The Biomineralization Conundrum of Calcified Extraradicular Deposits on the Apical Root Surface of Teeth: A Case Report

3.1. Microbial Implication in CEDARST Biomineralization

Microbe-mediated mineralization is a phenomenon ubiquitous in nature. All six taxonomic kingdoms contain members with the capability of forming minerals, including bacteria, fungi, viruses, and algae [22]. The mineralization processes in nature are calcification, silification, and iron mineralization [36-38], and the mechanisms involved can include extracellular and intracellular biomineralization [22].

Three types of bacterially mediated biomineralization processes have been described [39]: 1) biologically controlled mineralization, where specific cellular activity directs the nucleation, growth, morphology, and final location of minerals; 2) biologically induced biomineralization, where biological activity modifies chemical conditions in the environment, e.g., pH shifts or redox transformations; and 3) biologically influenced biomineralization, where passive mineral precipitation occurs in the presence of organic matter, such as extracellular polymeric substances (EPSs), which influence the crystal morphology and composition. Additionally, the term organo mineralization has been coined to define biologically influenced and biologically induced biomineralization [39]. Perhaps this latter definition is most suitable for CEDARST in teeth with AP, given the characteristics of this pathological mineralization, i.e., passive mineral precipitation by minerals coming from the saliva into the AP lesion, through a sinus tract (as most CEDARST case reports have been associated with sinus tracts) [15, 16]; as well as biological activity control from the bacteria established in the extraradicular biofilm, and, potentially, from the host. However, not every sinus tract leads to CEDARST, and other sources supplying the required minerals should be considered; specifically, inflammatory exudate from the periapical lesion, and periradicular tissue fluids saturated with minerals from bone solubilization, due to the AP inflammatory resorption process [15].

Although bacteria are believed to be only passively involved in the formation of dental calculus [27], it should be noted that some oral microorganisms have the ability to form intracellular and extracellular apatite crystals [40, 41] and so this kind of microbial calcification could eventually contribute to CEDARST formation. Neisseria flava, Aggregatibacter segnis, Streptococcus tigurinus, and Morococcus cerebrosus are bacteria that have been identified in vitro as contributors to the calcification of supragingival calculus [42]; it could be expected that these bacteria contribute in CEDARST formation, considering the saliva–sinus tract relationship. On the other hand, urease-producing bacteria, like Proteus spp., and the genera Providencia, Morganella, and Staphylococcus, have been associated with urinary calculi formation, through struvite biomineralization [25, 43]. Although struvite has not yet been described in CEDARST formation, there is a reasonable chance that this mineral contributes to this pathological mineralization, because Proteus spp [43], and the genera Providencia [45, 46], Morganella [47], and Staphylococcus [44] have all been identified in extraradicular biofilms, which are associated with cases of persistent AP.

Finally, microbial metabolism can provide a favorable environment for mineral precipitation [48]. Some researchers have demonstrated that microbial carbonic anhydrase significantly increases the rate of precipitation of carbonate minerals [49]. Additionally, on the host´s side, osteoclasts, the prime cells responsible for the periapical lesion formation, express carbonic anhydrase in the bone resorption process [50]. However, the carbonic anhydrase contribution in CEDARST has not yet been evaluated.

3.2. Role of EPSs in Biomineralization

Biofilms represent microbial communities that grow on many surfaces [51]. In such communities, microorganisms are encased in an organic matrix, in which the latter contains a variety of extracellular polymeric substances (EPSs) produced by the former, as well as other potential organic and inorganic compounds from the local environment [52]. Although the EPS composition varies with the structure and physiology of the component microorganisms, it may include a variety of capsule polysaccharides, extracellular DNA, secreted proteins, lipids, and—frequently—outer membrane vesicles (OMVs) [53].

Microorganisms can influence biomineralization by modifying milieu supersaturation levels through metabolic activity, as well as through the production of EPSs, which play an important role in the biomineralization process [39, 54]. EPSs can impact nucleation by stabilizing the first mineral seeds [39], possibly on account of their strong adsorption capacity (they are often anionic) for a large number of cations, and thereby act as a nucleation site in many environments [55]. It has also been evidenced that capsule polymers can adopt a secondary structure in the presence of some metal cations [52]; by such means, the former may influence mineral formation, considering their charge orientation and coordination chemistry [25]. Additionally, it has been stated that organic substances facilitate a decrease in the active energy of nucleation [56], so EPSs may also exhibit the same effect [57].

