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[目的]探讨软骨细胞外基质微环境对源于人脐带Wharton胶的间充质干细胞增殖的影响,观察该微环境促进干细胞向软骨细胞方向分化的潜能。[方法]①取3%(w/v)猪关节软骨细胞外基质和2.4%(w/v)海藻酸盐为原料,按体积比1∶1的比例充分混合,将其用带16 G针头的1 ml注射器匀速滴入氯化钙溶液制作软骨细胞外基质/海藻酸盐混合凝胶微球,通过扫描电镜观察该微球内部结构。②将人脐带Wharton胶中间质干细胞(WJMSCs)混于软骨细胞外基质/海藻酸混合液,滴入氯化钙溶液形成复合干细胞的混合凝胶微球进行培养,设为实验组;同理WJMSCs混合于单纯藻酸盐凝胶并形成微球作为对照组。③通过扫描电镜和FDA-PI染色观察上述两组微球内细胞的生长粘附情况,体外培养4周,经组织学和DNA测定观察干细胞的表型及增殖情况。[结果]①构建的软骨细胞外基质/海藻酸盐混合凝胶微球呈半透明乳白色,内部具有较多不规则的孔隙结构,具有三维立体培养细胞的环境特点。②比较实验组和对照组不同细胞培养时间段细胞增殖量,发现实验组细胞增殖率高于对照组,第28 d时两组细胞DNA含量的差异有统计学意义,(P﹤0.05)。③实验组中细胞呈圆形或椭圆形,形成明显的软骨陷窝样的结构,有大量细胞外基质分泌,对照组中细胞呈圆形,陷窝样的结构不明显。④组织学染色观察发现:实验组中培养的干细胞明显有向软骨细胞方向分化的趋势。[结论]软骨细胞外基质微环境不仅利于人脐带Wharton胶中间质干细胞的增殖,而且具有将WJMSCs向软骨细胞方向诱导分化的潜能。
[Objective] To investigate the effect of cartilage extracellular matrix microenvironment on the proliferation of mesenchymal stem cells derived from human umbilical cord Wharton glue and to observe the potential of the microenvironment to promote the differentiation of stem cells into chondrocytes. [Method] ① 3% (w / v) pig articular cartilage extracellular matrix and 2.4% (w / v) alginate were used as raw materials, mixed in a volume ratio of 1: 1, Of 1 ml syringe into calcium chloride solution to make chondrocyte extracellular matrix / alginate mixed gel microspheres. The microstructure of microspheres was observed by scanning electron microscopy. (2) The human umbilical cord Wharton glue-derived mesenchymal stem cells (WJMSCs) were mixed with the chondrocyte extracellular matrix / alginate mixture, mixed with calcium chloride solution to form composite stem cells mixed gel microspheres, and set as the experimental group; WJMSCs were mixed with simple alginate gel to form microspheres as a control group. (3) The growth and adhesion of cells in the two microspheres were observed by scanning electron microscopy and FDA-PI staining. The cells were cultured in vitro for 4 weeks. The phenotype and proliferation of stem cells were observed by histology and DNA assay. [Results] ① The constructed chondrocyte extracellular matrix / alginate mixed gel microspheres were semitransparent milky white with many irregular pore structures inside and had the environmental characteristics of three-dimensional cultured cells. (2) The proliferation of cells in experiment group and control group was higher than that in control group at the different cell culture time. The difference of DNA content between the two groups was statistically significant at the 28th day (P <0.05). In the experimental group, the cells were round or oval, forming a distinct cartilaginous lacuna-like structure with a large amount of extracellular matrix secretion. The cells in the control group were round, and the lacunar structure was not obvious. ④ Histological staining revealed that the stem cells cultured in the experimental group showed obvious tendency of differentiating into chondrocytes. [Conclusion] The cartilage extracellular matrix microenvironment is not only beneficial to the proliferation of human umbilical cord Wharton glue-derived mesenchymal stem cells, but also has the potential of inducing WJMSCs to differentiate into chondrocytes.