Gingival fibroblasts are better at inhibiting osteoclast formation than periodontal ligament fibroblasts

Abstract

Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast-like cells. Here, we investigated whether a second population of tooth-associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co-cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1alpha,25dihydroxycholecalciferol (dex + vit D(3)) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co-cultures with PLF than in GF-PBMC co-cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D(3). In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D(3). OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast-like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast-PBMC cultures with M-CSF and RANKL. Our results suggest that tooth-associated fibroblasts may trigger the formation of osteoclast-like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts.

The Src Inhibitor AZD0530 Reversibly Inhibits the Formation and Activity of Human Osteoclasts

“…For osteoclast activity analyses on bone slices, cocultures of osteoblasts and PBMCs in culture medium were supplemented with M-CSF and RANKL. In a previous coculture study and also in the present study (data not shown), we found that these molecules were pivotal for the newly formed osteoclasts to exert their osteoclastic activity (29). The cocultures in the present study were done for 3 weeks on bone slices, either in the absence or presence of 1 μmol/L AZD0530.…”

“…PBMCs from buffy coats (Sanquin) were isolated as described previously (29). Buffy coats were diluted 1:1 in HBSS containing 2% FCS.…”

“…Coculture experiments were done in 48-well plates as described (29). Osteoblasts and PBMCs were cultured in the presence of 10 −8 mol/L dexamethasone and 10…”

“…29. Real-time PCR primers were designed using the Primer Express software, version 2.0 (Applied Biosystems; Table 1).…”

“…Maximal numbers of multinucleated cells were usually seen after 3 wk in a PBMCs/osteoblasts coculture system (29). To allow for significant bone resorption, cocultures of PBMCs and osteoblasts were assessed for bone resorption after a culture period of 4 wk.…”