Bacteria have been detected as biofilm on the apical external root surface of teeth [12, 13, 45, 58, 59]; it can therefore be inferred that one or several components of EPSs in CEDARST are pivotal nucleation sites. It is also well established that bacterial biofilms are a common cause of persistent infections in the human body [51], as is the case in CEDARST.

Finally, it is important to note that, in general, fungi secrete large amounts of extracellular proteins with diverse functions including the reduction of soluble metal ions to their insoluble form and, eventually, to nanocrystals [60]. In this regard, as mentioned above, fungi are microorganisms that are potentially related to AP and might eventually contribute to the biomineralization role of EPSs in CEDARST. In addition, some filamentous fungi (Aspergillus niger, Aureobasidium pullulans, Cladosporium sphaerospermum and Exophiala jeanselmei) have been isolated from teeth with AP. These fungi are all able to produce melanin [10], which is a pigment and also a recognized virulence factor [61]. This melanin might contribute to the color appearance of CEDARST, which has been described as having a brownish-to-black color, as it was in the case described in this paper.

3.3. Roles of Nanobacteria and Nano-sized OMVs in Biomineralization

In 1998, a pioneering study described the presence of nanometer-scale objects—referred to as nanobacteria—and identified these structures as potential causative agents of urinary calculi [62]. However, there were some controversial issues with this work. First, the DNA isolated and sequenced in this study was identical to a common PCR contaminant [25]. Second, it was shown that aggregation of polysaccharides with Ca2⁺ could produce nanosized objects resembling nanobacteria [63]. Third, nanobacteria have been reported in environments in which normal-sized bacteria also reside [64]. In light of these concerns, it may be more likely that nanosized objects of < 250 nm diameter are microbial fragments, or small aggregates of minerals and organic matter, rather than independent lifeforms (nanobacteria) [65].

OMVs are nanosized (20–250 nm) spherical structures [66] that have been shown to be present in biofilms in both environmental [52] and medical [67] fields. They are produced by many Gram-negative bacteria and have been associated with microbial competition, predation, genetic exchange, quorum sensing, and interspecies communication [68]. OMVs contain peptides, nucleic acids, and, inherently, a surrounding membrane component consisting of lipopolysaccharides (LPSs) from the Gram-negative outer membrane [25, 67]. In morphology, they strongly resemble nanobacteria, which are locally influenced by mineral formation and solution chemistry [25]. Notably, some studies [25, 69, 70] have described a potential role of OMVs in the formation of urinary calculi. In this regard, it has been suggested that OMVs with crystal-promoting chemistry from some bacteria would enhance nucleation and growth, whereas OMVs from other bacteria, with crystal-inhibiting structures, would interfere with struvite nucleation [25], and that these opposing behaviors would be based on the LPS chemistry of OMVs and on other molecules from the local microenvironment [25]. This may suggest a potential mechanism in CEDARST formation, especially if we consider that Gram-negative bacteria have been observed predominantly among the extraradicular biofilm-forming bacteria associated with refractory AP [45]. However, further research is needed to confirm this hypothesis.

3.4. What Biominerals are Precipitated in CEDARST?

Although biomineralization of minerals induced by microorganisms has been widely investigated and reported [71, 72], its mechanisms and the characteristics of the biominerals precipitated in CEDARST need to be further explored. A recent CEDARST case report [16], using electron probe microanalysis, evidenced that calcium and phosphorus were the primary inorganic elements in the extraradicular deposit, showing that the molar Ca/P ratio of the deposit (1.25) was similar to that of dentine (1.32) and cementum (1.33), and confirming its calcified nature and similarity with the molar Ca/P ratio of dental calculus [73].

It has been evidenced that dental calculus involves four different crystals of calcium phosphate: brushite, octocalcium phosphate, whitlockite, and hydroxyapatite [74]. These minerals are initially formed as amorphous calcium phosphate, followed by intermediate calcium phosphate phases that finally transform to apatite [75]. Considering the calculus-like appearance of CEDARST, its location, and the fact that passive mineral precipitation is caused by minerals coming from the saliva into the apical lesion, as mentioned above, a similar crystal composition might be expected. However, until now, no study has confirmed the crystal composition of CEDARST. It should also be stressed that biomineralization is a process closely affected by such factors as pH, temperature, and ion concentration, as well as by enzymes that hydrolyze mineralization inhibitors [76]. All these determinants could influence the type of minerals precipitated in CEDARST.

Finally, it should be noted that minerals formed by biomineralization are not always strongly connected to cells, thus complicating the assessment of microbial-mediated minerals. In addition, the impact that biomineralization may have on microbial communities has yet to be addressed [77]. However, the rarity of CEDARST limits the potential for future studies of this issue.

