力学刺激联合诱导因子对组织工程软骨的影响  

EFFECT OF MECHANICAL STIMULATION COMBINED WITH INDUCTIVE FACTORS ON TISSUE ENGINEERED CARTILAGE

作　　者：王晖[1] 崔一民[2] 朱俊峰[2] 周贤德[2] 朱传敏[2] 陈晓东[1] 

机构地区：[1]上海交通大学医学院附属新华医院骨科,上海200092 [2]上海同济大学嘉定校区机械工程学院

出　　处：《中国修复重建外科杂志》2011年第11期1377-1382,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81171705);上海市教育委员会科研创新项目(08YZ37)~~

摘　　要：目的力学刺激与诱导因子是软骨组织工程中的重要诱导因素,研究力学刺激联合诱导因子对组织工程软骨的影响。方法 7日龄长枫杂交仔猪,体重3～6 kg,用于分离培养BMSCs,取第2代细胞以5×107个/mL密度接种于聚羟基乙酸-聚乳酸[poly(lactic-co-glycolic acid),PLGA]支架上制备细胞-支架复合物。实验随机分为5组,A、B、C组分别对细胞-支架复合物采用软骨诱导培养液诱导、单纯力学加载、力学加载联合软骨诱导液培养处理,其中力学加载参数为1 Hz、0.5 MPa、4 h/d;D、E组分别为单纯细胞-支架复合物阴性对照和猪自体软骨阳性对照。4周后各组分别取材行大体观察、组织学检测及实时荧光定量PCR检测。结果 C组细胞-支架复合物厚度、弹性模量及最大值负荷均显著高于A、B组(P<0.05)。A、B、C组组织切片HE染色均可见有软骨陷窝形成,番红O染色提示支架复合物的细胞外基质中有大量蛋白聚糖(glycosaminoglycan,GAG)形成,Ⅱ型胶原免疫组织化学染色均显示阳性结果,其中A组染色深度强于B组,C组强于A、B组,且最接近E组;C组GAG含量显著高于A、B组(P<0.05)。实时荧光定量PCR检测示,C组Ⅰ型胶原、Ⅱ型胶原及聚集蛋白聚糖mRNA表达量均显著高于A、B组,且A组各基因表达量均高于B组,差异均有统计学意义(P<0.05)。结论力学刺激联合软骨诱导因子能更好地促进细胞-支架复合物上的BMSCs产生更多胶原和GAG等细胞外基质,增强其力学特性,更有助于其向软骨细胞分化。Objective Mechanical stimulation and inductive factors are both crucial aspects in tissue engineered cartilage.To evaluate the effects of mechanical stimulation combined with inductive factors on the differentiation of tissue engineered cartilage.Methods Bone marrow mesenchymal stem cells（BMSCs） were isolated from newborn porcine（aged 7 days and weighing 3-6 kg） and expanded in vitro.The BMSCs at passage 2 were seeded onto a scaffold of poly（lactic-co-glycolic acid）（PLGA） in the concentration of 5 × 107/mL to prepare cell-scaffold composite.Cell-scaffold composites were cultivated in a medium with chondrocyte-inducted factors（group A）,in a vessel with mechanic stimulating only（group B）,or mechanic stimulating combined with chondrocyte-inducted factors（group C）（parameters of mechanics： 1 Hz,0.5 MPa,and 4 hours/day）.Cell-scaffold composite and auto-cartilage served as positive control（group D） and negative control（group E）,respectively.After 4 weeks of cultivation,the thickness,elastic modulus,and glycosaminoglycan（GAG） content of composites were measured.Additionally,BMSCs chondrogenic differentiation was assessed via real-time fluorescent quantitative PCR,immunohistochemistry,and histological staining.Results The thickness,elastic modulus,and maximum load in group C were significantly higher than those in groups A and B（P 0.05）.In groups A,B,and C,cartilage lacuna formation,GAG expression,and positive results for collagen type II were obsersed through HE staining,Safranin-O staining,and immunohistochemistry staining.The dyeing depth was deeper in group A than in group B,and in group C than in groups A and B;group C was close to group E.The GAG content in group C was significantly higher than that in groups A and B（P 0.05）.Real-time fluorescent quantitative PCR revealed that mRNA expressions of collagen type I,collagen type II,and GAG in group C were significantly higher than those in groups A and B（P 0.05）,and in group A than in group B（P 0.05）.Conclu

关 键 词：组织工程软骨 BMSCS 细胞外基质 力学刺激 支架材料 猪 

分 类 号：R318.01[医药卫生—生物医学工程]