studies, there are also other potential pitfalls that may weaken the usefulness of the results. These include the difficulty to standardize the size of the apical preparation because of the great natural variation in canal sizes, different total volume of the canals, variations in the microanatomy of the root canals, and differences in the quality and quantity of the microbial infection. In addition to this kind of natural variation, there does not seem to be any standard way of dealing with the smear layer or taking the microbiological samples. According to the treatment protocols, the smear layer has not been removed in a great number of the studies. This is likely to have an effect on the ability of several sampling methods to collect viable microorganisms left in the dentinal tubules or lateral canals behind the smear layer. In quantitative studies where dentin is sampled, it is difficult to collect equal amounts of dentin (sample) because of the natural variations in canal sizes and shapes and different types of instruments used.


Figure 2. A, Samples of Enterococcus faecalis from 10-fold dilution series after a brief contact with 2% chlorhexidine. Because of short contact time (2 seconds), no killing has taken place. Chlorhexidine (CHX) "carry-over" has prevented growth of the first two samples (arrows) on the plate (upper row, left) despite the presence of Tween-lecithin inactivator in the dilution series. Lower row: control sample with no CHX. If only the first two dilutions had been made, the result would have indicated complete killing of the microbes in just 2 seconds. B, The size of the initial inoculum and the length of the dilution series determine the depth of measurement of the colony forming units. In this example, killing effectiveness can by calculated to a level of about 99.99%. Upper row: medicament; lower row: control.0



QUALITATIVE OR QUANTITATIVE RESULTS; DETECTION LEVEL: MORE CHALLENGES?

Optimally, the goal of endodontic disinfection is complete eradication of the microbes from the root canal system of the affected tooth. Although difficult to achieve, this noble goal may have affected the study design of several studies as the results are often expressed qualitatively, only as "growth" or "no growth." This approach may in some cases hide differences in the effectiveness of different treatment protocols and chemicals used for disinfection. For example, two different treatments that cause 10% and 99.95% reduction in bacterial counts per canal, respectively, are reported as equal ("growth"Smile, unless the overall number of "no growth" cases is higher with one of the methods. It is possible that some of the recent poor results with Ca(OH)2 as a disinfecting agent can be partly explained by the fact that qualitative rather than quantitative approach has been used to measure the effectiveness of the disinfection. Quantitative measurement of the effect by endodontic disinfecting agents should therefore be preferred over qualitative approach. Accordingly, quantitative reporting has been used in some studies.32,44


However, there are a number of factors that create challenges for accurate and comparable counting of microbes in the samples. Residual medicament in dentin may result in a very low number of CFU per sample unless neutralizing agents are properly used. Dispersing the sample effectively may be required to detach the viable cells from the dentin chips, to increase the CFU numbers to correctly reflect the true number of microbes in the sample. Collection of small dentin samples is technically demanding and requires great care to secure a standardized yield from all samples. When the overall number of CFU in the sample is low, which is often the situation in infected and medicated dentin, it is also important not to lose the microbes during too vigorous dilution process.


Success in both qualitative and quantitative measurement of (surviving) microbes is dependent, among other factors mentioned above, on the design of the microbiological procedures that determine the detection level of the method. Detection level dictates whether the most effective killing can be announced, for example, on the level of 99%, 99.9% or 99.99% (see Figure 2 B). In other words, detection level tells how many cells in the test mixture must be alive so that growth can be detected by the culturing. Detection level is mainly dependent on the total number of cells in the initial reaction mixture/sample, proportion of the initial sample transferred into the dilution series, and the proportion of the mixture plated from each dilution. Theoretically, the detection limit can be easily calculated from the above numbers. In practice, detection limit is often reduced by carry-over of strong medicaments such as CHX or MTAD that may cause false-negative results at the beginning of the dilution series. Detection limit gives important information about the design and quality of the methods in studies of endodontic disinfecting agents. Unfortunately, detection limits are only rarely reported, making it more difficult to compare the results from different studies. One would hope that in the future, reporting the detection limit will be a routine requirement for studies on endodontic disinfection.


ANTIBACTERIAL IRRIGATING SOLUTIONS

The use of irrigating solutions is an important part of endodontic treatment. The irrigants facilitate removal of necrotic tissue, microorganisms and dentin chips from the root canal by a flushing action. Irrigants can also help prevent packing infected hard and soft tissue apically in the root canal and into the periapical area. Some irrigating solutions dissolve either organic or inorganic tissue. Finally, several irrigating solutions exhibit antimicrobial activity by actively killing bacteria and yeasts when in direct contact with the microorganisms. On the negative side, many irrigating solutions have shown cytotoxic activity and may cause severe pain reaction if they gain access into the periapical tissues.45


An optimal irrigant should have all or most of the positive characteristics listed above, but none of the negative or harmful properties. Presently, none of the available irrigating solutions can be regarded as optimal. However, with a combined use of selected products, irrigation will greatly contribute to successful outcome of treatment.


NaOCl

NaOCl is the most widely used irrigating solution. In water, NaOCl ionizes to produce Na+ and the hypochlorite ion, OCl-, that establishes an equilibrium with hypochlorous acid, HOCl. Between pH 4 and 7,


[Figure 3. A, Instrumented (upper part of the picture) and uninstrumented (lower part) root canal wall after irrigation with 5% sodium hypochlorite (NaOCl) for 10 minutes. Pulpal remnants and predentin have been effectively removed with NaOCl while the instrumented part (smear layer) seems relatively unaffected. Notice the typical calcospherites at the lower part of the image. B, Calcospherites on the uninstrumented canal wall after irrigation for 10 minutes with 2.6% NaOCl.]


chlorine exists predominantly as HClO, the active moiety, whereas above pH 9, OCl- predominates.46 It is the hypochlorous acid that is responsible for bacteria inactivation, the OCl- ion being less effective than the undissolved HOCl. Hypochlorous acid disrupts oxidative phosphorylation and other membrane-associated activities as well as DNA synthesis.47,48


NaOCl is used in concentrations varying from 0.5 to 7%. It is a very potent antimicrobial agent and effectively dissolves pulpal remnants and organic components of dentin (Figure 3). It is used both as an unbuffered solution at pH 11 in concentrations mentioned above and buffered with a bicarbonate buffer (pH 9.0) usually as a 0.5% (Dakin's solution) or 1% solution.46 Contradicting earlier statements, Zehnder et al.49 reported that buffering had little effect on tissue dissolution, and Dakin's solution was equally effective on decayed (necrotic) and fresh tissues. In addition, no differences were recorded for the antibacterial properties of Dakin's solution with an equivalent unbuffered hypochlorite solution.49


