The Bioactivity Assay Of The Recombinant Human Bone Morphogenetic Protein-2

Objective:Bone morphogenetic proteins (BMPs) regulate the differentiation and function of cells involved in bone and cartilage formation. Specifically, BMP-2 has been shown to be one of the most potent members of the BMP family for the induction of bone formation. In this study, we investigated a strategy to assay the bioactivity of the recombinant human bone morphogenetic protein-2 (rhBMP-2). The bioactivity of rhBMP-2 in vitro was assessed by determining its ability to stimulate the alkaline phosphatase (ALP) activity of rat C2C12 cells. In vivo, ectopic bone formation test by KM rats and repair bone defects in rabbit radius were processed.Methods:The rhBMP-2 protein has been expressed and purified in our laboratory previously. Several methods were processed to assay the bioactivity of rhBMP-2. In vitro, the osteoblastic to differentiation markers (ALP and mineralization rate) of C2C12 cells after induction of rhBMP-2 were assayed to identify the osteoinductive activity of rhBMP-2. Intramuscular implantation test was made in vivo, rhBMP-2 and collagen were compounded according to pharmacopeia at first. The prepared samples were implanted in muscles of rat's hind leg for two weeks. The amount of calcium deposited in the implants, X-ray and histotomy were measured to assay bioactivity of rhBMP-2. The bone-defect repairing ability of rhBMP-2 was verified by New Zealand rabbit radial bone repair trial after the composition of collagen and rhBMP-2.Result:In vitro, the ALP activity of C2C12 cells cultured with rhBMP-2 increased and the mineralization rate rose obviously contrast to control group. In intramuscular implantation test, new bone could be found in group of composition of collagen and rhBMP-2, whereas none could be found in collagen only group. The calcium deposition in the implants was higher than control group remarkable. Bone tissue could be observed by X-rays. The microtomy showed that rhBMP-2 compounded with collagen resulted in extensive bone formation compared to collagen only group, with the newly formed bone containing viable osteocytes, marrow, marrow cavity and vessel. In New Zealand rabbit radius repair test, the result revealed that rhBMP-2 repaired large bone defects and open up the bone marrow cavity by X-rays and biopsy assay.Conclusion:There were a series of experiments (ALP activity test in vitro, ectopic bone formation and repair bone defects in rabbit radius in vivo) carried out to assay the bioactivity of the rhBMP-2 in our study. The result suggested that rhBMP-2 could induce bone formation and promote bone repair observably.