We detected increased levels of HIF-1 and HIF-2 in the damaged pulp tissue; however, the manner in which damages modulate odontoclast regulatory factors is still unclear. molars, but OPG was dominantly expressed. High OPG expression was expected to have a negative regulatory effect on odontoclastogenesis; however, odontoclasts were not detected in the dental pulp of (mice. Relative ratio of RANKL/OPG in the damaged pulp was significantly higher than in undamaged control pulp. Pulp damages enhanced hypoxia inducible factor-1 and -2, reported to increase RANKL or decrease OPG. These results reveal that the relative ratio of RANKL/OPG is significant to pulpal odontoclastogenesis, and that OPG expression is not required for maintenance of pulp homeostasis, but protects pulp from odontoclastogenesis caused by damages. mice exhibit severe osteoporosis due to increased osteoclastogenesis in bone tissues28,29; however, the phenotypes in healthy or damaged dental pulp tissues of these mice have not been investigated. In this study, we assessed the potential regulatory mechanism of odontoclastic differentiation in dental pulp in mice, and explored the contribution of OPG in the regulation of damage-induced pulpal odontoclastogenesis using a tooth replantation surgery27. Our findings provide insights into the requirement of OPG for the maintenance of a steady-state in the normal pulp and the damaged pulp environment. Results Odontoclast regulatory molecules are expressed in dental pulp environment but anti-differentiation factor OPG is dominant RANKL and OPG were Rabbit polyclonal to NOTCH4 detected GSK-3326595 (EPZ015938) in both osteoblasts and osteocytes in mouse femora by immunohistochemical staining (Supplementary Fig. S1). We analyzed the expression pattern of GSK-3326595 (EPZ015938) these molecules in mouse dental pulp of maxillary first molars, and detected high expression of RANKL in odontoblasts but modest expression in dental pulp stromal cells (Fig.?1A, blue arrows: RANKL+ odontoblasts, blue arrowheads: RANKL+ pulp stromal cells). However, similar expression levels of OPG were observed in the entire dental pulp tissue, including odontoblasts and pulp stromal cells (Fig.?1B, red arrows: OPG+ odontoblasts, red arrowheads: OPG+ pulp GSK-3326595 (EPZ015938) stromal cells), but was undetectable in dental pulp tissues from mice. Real-Time PCR experiments revealed that the expression levels of and were significantly higher in the mouse maxillary first molars than in the mouse femora (Fig.?1C, left and right panels), with the expression consistent with a previous report27. Whereas, lower expression was observed in the molars that in the bone tissues (Fig.?1C, middle panel). Unlike molars, RNA obtained from femora are mostly derived from hematopoietic cells, which are not a major provider of osteoclast regulatory factors5. To exclude hematopoietic cells, we performed re-normalization of each molecule using the expression of (levels were comparable between bone and molars; however, the levels of and in molars remained significantly lower and higher than those in femora, respectively, in the re-normalized data (Fig.?1D). The relative ratio of to in molars was significantly lower than that in femora in both normalized conditions using ((Fig.?1E). Altogether, these results suggest that the odontoclast inducible factors, CSF-1 and RANKL are detectable in the healthy dental pulp, but high expression of OPG may be a negative regulator of odontoclastogenesis. Open in a separate window Figure 1 Odontoclast regulatory molecules are expressed but anti-differentiation factor OPG is dominant in the dental pulp environment. (A, B) Representative images of 6-week-old mouse maxillary first molars stained with anti-RANKL (A, left and middle panels) and anti-OPG (B, left and middle panels) antibodies. n?=?3. Blue arrows: RANKL+ odontoblasts, blue arrowheads: RANKL+ pulp stromal cells, red arrows: OPG+ odontoblasts, red arrowheads: OPG+ pulp stromal cells. Normal rabbit IgG (A, right panel) and sections from mice (B, right panel) were used as negative control, respectively. Right panels are magnified views of boxed areas. P: pulp, D: dentin. (CCE) Real-Time PCR analysis of and expression in 6-week-old mouse femora and maxillary first molars normalized with (C) or (D). Relative ratio of to normalized with (left panel) or (right panel) (E). Femora: n?=?5, Molars: n?=?5. *mice To evaluate whether OPG expression is indispensable for pulp environment in the healthy state, the presence of odontoclasts in maxillary first molars of mice were analyzed. TRAP and anti-cathepsin K staining revealed that the number of osteoclasts localized in the alveolar bone tissue were higher in mice than those in wild-type mice (Fig.?2A,B, red arrows: TRAP+ osteoclasts, red arrowheads: cathepsin K+ osteoclasts). However, odontoclasts were not observed in dental pulp tissues of both wild-type and mice (Fig.?2A,B, squares 1 and 3). In addition, RANKL expression levels were comparable between wild-type and mice indicating abundant availability of RANKL for odontoclastogenesis in the pulp environment (Supplementary Fig. S2). Open in a separate window Figure 2 OPG is not required to maintain the pulp environment in normal tooth. (A, B) Representative images.