3.5. Inhibitors of Mineralization

Although EPS, in the form of extraradicular biofilm, has been confirmed in teeth with AP by some authors [12, 13, 45, 58, 59], as mentioned above, clinically, not all teeth with AP develop CEDARST. For this reason, it could be speculated that inhibitors of mineralization from the host do not work efficiently and locally in some teeth affected by AP, thus contributing to CEDARST formation in some way. Calciprotein particles (CPPs) are dense membrane-less particles of mineral-binding proteins and calcium phosphate that are present in the blood and abundant in the extracellular fluids of mammals. An example is the hepatic glycoprotein fetuin-A, which forms nanoscale colloids with calcium phosphate [78, 79]. CPPs stabilize fluid phases and help maintain local mineral supersaturation during biomineral formation, solubilizing excess biominerals and thus preventing pathological mineral deposition in the extracellular space [79].

Mineral chaperones mediate transient solubility and stability of liquid mineral phases in situations of high supersaturation [80-83], complexing mineral ions and thus effectively lowering the availability of mineral solutes [78]. These issues are important, given the pivotal role of solubility phenomena in pathological mineralization processes [84]. For this reason, mineral chaperones have been cited as a prime explanation for pathological mineralization outside the skeleton, and designated as inhibitors of calcification [85]. Osteopontin [86] and fetuin-A [87] are examples of these chaperones, and these also play important roles in promoting bone destruction in AP [88] and periodontal disease [89], respectively. Because of this, it might be expected that when they do not work efficiently, they contribute to CEDARST generation.

Contrarily, bacterial destruction of saliva´s crystallization inhibitors (pyrophosphate, statherin, and proline-rich-peptides) has also been evidenced as a contributing factor in dental calculus formation, in light of their phosphatase and protease activities [75]. Some researchers have shown that bacterial pyrophosphatases contribute to dental calculus formation by hydrolyzing pyrophosphate [90, 91]; Corynebacterium matrucchoti, a bacteria found in dental plaque and calculus, has been proposed as one such contributor [90]. Additionally, it has been evidenced that bacterial protease, e.g., from Bacteroides loescheii, also contributes by degrading salivary protein inhibitors of calcium phosphate precipitation [92]. In this regard, it could be speculated that increased bacterial destruction of saliva´s crystallization inhibitors would contribute to CEDARST formation locally in the AP microenvironment. Finally, the above-mentioned potential inhibitory role of OMVs should also be recalled.

3.6. How Do Bacteria Circumvent the Immune System in CEDARST?

One of the puzzling issues raised by CEDARST is that, despite the plethora of innate and adaptive immune cells/molecules involved in AP [2], bacteria from this calcified biofilm can circumvent such immune mechanisms, so that CEDARST can persist even without treatment. For sure, bacteria have developed a variety of extremely sophisticated systems to evade host immune responses and establish infections in the body [93-95]. It can therefore be inferred that some of these systems/mechanisms are used by bacteria from CEDARST biofilm, especially given its calcified nature.

Notably, glycan-mediatedhost–pathogen interactions can benefit the pathogen, host, or both, given that glycans are highly diverse and ubiquitous in biological systems, being expressed on the surface of nearly all host and bacterial cells [96, 97]. Bacterial glycans play a vital role in bacterial colonization, invasion, and/or immune evasion. Some bacteria express cell-surface glycans that are pivotal for successful colonization on/in host cells; some express molecular mimicry of host glycans on their cell surfaces to subvert host immune surveillance; others cover their surface with layers of glycans to hamper host recognition of common pathogen-associated molecular patterns [96]. However, although host glycans and lectins play a crucial role in inducing bactericidal immune responses, many microorganisms have developed evasion mechanisms [97], and noncanonical variants of peptidoglycans [98, 99] or lipopolysaccharides [100, 101] can be expressed by some bacteria for immune evasion and/or successful colonization. Finally, many Gram-negative bacteria have evolved to express capsular polysaccharides for pathogen survival and/or immune evasion [102, 103].

For the purpose of immune system evasion, bacteria can also change their surface proteins or secrete proteases (intracellular and extracellular) that target pivotal host defenses, e.g., antibodies, complement factors, or antimicrobial peptides, thus allowing bacteria to remain undetected [104]. Furthermore, extracellular proteases are involved in biofilm adhesion, formation, modulation, and dispersion as well as in the breakdown of host extracellular matrix during colonization [105-107], and so these too could play a role in the CEDARST conundrum. However, the organic composition of the latter has not yet been characterized, and this is a study area worthy of further research.