不同浓度重组人BMP-7促进骨髓间充质干细胞复合组织工程支架材料TCP-COL体外构建组织工程软骨的研究

目的观察比较不同浓度重组人BMP-7对骨髓间充质干细胞复合组织工程支架材料TCP-COL体外构建组织工程软骨的影响。方法用梯度离心法获取人骨髓间充质干细胞（hMSCs）,将扩增的第3代hMSCs以1×10~7/ml的细胞浓度接种在支架材料TCP-COL上,分为三组进行培养：对照组（成软骨诱导液培养）,BMP-7（50）组（成软骨诱导液基础上加入50ng/ml的BMP-7）,BMP-7（100）组（成软骨诱导液基础上加入100ng/ml的BMP-7）。培养2周后进行扫描电镜观察（SEM）,苏木素伊红染色（HE）,阿尔新蓝染色,Ⅱ型胶原免疫组织化学染色（IHC）,荧光定量PCR（RT-PCR）,糖胺多糖定量（GAG quantification assay）研究。结果扫描电镜与苏木素伊红染色结果显示细胞在TCP-COL生物支架中分布均匀且紧密粘附在材料表面。阿尔新蓝染色,Ⅱ型胶原免疫组织化学染色,二型胶原（F=76.931,P〈0.05）,糖胺多糖（F=63.158,P〈0.05）荧光定量PCR,糖胺多糖定量（F=8.981,P〈0.05）结果显示BMP-7能有效促进人骨髓间充质干细胞复合支架材料TCP-COL体外成软骨,且100mg/ml的浓度应用效果大于50mg/ml。但在COL1基因的表达上,BMP-7（50）组和BMP-7（100）组都显著大于对照组（F=34.823,P〈0.05）。结论 BMP-7能有效促进人骨髓间充质干细胞复合支架材料TCP-COL体外成软骨,且100mg/ml的浓度应用效果大于50mg/ml,但应注意其促进骨质增生的作用。TCP-COL是一种有应用前景的生物支架材料。

Different doses of bone morphogenetic proteins-7 promote chondrogenesis of human mesenchymal stem cells in tricalcium phosphate-collagen scaffolds

Objective To investigate the effects of different doses of bone morphogenetic proteins-7 （BMP-7） on engineered cartilage formation. Methods Human mesenchymal stem cells （hMSCs） were isolated by density gradient centrifugation and then were seeded onto tricalcium phosphate-collagen scaffolds （TCP-COL）. hMSCs were cultured on the scaffold for two weeks and divided into three experimental groups： （1） control group： chondrogenic medium （without TGF-β）; （2） BMP-7 （50） group： chondrogenic medium （without TGF-β） with 50 ng/ml BMP-7; （3） BMP-7 （100） group： chondrogenic medium（without TGF-β） with 100 ng/ml BMP-7. The effect of BMP-7 on chondrogenesis was assessed by scanning electron microscopy （SEM） observation, hematoxylin-eosin （HE） staining, alcian blue staining, typeⅡ collagen immunohistochemical staining, RT-PCR and GAG quantification assay. Results BMP-7 could significantly induce the chondrogenic differentiation of hMSCs on the TCP-COL scaffold, and the BMP-7 （100） group had better effect than the BMP-7 （50） group （P〈0.05）. However, it is noteworthy that in the BMP-7 （50） group and the BMP-7 （100） group, a significant up-regulation of COL1 gene was detected compared with the control group, indicating that when implanted for cartilage repair, BMP-7 might lead to bone formation. Conclusions BMP-7 could significantly induce the chondrogenic differentiation of hMSCs on the TCP-COL scaffold, however, the dose of BMP-7 must be carefully regulated for that BMP-7 might lead to bone formation. TCP-COL scaffolds are potentially usable for cartilage tissue engineering.