Tissue Engineering Complex Graft Of Biomimetic Scaffold With Seeding Bone Marrow Mesenchymal Stem Cells And Coadministrated Bone Morphogenetic Protein-2

Objective:Bone transplantation is a promising method for the treatment of bone defects, however, which was intensively limited due to shortage of soure. Bone tissue engineering applied us an alternative way for obtaining the donor. The aim of this study was to clarify whether CS-Gel/HA - rhBMP-2 complex could be a good candidate for generating tissue engineered bone and being applied to repair bone defects.Methods:1. Induced expression of human BMP-2 in E.coli containing recombinant pBV220/cDNABMP-2 plasmid. Purified recombinant human BMP-2 from the inclusion body by the steps of inclusion body solublization and chromatography.2. Primary rabbit bone mesenchymal cell were in vitro cultured and its osteoblastic ability responded to rhBMP-2 treatment was evaluated by alkaline phosphotse activity and osteocalcin content determination.3. CS-Gel/HA-rhBMP-2 complex was prepared by rhBMP-2 modification. rhBMP-2 releasing assay was carried out to testify whether the complex can hold BMP-2 for a period and hence give seeded cells a long termed simulation.4. The establishment of rabbit bone defected animal models were conducted by creating an amputation of 15mm length in the middle region of bilateral forelimbs. In experimental group(right forelimbs) the defects were treated by the transplantation of BMC seeded CS-Gel/HA-rhBMP-2 complex which was being cultured 7 days prior to the operation. In scaffold group (left side), the implantation is CS-Gel/HA only. No internal or external fixation was applied, just sewed up the wound layer by layer. Evaluate the grade of repair by X-ray examination at 4,8 and 12 weeks after the implantation respectively. Evaluate the grade of repair by histological staining at 4, 12 weeks after the implantation respectively.Results:SDS-PAGE and western blot confirmed that the recombinant human BMP-2 was prepared successfully. rhBMP-2 could stimulate in vitro cultured BMC synthesis more osteocalcin and product more alkaline phosphotase activity. BMC showed good biocompatibility with the CS-Gel/HA-rhBMP-2 complex and could adhense to the complex at high rate. There are no significant differences of both X-ray grade and histology grade between experiment and control groups at 4 weeks after implantation, but the X-ray grade is better in experiment groups than in control groups at 8 and 12weeks after treatment, data are statistically significant(p<0.05). The histology grade is better in experiment groups than in control groups at 12weeks after treatment, data are statistically significant(p<0.05).Conclusion:Bioactive rhBMP-2 was successfully prepared by the means of bacterial expression and following purification. Which could induce in vitro cultured BMCs differentiate into osteoblastic cells. BMC seeded CS-Gel/HA-rhBMP-2 complex have good therapeutic effect on repairing bone defect.