NaOCl is best known for its strong antibacterial activity; it kills bacteria very rapidly even at low concentrations. Waltimo et al.50 showed that the resistant microorganism, Candida albicans, was killed in vitro in 30 seconds by both 5% and 0.5% NaOCl, whereas concentrations 0.05% and 0.005% were too weak to kill the yeast even after 24 hours of incubation. The high susceptibility of C. albicans to NaOCl was recently also verified by Radcliffe et al.33 Later, Vianna et al.51 contrasted these results partly, as 0.5% NaOCl required 30 minutes to kill C. albicans, whereas 5.25% solution killed all yeast cells in 15 seconds. In the latter study, however, organic material from the broth culture medium may have been present during the incubation with NaOCl which would explain delayed killing. Gomes et al.52 tested in vitro the effect of various concentrations of NaOCl against enterococcus. Enterococcus faecalis (Figure 4) was killed within 30 seconds by the 5.25% solution, while 10 and 30 minutes was required for killing all bacteria by 2.5 and 0.5% solutions, respectively. The higher resistance of E. faecalis to hypochlorite as compared with the yeast C. albicans was suggested also by Radcliffe et al.33 However, both of these studies are in contrast to the results reported by Haapasalo et al.37 who demonstrated rapid killing of E. faecalis by 0.3% NaOCl and by Portenier et al.53 who were able to show rapid killing of E. faecalis strains in logarithmic and stationary growth phase by even 0.001% NaOCl.


Experiments with Gram-negative anaerobic rods Porphyromonas gingivalis, Porphyromonas endodontalis, and Prevotella intermedia, often isolated from apical periodontitis, demonstrated high susceptibility to


[Figure 4. A scanning electron micrograph of growing Enterococcus faecalis cells.]


NaOCl, and all three species were killed within 15 seconds with 0.5 to 5% concentrations of NaOCl.51


The differences between in vitro and in vivo studies include the volume of the medicament available for killing, access to all microbes, and absence of other materials in the in vitro experiments that potentially protect bacteria in vivo. Many of the in vivo studies have failed to show a better antibacterial effect in the root canal by high concentrations of NaOCl as compared to low concentrations. Bystrom and Sundqvist54,55 studied root canals naturally infected, mainly with a mixture of anaerobic bacteria, and showed that although 0.5% NaOCl, with or without EDTA, improved the antibacterial efficiency of preparations compared with saline irrigation, all canals were not bacteria free even after several appointments. No significant difference in antibacterial efficiency in vivo between 0.5 and 5% NaOCl solutions was found in the study. Siqueira et al.56 using E. faecalis-infected root canals demonstrated the superior antibacterial affect against root canal bacteria of hypochlorite in comparison with physiological saline. However, no difference was detected between 1, 2.5, and 5% NaOCl solutions.


Many of the studies about the antibacterial effect of NaOCl against root canal bacteria are in vitro studies, in either "neutral" test tube conditions, in the root canals of extracted teeth, or in dentin blocks infected with a pure culture of one organism at a time. The in vivo studies, on the other hand, have focused on the eradication of bacteria from the root canals of teeth with primary apical periodontitis.


Peciuliene et al.57 studied the effect of instrumentation and NaOCl irrigation in failed, previously root-filled teeth with apical periodontitis. Old root fillings were removed with hand instruments, and the first sample was taken. No chloroform was used to avoid false-negative cultures. Bacteria were isolated in 33 of the 40 teeth before further instrumentation. E. faecalis was found in 21 teeth (in 11 as a pure culture), the yeast C. albicans in 6 teeth, Gram-negative enteric rods in 3 teeth, and other microbes in 17 teeth. The canals were then hand instrumented to size #40 or larger and irrigated with 2.5% NaOCl (10 mL per canal) and 17% buffered EDTA (pH 7 and 5 mL per canal). After instrumentation and irrigation, E. faecalis was still detected in six canals. Other microbes persisted in five canals after preparation. Enteric Gram-negative rods were no longer present in the second sample.


The disappearance of yeasts but not E. faecalis in the root canals may reflect their different susceptibilities to the described chemomechanical instrumentation or it may be a result of different biofilm characteristics and dentin penetration by these species.


The weaknesses of NaOCl include unpleasant taste, toxicity, and its inability to remove smear layer because of its lack of effect on inorganic material.58,59 The poorer antimicrobial effectiveness of NaOCl in vivo than in vitro is also somewhat disappointing. There are several possible reasons for the reduced in vivo performance. Root canal anatomy, particularly the difficulty to effectively irrigate the most apical region of the canal, is generally acknowledged as one of the main challenges. In addition, the chemical milieu in the canal is different from a test tube. Haapasalo et al.37 showed that the presence of dentin caused marked delays in the killing of E. faecalis by 1% NaOCl. The effect of other materials present in the necrotic or previously treated root canal, to the antimicrobial potential of NaOCl, has not been studied.


Studies measuring NaOCl cytotoxicity have indicated greater cytotoxicity and caustic effects on healthy tissue with 5.25% NaOCl than with 1.0 and 0.5% solutions.60,61 The fear of toxic and chemical complications is the main reason for that low concentrations 0.5 to 1% NaOCl solutions are used for canal irrigation instead of the 5.25% solution in many countries.45 However, more in vivo studies on persistent endodontic infections and retreatment are necessary for a deeper understanding of the relationship between NaOCl concentration and its antimicrobial activity against specific microorganisms, before final


[Figure 5. A, Smear layer on the surface of the root canal wall after hand instrumentation with H-files. B, Smear layer after instrumentation of the root canal before ethylenediamine-tetra-acetic acid irrigation.]


conclusions can be drawn regarding the optimal NaOCl concentration.


EDTA, Citric Acid and Other Acids

EDTA (17%, disodium salt, pH 7) has little if any antibacterial activity. On direct exposure for extended time, EDTA releases some of the bacteria's surface proteins by combining with metal ions from the cell envelope. This can cause even bacterial death. More importantly, EDTA is an effective chelating agent in the root canal.62,63 It removes smear layer (Figure 5) when used together (but not simultaneously) with NaOCl by acting on the inorganic component of the dentin (Figure 6). Therefore, by facilitating cleaning and removal of infected tissue, EDTA contributes to the elimination of bacteria in the root canal. It has also been shown that removal of the smear layer by EDTA (or citric acid) improves the antibacterial effect of locally used disinfecting agents in deeper layers of dentin.35,36 Niu et al.64 studied the ultrastructure on canal walls after EDTA and EDTA plus NaOCl irrigation by scanning electron microscopy (SEM): more debris was removed by irrigation with EDTA followed by NaOCl than with EDTA alone (Figure 7).


[Figure 6. A, Instrumented root canal wall after irrigation with 5% sodium hypochlorite (NaOCl) and 17% ethylenediamine-tetra-acetic acid (EDTA), each for 5 minutes. Smear layer has been completely removed. B, Close-up scanning electron micrograph of the root canal wall after removal of smear layer with NaOCl and EDTA.]


[Figure 7. Partial removal of smear layer by irrigation for 10 minutes with 17% ethylenediamine-tetra-acetic acid only. Mainly organic material has been left covering the surface.]


