Ⅱ型胶原糖胺聚糖复合支架对hUCMSCs成软骨诱导的实验研究  

Experimental research of type Ⅱ collagen composite glycosaminoglycan scaffold in hUCMSCs chondrogenic induction

作　　者：蔡第心 何鹏举[2] 谭洪波[2] 丁晶[2] 余开富[2] 张颖[2] 周田华[2] 杨军[2] 徐永清[2] 

机构地区：[1]昆明医科大学骨科,昆明650000 [2]成都军区昆明总医院骨科,昆明650000

出　　处：《重庆医学》2016年第21期2890-2893,共4页Chongqing medicine

基　　金：国家自然科学基金资助项目(81360274);第57批博士后科学基金面上资助项目(2015M572718);云南省应用基础研究自筹经费项目(2014F2106);成都军区昆明总医院院管项目(2012YG12)

摘　　要：目的探讨以人脐带间充质干细胞（hUCMSCs）作为软骨组织工程种子细胞,Ⅱ型胶原复合糖胺聚糖支架材料作为细胞载体及其细胞-支架复合体成软骨的可行性。方法制备Ⅱ型胶原复合糖胺聚糖多孔支架,电镜观察测定支架材料的孔径、孔隙率和亲水性,对支架材料进行相应的组织学分析。培养鉴定P3代的hUCMSCs,将hUCMSCs混悬液接种到Ⅱ型胶原复合糖胺聚糖支架上,不加诱导剂进行培养,3周后取出样品行甲苯胺蓝、番红精O染色,Ⅱ型胶原免疫组织化学染色及扫描电镜观察。结果第3代hUCMSCs高度表达CD29、CD105等间充质细胞标志,几乎不表达CD34等内皮细胞、造血细胞标志。Ⅱ型胶原复合糖胺聚糖支架材料呈白色多孔泡沫状,孔隙率为（91.8±2.17）%,孔径110-230μm,分布均匀,相互贯通。吸水膨胀率为（213.71±1.31）%,亲水性良好。甲苯胺蓝、番红精O及Ⅱ型胶原免疫组织化学染色呈阳性。细胞-支架复合体在培养过程中可观察到有软骨样组织形成并逐步增多,甲苯胺蓝、番红精O及Ⅱ型胶原免疫组织化学染色均呈阳性,电镜观察显示细胞在支架上增殖活跃,与材料黏附紧密,可见软骨样细胞及其周围大量交错连接的胶原纤维。结论 hUCMSCs与Ⅱ型胶原复合糖胺聚糖支架材料复合,可在体外不经诱导而初步构建组织工程软骨,为软骨损伤的修复奠定了一定的实验基础。Objective To investigate the chondrogenic feasibility of the human umbilical cord derived mesenchymal stem cells（hUCMSCs）as cartilage tissue engineering seed cells,typeⅡ collagen composite glycosaminoglycan scaffold as the cellular carrier and cell-scaffold complex.Methods The typeⅡcollagen composite glycosaminoglycan scaffolds was prepared.The pore diameter,porosity and hydrophilia of scaffold materials were observed and measured by electronic microscope.The corresponding histological analysis on the scaffold materials was performed.hUCMSCs of P3 generation were cultured and identified.The hUCMSCs suspension was inoculated in the typeⅡcollagen composite glycosaminoglycan scaffold for conducting culture without adding inducer.The samples were taken out after 3weeks and performed the toluidine blue and safranin O staining,typeⅡcollagen immunohistochemical staining and SEM scanning.Results hUCMSCs of P3 generation highly expressed the mesenchymal cell marker CD29 and CD105,while hardly expressed endothelial cells of CD34 and hematopoietic cell markers.The typeⅡcollagen composite glycosaminoglycan scaffold presented white porous foam like,the porosity was（91.8±2.17）%,the average pore diameter was 110-230μm,which was homogeneously distributed and had interpenetration.The scaffold showed good hydrophilicity with the water absorption expansion rate of（213.71±1.31）%.The scaffold staining of toluidine blue,safranin O and typeⅡ collagen was positive.The cartilage-like tissues were observed,and gradually increased in the surface of cell-scaffold complex along with culture,which were positive in Toluidine blue,safranin O and typeⅡ collagen staining,the electronic microscopic observation displayed that the cells were actively proliferated in the scaffold,closely adhered with the materials,the cartilage-like cells and a large number of peripheral collagen fibers with zigzag connection could be seen.Conclusion Compositing hUCMSCs and type Ⅱ collagen composite glycosaminoglycan scaffold could cons

关 键 词：人脐带间充质干细胞 种子细胞 糖胺聚糖 支架 组织工程软骨 

分 类 号：R68[医药卫生—骨科学] R318.08[医药卫生—外科学]