Endodontic biofilm, technology and pulpal regenerative therapy: where do we go from here?

In the past, scientific progress was considered as a series of steady and incremental developments taking place through the accumulation of accepted facts. This concept was then challenged by Kuhn (1962) who described an episodic model in which progress takes place in revolutionary steps when an accepted model undergoes sudden drastic changes or a paradigm shift. Microbial diseases, known under various names such as diseases of infection, infectious diseases, transmissible or communicable diseases, were considered to be caused by specific biological agents. Whilst that is true for classical infectious diseases such as tuberculosis and tetanus that are caused by single, specific aetiological agents, there are several chronic diseases that are caused by a consortium of microbial species living in an ecological habitat, known as biofilms. Apical periodontitis belongs to this group of diseases. The purpose of this editorial is to briefly but critically survey the physical and biological challenges that limit our ability to predictably cure apical periodontitis and to explore the emerging opportunities in endodontic microbiology, cell biology and new generative medicine. Apical periodontitis is an inflammatory disorder of periradicular tissues caused by aetiological agents of endodontic origin. Although Antony van Leeuwenhoek (Dobell 1960) described several kinds of microorganisms (animalcules) in the dead pulps of carious human teeth in 1697, and two centuries later, the presence of several kinds of bacteria was rediscovered (Miller 1894) in necrotic pulps within root canals of diseased human teeth, the role of microorganisms in the causation of apical periodontitis remained uncertain until 1965 when Kakehashi and others convincingly demonstrated (in a rat model) the essential role of microbes in apical periodontitis (Kakehashi et al. 1965). In the mid-1970s, using advanced anaerobic technology, Sundqvist extended the aetiological role of microbes to apical periodontitis associated with human teeth containing necrotic pulps (Sundqvist 1976). In 1987, using the technique of correlative light and transmission electron microscopy, Nair presented the ultrastructural visualization of intracanal microbial flora and described it as embedded in ‘…extracellular matrix of bacterial origin’ and existing as sessile ‘… intricate communities promoting a symbiotic relationship’ (Nair 1987), what is now known as endodontic biofilms. Today, the confocal laser scanning microscopy (CLSM) has become the foremost imaging tool for biofilm research. The CLSM enables optical serial sectioning in different planes of fluorescence labelled living and fixed biofilm specimens. The CLSM contributed much to our understanding of biofilms in general and also of endodontic biofilms – the latter mostly by extrapolation of information from general and medical microbiology or by imaging biofilms extirpated from root canals. To the author's knowledge, there has been only one attempt to apply CLSC to endodontic biofilms in vivo (Schaudinn et al. 2009), and it has not yet been applied successfully to obtain in situ visualization of biofilms in root canals of diseased teeth, with the accuracy, strength of evidence and clarity of information that was achieved repeatedly (Nair 1987, Nair et al. 1990a, Nair et al. 2005) with the precision of correlative light and transmission electron microscopy (Fig. 1). (From Nair, Periodontology 2000 13, 121–148, 1997) To my knowledge, the term ‘biofilm’ was coined for the first time by Poul Harremoes, a Danish physicist, in a paper dealing with the diffusion kinetics of fluids into slimy biofilters that he called ‘biofilms’. Recognizing the crux of the problem he wrote, ‘… little attention has been given to … the fact that organisms are gathered in folks into which the substrate has to diffuse. This phenomenon is of particular significance for attached biofilms’ (Harremoes 1977). A biofilm (Costerton et al. 1995) is a polymicrobial community, with a biofilm growth pattern, living embedded in a self-produced matrix of highly hydrated extracellular polymeric substances (EPS). The microbes in a biofilm adhere to each other and/or to a moist surface as against planktonic organisms, which are free-swimming/floating single microbial cells in an aqueous environment. The intracanal microbes living in a biofilm (Fig. 1) can advance or their products can egress into the periapex. Consequently, the body mounts an array of defence consisting of several classes of cells, of intercellular messengers, and of effector molecules (Nair 2004). In spite of this formidable defence, the body cannot remove the microbes, well entrenched as biofilms, in the sanctuary of the necrotic pulp within root canals. In essence, apical periodontitis is not self-healing. The biofilm microbes and the host defence clash, and