The cellular and molecular mechanisms underlying tooth impaction remain poorly understood. It is assumed that an imbalance between the osteosynthesis and bone resorption is one of the factors playing an important role in the development of tooth eruption abnormalities . A key role in controlling the process of bone remodeling belongs to cytokine signaling systems. The regulatory protein osteocalcin, which is produced by osteoblasts , is involved in the process of osteosynthesis and considered a valid marker of bone formation. In turn, bone resorption is regulated by the osteotropic system RANKL/RANK/OPG .
The role of the RANKL/RANK/OPG signaling pathway in tooth eruption has been demonstrated in animal models to a greater extent . Nevertheless, there is a lack of experimental studies dedicated to the role of the RANKL/RANK/OPG axis in the tooth eruption process in humans under the normal and pathological conditions due to the complexity of sampling and obligatoriness of confirmation from patients and ethics committee approval. The small number of studies dealing exclusively with the changes in the state of the cytokine system RANKL/RANK/OPG in bone tissue of patients with impacted teeth is available . Therefore, the purpose of our research was to determine the levels of its key components as indicators of the processes of bone resorption and bone formation.
The current findings showed no significant difference in RANK and OC levels between the bone tissue samples of healthy persons and samples taken near the healthy tooth in patients with tooth impaction. A slight tendency to decreased OPG level in the bone tissue near the healthy tooth in patients with tooth impaction compared with the control was observed on a background of a significant decrease in the content of membrane-bound and soluble forms of RANKL. These findings clearly indicate a slight deviation in the production of cytokines by osteoblasts. Nevertheless, since we demonstrated a simultaneous decrease in the content of both cytokines RANKL and OPG on the background of the unchanged level of the preosteoclast marker RANK and marker of osteosynthesis osteocalcin, in general, this ratio of locally synthesized cytokines ensured a normal process of tooth eruption.
Interestingly, we obtained completely different results concerning the state of the RANKL/RANK/OPG system in the area of impacted tooth. The findings of the present study demonstrated a significant increase in the relative content of RANK, OPG, both forms of RANKL and osteocalcin in the IT area vs. control. Based on these observations we can assume an accumulation of osteoclast precursors as well as active osteoblastic cells, which excessively produce cytokines in the area of the impacted tooth. In addition, since RANKL is produced not only by osteoblasts, but by activated by B and T cells as well , high RANKL level may be explained by the recruitment of immunocompetent cells and the development of the local proinflammatory reaction near the impaction area – the formation of mononuclear infiltrations, accumulation of preosteoclasts, preosteoblasts and fibroblasts, that actively produce cytokines. Nevertheless, despite the high levels of cytokines tooth eruption does not occur in this area.
Therefore, it may be important to consider the ratios of the components of the RANKL/RANK/OPG system that are determined its physiological effects on the bone tissue metabolism. We used two indices for assessing the state of local bone remodeling in the areas near the healthy and impacted teeth in patients with tooth impaction: RANK/RANKL mainly as an indicator of bone resorption and RANKL/OPG that reflects the balance between bone formation and resorption. In the area of the impacted tooth, we found a decreased RANK/RANKL and increased RANKL/OPG ratios compared with the healthy tooth that may indicate impaired bone remodeling process and, as a result, disturbances in tooth eruption process. Moreover, it has been reported earlier an increase in RANKL/OPG ratio in periodontal tissues under inflammatory pathological conditions, such as periodontitis . This confirms our assumption that an elevated RANKL/OPG ratio also indicates the development of the local proinflammatory reaction near the area of impacted teeth that may lead to the manifestation of such symptoms as pain and swelling.
This method, based on comparison of the RANK/RANKL and RANKL/OPG ratios during the normal and pathological eruption process, allows to estimate the intensity of the processes of bone resorption and bone formation in patients and may be recommended for implementation in orthodontic practice for the detection of local cytokine dysregulation in the impaction area and for choosing an effective method for correction of this pathological condition.