Citric acid can also be used for irrigation of the root canal and for removal of the smear layer.62,65,66 Similar to EDTA, complete removal of smear layer requires also irrigation with NaOCl before or after citric acid irrigation (Figure 8). Concentrations ranging from 1 to 50% have been used.65 In comparison with ultrasound, 10% citric acid has been shown to remove the smear layer more effectively from apical root-end cavities than ultrasound.67 In another study, powdered dentin-resin mixture was more soluble in a 0.5, 1, and 2 M citric acid than in 0.5 M EDTA.68 Contrary to this, Liolios et al.69 reported better removal of smear layer by commercial EDTA preparations than with 50% citric acid, while other studies have found only small or no difference between citric acid and 15% EDTA in their capacity to remove the smear layer.70,71 A comparative study showed that 10% citric acid was more effective than 1% citric acid, which was more effective than EDTA in demineralizing dentin.72 Takeda et al.73 reported that irrigation with 17% EDTA, 6% phosphoric acid, and 6% citric acid did not remove the entire smear layer from the root canal system. This is not unexpected, however, as it is known that NaOCl is also required for complete removal of the smear layer. The acids demineralized the intertubular dentin making the tubular openings larger than by EDTA. The authors also showed that CO2 laser was useful in removing the smear layer and that the Er:YAG laser was even more effective than the CO2 laser in smear layer removal.


Smear layer removal facilitates penetration of sealers into the dentinal tubules. It also enhances disinfection of the root canal wall and deeper layers of dentin. Both EDTA and citric acid can effectively remove the smear layer when used together with NaOCl. Citric acid and EDTA may have weak antimicrobial activity as stand-alone products. However, their antimicrobial effectiveness has not been extensively documented and appears to be of minor importance.


[Figure 8. A, Instrumented root canal wall after irrigation for 10 minutes with 50% citric acid only. Similar to ethylenediamine-tetra-acetic acid, sodium hypochlorite irrigation is needed in addition for complete removal of the smear layer. B, Uninstrumented part of the root canal wall after irrigation for 10 minutes with 50% citric acid. Citric acid alone cannot dissolve the organic tissue within a reasonable time.]


Hydrogen Peroxide

Hydrogen peroxide (H2O2) is a biocide that has been widely used for disinfection and sterilization.46 However, in endodontics, H2O2 has not been very popular, and presently its use is generally in decline. H2O2 is a clear and colorless liquid. Concentrations from 1 to 30% have been used. H2O2 produces hydroxyl-free radicals (HO.) that attack microbial components such as proteins and DNA.46 H2O2 is nonproblematic from an environmental point of view because it degrades into water and oxygen. H2O2 is relatively stable in solution, but many products contain stabilizers to prevent decomposition. H2O2 has antimicrobial activity against various microorganisms including viruses, bacteria, yeasts, and even bacterial spores.74 It is more effective against Gram-positive than Gram-negative bacteria. Catalase or superoxide dismutase produced by several bacteria can provide them partial protection against H2O2.


Use of H2O2 in endodontics has been based on its antimicrobial and cleansing properties. Thirty percent H2O2 (Superoxol) has been recommended as the first step in tooth surface disinfection after mechanical cleaning.75 H2O2 acts on the organic matter on the tooth making other disinfectants, such as iodine, more effective. It has been widely used earlier for cleaning the pulp chamber from blood and tissue remnants. It has also been used in canal irrigation, but evidence supporting the effectiveness of H2O2 as a root canal irrigant is scarce. On the contrary, Siqueira et al.76 reported that a combination of NaOCl and H2O2 gave no advantage over NaOCl alone against E. faecalis in contaminated root canals ex vivo. Heling and Chandler77 found strong synergism between H2O2 and Chlorhexidine (CHX) in disinfection of infected dentin: a combination of the two medicaments at low concentration was far more effective in sterilizing dentin than these or any other medicament alone. The synergistic mode of action between CHX and H2O2 was later documented also by Steinberg et al.78 In a recent study, 10% H2O2 was used as part of the irrigating protocol in monkey teeth.79 A total of 186 root canals in 176 teeth were inoculated with a known mixture of bacteria for several months. One group consisted of anaerobic bacteria and streptococci; the second group was identical to group one except with added E. faecalis. The root canals were sampled and instrumented manually to size #40 to #60 and irrigated with buffered 1% NaOCl, followed by 10% H2O2. Final irrigation was with NaOCl. Sodium thiosulpfate was used to inactivate hypochlorite before sampling, and a second bacteriological sample was taken. In group 1 (160 canals), bacteria were found in 98% and 68% of the canals in samples 1 and 2, respectively. In group 2, with E. faecalis, the corresponding frequencies were 100% and 88%. Although the bacterial counts were greatly reduced, the study indicated the difficulty to completely eradicate bacteria from infected root canals in monkey teeth.79


Despite the long history of use of H2O2 in endodontics, evidence supporting its use is at best weak. However, it still has a role as part of the tooth surface disinfection protocol. Furthermore, the potential benefit of the suggested synergistic effect with CHX in deep dentin disinfection remains to be evaluated clinically.


CHX

Chlorhexidine digluconate (CHX) is widely used in disinfection because of its excellent antimicrobial activity.80-82 It has gained increased popularity in endodontics as an irrigating solution and as an intracanal medicament. Unlike NaOCl, CHX does not have bad smell, it is not equally irritating to periapical tissues, and neither does it cause dramatic spot bleaching of the patients clothes. Its antimicrobial effectiveness is well documented in endodontics. However, it completely lacks tissue dissolving capability, an important reason for the popularity of NaOCl (Figure 9).


CHX is perhaps the most widely used antimicrobial agent in antiseptic products. It permeates the cell wall or outer membrane (Gram-negative cells) and attacks the bacterial cytoplasmic or inner membrane of the yeast plasma membrane. In high concentrations, CHX causes coagulation of intracellular components.46 CHX gluconate has been in use for some time in dentistry because of its antimicrobial properties, its substantivity (long-term continued effect), and its relatively low toxicity compared to some other agents. However, the activity of CHX is dependent on the pH and is greatly reduced also in the presence of organic matter.82


CHX is effective against both Gram-positive and Gram-negative bacteria as well as yeasts, although activity against Gram-negative bacteria is not as good as against Gram-positive bacteria.83-85 Mycobacteria and bacterial spores are resistant to CHX.80,81 Therefore, CHX is not as suited to chairside sterilization of gutta-percha cones as NaOCl.86,87 Studies indicating equally good performance by CHX and NaOCl in gutta-percha disinfection have not used bacterial spores in testing.88 CHX is not very effective against viruses, its activity being limited to viruses with a lipid envelope.89 CHX is cytotoxic in direct contact with


[Figure 9. A, Instrumentation smear layer seems intact after irrigation for 10 minutes with 2% chlorhexidine. B, Uninstrumented area in the root canal after irrigation with 2% chlorhexidine only. Chlorhexidine has no tissue dissolving capability.]


human cells. A study using fluorescence assay on human periodontal ligament (PDL) cells showed no difference in cytotoxicity by 0.4% NaOCl and 0.1% CHX.61 The potential benefits of using CHX in endodontics have been under active research over the last several years. Several studies have compared the antibacterial effect of NaOCl and CHX against intracanal infection.