strike an equilibrium that allows the persistent biofilms in the root canal and limited host response leading to various categories of lesions that are given the overarching name of apical periodontitis (Nair 1997). Because intracanal biofilm is the essential aetiological agent of primary apical periodontitis, the logical treatment for the disease consists of mechanically disrupting the biofilm, removing or at least reducing substantially the intracanal microbial load with an irrigating solution and then preventing reinfection by root filling. Currently, this is achieved by root canal instrumentation, irrigation with sodium hypochlorite (NaOCl) solution, rinsing with EDTA and, in multiple visit treatments, application of a microbicide dressing. Investigations (Nair et al. 2005), however, show that the infectious agents are mostly located in uninstrumented recesses of the main canals, the isthmus communicating between them and in accessory canals. The microbes in these untouched locations exist primarily as biofilms that are not removed predictably by instrumentation and irrigation with NaOCl in one treatment session (Nair et al. 2005), nor after two-visit treatment protocols (Vera et al. 2012). The application of two-visit protocols did result in microbial reduction in the treated root canals as compared to one-visit protocols, but even so, microbes remained in isthmuses and other inaccessible areas of the canal system. An important point regarding the effectiveness of root canal treatment is whether or not residual microorganisms really matter? The long-term impact of residual biofilm depends on several factors such as its location within the root canal system, the size and composition of the microbial population in the biofilm, and the availability of nutrients for the microbes. Root canals containing microbes in ‘subcritical’ numbers or situated in locations where they or their products are inaccessible to periapical tissues can remain ‘harmless’. Depending on the factors outlined above and those that have been dealt with at length (Sundqvist & Figdor 2003, Nair et al. 2005); remaining microbes may or may not pose an immediate obstacle for healing. It will be a challenging task to find out which residual infections in what composition, quantity and location will persist and thus impair apical healing. However, the occurrence of post-treatment apical disease some years after initial root canal treatment that appeared to heal initially (radiographically) may be an indication that residual biofilms (or possibly renewed infection through coronal microleakage) are of concern for apical healing. Therefore, the problem of microorganisms surviving in the inaccessible, remote areas of the canal system beyond the reach of contemporary treatment is a potential risk that there may result over time an unfavourable apical healing response (Sjögren et al. 1997). When the treatment is performed to a sufficient standard, healing of the periapical lesion usually occurs with osseous regeneration, which is characterized by gradual reduction and resolution of the radiolucency on subsequent follow-up radiographs. For various reasons, complete bone healing of a lesion or a reduction in the volume of a lesion may not occur in all root filled teeth. Such nonhealing post-treatment periapical radiolucencies are also known as persistent apical periodontitis or simply as endodontic failures. Taken together, there are six biologic factors that contribute to persistent post-treatment apical radiolucencies (Nair 2006). They are as follows: (i) residual intraradicular biofilm in the complex apical root canal system (Nair et al. 1990a) (Figs 2 and 3), (ii) extraradicular infection, generally in the form of periapical actinomycosis (Nair & Schroeder 1984), (iii) extruded root canal filling or other exogenous materials that cause a foreign body reaction (Nair et al. 1990ab), (iv) accumulation of endogenous cholesterol crystals that irritate periapical tissues (Nair et al. 1993, 1998), (v) true cystic lesions (Nair et al. 1993) and (vi) scar tissue healing of the lesion (Nair et al. 1999). (Reprinted from Nair, Int Endod J, 2006, 39, 241–81) (Reprinted from Nair, Int Endod J, 2006, 39, 241–81) There has been considerable interest as to whether the apical root canal system contains microbes immediately after completing root canal treatment. It has been shown that 14 of 16 instrumented and root filled canals in mandibular molars with apical radiographic lesions had residual biofilm in mesial roots when the treatment was completed in one-visit (Nair et al. 2005). Further, microcomputer tomography (μCT) has detected inadequacies in root canal instrumentation techniques. For example, approximately 40% of the walls of root canals remained untouched by instruments (Peters et al. 2001, 2003, Huebscher et al. 2003). The great anatomical complexity of the root canal system (Hess 1921, Perrini & Castagnola 1998) and