重要的概念:关节软骨缺损的修复是近年组织工程涉及到软骨和软骨下骨的双重修复，众多研究者从支架设计和种子细胞选择方面都进行了研究，重点在于选择合适的细胞载体解决长期修复缺损时的双层明显分层及断裂的难题，选择合适的种子细胞来匹配双层不同组织结构的需求问题。

应用要点：实验采用单个整体层状修复体模拟正常的关节软骨结构，修复体的上层可以接种软骨细胞，下层可接种成骨细胞；或整体接种骨髓间充质干细胞然后使其再分别诱导分化为软骨细胞和成骨细胞，在体外或体内共培养形成组织工程骨软骨复合物，以达到修复关节软骨的目的。

偏倚或不足：实验中发现修复体表面细胞数明显多于内部细胞数，原因可能是修复体表面的氧气和营养比内部更充足，使细胞有一种向外贴附生长的倾向性，此情况可望经过微重力旋转仪培养或灌注培养来解决。

摘要
目的：观察胶原/壳聚糖/β-磷酸三钙层状梯度修复体对经诱导骨髓间充质干细胞的吸附作用及对细胞生物学性状的影响，评价其作为关节软骨工程支架的可行性。
方法:实验于2007-09/2008-03在广东省组织工程构建与检测实验室进行。①实验方法：以胶原、壳聚糖、β-磷酸三钙制备复合支架，孔隙率≥ 90%，孔径100 μm；体外培养新西兰大白兔骨髓间充质干细胞并成骨诱导3 周，经检测诱导成功后，使之吸附于多孔胶原/壳聚糖/β-磷酸三钙层状梯度修复体上三维立体培养4周。②观察指标：通过倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测修复体三维立体培养对成骨细胞的表型、增殖及功能的影响。
结果：①骨髓间充质干细胞经成骨诱导，检测碱性磷酸酶染色阳性，钙结节染色阳性，证实诱导成功。②胶原/壳聚糖/β-磷酸三钙复合支架亲水性好，经诱导的骨髓间充质干细胞吸附于支架表面24 h后,顺孔隙迁徙至支架内部，倒置显微镜和扫描电镜观察显示细胞在孔壁贴附良好。③组织学及免疫组织化学检测表明成骨细胞在复合支架上增殖和表型维持稳定，能分泌细胞外基质，Ⅰ型胶原免疫组织化学染色阳性。　
结论：胶原/壳聚糖/β-磷酸三钙层状梯度修复体与经成骨诱导的骨髓间充质干细胞相容性良好，为修复关节软骨下层骨的进一步研究提供了实验依据。
关键词：胶原；壳聚糖；磷酸三钙；关节软骨；细胞相容性；组织工程；生物材料

朱书涛，朱立新，余磊，严乐平，于巧莲，黄锐，张世浩，王九限胶原/壳聚糖/β-磷酸三钙层状修复体负载骨髓间充质干细胞体外培养[J].中国组织工程研究与临床康复，2008，12(10):1815-1818
[www.zglckf.com/zglckf/ejournal/upfiles/08-10/10k-1815(ps).pdf]

In vitro culture of bone mesenchymal stem cells on the layered collagen-chitosan-beta-tricalcium phosphate composite scaffold

Abstract

AIM：To evaluate the feasibility of the layered cylindric collagen-chitosan-β-tricalcium phosphate composite as a scaffold for the articular cartilage tissue engineering after an observation of how it absorbs the induced bone mesenchymal stem cells and affects the cell biological behaviors.
METHODS: The experiment was carried out in the Key Laboratory of Tissue Construction and Detection of Guangdong Province from September 2007 to March 2008.①The collagen-chitosan-β-tricalcium phosphate composite was prepared in a pore diameter of 100 μm and porosity ≥ 90%. Rabbit bone mesenchymal stem cells were cultured and induced in vitro, then the cells were seeded onto the porous collagen-chitosan-β-tricalcium phosphate composite scaffold and were cultured in a three-dimensional environment for 4 weeks.②The effects of the composite scaffold on the phenotype, proliferation, and function of osteoblasts were observed by the inverted phase contrast microscopy, histology, scanning electron microscopy, and immunohistochemistry.
RESULTS: ①The induced bone mesenchymal stem cells were positive for both alkaline phosphatase stain and calcification tubercles stain, which all indicated successful induction.②Due to good hydrophilicity of collagen-chitosan-β-tricalcium phosphate composite scaffold, the induced bone mesenchymal stem cells adhered to the surface of the scaffold by the inverted phase contrast microscopy and scanning electron microscopy, then cells migrated into the scaffold 24 hours later.③The results of histology and immunohistochemistry revealed that osteoblasts proliferated on the composite scaffold, maintained a rounded morphology and could secrete the extracellular matrix. The immunohistochemistry stain of collagen Ⅰ was positive.
CONCLUSION: The layered cylindric collagen-chitosan-β-tricalcium phosphate composite scaffold has a good compatibility with the induced bone mesenchymal stem cells, and it will provide experimental evidences for the further study of article cartilage repair.

Zhu ST, Zhu LX, Yu L, Yan LP, Yu QL, Huang R, Zhang SH, Wang JX. In vitro culture of bone mesenchymal stem cells on the layered collagen-chitosan-beta-tricalcium phosphate composite scaffold.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(10):1815-1818 [www.zglckf.com/zglckf/ejournal/upfiles/08-10/10k-1815(ps).pdf]