While many studies show little or no difference between their antimicrobial effectiveness,77,90-92 their mode of action may indicate important differences. The effects of 15 minutes of irrigation of experimental biofilms by mixtures of endodontic bacteria on dentin blocks have been evaluated by SEM and by culturing.93 Six percent NaOCl was the only irrigant that completely eliminated (removed) the biofilm as verified by SEM observations and killed all bacteria. Two percent CHX was equally effective in bacterial killing, and no growth was detected in any of the samples after the CHX treatment. However, an important difference was observed in how the biofilm was structurally affected by irrigation with these two solutions: 6% hypochlorite completely removed the biofilm while the CHX solution had no effect on the biofilm structure.93 Although the bacteria were killed, the result indicates that as the biofilm remains in the canal after CHX irrigation, it may continue to express its antigenic potential if allowed the possibility to communicate with living periapical tissue. Moreover, such residual organic tissue may have a negative impact on the quality of the seal of the permanent root filling. Different types of biofilm are shown in Figure 10.


Some studies have indicated differences in the killing of certain endodontic microbes by hypochlorite and CHX. An in vitro study demonstrated differences in the killing of enterococci by CHX and NaOCl. While 5.25% NaOCl killed E. faecalis within 30 seconds, lower concentration (4.0 to 0.5%) of hypochlorite required 5 to 30 minutes for complete killing of the bacteria.52 In the same study, 0.2 to 2% CHX killed the E. faecalis cells in 30 seconds or less in all concentrations tested. The result was later supported by two other studies using E. faecalis and Staphylococcus aureus as test organisms.51,94 However, the results of these studies have been contradicted by some other studies with regard to the effectiveness of NaOCl.37,53


The antifungal effectiveness of CHX has been shown in several studies.50,95-97 In a study of the effectiveness of various endodontic disinfecting agents, it was found that combinations of disinfectants were equally or less effective against fungi than the more effective component alone.50


An interesting, but not yet fully understood synergism has been reported between CHX and H2O2.77 In


[Figure 10. A, Densely packed natural biofilm (in vivo) on the root canal wall of a tooth with apical periodontitis. The film consists of bacteria of different types and shapes and remnants of necrotic pulp tissue. B, Cocci and rod-shaped bacteria in necrotic pulp tissue of a tooth with apical periodontitis. C, Early stages of biofilm formation in vitro on root dentin by a mixture of oral bacteria. Binding of coccoid cells on long rod-shaped bacteria can be seen. D, More advanced stage of biofilm formation in vitro on root dentin by a mixture of oral bacteria. Many of the bacterial cells in the larger aggregate are embedded in extracellular matrix. E, Biofilm by a mixture of oral bacteria grown under increased CO2 atmosphere in vitro on root dentin.]


vitro studies using the dentin block model indicated strong synergism between these two agents against E. faecalis infection in dentinal tubules. Complementary in vitro experiments by Steinberg et al.78 demonstrated that the combination of CHX and H2O2 completely eradicated E. faecalis in concentrations clearly lower than required when the compounds were used alone. It is possible that CHX, as a membrane active agent, makes the bacterial membranes more permeable to H2O2, that can more easily penetrate the cells and cause damage to the intracellular organelles.78 Although NaOCl and H2O2 are occasionally used together in root canal irrigation, such synergistic effect has not been detected between them in the dentin block model.77 The CHX-H2O2 synergism has been demonstrated also in a study of antiplaque mouth rinse.98 Surprisingly, so far there are no published studies of the clinical performance of the combinations of CHX and H2O2. Combinations of CHX and carbamide peroxide have been shown to be additive in their cytotoxicity,99 but corresponding experiments with CHX and H2O2 are lacking.


Inhibition of the antimicrobial activity of endodontic irrigants, by substances present in the root canal, has recently been discussed in a number of studies.37-39 Haapasalo et al.37 showed in an in vitro study that the effect of CHX is reduced or delayed, by the presence of dentin. In following studies by the same group, Portenier et al.38 detected loss of CHX antimicrobial activity by high concentration (18%, v/w) of bovine serum albumin. In a clinical situation, inflammatory exudate rich in proteins may thus have a negative impact on the effectiveness of CHX. A subsequent study showed that organic dentin matrix and heat-killed microbial cells were effective inhibitors of CHX activity.39 In a study by Sassone et al.,100 CHX was incubated together with low concentration (0.5%, v/w) albumin, and no inhibition of the CHX activity could be detected. Albumin concentration in human serum is approximately 2 to 3% and the total protein concentration is approximately 7%.101


During the last few years, several studies have measured the activity of CHX gel against root canal bacteria. Vianna et al.51 found that CHX in a gel form required a longer time to kill E. faecalis than the corresponding concentration in a liquid. Oliveira et al.102 reported that 2% CHX gel and 5.25% NaOCl showed excellent activity against E. faecalis. When diluted to 1.5% solution, NaOCl reduced the E. faecalis counts initially, but the bacterial counts increased during the 7-day follow-up period to the level comparable to the control group.


Because CHX lacks the tissue dissolving activity of NaOCl, there have been efforts to simplify the clinical work by combining the two solutions to obtain combined benefits from both. However, CHX and NaOCl are not soluble in each other and a brownish-orange precipitate is formed (Figure 11). Although the antimicrobial and other characteristics of the precipitate and the liquid phase have not been thoroughly examined, the precipitate prevents clinical use of the mixture. Marchesan et al.103 showed that the precipitate was soluble to 0.1 mol/L acetic acid, but the brown/orange color of the solution remained. Atomic absorption spectrophotometry indicated that the precipitate contained iron which may be the reason for the color.


Despite of some shortcomings, there is increasing evidence that CHX gluconate, as a 2% solution (liquid or gel), may offer a good alternative for root canal


[Figure 11. Mixing sodium hypochlorite with chlorhexidine causes a brown/orange precipitate. The color may be due to iron impurities in hypochlorite. The mixture should not be used for irrigation of the root canals.]


irrigation. However, one should bear in mind that the majority of the research on the use of CHX in endodontics is done using in vitro and ex vivo models and Gram-positive test organisms, mostly E. faecalis. The possibility cannot be excluded that the experimental designs give a biased (too positive) picture of the usefulness of CHX as an antimicrobial agent in endodontics. More research is needed to identify the optimal irrigation regimen for various types of endodontic treatments. CHX is presently marketed as a water-based solution, as a gel (with Natrosol), and as a liquid mixture with surface active agents. Future studies of the various CHX preparations will establish the comparative effectiveness of the various CHX combinations in vivo.