the ecological organization of the flora into biofilms (Costerton et al. 1995, 2003, Costerton & Stewart 2000) make the goal of achieving a microbe-free canal by root canal treatment currently unattainable. Thus, biology sets limits to technology in endodontics. Then, the question arises as to why a large number of lesions of apical periodontitis heal after conventional root canal treatment. This is because the root canal treatment results in the following: (i) a substantial reduction in the intracanal microbial load to a subcritical level to sustain the inflammation of the periapex (Nair et al. 2005), (ii) the drastic disturbance to the delicate microbial ecosystem, (iii) destruction of the microbial habitat, (iv) the robust host defence contributes to the healing of apical radiolucent lesions after root canal treatment and further, (v) it also depends on access of the biofilm microbes to the periapical tissues (Sundqvist & Figdor 2003). As there are physical and biological limits to technological innovations in endodontics, further progress in Endodontology lies with advances in endodontic microbiology and cell biology. The new frontiers of the chemo-mechanical landscape of endodontic treatment will be drawn by control of the biofilm. In the short term, it should be possible to develop quick acting, low viscous, deep diffusing and body compatible microbicides. Microbes in biofilms are more than a thousand times resistant to antimicrobial agents as against the same organisms in free-floating (planktonic) form (Costerton et al. 1995, Wilson 1996). Therefore, the reported positive effects of microbicides of root canal disinfectants based on short-term in vitro experiments (Sirén et al. 2004) may not be clinically valid! Further, the biofilm is a massive genetic lending library for microbes. This is because the EPS matrix is flooded with the components of lysed microbial cells, which include DNA fragments for lateral or horizontal gene transfer. Therefore, caution must be exercised in the use of intracanal antibiotics so as to avoid horizontal gene transfer, allowing pathogenic bacteria, to share gene resistance to drugs. Understanding the complexities of biofilms is the key for further progress in root canal treatment. It should be possible to develop biofilm inhibitors and agents that weaken the biofilm structure, particularly the EPS. Microbes in the biofilm communicate with each other through a biochemical signalling language known as the quorum sensing, which literally means to judge the population size or density of the microbes in the biofilm. If the population size is small or subcritical for them to advance into the host tissue, they may be able to ‘lie low’ by ‘switching off’ offensive genes by the mechanism known as gene silencing (Fire et al. 1998). As in human wars, an army with compromised lines of communication is vulnerable to isolation and defeat, disrupting the microbial communication lines should help to control microbes in root canal biofilms. Also known as RNA interference (RNAi) (Fire et al. 1998), gene silencing was discovered in 1998, a discovery that won the Nobel Prize for Andrew Fire and Craig Mello in 2006. In gene silencing, certain molecules trigger the inactivation of the messenger RNA from a particular gene, so that no related protein is produced. Thus, the gene is silenced, inactivated, blocked or turned off by a cellular mechanism that is distinctly different from genetic modification by chromosomal changes and gene mutation. Gene editing is another fast emerging cell-based gene therapy to restore or modify gene function by correcting the disease-causing gene by removing it/or replacing it with a normal or modified version of the gene. It is ‘cut and paste’ (Komiyama 2013) molecular technology or genetic ‘surgery’ using molecular ‘scissors’! Could gene silencing and gene editing lead to new treatments? It has been shown in monkeys that blood cholesterol levels can be lowered by suppressing a gene (Soutschek et al. 2004). Thus, attaining the capability to correct or block disease-promoting microbial genes should lead to new treatment lines against microbes in the biofilm. Thus, there is considerable interest to develop antibiofilm strategies that can be clinically applied to treat biofilm diseases. These include (i) improving methods for physical debridement of biofilm, ii) application of chemical microbicides, (iii) developing biological strategies that change the genotype, phenotype and/or behaviour of microbes so as to make them poor biofilm builders and (iv) that weaken the EPS. In a nutshell, our contemporary chemo-mechanical treatment of root canals needs to evolve into broader mechano-chemo-biological procedures. The problem of the currently intractable residual biofilm in root filled teeth has important implications in other areas of endodontics, for instance, on pulpal regenerative treatments designed to replace ‘damaged root