The Experimental Study Of Recombinant RANK On Inhibiting Osteoclasts And Preventing Osteoporosis

The identification of the OPG/RANKL/RANK system as the dominant, final mediator of osteoclastogenesis represents a major advance in osteoclast signal transduction field. RANKL interacts with its receptor RANK on the surface of osteoclasts precursors and triggers the differentiation, activation and maturation of osteoclasts. OPG competes with RANK for binding to RANKL, thus blocking several biological activities of osteoclasts. Especially the change of OPG/RANKL ratio may immediately influences osteoclast's growth, thus affects bone's metabolism. This soluble form of recombinant RANK protein can also bind to RANKL but it can neither mediate the differentiation of osteoclast precursors nor promote the aggregation and migration of osteoclast precursors, its effects on the inhibition of osteoclasts are similar to those of OPG. Recombinant RANK protein will bring a new method on the treatment of osteoporosis.ObjectiveTo study the inhibitory effects of recombinant RANK protein on osteoclasts and loss of bone mass in ovariectomized mice or rats in vivo and in vitro.Methodâ‘ Osteoclast like cells from SD rats were harvested and cultured in DMEM with 10% FBS. After 6 d coculture,10-6 g/L,10-5 g/L,10-4 g/L recombinant RANK Protein were added to these coculture systems respectively. TRAP stain positive cells counting and resorption pits counting were preformed in the following the 3 rd.â‘¡Murine osteoblasts were co-cultured with RAW264.7 cells at a ratio of four to one for 6 days, and then murine recombinant RANK protein was added to the co-cultures at concentrations of 10-6 g/L,10-5 g/L,10-4 g/L. A blank control was also included. Three days after treatment, the morphology and number of osteoclasts were examined. The number of TRAP-positive cells per unit area was calculated and the numbers of resorption pits in bone slices were counted.â‘¢Female Wistar rats were ovariectomized bilaterally and were treated with recombinant RANK protein at 5 mg/kg body weight for 3 months, then bodyweight, biochemical markers of bone metabolism, bone mineral density, and bonemorphology were examined.â‘£Female KM mice were ovariectomized bilaterally and were treated withmurine RANK protein at 5 mg/kg body weight. Three months later, body weight,biochemical markers of bone metabolism, bone mineral density, Micro CT scanand bone morphology were examined.Resultâ‘ In the 3 rd after the addition of Rank protein,TRAP stain positive cellscounting decreased significantly in the Rank protein group than in the controlgroup, and the Rank protein group (10-4 g/L) decreased most significantly whencompared with the control group. Also bone resorption pits counting in theexperimental groups were significantly reduced after 3 days coculture withrecombinant RANK protein.â‘¡After co-culture of murine osteoblasts with RAW264.7 cells, many maturemultinucleated osteoclasts appeared, as confirmed by TRAP staining. Three daysafter RANK treatment, the numbers of TRAP-positive osteoclasts and resorptionpits in bone slices decreased significantly in each treatment group compared to theblank control group, with the most significant decrease observed in the 10-4 g/Lgroup.â‘¢In the ovariectomized rats experiments, RANK significantly inhibitedovariectomy-induced loss of bone mass. Compared to the control group, theRANK-treated group exhibited higher bone mineral density and nearly completeinhibition of TRAP-positive osteoclasts in bone slices.â‘£In the ovariectomized mice experiments, RANK significantly inhibitedovariectomy-induced loss of bone mass. Compared to the control group, theRANK group showed increased distal femur BMD and decreased trabecularspacing (Tb.Sp), whereas the OVX group had significantly decreased distal femurBMD, significantly decreased Tb.Th, and increased Tb.Sp. There was a significantdifference in bone volume fraction (BV/TV%) between the groups. TheTRAP-positive osteoclasts in distal femur bone slices were nearly completeinhibited.Conclusion â‘ In vitro, recombinant RANK protein can effectively inhibit osteoclast differentiation and resorption activity.â‘¡In vivo, recombinant murine RANK protein effectively inhibits the activity of osteoclasts and the resulting bone resorption.