| Background：The repair of bone defects remains a major orthopedic clinical problem. In recent years, asthe development of tissue engineering, tissue-engineered bone has become a research focus inthe treatment of bone defects. Bone regeneration is a complex process in which multi-factorsinteract with each other. Lots of growth factors play an important regulatory role in thisprocess. Among them, bone morphogenetic protein (BMPs) has a close relationship withosteogenesis. Among the BMP family, BMP2has the highest osteoinduction activity, andBMP7could promote the proliferation of osteoblast and accelerate the new bone growth.Therefore, we speculate that co-expression of BMP2and BMP7in ADSCs may be helpful forcell transformation to osteoblasts, thus promoting the growth and healing of bone tissues.Objective：The purpose of this study was to investigate the feasibility and advantages ofconstructing a novel tissue-engineered bone, using β-tricalcium phosphate (β-TCP) andadipose derived stem cells (ADSCs), which were modified with human bone morphogeneticprotein2gene (BMP2) and human bone morphogenetic protein7gene (BMP7), throughlentivirus transfection.Methods：(1) Rat ADSCs were isolated and expanded in vitro, and detected the cell surfacemarkers and multilineage differentiation potential.(2) The lentiviral expression vectors with green fluorescent protein (GFP) gene carryingBMP2and lentiviral vectors with red fluorescent protein (RFP) gene carrying BMP7werepackaged and produced, named Lv-BMP2and Lv-BMP7, respectively. ADSCs weretransfected with Lv-BMP2(Lv-BMP2group), Lv-BMP7(Lv-BMP7group), co-transfected with Lv-BMP2and Lv-BMP7(Lv-BMP2+Lv-BMP7group), or transfected with no virus(control group). Then the expression of BMP2and BMP7was determined by realtime PCRand Western blot. The expression of osteocalcin (OCN) and Collagen I protein in ADSCs14days after transfection was detected by Western blot in each group. Alkaline phosphatase(ALP) activity of ADSCs was also measured.(3) ADSCs were transferred by lentivirus vector with BMP2and BMP7genes.PicoGreen dsDNA assay evaluated cell growth. Alkaline phosphatase (ALP) activity ofADSCs was measured. Alizarin red S staining and real-time PCR were used to test theosteoblast activities of ADSCs.(4) Preparing rat bone defect models and implantations of ADSCs/β-TCP,BMP2/ADSCs/β-TCP, BMP7/ADSCs/β-TCP, BMP2+BMP7/ADSCs/β-TCP. Radiographsand histomorphology of rat femurs were examined at2,4and6weeks after the surgery.Results：(1) ADSCs were obtained from subcutaneous adipose tissue in the inguinal groove of4-week-old Sprague-Dawley rats. The cells were separated and cultured. The expression ofCD29and CD44was positive in cells surface, while CD34, CD45and CD11b were negative.ADSCs could differentiated into osteoblast and adipoblast.(2) Lentiviral vectors overexpressing BMP2or BMP7were correctly constructed. Theexpression of OCN and Collagen I protein in co-transfected group was significantly higherthan that in other groups. The ALP activity in co-transfected group was significantly higherthan that in other groups.(3) After seeding ADSCs into the β-TCP scaffolds, PicoGreen dsDNA assaydemonstrated that ADSCs proliferation was well in each group.14days after cell seeding, theALP activity was higher in the co-transfection group than that in one gene modified group.More ADSCs mineralization and more mRNA relative expression of osteogenic marker genessuch as ALP, OPN and OC were also observed in co-transfection group.(4) In anamal assays, ectopic osteogenesis was observed in co-transfection group andone gene transfection group. At6weeks post-operation, co-transfection group exhibit betterbone formation than one gene transfection group in radiographs and histological observation.Conclusions：We successfully constructed tissue-engineered bone by using β-TCP scaffolds combined with hBMP2and hBMP7modified ADSCs. Then we implanted the engineered-bone to SDrats to examine the bone formation in vivo. We conclude that both BMP2and BMP7genecould be co-expressed in ADSCs through lentivirus mediated co-transfection of both genes.Furthermore, we found that co-expression of BMP2and BMP7gene would be helpful forosteogenesis compared with single gene. |
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