structures as well as the pulp–dentine complex’ (Murray et al. 2007). The complexity of the issues involved in endodontic regenerative therapy is being debated (Spångberg 2009, Hargreaves & Law 2010, Andreasen & Bakland 2012, Fouad & Nosrat 2013, Lin et al. 2013). On the other hand, there is a consensus that many teeth with infected and necrotic pulps cannot be made completely microbe-free using contemporary treatment procedures (Nair et al. 2005, Vera et al. 2012). Further, biofilms may be present even in root filled teeth with radiographically healed apical lesions. These pockets of residual biofilm pose some risk of apical healing around a root filled canal, but have significant potential to perturb regeneration of a healthy pulp. Nevertheless, many clinicians and researchers seem to believe that therapeutic replacement of damaged dental body parts is within practical reach in spite of the unpredictable and variable outcomes of pulp regenerative procedures (Kahler et al. 2014). Even if an immature tooth with pulp disease but with a vital apical growth region may undergo ‘revascularization’ and may become to some extent stronger, longer and be preserved in a better state than if untreated (Cotti et al. 2008), there remains the threat of subsequent breakdown under microbial activity (Lin et al. 2014). It has been claimed that application of the principles of tissue engineering can result in the ‘regeneration of dental pulp with newly deposited continuous layer of dentine’ (Huang et al. 2010) in human root segments implanted into immunocompromised mice. Leaving aside the question as to which type of new hard tissue and under what experimental condition was formed inside the hollowed root segments (Huang et al. 2010), it will suffice here to say that the experimental model used was sterile. It remains to be shown that regenerative cells establish themselves in an infected root canal and recreate the pulp–odontoblast–dentine complex. The enthusiasts of pulpal regenerative therapy do not see the ‘elephant in the room’. There is no evidence yet for the much celebrated claim that a ‘paradigm shift is taking place’ (Huang 2008) in the clinical management of neither immature nor mature teeth with infected and totally necrotic pulps (Nair 2014). This situation is reminiscent of what happened in dental research during the 1970s. A huge sum of money was then spent on developing vaccines against caries and periodontal diseases. In retrospect, with the benefit of hindsight, we know that those investments in money, materials and efforts did not lead to the desired results. The infatuation with pulp regenerating therapy is understandable but may turn out to be ‘barking up the wrong tree’. This is because the breakthrough in research is more to in dental from the new generative (Nair 2014). The of in et al. 1997) demonstrated that a cell recreate a The experimental for the of was out in the on or tissue be from a than a after the of researchers & that cell can be to become cells The Nobel Prize in and for was to and for their In 2013, human cells were to cells using the technique of cell et al. 2013). There is an to develop and methods to cells to It was reported in that blood cells from to a solution for in the cells and were able to form different of cells et al. 2014). The experimental called of is yet to be successfully by other for It is that advances in cell research undergo and by other so should in pulp regeneration to that experimental and clinical protocols are by other and clinicians in the endodontic In the of new replacement teeth, the of a et al. 2013) particular The researchers cells from human The cells were in vitro and with A of the cells was implanted a and teeth with Taken together, the developments in cell and new generative technology that the regarding the availability of and cells are It is the author's that there is potential for a new tooth in a healthy compared with to one that may pockets of This is because of the limits of treatment to predictably remove all residual biofilms in teeth with necrotic In to microbes living in a biofilm have different from planktonic organisms of the same The in the EPS enables them to and in various The EPS and the layer of cells the of the biofilm In to the physical the lateral gene biofilm microbes more resistant to microbicides and antibiotics (Costerton et al. 1995, Wilson 1996). The biological challenges of dealing with residual root canal biofilms have so compromised our ability to predictably cure apical These the to such compromised and teeth be by replacement teeth and in vivo using In other … and dental will become as new teeth be in using the cell (Nair 2014). This is to of a damaged and it based on the of to restore it by A new generative treatment for diseased teeth is on the of the regenerative (Nair 2014). The of Endodontology than This editorial is based on two given by the at the of the of in and the in in 2013,