The biofilm concept recognizes biofilm formation as a key mechanism linked to microbial survival, and its application in endodontics has led to the understanding of their involvement in the pathogenesis of endodontic infections [1–3]. In general, biofilm formation reflects an essential mechanism of microbial adaptation to environmental conditions. Bacteria in biofilms are surrounded by a matrix of bacterial exopolysaccharides and exogenous substances (polysaccharides, proteins, mineral crystals, extracellular DNA) [10, 11] that protect them from the host’s immune defences. Antibodies and phagocytes have difficulties to penetrate into the biofilm and may even undergo deactivation whilst inside the matrix [10, 11]. Bacteria in biofilms are also less susceptible to the action of antibiotics, which may contribute to the development of chronic infections and relapses [12, 13].

Several studies have described the presence of biofilms formed in infected root canals [14–16]. Biofilm structures have been reported to be formed alongside the canal walls, inside dentinal tubules, apical deltas and periapical areas [1–3]. The presence of these microbial structures has been associated with different clinical states including post-treatment endodontic infections [14–16].

Of importance is to understand the biological basis of biofilm formation as it is possible that various microbial genetic regulatory pathways involved may also play a crucial role in mechanisms of resistance to host immune defences and antimicrobial treatment [12]. Notwithstanding the characterization of biofilms in infected root canals, the mechanisms behind their formation in root canals have not been well established. As most of the species found in root canals are also found in the oral cavity, it is reasonable to speculate that the formation of microbial biofilms in root canals may have similar mechanisms as oral biofilms. Figure 3.2 depicts the main events occurring during the formation of a biofilm.

Contrary to the traditional view of extra-radicular tissues being always free of bacteria, compelling clinical evidence now exists on bacteria forming biofilms on extra-radicular surfaces [33–36]. Although most of the studies are described as case reports, it is reasonable to conclude from the available information that the formation of extra-radicular biofilms occurs with relative frequency.

Although still unclear, the formation of extra-radicular biofilms seems to be a consequence of massive infection of the root canal system associated with prolonged exposure of the canal space to the oral environment [34]. Of interest is, however, that most cases presenting extra-radicular biofilms are associated with sinus tracts which may indicate inclusion of oral fluids during biofilm formation. The latter hypothesis is sustained with the finding of calculus-like extra-radicular biofilms [33, 34, 36]. Figure 3.3 shows a case of maxillary right central and lateral incisors with deficient root canal treatments and presenting calculus-like material covering the apexes of roots [33]. Upon clinical inspection, an open fistula was detected in the apical area of teeth 11 and 12. Both teeth were sensitive to percussion, and the apical mucosa was sensitive to palpation. Radiographic examination showed a large periapical lesion with a thick layer of radiopaque material covering both root tips. Treatment included orthograde retreatment followed by apical surgery. As it is visualized in the clinical photograph, during surgery both root tips presented a calculus-like material covering the root surfaces. Examination by scanning electron microscopy (SEM) of the resected specimens confirmed the presence of mineralized biofilms in the apexes, where cells were embedded in mineralized matrix in a very similar fashion as supra- or sub-gingival calculus. One of the possible explanations for the occurrence of these mineralized structures is the long-standing sinus tract which may have allowed passage of fluids from the oral environment, including minerals and salts that could form these solid mineralized masses.

The presence of biofilms in root-filled teeth leads to a chronic inflammatory reaction in the periapex which is characterized by the proliferation of macrophages, lymphocytes and plasma cells [37, 38]. Chronic apical lesions become encapsulated in collagenous connective tissue which is stimulated by the upregulation of connective tissue growth factors (TGF-β) [37, 38]. In this chronic phase which can remain symptomless for long periods of time, activated T cells produce cytokines that downregulate the output of pro-inflammatory cytokines (IL-1, IL-6 and TNF-α), leading to the suppression of osteoclastic activity and reduced bone resorption [37, 38]. Upon a secondary invasion of microorganisms, the lesion can spontaneously turn into an acute inflammatory reaction by rapid recruitment of PMNs , a feature that is characterized by a rapid restitution of apical bone resorption. And the previous silent clinical situation may suddenly turn into a symptomatic phase.

The chronic inflammatory lesion associated to failed-root canal treatments is in many cases associated to the presence of well-developed fibrous capsules consisting of dense collagenous fibres that are firmly attached to the root surface [37, 38]. These chronic lesions, also known as granulomas , do not normally harbour microorganisms but only in special cases: (a) acute inflammatory phase [39], (b) periapical actinomycosis [40–42], (c) transient contamination during root canal instrumentation [43, 44] and (d) infected periapical cysts with cavities open to the root canal [45]. The main function of the apical granuloma is thus to contain and encapsulate the advancement of the infection. In the lumen of the granuloma, macrophages, including blood-derived macrophages, epithelioid cells and multinucleated giant are aimed to kill bacteria. However, complete eradication of bacteria does not always occur. As it has been described in few case reports [40–42], species of Actinomyces and Propionibacterium (formerly Arachnia) have been found forming clusters within the lumen of the granulation mass. Although the mechanisms behind clustering formation are not clear, it may seem that clustering occurs as a microbial strategy to persist within the granuloma and perhaps to reactivate and escape under special circumstances.

The survival of Mycobacterium tuberculosis in granulomatous tissues is a good example for understanding the mechanisms behind bacterial survival within a granuloma. It has been proposed that the unfavourable conditions inside the granuloma, such as nutrient limitation and low oxygen tension, trigger the metabolic downshift of M. tuberculosis into dormancy [46]. Of critical interest is, however, that under specific circumstances M. tuberculosis re-establishes its metabolic and replicative activity by the activation of a complex cascade of enzymes regulated by resuscitation-promoting factors (Rpf) [46]. Although it has not been established if Rpf orthologs are present in Actinomyces or other endodontic pathogens, the reactivation from a dormant state seems to be an interesting hypothesis to clarify the occurrence of exacerbations of chronic infections. This hypothesis was tested in an experimental study, where biofilm cultures of S. anginosus and L. salivarius were forced to enter a state of dormancy by exposing them to nutrient deprivation [47]. Dormant cells were then forced to reactivate by exposure to fresh nutrients, but even after 96 h the cells remained metabolically inactive. This observation highlights the null physiological response of dormant cells even in the presence of fresh nutrients, which may act as a mechanism to resist further disturbances.

The increased survival rate of bacteria is one of the fundamental causes of endodontic treatment failure and because chronic infections present as a complicated challenge [1–3]. In order to understand the mechanisms by which bacteria survive, it is important to differentiate two main concepts: resistance and tolerance. As it is illustrated in Fig. 3.4, resistance comprises the mechanisms that are specifically exerted by bacteria in the presence of antimicrobials and that are aimed to inactivate them. Common resistance mechanisms include physical prevention of the antimicrobials from reaching its target (e.g. low diffusion through the biofilm matrix), alteration of the target such that it is no longer recognized by the antimicrobial (e.g. modification of cell receptors) and inactivation of the antibiotic properties to obstruct its ability to interact with its target [48]. Tolerance is fundamentally different as it does not affect the ability of the antimicrobial to interact with its target. Although the molecular events that lead to antimicrobial tolerance in bacteria are not yet clear, the mechanisms that are involved seem to be mainly controlled by phenotypic adaptive processes (e.g. metabolic downregulation or adaptation) [48]. Phenotypic tolerance is elicited as a result of environmental factors (such as nutrient deprivation and pH changes) that affect antimicrobial-induced killing, whereas genotypic tolerance can arise from specific genetic changes within the tolerant bacteria [48].