Construction And Investigations Of The Osteogenic Potential Of The HBMP2 Gene Activated Nanobone Putty

Objective: To clone human bone morphogenetic protein 2 (hBMP2) gene and construct the gene's eukaryotic expression vector; To investigate the osteogenic potential of hBMP2 gene activated nanobone putty in inducing ectopic bone formation and to investigate the effects of the hBMP2 gene activated nanobone putty on repairing bone defects.Method: The total RNA was extracted from human osteoma cells, the human BMP2 cDNA was amplified by RT-PCR and inserted into pGEM-T vector. The positive clones were screened out, then the recombinant plasmid was confirmed by restriction enzyme digestion, PCR and the analysis of nucleotide sequence. The BMP2 cDNA in the pGEM-T cloning vector was inserted into the pcDNA3.1 eukaryotic expression vector; Twenty-four Kunming mice were used in this experiment. The mice were divided into two groups randomly. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 (control side 1) or group 2 (control side 2) respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis in 2 and 4 weeks after operation; Fifty-four rabbits were used in this experiment. Forty-eight of the rabbits were made bilateral 15mm radial defect models and were divided into three groups randomly. The groups were treated with different materials: group A, nanobone putty+hBMP2 plasmid; group B, putty+blank plasmid and group C, nanobone putty only. 6 rabbit were made left radial defects as a blank control. The effect of bone repairing was evaluated by radiography, histology, molecular biology and biomechanics analysis in 4, 8 and 12 weeks after operation.Result: The cDNA sequences which was inserted into pGEM-T and pcDNA3.1 plasmids was human BMP2; All the animals recovered well from the operation. The tissue of the experiment side had the expression of hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed in 2 weeks after operation and there were mass mature bone in 4 weeks. No bone formation was observed in the control side. The ALP activity in the experiment side was obviously higher than that in the control side; All the animals recovered well from the operation. The tissue of group A has the obviously expression of hBMP2 protein and higher ALP level. New bone formation rate and antibending strength of group A was significantly higher than that of group B and C. The defects in blank control were not healed.Conclusion: The pcDNA3.1-hBMP2 eukaryotic vector is successfully constructed; The hBMP2 gene activated nanobone putty has a obviously activity in inducing ectopic bone formation; The hBMP2 gene activated nanobone putty has strong osteoinductive ability and the repair capability of bone defects is more effective than nanobone putty only.