实验室介绍：实验在口腔生物医学工程教育部重点实验室完成。该实验室建筑面积1 800平方米，总资产 2 500万元人民币，拥有一批特色和大型仪器设备,是全国口腔医学院校重要的研究基地之一，主要承担国家和湖北省有关的科研项目，具有完成细胞生物学实验的良好条件。

偏倚或不足：实验未将三维立体诱导分化的软骨细胞与壳聚糖材料结合而形成的组织工程软骨在动物体内进行组织学、生物力学等性能的检测。其次，应检测软骨细胞和壳聚糖复合支架的体外培养的合适期限，以缩短不必要的体外培养时间。

同行评价：作者采用转化生长因子β3和胰岛素样生长因子I联合定向诱导藻酸钠微球包被的骨髓基质干细胞诱导成软骨。实验结果显示诱导后细胞具有软骨细胞的特征，不会发生去分化等。该方法能较好地解决软骨种子细胞来源问题。课题具有创新性，对软骨组织工程种子细胞研究具有指导意义。

摘要
背景：传统的体外诱导干细胞向软骨细胞定向分化的方法有高密度细胞团块培养和平面培养。细胞团块诱导方式培养过程易损害细胞活性，而平面培养获得的细胞数量有限。
目的：观察藻酸钠微球中骨髓间充质干细胞向软骨细胞的定向分化潜力，及其在壳聚糖复合支架上的生长情况。
设计、时间及地点：对比观察实验，于2007-03/10在武汉大学口腔医学院口腔生物医学工程教育部重点实验室完成。
材料：SPF级1月龄雄性Wistar大鼠7只。壳聚糖复合支架材料由武汉大学资源与环境学院研制。藻酸钠盐、转化生长因子β3、胰岛素样生长因子Ⅰ均为Sigma公司产品。
方法：取Wistar大鼠股骨和胫骨骨髓，体外扩增培养骨髓间充质干细胞。实验组用转化生长因子β3和胰岛素样生长因子Ⅰ联合定向诱导藻酸钠微球包被的骨髓间充质干细胞3周，对照组培养条件中无转化生长因子β3和胰岛素样生长因子Ⅰ。诱导3周后，免疫组织化学方法和反转录-聚合酶链反应法检测Ⅱ型胶原蛋白和aggrecan的表达。从微球中释放诱导的软骨细胞并接种于壳聚糖复合支架，扫描电镜观察其生长情况。
主要观察指标：软骨特异性的Ⅱ型胶原、aggrecan的表达以及软骨细胞的三维形态。
结果：实验组藻酸钠微球中的骨髓间充质干细胞表达的Ⅱ型胶原和aggrecan明显高于对照组(P < 0.05)，与原代软骨细胞比较差异无显著性意义(P > 0.05)。壳聚糖复合支架支持分化软骨细胞的贴壁、伸展、增殖和迁徙，并呈典型的软骨细胞生长形态。
结论：转化生长因子β3和胰岛素样生长因子Ⅰ能诱导藻酸钠微球中的骨髓间充质干细胞定向分化成软骨细胞，并与壳聚糖复合支架表现出良好的组织相容性。
关键词：骨髓间充质干细胞；软骨分化；壳聚糖；组织工程；生物材料

雷鸣，刘世清，刘宇兰，彭昊，汪喆.藻酸钠微球中骨髓间充质干细胞向软骨细胞的定向分化[J].中国组织工程研究与临床康复，2008，12(19):3659-3662 [www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3659(ps).pdf]

Chondrogenesis of bone marrow mesenchymal stem cell in alginate microspheres

Abstract
BACKGROUND: Conventionally, the stem cells are induced to differentiate into chondrocytes in vitro by means of high-density cell aggregation culture or plane culture. But the cell aggregation is prone to damage the cell activity, and plane culture is limited in the number of harvesting cells.
OBJECTIVE: To investigate the differentiation potential of bone marrow mesenchymal stem cells (MSCs) to chondrocytes in alginate microspheres, and observe the growth of chondrocytes on chitosan composite scaffolds.
DESIGN, TIME AND SETTING: An observation study was carried out in the Key Laboratory of Biomedical Engineering of the State Education Bureau, School of Stomatology, Wuhan University (Wuhan, Hubei, China) from March to October in 2007.
MATERIALS: Seven Wistar rats of SPF grade and one month old were used in this study. Chitosan composite scaffolds were provided by College of Resource and Environment in Wuhan University (China). Transforming growth factor (TGF) β3 and insulin-like growth factor (IGF) Ⅰ were the products of Sigma Company (USA).
METHODS: Bone marrow was harvested from rat femur and tibia, and then were amplified to culture MSCs in vitro. MSCs capsulated in alginate microspheres were induced by TGF-β3 and IGF-I, serving as the experiment group. While those cultured without TGF-β3 and IGF-I were taken as controls. Three weeks later, the expressions of collagen type II and Aggrecan were evaluated by immunohistochemistry staining and reverse transcriptase-polymerase chain reaction assay. After the induced chondrocytes were seeded on the chitosan composite scaffolds, their morphology was evaluated by scanning electron microscope.
MAIN OUTCOME MEASURES: The expressions of collagen type II and aggrecan, together with the morphology of chondrocytes were all observed.
RESULTS: The MSCs capsulated in alginate microspheres were induced to express the higher levels of collagen type II and aggrecan by TGF-β3 and IGF-I than those without any growth factor (P < 0.05). No significant differences were found in the experiment group compared to the primarily cultured chondrocytes (P > 0.05). The chitosan composite scaffolds supported the adhesion, proliferation and migration of the induced chondrocytes, which exhibited typical growth of chondrocytes.
CONCLUSION: Combination TGF-β3 with IGF-I can induce the MSCs capsulated in alginate microspheres to differentiate into chondrocytes, and chitosan-based scaffold is biocompatible with the induced chondrocytes.

Lei M, Liu SQ, Liu YL, Peng H, Wang Z.Chondrogenesis of bone marrow mesenchymal stem cell in alginate microspheres.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(19):3659-3662
[www.zglckf.com/zglckf/ejournal/upfiles/08-19/19k-3659(ps).pdf]