Iodine Potassium Iodide (IPI)

Iodine compounds are among the oldest disinfectants still actively used. They are best known for their use on surfaces, skin, and operation fields. Iodine is less reactive than the chlorine in hypochlorite. However, it kills rapidly and has bactericidal, fungicidal, tuberculocidal, virucidal, and even sporicidal activity.101 Molecular form, I2, is the active antimicrobial component.104 Poor stability of iodine in aqueous solutions motivated the development of iodophors ("iodine carriers"Smile: povidone-iodine and poloxamer-iodine. Iodophors are complexes of iodine and a solubilizing agent that gradually releases the iodine.104 Iodophors are less active against some yeasts and bacterial spores than are the alcoholic iodine solutions (tinctures). Iodine penetrates rapidly into the microorganisms and causes cell death by attacking the proteins, nucleotides, and other key molecules of the cell.104,105


Iodine potassium iodide (IPI) has been successfully used in tooth surface disinfection.75 Potassium iodide is used to dissolve iodine in water, but the antimicrobial activity is carried by the iodine, while potassium iodide has no activity against the microbes.


The effectiveness of 2.5% NaOCl and 10% iodine for disinfection of the operation field (tooth, rubber dam, and the clamp) has been compared by bacterial culturing and polymerase chain reaction.106 The operation field was treated with 30% H2O2 and either by 10% iodine or 2.5% NaOCl. No significant difference in the recovery of cultivable bacteria from various sites in either group was detected. However, bacterial DNA was detected significantly more frequently from the tooth surfaces after iodine treatment (45%) than after NaOCl (13%) treatment.106


Molander et al.107 suggested that irrigation with 5% IPI before Ca(OH)2 medication did not have an effect on the overall antimicrobial power. However, it is possible that IPI reduces the frequency of persisting strains of E. faecalis. Peciuliene et al.57 studied the effect of iodine irrigation in 20 teeth with previously root-filled canals and apical periodontitis. The results showed that when used after normal chemomechanical preparation, IPI increased the number of culture negative canals.


In the root canal, iodine compounds come in contact with a variety of substances such as dentin and various proteins. Studies of the interaction of IPI with the chemical environment of the necrotic root canal have shown that dentin can reduce or even abolish the effect of 0.2/0.4% IPI against E. faecalis.37,38 However, pure hydroxyl apatite or bovine serum albumin had little or no effect on the antibacterial activity of IPI. Portenier et al.39 have shown that dentin matrix (mostly dentin collagen) and heat-killed cells of E. faecalis and C. albicans inhibit the antibacterial activity of IPI. These studies indicate that inactivation of iodine compounds is one factor explaining the difficulty in obtaining sterile root canals.


Figure 3. A, Instrumented (upper part of the picture) and uninstrumented (lower part) root canal wall after irrigation with 5% sodium hypochlorite (NaOCl) for 10 minutes. Pulpal remnants and predentin have been effectively removed with NaOCl while the instrumented part (smear layer) seems relatively unaffected. Notice the typical calcospherites at the lower part of the image. B, Calcospherites on the uninstrumented canal wall after irrigation for 10 minutes with 2.6% NaOCl.0

Figure 4. A scanning electron micrograph of growing Enterococcus faecalis cells.0

Figure 5. A, Smear layer on the surface of the root canal wall after hand instrumentation with H-files. B, Smear layer after instrumentation of the root canal before ethylenediamine-tetra-acetic acid irrigation.0

Figure 6. A, Instrumented root canal wall after irrigation with 5% sodium hypochlorite (NaOCl) and 17% ethylenediamine-tetra-acetic acid (EDTA), each for 5 minutes. Smear layer has been completely removed. B, Close-up scanning electron micrograph of the root canal wall after removal of smear layer with NaOCl and EDTA.0

Figure 7. Partial removal of smear layer by irrigation for 10 minutes with 17% ethylenediamine-tetra-acetic acid only. Mainly organic material has been left covering the surface.0

Figure 8. A, Instrumented root canal wall after irrigation for 10 minutes with 50% citric acid only. Similar to ethylenediamine-tetra-acetic acid, sodium hypochlorite irrigation is needed in addition for complete removal of the smear layer. B, Uninstrumented part of the root canal wall after irrigation for 10 minutes with 50% citric acid. Citric acid alone cannot dissolve the organic tissue within a reasonable time.0

Figure 9. A, Instrumentation smear layer seems intact after irrigation for 10 minutes with 2% chlorhexidine. B, Uninstrumented area in the root canal after irrigation with 2% chlorhexidine only. Chlorhexidine has no tissue dissolving capability.0

Figure 10. A, Densely packed natural biofilm (in vivo) on the root canal wall of a tooth with apical periodontitis. The film consists of bacteria of different types and shapes and remnants of necrotic pulp tissue. B, Cocci and rod-shaped bacteria in necrotic pulp tissue of a tooth with apical periodontitis. C, Early stages of biofilm formation in vitro on root dentin by a mixture of oral bacteria. Binding of coccoid cells on long rod-shaped bacteria can be seen. D, More advanced stage of biofilm formation in vitro on root dentin by a mixture of oral bacteria. Many of the bacterial cells in the larger aggregate are embedded in extracellular matrix. E, Biofilm by a mixture of oral bacteria grown under increased CO2 atmosphere in vitro on root dentin.0

Figure 11. Mixing sodium hypochlorite with chlorhexidine causes a brown/orange precipitate. The color may be due to iron impurities in hypochlorite. The mixture should not be used for irrigation of the root canals.0.015625



ANTIBIOTIC-CONTAINING IRRIGATION SOLUTIONS

MTAD and Tetraclean

MTAD [a mixture of tetracycline isomer, acid, and detergent (Biopure, Dentsply, Tulsa, OK)] is a new generation combination product for root canal irrigation.105,106 Tetraclean (Ogna Laboratori Farmaceutici, Muggio, Italy) is another combination product similar or close to MTAD.108 MTAD has a low pH (2.15) because it contains citric acid, it removes smear layer after NaOCl irrigation, and it has antibacterial activity against endodontic microbes. The main potential benefits of MTAD are that it makes irrigation simpler by combining smear layer removal activity with antimicrobial effect and that it may be "gentler" with dentin than EDTA.109 The authors who introduced MTAD have recommended the use of 1.3% NaOCl during instrumentation, followed by MTAD to remove the smear layer.110 However, 1.3% NaOCl may not be strong enough to completely clean the uninstrumented parts of the root canal (Figure 12). Beltz et al.111 found that MTAD solubilizes dentin, whereas organic pulp tissue is unaffected by it. Zhang et al.112 showed that MTAD is less cytotoxic than eugenol, 3% H2O2, Ca(OH)2 paste, 5.25% NaOCl and EDTA, but more cytotoxic than 2.63% NaOCl.


