Study Of Biological Activities Of RhBMP-2 And It's Osteoinductivity Combined With Xenologous Bone Implanted Ectopicly In Vivo

Objective:(1)To investigate a new grafting material of bone xenograft with strong bone inductive and conductive capacity. (2) To observe osteoinductivity and the dose-effect relationship with administration of rhBMP-2/DBCB reconstituted bone xenograft.(3)To study the expression of Collagen I , II and alkaline phosphatase in osteogenesis induced by rhBMP-2 in vivo.Methods:A new xenograft was made by combining recombinant human bone morphogenetic photein-2(rhBMP-2) with antigen-free partial demineralized bovine cancellous bone (DBCB).Experiment I :144 male BALB/C mice aged 6 weeks were divided into four groups at random (study group A and B, control group I and II),36 in each group. rhBMP-2/DBCB1 was implanted in the right thigh muscle pouch in the study group A and rhBMP-2/DBCB2 in the study group B,DBCB in the control group I and rhBMP-2 0.1 mg in the control group II .The mice were sacrificed at 7d, 14d and 21d after implantation. In order to investigate osteoinduction and osteogenesis,radiographs of new bone in thigh pouches were taken 21 days after the implantation.Histological analysis was performed and ALP activity,calcium content were measured 7,14,21 days after the implantation.Exeperiment II :Composites of rhBMP-2(0.4mg) and cancellous bone carrier rhBMP-2/DBCB1 were implanted in the right thigh pouches of BALB/C mice. In order to investingate expression of Collagen I , II and alkaline phosphatase in osteogenesis induced by rhBMP-2, immunohistochemistry analysis was performed 3, 5, 7, 9, 11, 14, 17 and 21 days after the implantation; meanwhile alkaline phosphatase activity was measured 7, 14 and 21 days after the implantation.Results: (1) Histological examination showed that rhBMP-2/DBCB1 (group A) induced chondrogenesis on the 7th day, with wovenbone formed on the 14th day, and lamellar bone and marrow on the 21st day .Meanwhile,histological examination showed hypertrophied mesenchymal cells and few chondrocytes on the 7th day in rhBMP-2/DBCB2(group B);with cartilage and partial woven bone formed on the 14th day;and scant cartilage island,partial lamellar bone on the 21st day.The quality and quantity of bone-forming of the study group B were inferior to those of group A.While DBCB(control group I) and rhBMP-2 0.1 mg (control group II)failed to induce chondrogenesis or osteogenesis on the 7th ,14th and 21st days. (2)The alkaline phosphatase activities and calcium content in study group(group A and group B) were higher than those in control group (group I and group II )with significant difference (p<0.05);meanwhile, the ALP activities and calcium in study group A were higher than those in study group B. (3)The appearance of type II Collagen was accompanied by that of chondroblasts and chondrocytes. Type I Collagen and ALP, osteoblast differentiation markers, were expressed at the early stage of chondrogenesis, but type I Collagen was still expressed at high level, ALP decreased gradually with the appearance of osteoblasts and osteogenesis.Conclusion:(1) rhBMP-2/DBCB is a highly osteoinductive grafting material with the treated bovine cancellous bone being a good slow-delivery carrier which can enhance the osteoinductive capacity of rhBMP-2. (2)The osteoinductivity of rhBMP-2/DBCB reconstituted bone xenograft is ratioed to the close of rhBMP-2. (3)rhBMP-2 stimulated expression of rhBMP-2 I , II and alkaline phosphatase in osteogenesis in vivo.