In vitro and ex vivo microbial leakage assessment in endodontics: A literature review.

The aim of this study was to perform a literature review of published in-vitro and ex-vivo studies, which evaluated microbial leakage in endodontics in the past 10 years. A comprehensive electronic literature search was carried out in PubMed database for English articles published from 2005 to 2016 using the keywords "endodontics," "in vitro," "ex vivo," "microbial leakage," "microbial penetration," "saliva," "Enterococcus faecalis," "E. faecalis," "endodontic sealers," "temporary filling material," "apical plug," "mineral trioxide aggregate," and "MTA." The keywords were combined using Boolean operators AND/OR. Based on our search strategy, 33 relevant articles were included in the study. There are three main methods for assessment of bacterial microleakage, namely, (A) the dual-chamber leakage model, (B) detection of bacteria using a scanning electron microscope (SEM), and (C) polymerase chain reaction. All bacterial leakage models have some limitations and may yield different results compared to other microleakage evaluation techniques (i.e., dye penetration, fluid filtration, or electrochemical tests). The results of SEM correlated with those of microbial leakage test in most studies. Microbial leakage test using saliva better simulates the clinical setting for assessment of the leakage of single or mixed bacterial species.

INTRODUCTION

The ultimate goal of endodontic treatment is to eliminate the diseased pulpal tissue from the root canal system, provide a suitable environment for healing, and prevent apical periodontitis. Microorganisms are the main cause of pulpal and periapical diseases.[1] Well-packed root canal filling material and a hermetic apical seal allowing no leakage are crucial for successful endodontic treatment.[2] Evidence shows that apical periodontitis is caused by intracanal bacteria.[3] Apical periodontitis is treated by chemomechanical cleaning and disinfection of the root canal system followed by filling of the root canal and providing apical and coronal seal to prevent reinfection.[4] However, many studies have reported bacterial penetration through the entire length of the root canal within a few days following root canal filling with gutta-percha, which indicates that a perfect seal is hard to achieve in endodontic therapy.[56]

In this regard, different methods have been designed for microbial leakage assessment in endodontics. The aim of this study was to evaluate the different techniques proposed for microbial leakage assessment in endodontics by reviewing the relevant articles published in the past 10 years.

A comprehensive electronic literature search was carried out in PubMed database for English articles published from 2005 to 2016 using the keywords “endodontics,” “in vitro,” “ex vivo,” “microbial leakage,” “microbial penetration,” “saliva,” “Enterococcus faecalis,” “E. faecalis,” “endodontic sealers,” “temporary filling material,” “apical plug,” “mineral trioxide aggregate,” and “MTA.” The keywords were combined using Boolean operators AND/OR. Based on our search strategy, 33 relevant articles were included in the study [Table 1].

Table 1

List of included studies on microbial leakage published from 2005 to 2016 in PubMed-indexed journals

Search of the literature yielded 33 studies, which met our inclusion criteria. Information regarding the authors, titles, microbial leakage model used, and the results of the 33 studies are presented in Table 1.

Three main methods are available for assessment of bacterial microleakage, namely, (A) the dual-chamber leakage model, (B) detection of bacteria using a scanning electron microscope (SEM), and (C) polymerase chain reaction (PCR). In the dual-chamber leakage model, there is a split chamber with a connection path through the root canal of the teeth fixed at the center. The upper chamber contains bacterial species cultured in brain heart infusion broth and the lower chamber contains the brain heart infusion broth. The entire root is covered with a sealing material while the root tip (apex) is left uncovered. In case of occurrence of bacterial leakage, the culture medium in the lower chamber becomes turbid.[172327] In the SEM and PCR techniques, bacteria can be directly visualized or detected in the root canal or dentinal tubules.[2633] Karagenc et al.[34] reported a poor correlation between the results of microbial leakage test and fluid filtration, electrochemical test, and dye penetration. Nawal et al.[15] showed that the results of SEM correlated with those of microbial leakage test. In the PCR method, DNA extracted from the specimens is amplified and then identified by the OCEAN technique.[16]

In the dual-chamber or split chamber model, the upper chamber may contain a single species (E. faecalis, S. mutans, P. mirabilis, or S. epidermidis),[7172335] multiple species,[827] or saliva.[1018] Timpawat et al.[39] demonstrated that bacterial leakage model (mainly coronal) better simulated the clinical and biological setting than the dye penetration method. According to their study, most endodontic cements have adequate antibacterial activity to stop the ingress of bacteria. Microbial leakage studies cannot estimate the time of occurrence of periradicular infection because it depends on several factors such as the virulence of microorganisms, defense capacity of the periradicular tissues, nutritional status, and bacterial interactions. However, chronic or acute infections may occur when microorganisms are present at the periapex.[4041] The usage of human saliva is advantageous because it highly simulates the clinical setting. However, it cannot simulate the alterations in the oral environment such as thermal changes or the effect of dietary regimen on the salivary flow.[101819] Verissimo et al.[42] showed that the evaluation of coronal leakage by use of bacteria provided more biologically significant and clinically relevant data than other methods.

Assessment of the sealing ability of gutta-percha obturation using saliva leakage method is based on the activity of salivary hydrolytic enzymes and their ability to break the seal.[18] Microbial products cause disintegration of gutta-percha and compromise the adaptation of gutta-percha to root canal walls, thus impairing the seal. In a study by Maniglia-Ferreira et al., decomposition and destruction of polyisoprene (the main substance of gutta-percha) produced high amounts of carboxyl and hydroxyl radicals during thermomechanical compaction and thermoplastic techniques, which resulted in molecular weight reduction and a decrease in the stability and sealing ability of the filling material and increased coronal microleakage.[43] In this review study, we found 31 studies that used split chamber technique (25 single species, 2 multiple species, and 4 saliva), of which 3 studies had used SEM and 1 study had used PCR technology.

CONCLUSION

All bacterial leakage evaluation techniques have some limitations, and may yield different results compared to other microleakage assessment methods (i.e., dye penetration, fluid filtration, or electrochemical tests). In most reviewed studies, the results of SEM correlated with those of the microbial leakage test. Microbial leakage test using saliva better simulates the clinical setting in assessment of leakage of single or mixed bacterial species. The greatest advantage of the PCR technique is its high specificity for detection of target microorganisms and decreasing the false positive results, which refer to the presence of residual bacteria within the root canal system before obturation.

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Conflicts of interest

There are no conflicts of interest.