The antibacterial activity of MTAD is of particular interest as it contains doxycycline (tetracycline) in


[Figure 12. Uninstrumented root canal wall irrigated for 5 minutes with 1.3% sodium hypochlorite (NaOCl) followed by 5-minute irrigation with MTAD (a mixture of tetracycline isomer, acid, and detergent). Although this combination produces excellently clean canal walls in the instrumented areas (smear layer), the low concentration NaOCl may be too mild to thoroughly clean the uninstrumented areas if the irrigation time is not long enough. In this figure, some of the predentin is still left to cover the mineralized dentin and the calcospherites.]


high concentration. Shabahang et al.113 and Shabahang and Torabinejad114 investigated the effect of MTAD on root canals contaminated with either whole saliva or E. faecalis of extracted human teeth and reported good antibacterial activity. Portenier et al.32 showed that MTAD killed E. faecalis in vitro in less than 5 minutes. In an ex vivo study, Kho and Baumgartner115 compared the antimicrobial effectiveness of NaOCl/EDTA and NaOCl/MTAD in extracted roots infected for 4 weeks with E. faecalis. After chemomechanical preparation and irrigation, the roots were pulverized in liquid nitrogen and viable bacteria were counted. No difference was measured between the two irrigation regimens. Another ex vivo study using roots of 26 matched pairs of teeth compared the same irrigation regimens with a different type of sampling procedure.116 In this study, NaOCl/EDTA (5.25%/15%) irrigation resulted in 0/20 culture positive samples in both sample 1 (directly after irrigation) and sample 2 (after instrumenting the canals 2 instrument sizes wider) using a sensitive sampling protocol. However, in the other experimental group of 1.3% NaOCl/MTAD, the corresponding number of culture positive samples was 8 and 10 out of the total of 20 samples in each group. It should be noted that in the absence of negative control (e.g. water irrigation), it is not possible to know what may have been the reduction of bacterial CFU in the NaOCl/MTAD group. Nevertheless, the result indicates better performance by NaOCl/EDTA than by NaOCl/MTAD irrigation. The result is interesting because EDTA alone lacks antimicrobial activity against E. faecalis. The mechanisms of action of sequential use of NaOCl and EDTA on bacterial viability have not been thoroughly studied. There are no reports so far on the antibacterial effectiveness of Tetraclean.


The antibacterial effect of MTAD may be based not only on the antibiotic component (doxycycline) but also on the combined effect of doxycycline and the other ingredients (Tween 80, citric acid) on the integrity and stability of the microbial cell wall. However, there is no specific information available on such effects regarding MTAD.


Figure 12. Uninstrumented root canal wall irrigated for 5 minutes with 1.3% sodium hypochlorite (NaOCl) followed by 5-minute irrigation with MTAD (a mixture of tetracycline isomer, acid, and detergent). Although this combination produces excellently clean canal walls in the instrumented areas (smear layer), the low concentration NaOCl may be too mild to thoroughly clean the uninstrumented areas if the irrigation time is not long enough. In this figure, some of the predentin is still left to cover the mineralized dentin and the calcospherites.
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Re: Irrigants and Intracanal Medicaments
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Physical Means of Canal Cleaning and Disinfection


ULTRASONIC CLEANING

Endodontic ultrasound has become an important tool in modern endodontic work. Ultrasound is used in a variety of tasks including finishing access cavity preparation, removing pulp stones, locating canal orifices, opening calcified canals, fractured instrument removal, placing cements and root filling materials, and retro canal preparation during surgical procedures.117 The use of ultrasonic energy for cleaning of the root canal and to facilitate disinfection has a long history in endodontics. The comparative effectiveness of ultrasonics and hand instrumentation techniques has been evaluated in a number of earlier studies.117-121 The majority of these studies concluded that the ultrasonics, together with an irrigant, contributed to a better cleaning of the root canal system than irrigation and hand instrumentation alone. Cavitation and acoustic streaming of the irrigant contribute to the biological chemical activity for maximum effectiveness.122 Analysis of the physical mechanisms of the hydrodynamic response of an oscillating ultrasonic file suggested that stable and transient cavitation of a file, steady streaming, and cavitation microstreaming all contribute to the cleaning of the root canal.123


Several different methods have been used to study the effect of ultrasound on the cleanliness of the canal. These include bacteriological, histological, and microscopic techniques.124-128 Studies focusing on the ability of ultrasound to remove smear layer have shown contradictory results. This is not, in fact, surprising because it is generally known that smear layer can be removed primarily by chemical means only, or by appropriate laser treatment.129,130 It has also been shown that to work effectively, ultrasonic files must be free in the canal without contact with the canal walls.131


Among the areas that are particularly difficult to clean are anastomoses between double canals, isthmuses and fins. Several studies have indicated the importance of ultrasonic preparation for optimal debridement of the root canals and isthmuses.130,132-134 Ultrasonics also eliminated bacteria from canals more effectively than hand instrumentation.134-136 However, not all studies have supported this finding.137 It has been proposed that canal anatomy is more important than ultrasound for effectiveness of the cleaning procedures.138


The direct bactericidal effect by ultrasonic energy seems to be very limited at best.139,140 Ultrasonics seems to exert its antimicrobial effect when used together with irrigants, perhaps via the physical mechanisms of cavitation and acoustic streaming. It is also possible that ultrasonics helps irrigants penetrate into areas, in complex canal systems, not easily reached by normal irrigation.


In a series of studies on the effect of canal shape (taper) and instrument design, Lee et al.141 demonstrated in simulated plastic root canals that the diameter and taper of root canal influenced the effectiveness of ultrasonic irrigation to remove artificially placed dentin debris. After ultrasonic irrigation, the amount of debris in the #20/04 taper group was significantly higher than that for the size #20/06 group and the size #20/08 group. Van der Sluis et al.142 using roots from human teeth ex vivo reported that there was a tendency for ultrasonic irrigation to be more effective in removing artificially placed dentin debris from simulated canal extensions from canals with greater tapers.


A study using a split tooth technique found that ultrasonic irrigation ex vivo was more effective than syringe irrigation in removing artificially created dentin debris placed in simulated uninstrumented extensions and irregularities in straight, wide root canals.141 In another study, passive ultrasonic irrigation with 2% NaOCl was more effective in removing Ca(OH)2 paste from artificial root canal grooves than syringe delivery of 2% NaOCl or water as an irrigant.143


Ultrasonic instrumentation can also have an impact on canal dimensions and in some cases can cause unwanted complications such as straightening of the canal (transportation), perforations, and extrusion of infectious material beyond the apex.131,144-147 A histobacteriological study of teeth with non-vital pulps showed compacted debris and bacteria in the apical region and in the dentinal tubules after ultrasonic instrumentation.148 In one study, the step preparation technique was shown to cause more extrusion than the standard technique, whereas the least extrusion was detected with the crown down and ultrasound techniques.149 Interestingly, Van der Sluis et al.142 suggested that a smooth wire during ultrasonic irrigation is as effective as a size 15 K-file in removal of artificially placed dentin debris in grooves in simulated root canals in resin blocks. It is possible that using an ultrasonic tip with a smooth, inactive surface, preparation complications are less likely to occur.


