Bone marrow mesenchymal stem cells modified by hTGF-β1 combined with calcium aiginate gel in three dimensional condition to construct tissue engineering cartilage / 中华显微外科杂志

ABSTRACT

ObjectiveTo investigate the feasibility of hTGF-β1 transfected bone mrrow mesenchymal stem cell (BMSCs) combined with calcium alginate gel in three dimensional condition to construct tissue engineering cartilage.MethodsRats BMSCs were obtained and cultured by whole bone marrow method,and then the third-generation cells were seeded into cell culture plate,and were divided into 3 groupsAdhTGF-β1 transfected group, Ad-EGFP transfected group and control group. The control group was added in common medium without any treatment while the other 2 groups were respectively added in serum-free medium containing Ad-hTGF-β1 or that containing Ad-EGFP.Seven days later,real-time fluorescent quantitation PCR and Western blot were employed for detecting the expression of TGF-β1.Then,the BMSCs which successfully transfected by Ad-hTGF-β1,were continually cultured in vitro.Andthe confound of cells-calcium alginate gel,the cell density was 1.0 × l07 per ml,were prepared and cultuered in constant temperature incubator.Ten days later,examine the morphous and proliferation of cell.Last,paraffin slice of the cell-gel confound was stained by HE,toluidine blue and Masson staining,while immunohistochemical for the secretion of collagen Ⅱ.ResultsSeven days after the transfection, real-time fluorescent quantitation PCR revealed that the average relative expression of TGF-β1 wasAd-hTGF-β1 group 0.863,and Ad-EGFP group 0.183, the control group 0.180, and the expression difference of TGF-β1 was statistically diffence(P ＜0.05). Western blot proved strong TGF-β1 expression in Ad-hTGF-β1 group while it was detected a little in the other two groups. Globose cells were observed through inverted microscope in the calcium alginate gel.MTT proved the amount of cells were not statistically diffence (P ＞ 0.05) at different time point.HE staining proved amount cartilage lacuna formation in the gel, while the secretion of cartilage matrix were proved by toluidine blue and Masson,and immunohistochemical proved the expression of collagen Ⅱ.Conclusion BMSC trnsfected by hTGF-β1 could be successfully induced to chondrocyte, as the cells morphous maintained.This three dimensional condition could preferably mimicry cell growth pattern as in vivo.