Osteoclastogenesis induced by sRANKL and M-CSF from the non-adherent cell population of human bone marrow is inhibited by rhBMP-2 alone or together with rhVEGF

During bone development and repair, angiogenesis, osteogenesis and bone remodelling are closely associated processes that share some common mediators. In the present study non-adherent human bone marrow mononuclear cells under the induction of sRANKL and M-CSF, differentiated into osteoclasts with TRAP positive staining, VNR expression, and Ca-P resorptive activity. The effects of various combinations of rhBMP-2 (0, 3, 30, 300 ng/ml) and rhVEGF (0, 25 ng/ml) on osteoclastogenesis potentials were examined in this experimental system. The percentages of TRAP-positive multiple nucleated cells represent osteoclast differentiation potential and the percentages of resorptive areas in the Ca-P coated plates resemble osteoclast resorption capability. The presence of rhBMP-2 at 30 and 300 ng/ml showed inhibitory effects on osteoclast differentiation and their resorptive capability in the human osteoclast culture system. rhVEGF (25 ng/ml) enhanced the resorptive function of osteoclast whenever it was used alone or combined with 3 ng/ml rhBMP-2. However, rhVEGF induced resorptive function was inhibited by 30 ng/ml and 300 ng/ml rhBMP-2 at a dose-dependent manner. Statistical analysis demonstrated that an interactive effect exists between rhBMP-2 and rhVEGF on human osteoclastogenesis. These findings suggested that an interactive regulation may exist between BMPs and VEGF signaling pathways during osteoclastogenesis, exact mechanisms are yet to be elucidated.

Osteoprotegerin expression by periodontal ligament fibroblasts

Objectives: Receptor Activator of NF-kappaB ligand (RANKL), through binding to its receptor (RANK), plays an important role in osteoclast differentiation and activation. Conversely, osteoprotegerin (OPG), a decoy receptor for RANKL, inhibits osteoclastogenesis and subsequent bone turnover. Little is known about the role of resident periodontal ligament fibroblasts in regulating bone turnover. The aim of this study was to determine (i) if periodontal ligament fibroblasts produced OPG in vitro and (ii) the effects of IL-1b and TGF-b1 on OPG expression. Methods: Three human periodontal ligament fibroblast populations, developed by explant culture, were grown to confluence in 6-well plates in DMEM supplemented with 10% FCS. Cells were washed in HBSS and then cultured for an additional 48 hours in serum-free media supplemented with IL-1b or TGF-b1 at 10ng/ml. OPG expression levels in the conditioned medium were determined by ELISA (R&D Systems, UK) and confirmed by Western blot. Results: All three fibroblast strains produced quantifiable levels of OPG. Both IL-1b and, to a lesser extent, TGF-b1 significantly stimulated OPG expression in all fibroblast strains (p<0.05). Pre-incubation of samples with N-glycosidase F prior to Western blots indicated glycosylation of expressed OPG. Conclusions: These data indicate that periodontal ligament fibroblasts can regulate osteoclast activation via the RANK/RANKL signalling pathway. These fibroblasts may play an important role in regulating bone turnover both in periodontal disease and orthodontic tooth movement.