Recently, serious damage to paradental tissues was reported in a case where dental cement, used for cementing a post, was removed by ultrasound from a maxillary incisor.150 After "several minutes" of ultrasound treatment, the patient complained of discomfort. During the following days and weeks, a large necrotic zone developed, affecting the bone and soft tissue around the tooth, resulting in the loss of the tooth. In controlled use, ultrasound together with irrigation, however, is not likely to cause harmful temperature rise.151


LASERS

Lasers in endodontics are dealt with in Chapter 26E, "Laser in Endodontics". However, their role in endodontic disinfection will be summarized briefly in the following sections. The potential of different endodontic lasers in eradicating root canal microbes has been a focus of interest for many years. Early comparative studies indicated, however, that the antibacterial effectiveness of lasers in the root canal was inferior to NaOCl irrigation.152-154 Excellent antibacterial efficiency against E. faecalis was reported by Gutknecht et al.;155 using a holmium:yttrium-aluminum-garnet (Ho:YAG) laser on root canals infected with this species in vitro, 99.98% of the bacteria were eliminated. However, Le Goff et al.156 obtained only an 85% decrease in the bacterial counts by a CO2 laser, clearly less than by irrigation with 3% NaOCl. Contrary to the main stream of results with laser treatment, Kesler et al.157 indicated that complete sterility of the root canal can be obtained with a CO2 laser microprobe coupled onto a special hand piece attached to the delivery fiber. Schoop et al.158 in subsequent studies indicated that the effect of laser is dependent on the applied output power and specific for different bacteria. Complete sterility still seems to remain a challenge with laser treatment.159,160 Another important aspect of laser radiation in endodontics is the effect of lasers on the smear layer, which may also facilitate effective disinfection of the canal.


New Developments in Root Canal Disinfection


Several new technologies have been introduced during the last few years to improve the effectiveness of root canal disinfection. Increasing attention has been focused on the use of ozone, photoactivated disinfection with low-energy laser, electrochemically activated water, and electric current.161-165 In a comparative study, 3% NaOCl was more effective than electrochemically activated water in eradicating E. faecalis in an ex vivo model.162 Nagayoshi et al.164 reported relatively equal effectiveness in killing E. faecalis with ozonated water and 2.5% NaOCl, when the specimen was irrigated with sonication. However, NaOCl was superior to ozonated water in killing E. faecalis in broth culture and in biofilms.166 Estrela et al.167 studied root blocks infected for 60 days with a strain of E. faecalis and were unable to eradicate all bacteria with any of the methods used, including ozonated water, gaseous ozone, 2.5% NaOCl, and 2% CHX. Many of the new methods offer a biological approach to canal disinfection. However, available evidence so far has failed to show that these methods would be superior or sometimes even equal to existing ones with regard to their antimicrobial effectiveness in the infected root canal.


One of the latest new developments for canal disinfection are bioactive materials such as bio(active) glass.168 In vitro studies have shown that bioglass has antimicrobial activity against a range of microbes and that this activity is, surprisingly, potentiated by dentin.169,170 However, studies demonstrating better antimicrobial effect when compared to Ca(OH)2 in vivo so far are lacking. Recent experiments with nanometric bioactive glass indicated excellent antimicrobial effect in a human dentin model.171 More research will be needed to evaluate the value of bioglass in root canal disinfection.


Intracanal Interappointment Medicaments


INTRODUCTION


In the treatment of teeth with a vital pulp, there is no need for intracanal medication. However, if time does not allow completion of the treatment in one appointment, it is generally recommended that the root canal should be filled between appointments with an antibacterial dressing, for example, Ca(OH)2, to provide sterility in the canal space until a permanent root filling is placed. However, there are no studies comparing the bacteriological status of the root canals following pulpectomy, when the canals have been left empty or filled with an antibacterial dressing.


The question of the role of intracanal medicaments becomes more relevant, and complex, in the treatment of pulpal necrosis and apical periodontitis. There is overwhelming evidence in the literature that many if not most root canals contain viable microorganisms after the completion of the chemomechanical preparation at the end of the first appointment.8-10,54,55,57,172-175 Therefore, a variety of intracanal medicaments have been used between appointments to complete disinfection of the root canal. In addition to killing bacteria, intracanal medicaments may have other beneficial functions. Ca(OH)2 neutralizes the biological activity of bacterial lipopolysaccharide176,177 and makes necrotic tissue more susceptible to the solubilizing action of NaOCl at the next appointment. Another aspect in using intracanal medicaments may be that a more thorough instrumentation is achieved because of the longer overall time used for the treatment. On the other hand, several appointments can also increase the risk for aseptic complications, for instance, through a leaking temporary filling and poor patient compliance.178


Several studies have indicated a poorer prognosis of the treatment of apical periodontitis if viable bacteria are residing in the root canal system at the time of filling.5-7 Other studies, however, have contradicted these results and reported no significant differences in healing between teeth filled after positive or negative cultures from the root canal,8 or between treatments performed in one or two appointments.8,9 It has also been suggested that "intracanal sampling techniques suffer from deficiencies that limit their predictive value."179 A permanent root filling of high quality using endodontic cements with antibacterial activity can effectively seal and entomb residual microorganisms in the canal and prevent communication with periradicular tissues. Continued killing of the micro-organisms could take place due to the antibacterial activity of the root-filling materials44,180 and unavailability of nutrients.


CA(OH)2

Ca(OH)2 has a special position in endodontics. Indications for the use of Ca(OH)2 in the prevention and treatment of various pulpal and periapical conditions have been numerous. In addition to endodontic infections, use of Ca(OH)2 has been widely advocated in dental traumatology and in the treatment of resorptions. The classical studies in the 1970s and 1980s at the university of Umea, Sweden, were a strong stimulus for the wide spread use of Ca(OH)2 as a local disinfecting medicament in the root canal for the treatment of apical periodontitis. Bystrom et al.6 reported that Ca(OH)2 was an effective intra-canal medicament, rendering 34 out of 35 canals bacteria free after a 4-week period. The effectiveness of interappointment Ca(OH)2 was also reported by Sjogren et al.,181 who demonstrated that a 7-day dressing with Ca(OH)2 eliminated all bacteria in the root canal. However, these pioneer studies have been challenged by others who reported a residual flora in 7 to 35% of teeth after 1 or more weeks with Ca(OH)2 in the canal.21,182-184 Kvist et al.185 reported the antimicrobial efficacy of endodontic procedures performed in a single visit (with 10-minute iodine irrigation), compared with a two-visit procedure, including an interappointment dressing with a Ca(OH)2 paste. Residual microorganisms were detected in 29% of the one-visit teeth and in 36% of the two-visit-treated teeth, with no statistically significant differences between the groups.


