三明治法构建兔间充质干细胞-脱细胞真皮基质定向分化成软骨培养的研究  

Preliminary experimental research on sandwich structure culture of mesenchymal stem cells-acellular dermal matrix directed differentiation into chondrocytes in rabbit model

作　　者：王立勋[1] 姜建元[1] 吕飞舟[1] 马晓生[1] 夏新雷[1] 

机构地区：[1]复旦大学附属华山医院骨科,上海200040

出　　处：《中华实验外科杂志》2013年第9期1942-1945,共4页Chinese Journal of Experimental Surgery

摘　　要：目的 探索天然的脱细胞真皮基质（ADM）的制备方法；观察ADM的孑孔隙率和生物力学特性；探讨间充质干细胞（MSCs）结合ADM支架材料的生物学特性.方法 选用新西兰兔的皮肤作为原料,切取厚度为500 μm的真皮层,切成1.5 cm×1.5 cm的正方形小块,采用化学、物理和生物综合技术制备ADM支架材料,并对其进行孔隙率和生物力学检测.采用三明治法将MSCs与ADM复合培养并定向诱导分化成软骨,然后分别进行扫描电镜观察、激光扫描共聚焦显微镜观察和成软骨分化检测等分析比较.结果 ADM的孔隙率为80.26％.ADM的拉伸强度为4.52 Mpa.将sC-MSCs种植在ADM上,经过21 d的体外培养,硫酸糖胺聚糖（sGAG）的平均值为（4.6±0.9）μg/mg,与第3天的sGAG值（1.1±0.2）μg/mg比较,差异有统计学意义（P＜0.05）.Ⅱ型胶原的含量第21天也比第3天增加[（6.2 ±0.9）μg/mg比（2.4 ±0.6）μg/mg],差异有统计学意义（P＜0.05）.扫描电镜观察和激光扫描共聚焦显微镜观察结果显示,ADM为MSCs的增殖和分化提供了充足的空间,MSCs种植在ADM支架中分布均匀,并且长期存活,具有良好的生物细胞相容性.结论 ADM支架材料未发现有细胞毒性,同时具有较为合适的孔隙率,更利于植入细胞的黏附与增殖；在处理过程中,能保持ADM的生物力学性能.用三明治法构建MSCs-ADM具有良好的成软骨定向分化的能力.Objective To prepare a type of acellular dermal matrix （ADM） and analyze its properties.Methods ADM was prepared by means of combined treatments with physical,chemical and biological methods,and its structure,composition and properties were evaluated Mesenchymal stem cells （MSCs）were seeded onto ADM scaffold and MSC-loaded ADM grafts were analyzed by confocal microscopy and scanning electron microscopy.Results The porosity of ADM reached to 80.26％ and the tensile strength was about 4.52 MPa.The MSCs were seeded on ADM and cultured for 21 days,the average amount of sulfated glycosaminoglycans （sGAG） and type Ⅱ collagen protein were increased.Thus,prolonged MSCs-ADM scaffold interactions favored enhanced chondrogenic potential.The results of scanning electron microscopy and confocal fluorescence microscopy showed that MSCs can survive in ADM grafts.Conclusion We used natural rabbit acellular dermal matrix as scaffold that presents a more natural microenvironment to support growth and proliferation of MSCs than synthetic scaffolds.Ultimately,it will be directed towards developing a clinically feasible strategy for use of MSC-ADM in the reconstruction of articular cartilage.

关 键 词：间充质干细胞 脱细胞真皮基质 软骨修复 成软骨诱导 兔 

分 类 号：R68[医药卫生—骨科学]