Bone resorptive activity in symptomatic and asymptomatic apical lesions of endodontic origin

Apical periodontitis originates from host’s immune response to a dominant Gram-negative anaerobic biofilm localized inside the root canal system of the tooth and their respective by-products. Given the inability to eliminate bacteria, the host attempts to localize the infection and prevent further dissemination at the expense of apical tissue breakdown that results in the formation of an osteolytic apical lesion (AL), the hallmark of chronic forms of apical periodontitis [1].

AL is heterogeneous from a clinical point of view depending on its association with clinical symptoms, being either symptomatic apical periodontitis (SAP) or asymptomatic apical periodontitis (AAP) [2]. This clinical variability is expected to depend on the dynamic balance between bacterial consortia and the host’s response [3‐5]. Recent studies support that symptomatic apical periodontitis associates with changes in bacterial load and diversity [4], as well as host’s immune response, involving interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, and matrix metalloproteinase (MMP)-9, respectively [6‐8]. Although the clinical diagnosis of symptomatic apical periodontitis is straightforward, its biologic significance remains to be known.

Osteoclasts are the final cellular effectors of bone resorption, determining progression versus healing processes in AL. Osteoclast differentiation and activation from its monocytic precursors is regulated in part through the balance between the receptor activator of nuclear factor κB (RANK), its ligand (RANKL), and its decoy receptor, osteoprotegerin (OPG). Accordingly, significantly higher RANKL levels have been reported in AL compared with healthy tissues [9]. RANKL/OPG ratio has also been proposed as an indicator of AL progression [10, 11]. Tartrate-resistant acid phosphatase (TRAP)-5 on the other hand, is an enzyme released along with bone matrix degradation products by active osteoclasts, representing a direct biomarker of osteoclastic activity and bone resorption [12‐17].

Higher levels of TRAP-5 are associated with the progression of bone destructive diseases; there is a current need to identify biomarkers for AL progression [18]; however, up to now there are no clinical studies linking TRAP-5 with apical periodontitis [19, 20]. Furthermore, bone resorptive dynamics in symptomatic and asymptomatic states of apical periodontitis remain unknown [7]. We aimed to assess the levels and diagnostic accuracy of a set of bone resorption biomarkers in AL from patients with clinical diagnoses of SAP, AAP, and healthy periodontal ligaments as controls, including TRAP-5, RANKL, and OPG.

Controls, asymptomatic apical periodontitis and symptomatic apical periodontitis groups had a mean age of 13.7, 50.1, and 46.5 years and 7, 12, and 11 were women, respectively. No smokers were reported in the controls, whereas there were six smokers in each apical periodontitis group. Regarding biomarker levels (Fig. 1), significantly higher levels of TRAP-5 were observed in SAP in comparison to AAP and healthy controls (p < 0.05), and with no differences between the latter two groups. The levels of RANKL, OPG, and the RANKL/OPG ratio were significantly higher in AAP and SAP groups compared with healthy controls (p < 0.05), and no differences were found between both apical periodontitis groups.

The diagnostic performance of biomarkers is illustrated with ROC curves (Fig. 2a, b, c) and their respective values are presented in Table 1a, b, c. RANKL, OPG, and RANKL/OPG demonstrated statistically significant (p < 0.05) diagnostic accuracy to identify AAP versus healthy controls, where the highest performance corresponded to RANKL (area under the curve =0.77, 95% CI 0.65–0.89), [Fig. 2a and Table 1a ]. Optimal cut-off points were obtained for each marker by using Youden’s index. OPG showed the best performance, with a sensitivity of 80% and a specificity of 69% at a cut-off point of 50 pg/mL, followed by RANKL and RANKL/OPG ratio.

In the construction of ROC curves for the diagnosis of SAP versus healthy controls [Fig. 2b and Table 1b], TRAP-5 (AUC = 0.83, 95% CI 0.68–0.98), and RANKL (AUC = 0.83, 95% CI 0.71–0.95) were the markers with the highest accuracy, followed by RANKL/OPG ratio and OPG (p < 0.05). According to Youden’s index, TRAP-5 showed a sensitivity of 71%, a specificity of 100%, at a cut-off point of 12.48 ng/mL, followed by RANKL with a sensitivity of 77% and a specificity of 69% at a cut-off point of 90.64 pg/mL.

TRAP-5 (AUC = 0.71, 95% CI 0.54–0.89) was the only biomarker that showed potential to discriminate between SAP and AAP [Fig. 2c) and Table 1c, p < 0.05]. TRAP-5 showed a sensitivity of 73% and a specificity of 80% at a cut-off point of 12.54 ng/mL.

AL results from an imbalanced osteoclastic activity induced by the immuno-inflammatory response to endodontic bacterial infection [24]. Clinically symptomatic ALs have been suggested to represent an immunologically active stage of the disease [7, 8], but up to now, bone resorptive activity had not been established. In the present study, we assessed the levels of bone resorption markers in symptomatic and asymptomatic AL and healthy periodontal ligament as controls and found statistically significant higher levels of RANKL and OPG in both types of apical lesions compared to healthy periodontal ligament, while higher TRAP-5 levels were found only in symptomatic AL, suggesting that the later represent progressive lesions. TRAP-5 had diagnostic potential for SAP, representing a potentially useful candidate biomarker for apical periodontitis progression.

