提取并部分纯化bBMP促进节段性骨缺损愈合的定量组织学研究

目的 从小牛骨基质中提取并部分纯化bBMP ,以促进节段性骨缺损的愈合。方法 通过SDS PAGE电泳及生物活性鉴定实验 ,证实bBMP保留了其活性蛋白成分 ,具有良好的骨诱导活性。在此基础上 ,合成一种新型复合人工骨材料即HA bBMP Co ,用以整复家兔尺骨中段 1.5cm节段性骨缺损 ,并对缺损区新骨生长量进行定量组织学测定。结果 HA bBMP Co复合人工骨植入整复节段性骨缺损 ,其新骨生长量明显高于同期HA Co复合人工骨组 (P <0 .0 1)。结论 HA bBMP Co复合人工骨能有效地促进节段性骨缺损的愈合 。

SPECIFIC BINDING OF HUMAN BONEMORPHOGENETIC PROTEIN （2A） WITH MOUSEOSTEOBLASTIC CELLS

Human bone morphogenetic protein 2A (hBMP2A) cDNA terminal 567 nucleotides were cloned and expressed in a phage display vector pCSM21. Human BMP2A C-terminal peptide displayed on the surface of the phage can bind specifically to the surface of mouse osteoblastic cell (MC3T3) membrane. ELISA assay showed a positive signal of the binding by using antibody against M13 phage gene 8 protein. After labeling with 3HTdR,the counts of the binding groups were 3 to 10 times higher than the control groups. It suggests that the surface of MC3T3 cells exist the receptor for hBMP2A.

EXPRESSION OF BONE MORPHOGENETIC PROTEIN-1 IN FETAL HUMAN CARTILAGE AND BONE DURING ENDOCHONDRAL OSSIFICATION

The expression of mRNA for bone morphogenetic protein-1 (BMP-1) was investigated in fetal human cartilage and bone during the developing endochondral ossification by using in situ hybridization. BMP-1 mRNA was localized in the hypertrophic chondrocytes, the cells of perichondrium, osteoblasts and marrow cells. The results suggest that BMP-1 may involve in cartilage and bone formation, and in regulation of calcification of growth plate.As a comparative study, the expression of TGF-β1 protein was investigated in cartilage and bone using immunohistochemical staining, correlated expression of TGF-β1 and BMP-1 was found. The results suggest that BMP-1 and TGF-β1 act synergistically to influence endochrondral ossification.

CO-TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS WITH HUMAN BMP2 AND VEGF165 GENES

Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.

Ectopic bone formation of human bone morphogenetic protein-2 gene transfected goat bone marrow-derived mesenchymal stem cells in nude mice

Objective: To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs). Methods: Goat bone marrow- derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene(Group 1), Adv-beta gal transfected MSCs (Group 2)and uninfected MSCs(Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals. Results: Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups. Conclusions: BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.

Calcium citrate： a new biomaterial that can enhance bone formation in situ

Objective： To investigate the effect of a new biomaterial combining calcium citrate and recombinant human bone morphogenetic protein-2 （rhBMP-2） on bone regeneration in a bone defect rabbit model. Methods： Totally 30 male New Zealand white rabbits were randomly and equally divided into calcium citraterhBMP-2 （CC-rhBMP-2） group and rhBMP-2 only group. Two 10 ram-long and 5 ram-deep bone defects were respectively created in the left and right femoral condyles of the rabbits. Subsequently 5 pellets of calcium citrate （10 mg） combined with rhBMP-2 （2 rag） or rhBMP-2 alone were implanted into the bone defects and compressed with cotton swab. Bone granules were obtained at 2, 4 and 6 weeks after procedure and received histological analysis. LSD t-test and a subsequent t-test were adopted for statistical analysis. Results： Histomorphometric analysis revealed newlyformed bones, and calcium citrate has been absorbed in the treatment group. The percent of newly formed bone area in femoral condyle in control group and CC-rhBMP-2 group was respectively 31.73%±1.26% vs 48.21%±2.37% at 2 weeks; 43.40%±1.65% vs 57.32%±1.47% at 4 weeks, and 51.32%±7.80% vs 66.74%±4.05% at 6 weeks （P〈0.05 for all）. At 2 weeks, mature cancellous bone was observed to be already formed in the treatment group. Conclusion： From this study, it can be concluded that calcium citrate combined with rhBMP-2 signifcantly enhances bone regeneration in bone defects. This synthetic gelatin matrix stimulates formation of new bone and bone marrow in the defect areas by releasing calcium ions.