An investigation into the role of RANKL and sclerostin in dentoalveolar ankylosis

Abstract

Background: Dentoalveolar ankylosis may occur spontaneously or as a sequela to dental trauma and results in an area of bone fusing to the tooth (Kurol 1981). Infra-occlusion following dentoalveolar ankylosis results in a number of significant orthodontic sequelae. These include overeruption of the opposing tooth, tipping of adjacent teeth towards the ankylosed tooth, a loss of arch length, space loss and a shift of the dental midline (Messer and Cline 1980; Andlaw 1974; Ponduri et al. 2009). Vertical alveolar growth may also be hindered (Kjaer et al. 2008). The understanding of the biological processes behind the formation and repair of the ankylotic lesion is incomplete. Following dental trauma, the periodontal ligament (PDL) may be the source of the cells that repopulate a tooth root defect that determine whether ankylosis occurs (Erausquin and Devoto 1970; Lin et al. 2000; Melcher 1970; Line et al. 1974). When the PDL space is repopulated by cells from a source outside the true PDL tissues (such as the alveolar bone) healing may occur by way of dentoalveolar ankylosis. Research in the field of bone biology has recently focused on the role of the osteocyte. This cell, with its unique location embedded in bone, may have an essential role in bone metabolism. Osteocytes produce sclerostin, a protein that inhibits bone formation. There is also evidence that the osteocyte may be a major source of receptor activator of NF-κβ (RANKL) which is essential for osteoclastogenesis. This project aims to investigate the expression of RANKL and sclerostin in a rat model of dentoalveolar ankylosis induced by a hypothermal insult. The null hypothesis is that an applied cold insult and subsequent ankylosis does not affect the expression of RANKL and sclerostin within the dentoalveolar complex. Methods: Dentoalveolar ankylosis was induced in fifteen, eight week old, male, Sprague-Dawley rats (5 groups of 3 rats each) by application of dry ice to the upper right first molar tooth. An additional 3 rats served as untreated controls and the experimental rats were sacrificed at days 0, 4, 7, 14 and 28. Immunohistochemical detection of RANKL and sclerostin was performed and the number of RANKL and sclerostin positive and negative cells as well as the number of empty lacunae representing dead osteocytes were calculated and compared between groups. Results: The cold insult resulted in dentoalveolar ankylosis, with the periodontal ligament (PDL) almost completely replaced by bone in the furcation region of the root 14 days after injury with regeneration of the PDL evident after 28 days. Resorption of the ankylotic bone and cementum was evident in the furcation region. There was also a statistically significant increase in the number of empty lacunae due to osteocyte death that coincides with the incidence of maximal ankylosis. RANKL was detected in bone marrow stromal cells, osteoblasts and bone lining cells, osteoclasts, endothelial-like cells lining vessels, epithelial cells, odontoblasts and periodontal fibroblasts. However, clear staining in osteocytes was not evident. Epithelial rests of Malassez showed strong expression of RANKL. When ankylosis was present, there was a statistically significant difference in sclerostin expression between the areas of bone closest to, and farthest away, from the furcation area. There was a non-statistically significant trend towards reduced sclerostin expression at days 7 and 14 followed by a slight increase in expression at day 28. The slight increase in sclerostin expression at day 28 may indicate the establishment of a healing response. In considering these results, it should be noted that this experiment uses a model of ankylosis in which the ankylotic lesion develops following a thermal insult. The factors that initiate ankylosis in a clinical situation are incompletely understood and may differ from this model. Conclusions: Whilst RANKL was not detected in osteocytes in this model of ankyloses there was strong expression of RANKL by ERM in the PDL and a significant change in sclerostin expression near the area of ankylosis. This may contribute evidence that RANKL, sclerostin and the osteocyte might have a role in influencing the regeneration of the PDL following dentoalveolar ankylosis. The null hypothesis that an applied cold insult and subsequent ankylosis does not affect the expression of RANKL and sclerostin within the dentoalveolar complex is rejected.