In the last decades, considerable progress has been made in understanding the molecular basis of osteoclastogenesis. It has been discovered that the process of osteoclast formation requires the involvement of NF-κB signaling, which is activated in response to several major “osteoclastogenic” cytokines, in particular, RANKL and macrophage colony-stimulating factor 1 (M-CSF1) . RANKL and other proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), activate NF-κB-associated signaling pathways, inducing c-Fos and nuclear activated T-cell factor (NFATc1), and also inhibit NFATc1 repressors, thereby positively regulating formation and activation of osteoclasts .
In addition to the importance of the balance between osteosynthesis and bone resorption, it is known that reorganization into the periodontal membrane is also required for an adequate course of tooth eruption . This process involves the activation of apoptosis of the inner cell layer of the periodontium and the formation of a moving layer of apoptotic cells. Recently, it has been suggested that inappropriate process of apoptosis of osteoblasts/osteocytes accounted for, at least in part, the imbalance of bone remodeling. Accumulated evidence suggests that caspase-3 is a critical enzyme for apoptosis and cell survival; however, the exact role of caspase-3 in bone remodeling and development of skeletal diseases is largely unknown. To clarify why despite the high osteoclastogenic potential of cells in the impaction area, tooth eruption does not occur, the next step was to estimate the state of NF-κB signaling as a trigger factor for apoptosis in bone tissue of patients with impacted teeth.
Since the RANKL level was declined in the bone tissue selected near the healthy tooth of patients with tooth impaction compared with control patients, it was logical to observe a slight decrease in the NF-κB level and, as a result, in the NFATc1 and procaspase-3 (32 kDa) levels. Nevertheless, the content of caspase-3 subunit p17 was significantly higher thereby ensuring the reorganization into the periodontal membrane and normal course of the tooth eruption.
In the present study, we found that there was no difference in the NF-κB and NFATc1 levels compared with the control group despite the established activation of the RANKL/RANK/OPG signaling axis in the impaction area. Although an accumulation of osteoclast precursors in the IT area was observed, our results indicate a blocked or delayed process of osteoclastogenesis in the bone tissue near the impacted tooth. These data indicate that there is no adequate maturation and activation of osteoclasts, therefore resorption in the area of the impacted tooth does not normally occur, which may explain the tooth impaction and/or delayed tooth eruption. Understanding the molecular basis of impaired bone resorption in the IT area is required for further investigation.
While assessing the process of apoptosis we detected an increase in caspase-3 p17 subunit level on the background of declined procaspase-3 content in the area of the healthy tooth. At the same time, we obtained different results in the area of the impacted tooth and found a simultaneous elevation of both procaspase-3 and caspase-3 p17 subunit. According to these findings, a ratio between subunit p17 and procaspase-3 was lower in the area of the impacted tooth compared with the bone tissue, which was taken near the healthy tooth in patients with tooth impaction. These observations indicate the enhanced cleavage of procaspase-3 and its activation in the area of healthy tooth of patients with IT, that promote the tooth eruption process. We have to emphasize that the impairment of caspase-3 activation and the accumulation of inactive form in the area of tooth impaction may lead to tooth eruption failure. Another evidence of impaired or even inhibited apoptosis in the IT area may be elevated OPG level, since it has been proven that OPG inhibits apoptosis . In addition, it may be assumed that elevated caspase-3 level in the impaction area may possibly indicate the death of functional cells – preosteoclasts and osteoblasts, and explain the absence of NF-κB activation after the observed activation of the RANKL/RANK/OPG system near the IT.
The obtained experimental results may be beneficial for practical medicine, especially for the new field of molecular orthodontics, as these data extend the current understanding of the cellular and molecular mechanisms of tooth impaction in humans and emphasize the role of the NF-κB-associated RANKL/RANK/OPG signaling pathway in disturbances of tooth eruption. Based on our data, therapeutic approaches for patients with failed tooth eruption should be directed to restoring the normal course of the resorption process in order to facilitate tooth eruption. Thus, to improve the functioning of local cytokine pathways responsible for osteoregulation in patients with impacted teeth, mechanical stimulation for local therapy may be beneficial. A removable orthodontic apparatus, developed earlier by Flis et al.  allows improving blood circulation within the area of the impacted tooth that may influence the state of molecular pathways involved in bone remodeling. The therapeutic use of this apparatus could stimulate mechanically the formation of an eruptive pathway, set the direction for a mature impacted tooth with high cellular potential, and facilitate its movement to the oral cavity.