Immobilized-OPG-Fc on Titanium Inhibits RANKL-dependent Osteoclast Differentiation

Successful bone remodeling in surrouding bone tissues of dental implants requires coordinated activities of both osteoblasts and osteoclasts. The Receptor Activator of NF-kB Ligand (RANKL)-RANK pathway, and Osteoprotegerin (OPG), an antagonist of RANKL function, are essential for osteoclast differentiation and its development. Therefore, exogenous OPG; i.e. supplementation of soluble OPG or immobilization of OPG on titanium, would be expected to improve the artificial imbalance in the RANKL/RANK/OPG system which triggers the disruption of the coupling mechanism between bone formation and bone resorption. Objectives: The purpose of the present study was to examine the effect of OPG-Fc immobilized on titanium, on the initial differentiation of osteoclast precursor RAW264.7 cells. Methods: The RAW264.7 cells were cultured on titanium specimens on which OPG-Fc was immobilized, according to our previous studies. In brief, titanium disks were immersed in g-aminopropyltriethoxysilane and, subsequently, in glyoxylic acid monohydrate. Then the surfaces of the specimens were treated with sodium borohydride, to reduce the imine to amine groups. The carboxyl groups were activated with N-hydroxylsuccinimide (NHS) / N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC), and treated with OPG-Fc, to immobilize OPG-Fc on the surface (OPG-Fc-titanium). OPG-Fc-free titanium was prepared by the treatment with ethanolamine-HCl, just after carboxyl groups were activated by NHS/EDC treatment. Untreated-titanium specimens were used as control titanium. Results: The enhancement of TRAP and cathepsin K mRNA expression in RAW264.7 cells exposed to RANKL stimulation on OPG-Fc-immobilized titanium was significantly lower than that in RAW264.7 cells exposed to RANKL on either OPG-Fc-free-titanium or control titanium (ANOVA, p<0.01). Conclusions: These results, taken together, suggested that immobilization of OPG-Fc on a titanium surface effectively inhibited the differentiation of RAW264.7 cells induced by RANKL stimulation, which will be expected to improve the prognosis of dental implants. Supported by grant No. 18689046 from the Japan Society for the Promotion of Science.