Zerella et al.42 compared the antibacterial activity of Ca(OH)2 mixed either with water or with 2% CHX in vivo. Pure Ca(OH)2 completely disinfected 12 out of 20 teeth, while the Ca(OH)2-CHX paste disinfected 16 of 20 teeth. The difference, however, was not statistically significant because of small sample size. Siqueira et al.186 examined bacterial reduction in teeth with apical periodontitis, after instrumentation and irrigation with 0.12% CHX solution and after 7 days of intracanal medicament with a Ca(OH)2-CHX (0.12%) mixture. After finishing the chemomechanical preparation, 7 of the 13 cases still showed growth, while after the Ca(OH)2-CHX treatment only one of 13 teeth was culture positive.


Vivacqua-Gomes et al.40 examined the benefit of interappointment Ca(OH)2 medicament in root canals in an ex vivo model. The premolar teeth were infected with E. faecalis for 60 days, and the canals were instrumented using rotary instruments. Irrigation, interappointment medication, and root filling were performed following five different protocols, either in single visit or in multiple visits. A second bacteriological sample was obtained 60 days after the root filling (the root fillings were removed and samples taken). Bacteria were found in 3 of 15 teeth (20%) irrigated with 2% CHX gel and filled in single visit and in 4 of 15 teeth (25%) irrigated with CHX and filled with Ca(OH)2 for 14 days before the gutta-percha/sealer root filling was placed. Teeth that were left empty for 1 week after irrigation and before root filling, or irrigated with saline only instead of CHX, or filled without sealer showed bacteria in 40 to 100% of the teeth 60 days after the root filling was placed. Because of the small size of the experimental groups, far-reaching conclusions cannot be made. However, the result supports the finding of other studies indicating that interappointment Ca(OH)2 may not add to the antibacterial effectiveness of the treatment. Moreover, the results emphasize the importance of not leaving the root canal empty (no medicament, no root filling) as well the role of sealer in the joined effort to combat infection.


CHLORHEXIDINE DIGLUCONATE

CHX is used as an irrigating solution during or at the end of instrumentation. However, CHX has also been used as an intracanal medicament between the appointments. Recently, interest has been focused on the effectiveness of CHX in gel form or as a mixture with Ca(OH)2 as an intracanal interappointment dressing.187,188 The information available is based mostly on in vitro and ex vivo experiments in which several intracanal medicaments have been compared for their activity against induced dentin infection. Siren et al.188 using a bovine dentin block model reported that Ca(OH)2 mixed with CHX was much more effective in disinfecting dentin infected with E. faecalis that pure Ca(OH)2. Ercan et al.187 reported 2% CHX gel was significantly more effective than Ca(OH)2 combined with 2% CHX, or Ca(OH)2 alone, against root dentin infected with E. faecalis and the yeast C. albicans after 7, 15, and 30 days of incubation. Similarly, it has been reported that 2% CHX gel alone completely inhibited the growth of E. faecalis after 1, 2, 7, and 15 days in the root canal whereas Ca(OH)2 allowed some microbial growth at all experimental times.41 Interestingly, in this study, the combination of the CHX gel and Ca(OH)2 had killed all bacteria in the 1- and 2-day samples, but failed to secure sterility in the 7- and 15-day samples. The antibacterial efficacy of intracanal medication with Ca(OH)2, 2% CHX gel, and a combination of both was assessed in a clinical study in teeth with chronic apical periodontitis.43 Bacterial samples were taken before and 7 days after filling the canals temporarily with the medicaments. CHX and Ca(OH)2, alone as well as their mixture, all performed equally well, and no statistically significant differences could be detected in their antibacterial effectiveness.


OTHER ALTERNATIVES FOR INTERAPPOINTMENT MEDICAMENTS

Intracanal Medicaments Containing Antibiotics

Throughout the history of endodontics, there have been time periods with increased interest in the use of local antibiotics as temporary canal dressings for root canal disinfection.189-196 Locally used antibiotics have not, however, become an established part of root canal disinfection and eradication of the infection. The reasons for the failure of antibiotics to overtake endodontic infection control are many. Many of the antibiotics tested are bacteriostatic, which may not be a good strategy for treating endodontic infections. Generally, bacteriostatic antibiotics prevent the growth of the microorganisms without killing them, giving the host defense a possibility to deal with the infection. In the necrotic root canal, however, there is no host defense because of the lack of the circulation. Therefore, the antibacterial effect of such antibiotics in the root canal may be only temporary. On the other hand, it is possible that some bacteriostatic antibiotics can have a bactericidal effect when used in high concentrations, usually the case with locally used antibiotics. However, information about this is scarce and presently there is no direct evidence of bacteriostatic antibiotics used in the root canal, although one study indicated that mixing erythromycin with Ca(OH)2 improved the effectiveness against E. faecalis as compared to Ca(OH)2 alone.197


With bactericidal antibiotics, the potential problem in the root canal may be the metabolic and physiological state of the microorganisms. Many bactericidal antibiotics are most effective when the microbial cells are in active growth phase, which may not be the case in the necrotic root canal with only limited nutrients available. In general, specific information about the effectiveness of intracanal antibiotics in infection control in endodontics is limited. Recently, an interesting new development has taken place with the use of antibiotic cocktails in the treatment of teeth with immature apex and apical periodontitis.198,199 It is possible that better circulation and survival of some pulpal cells in the apical root canal are among the key factors for the promising results reported so far. Future research will show whether this approach can be extended to the treatment of teeth with closed apex and apical periodontitis.


Phenol Compounds

Chemicals of the phenol group such as phenol, formocreosol, cresatin, parachlorophenol (monoparachlorophenol), camphorated phenol, and camphorated parachlorophenol have a long history in endodontics as locally used root canal disinfecting agents. They have been applied into the pulp chamber in a moist cotton pellet (vapor effect), or the whole canal has been filled with liquid with various concentrations of the phenol compound.200-208 The rationale of using phenol compounds for root canal disinfection has its roots in their role as general disinfecting agents in the past. However, emphasis of safety in addition to effectiveness has resulted in dramatic decline in their use generally. Also in endodontics, concerns have been raised regarding the toxicity and possible mutagenicity of the disinfecting agents of the phenol group.209-213 There are several demonstrations of their cytotoxicity,209,214,215 however, recent studies indicate that the risk of genotoxicity by the various phenol compounds used in endodontics is small.211-213 Comparative studies of the antimicrobial effectiveness of the phenol compounds have not been able to show superiority of the substances over the other. On the contrary,200,203,205,208,209 Bystrom et al.203 reported that Ca(OH)2 was superior to camphorated parachlorophenol (CMCP) by its antibacterial potential when used for 4 weeks as the local intracanal medicament. Several studies have indicated relatively rapid loss of activity of CMCP in the canal, although the results show variation.206,216 In the balance of the benefits and the demonstrated and potential weaknesses of phenol compounds, it can be predicted that they will be increasingly replaced by other, more biological disinfecting agents.


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