4 Effect of SFRP-1 on osteoclastogenesis in rat bone marrow cell cultures. protein-1 (SFRP-1), which was found to be down-regulated at days Oligomycin 3 and 9. Using rat bone marrow cultures to conduct in vitro osteoclastogenic assays, it was demonstrated that SFRP-1 inhibited osteoclast formation in a concentration-dependent fashion. However, with increasing concentrations of SFRP-1, the number of TRAP-positive mononuclear cells increased suggesting that SFRP-1 inhibits osteoclast formation by inhibiting the fusion of mononuclear cells (osteoclast precursors). Co-culturing bone marrow mononuclear cells and dental follicle cells demonstrated that the Oligomycin dental follicle cells were secreting a product(s) that inhibited osteoclastogenesis, as measured by counting of TRAP-positive osteoclasts. Adding an antibody either to SFRP-1 or OPG partially restored osteoclastogenesis. Adding both anti-SFRP-1 and anti-OPG fully negated the inhibitory effect of the follicle cells upon osteoclastogenesis. These results strongly suggest that SFRP-1 and OPG, both secreted by the dental follicle cells, use different pathways to exert their inhibitory effect on osteoclastogenesis. Based on these in vitro studies of osteoclastogenesis, it is likely that the down-regulation of SFRP-1 gene expression in the dental follicle at days 3 and 9 is a contributory factor in allowing the major and minor bursts of osteoclastogenesis to occur. Thus, inhibition of SFRP-1 gene expression in combination with inhibition of OPG gene expression likely are critical events in enabling alveolar bone resorption to occur such that teeth will erupt. strong class=”kwd-title” Keywords: Dental follicle, Microarray, Osteoclastogenesis, SFRP-1, Tooth eruption Introduction Osteoclastogenesis and subsequent bone resorption are major requirements for tooth eruption [see review by 1]. To accomplish this requires the presence of the dental follicle, a loose connective tissue sac that surrounds the unerupted tooth. Specifically, mononuclear cells (osteoclast precursors) are recruited to the follicle [2, 3] where they fuse to form osteoclasts for alveolar bone resorption of the eruption pathway. In the first mandibular molar of the rat, the maximal number of mononuclear cells and osteoclasts are seen at day 3 [3, 4] and this correlates with the maximal gene expression of 2 chemotactic molecules in the follicle C colony stimulating factor-1 (CSF-1)  and monocyte chemotactic protein-1 (MCP-1) . These molecules are chemotactic for monocytes in vitro and they are secreted and chemotactically active by the dental follicle . Prior to the time Oligomycin of eruption of the 1st mandibular molar in the rat at day 18 postnatally, there is a major burst of osteoclastogenesis at day 3 [3, 4] and a minor burst at day 10 . A ratio of receptor activator of nuclear factor kappa B ligand (RANKL) to osteoprotegerin (OPG) that reflects more RANKL will promote osteoclastogenesis [e.g., see review by 7]. Thus, for the major burst of osteoclastogenesis at day 3 we have shown that the gene expression of OPG in the follicle is downregulated by the CSF-1 produced in the follicle to promote a favorable ratio for osteoclastogenesis . At day 10, the expression of OPG in the follicle is higher because of the reduced expression of CSF-1 but the gene expression of RANKL in the follicle is elevated . Consequently, a slightly favorable ratio of RANKL/OPG likely is established to allow a minor burst of osteoclastogenesis. Based on the above, the first aim of this study was to determine if other molecules might be involved in the recruitment of osteoclast precursors and/or the subsequent osteoclastogenesis needed for tooth eruption. DNA microarray studies were conducted to determine which chemokines and their receptors were expressed chronologically in the dental Oligomycin follicle in vivo. These studies suggested that another gene, secreted frizzled-related protein-1 (SFRP-1) had a reduced expression at both day 3 and day 9; i.e., times that correspond to the osteoclastogenic bursts. Thus, the second aim of this study was to determine the role of SFRP-1 in osteoclastogenesis, as determined by in vitro osteoclastogenesis assays. Materials and methods Mouse monoclonal to CD3E Dental follicle isolation and cell culture Dental follicles were surgically isolated from the first mandibular molars of rats (Harlan Sprague-Dawley) at postnatal days 1, 3, 5, 7, 9 and 11 for RNA isolation. For cell culture, the dental follicles of the first mandibular molar were isolated from 5C6 day old rats and trypsinized to obtain the dental follicle cells, as previously described . The dental follicle cells were cultured in minimum essential medium (MEM) (Sigma-Aldrich, St Louis, MO, USA) plus 10% (v/v) newborn calf serum, 1 mM sodium pyruvate, 1% penicillin/streptomycin and 0.2% fungazone, in an incubator at 37 C with 5% CO2 until passage 5C6. Follicle cells of passages 6C9.
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