Bone resorption is a multistep process where RANK/RANKL/OPG pathway and TRAP-5 play key roles. Through binding RANK, RANKL is among the key osteolytic cytokines that promote osteoclast maturation and activation. OPG on the other hand, is a decoy receptor that prevents the coupling of RANK to RANKL [10]. Unlike the RANKL/RANK axis, which might either result or not in osteoclast differentiation depending on the OPG levels, TRAP-5 is an enzyme released along with bone matrix degradation products by active osteoclasts representing a direct biomarker of osteoclastic activity and bone resorption [12‐14, 19, 20].

In the present study the levels of RANKL, OPG and RANKL/OPG ratio were significantly higher in symptomatic and asymptomatic apical lesions versus healthy controls, whereas no significant differences were found between both lesion types. Previous reports associate high levels of RANKL and OPG with apical lesions [10, 17, 25]. In line with our results, a recent report showed no differences in RANKL and OPG levels between asymptomatic and symptomatic apical lesions, whereas a significantly higher number of gram-negative bacteria along with a positive correlation with OPG were found in the later [26]. Whereas the participants showed an homogeneous distribution by gender, an intrinsic methodologic drawback for including healthy periodontal ligament as controls is the age difference among healthy and AP groups, although it represents the closest histophysiologic counterpart for ALs in humans [22]. Accordingly, higher mRNA expression levels were found for RANKL and OPG in apical granulomas versus periodontal ligaments [10]. Recently, RANKL was identified at higher levels in apical exudates from asymptomatic apical periodontitis compared to irreversible pulpitis, whereas OPG levels remained mostly undetectable [27]. So far, there are no previous studies that associate the RANKL/OPG levels with the clinical status in AP. The increased levels of RANKL, OPG, and RANKL/OPG ratio in ALs suggest that these lesions display an imbalance towards osteoclast differentiation, though it might not necessarily result in higher osteolysis [10, 27]. Based on our results, these markers did not differentiate between symptomatic and asymptomatic apical lesions.

TRAP-5 levels were significantly higher in symptomatic apical lesions compared with asymptomatic lesions and healthy controls, while no differences were found between asymptomatic lesions and controls. TRAP-5 has been proposed to be the most reliable marker of bone resorptive activity. Clinical studies, as well as animal and in vitro experimental models, demonstrate that elevated levels of TRAP-5 can be more specifically explained by an increase in the activity of osteoclasts [12‐15, 28]. Accordingly, studies conducted in animal models demonstrate that bone resorption rate is significantly higher during the acute stage of apical periodontitis, compared to its chronic phase, where it becomes slower to finally achieve stabilization [29].

Bone resorption and RANKL production by several cell types is synergistically induced by IL-1, IL-6, and TNF-α. In line with our results, clinical studies report significantly higher levels of these cytokines in apical lesions versus pulp tissue controls and symptomatic versus asymptomatic apical lesions [7, 8, 23]. Despite the RANKL/OPG ratio has been proposed as an indicator of progression or stability of asymptomatic lesions [10], osteolytic cytokines are redundant. TNF-α and IL-1 can also stimulate osteoclast differentiation via RANKL/RANK-independent mechanisms [30, 31], implicating that osteoclastogenesis is only partially accounted by the RANKL/RANK axis. Altogether, these antecedents suggest that inflammation enhances osteoclast differentiation in periapical lesions via RANKL/OPG, but only symptomatic lesions might truly represent an active stage, as reflected by concomitantly elevated RANKL/OPG ratio and TRAP-5 levels. Based on previous reports, it is possible that activation pathways other than RANKL might be induced in symptomatic lesions, resulting in exacerbated bone resorption. The detection of RANKL, OPG, and TRAP at lower levels in healthy periodontal ligament on the other hand might reflect bone homeostasis.

The diagnostic accuracy of OPG, RANKL, and RANKL/OPG ratio showed potential to identify apical lesions versus healthy controls, but failed to discriminate between AAP and SAP. Thus, OPG and RANKL could be useful in the diagnosis of apical periodontitis; however, they do not differentiate between its clinical forms. TRAP-5 on the other hand proved to have the highest diagnostic accuracy to discriminate SAP from AAP and healthy controls, presenting potential as a marker for progressive lesions. Accordingly, TRAP levels also demonstrated diagnostic precision to identify marginal chronic periodontitis in a recent study [15]. Currently, apical diagnosis is based solely on clinical and radiological parameters, but they fail to predict treatment outcome in the short term. Thus, TRAP-5 screening in a noninvasive approach might have potential as biomarker for progressive AL to aid clinical decisions in endodontic and restorative procedures in oral fluid samples, such as gingival crevicular fluid, saliva, or in apical exudates during endodontic treatment [15, 32]. In fact, TRAP levels have already shown a good performance to identify chronic periodontitis in gingival crevicular fluid [15]. Future prospective studies are needed to assess its usefulness as a predictive marker.

Apical lesions showed higher RANKL and OPG levels than healthy tissues and TRAP-5 levels were the highest in symptomatic apical lesions, suggesting that the later represents a progressive disease state that can be identified by TRAP-5.