Molecular Mechanisms Of Osteoclastgenesis And Local Modulation Of Osteoclast Differentiation And Activity In Osteolytic Diseases

Part one Molecular mechanisms of osteoclastgenesisOBJECTIVE: Bone resorption occurs continuously throughout life to maintain skeletal mass and calcium balance. This involves coupled bone formation and resorption, which are carried out by osteoblasts and osteoclasts respectively. Osteoclast is a multinucleated cell specialised to carry out lacunar resorption. Osteoclasts are not commonly seen in normal adult bone but are often found at sites of osteolysis in diseases affecting bones and joints. Disorder of osteoclast differentiation and activity plays key roles in the process of osteolytic diseases. Great progress in osteoclast biology has been made in recent years. However, the molecular mechanisms of osteoclastgenesis are still widely controversial. This study was devised to investigate whether there are other mechanisms of osteoclastgenesis besides the RANKL-induced osteoclastgenesis pathway.METHODS: Human and mouse peripheral blood monocytes (PBMCs) and RAW264.7 cell line were cultured on glass coverslips and dentine slices in vitro. Macrophage-colony stimulating factor (M-CSF), tumor necrosis factor alpha (TNFα) and LIGHT (homologous to lymphotoxins, exhibits inducible expression, andcompetes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes) with different concentration were added to the culture respectively. The inhibitors of RANKL, TNFαand LIGHT were also added respectively in certain groups. The cultures were stopped at different time point and osteoclast formation were determined by the expression of tartrate resistent acid phosphatase (TRAP), vitronectin receptor (VNR) and F-actin ring and the formation of multi-nucleated cells MNCs and lacunar resorption on dentine slices.RESULTS: In the presence of M-CSF, large numbers of TRAP+ and VNR+ multinucleated cells capable of lacunar resorption were noted in the cultures of human and mouse PBNCs and RAW264.7 cell line when TNFαand LIGHT were added respectively. Addition of RANKL inhibitor (OPG) did not inhibite the extent of osteoclast formation, while addition of neutralizing antibodies to TNFαreceptor I and II abolished the formation of TRAP+ MNCs and lacunar resorption induced by TNFαand the addition of LIGHT decoy receptor (DcR3) and neutralizing antibody to TNFαreceptor II respectively inhibited the formation of TRAP+ MNCs and lacunar resorption pits on dentine slices.CONCLUSION: In the presence of M-CSF, TNFαand LIGHT can directly induce osteoclast differentiation independent of RANKL. OBJCETIVE: Osteolytic focus presents in variable diseases, such as aseptic loosening of prosthesis and Paget's disease. Osteoclast, which works as the executive cells of bone resorption in vivo, plays key roles in the formation of osteolytic focus. This study was devised to investigate the local factors which modulate osteoclast formation and activity in osteolytic diseases.METHODS: Macrophages were isolated from periprosthetic pseudomembrane and cultured in the present and absence of M-CSF, RANKL and TNFα, and the formation of osteoclast was determined by the expression of TRAP and VNR and the formation of lacunar resorption pits on dentine slices. Fibroblast-like stromal cells (FLSCs) were isolated from from human periprosthetic pseudomembrane, Paget's disease focus and Paget's osteosarcoma specimens. The FLSCs were cultured in vitro and passaged for two times. Then parts of the FLSCs were co-cultured with human PBMCs in a contact and none-contact system in the presence and absence of M-CSF. The inhibitors of RANKL and TNFαwere also added to the cultures respectively. The formation of osteoclast formation was observed at the end of culture. The other parts of FLSCs were used for RT-PCR and ELISA for the expression of RANKL and TNFαmRNA and protein.RESULTS: In the presence of M-CSF and RANKL, large numbers of TRAP+ and VNR+ multinucleated cells capable of lacunar resorption were noted in the culture of macrophages derived from periprosthetic pseudomembrane. TRAP+ MNCs and lacunar resorption pits on dentine slices were also noted in co-cultures of PBMCs and FLSCs derived from periprosthetic pseudomembrane and from Pagetic bone and Paget's sarcoma in the presence of M-CSF. In the co-cultures of PBMCs and FLSCs derived from periprosthetic pseudomembrane, the addition of OPG and neutralizing antibody to TNFαalone significantly reduced but did not abolish the extent of osteoclast formation, whereas the addition of both together abolished the formation of TRAP+ MNCs and lacunar resorption. In the co-culture of PBMCs and FLSCs derived from Pagetic bone and Paget's osteosarcoma, the addition of OPG abolished the formation of TRAP+ MNCs and lacunar resorption pits, whiled the addition of neutralizing antibody to TNFαinhibited the extension of osteoclast formation. FLSCs of different origins all expressed RANKL and TNFαmRNA and protein.CONCLUSION: Our results indicate that macrophages derived from periprosthetic pseudomembrane are capable of differentiating into mature osteoclast through RANKL-dependent pathway pathway. FLSCs derived from periprosthetic pseudomembarne are capable of inducing the differentiation of normal human peripheral blood mononuclear cells to mature osteoclasts by mechanisms that involve both RANKL and TNFα. FLSCs derived from Paget's disease and Paget's osteosarcoma can express RANKL and support osteoclast formation. Suppression of both RANKL and inflammatory cytokines is likely to be required to control osteolytic focus.