中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (34): 6308-6312.doi: 10.3969/j.issn.1673-8225.2010.34.010

• 生物材料基础实验 basic experiments of biomaterials • 上一篇    下一篇

胶原支架复合兔骨髓间充质干细胞体外模拟的微重力培养

陈  辉1，李  斌1，王  健1，李  涛1，张  伟1，林  涛2，杨  光1，满振涛1   

  1. 1山东大学附属省立医院关节外科，山东省济南市 250021；2山东大学附属济南市中心医院，山东省济南市  250013
  • 出版日期:2010-08-20 发布日期:2010-08-20
  • 通讯作者: 王健，硕士生导师，教授，主任医师，博士，山东大学附属省立医院关节外科，山东省济南市 250021 wangjian2@ medmail.com
  • 作者简介:陈辉★，男，1986年生，山东省菏泽市人，回族，山东大学在读硕士，主要从事骨关节外科研究。 ch_hp112@126.com
  • 基金资助:

    山东省医药卫生科研计划项目(2005-97)，山东省科技攻关计划资助项目(2006GG2302049)。

Culture of rabbit bone marrow mesenchymal stem cells and collagen scaffolds under the mimic microgravity environment in vitro

Chen Hui1, Li Bin1, Wang Jian1, Li Tao1, Zhang Wei1, Lin Tao2, Yang Guang1, Man Zhen-tao1   

  1. 1 Department of Joint Surgery, Provincial Hospital Affiliated to Shandong University, Jinan  250021, Shandong Province, China; 2 Jinan Central Hospital Affiliated to Shandong University, Jinan  250013, Shandong Province, China
  • Online:2010-08-20 Published:2010-08-20
  • Contact: Wang Jian, Master’s supervisor, Professor, Chief physician, Doctor, Department of Joint Surgery, Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China wangjian2@medmail.com
  • About author:Chen Hui★, Studying for master’s degree, Department of Joint Surgery, Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China ch_hp112@126.com
  • Supported by:

     the Scientific Research Program of Medical Science of Shandong Province, No. 2005-97*; the Key Program in Science and Technology of Shandong Province, No. 2006GG2302049*

PDF

505

被引次数

15

摘要：

背景：骨髓间充质干细胞向软骨细胞诱导的方法包括体外高密度微团培养、体外单层细胞培养、体外三维支架环境诱导、与软骨细胞体外共培养诱导和基因转染诱导培养等。
目的：验证模拟微重力条件下诱导后的兔骨髓间充质干细胞在Ⅰ、Ⅱ型胶原支架上黏附、伸展和增殖情况。
方法：对兔骨髓间充质干细胞体外进行原代和传代培养，按加入诱导条件不同分为2组：实验组(转化生长因子β1+ 胰岛素样生长因子1诱导)组、空白对照组。3周后分别作MTT比色试验、糖胺聚糖检测和免疫组织化学染色。将诱导后的实验组细胞分别种植于Ⅰ、Ⅱ型胶原支架，分为4组培养：复合Ⅱ型胶原支架静置培养、复合Ⅱ型胶原支架模拟微重力培养组、复合Ⅰ型胶原支架静置培养、复合Ⅰ型胶原支架模拟微重力培养组，1周后作苏木精-伊红、甲苯胺蓝染色。
结果与结论：实验组MTT吸光度值和糖胺聚糖水平检测结果均大于空白对照组，且Ⅱ型胶原免疫组织化学检测阳性。苏木精-伊红、甲苯胺蓝染色显示，Ⅱ型胶原支架复合细胞的数量明显高于Ⅰ型胶原支架；模拟微重力培养条件下胶原支架复合细胞的数量明显高于静置培养组。结果说明微重力培养环境有利于高密度细胞的黏附、增殖，有利于细胞之间的信号传递，为维系细胞的生长和代谢提供适宜的微环境；作为诱导后骨髓间充质干细胞的支架材料Ⅱ型胶原支架优于Ⅰ型胶原。

关键词: 骨髓间充质干细胞, 软骨细胞, 转化生长因子β1, 胰岛素样生长因子1, Ⅰ型胶原支架, Ⅱ型胶原支架, 模拟微重力

Abstract:

BACKGROUND: The method of differentiating from bone marrow mesenchymal stem cells (BMSCs) into chondrocytes included in vitro high-density micelle culture, in vitro monolayer cell culture, in vitro three dimensional stent induction, in vitro co-culture induction with chondrocytes and gene transfection.
OBJECTIVE: To study adhesion, extension and proliferation of rabbit BMSCs in type Ⅰ and Ⅱ collagen scaffolds under the mimic microgravity environment in vitro.
METHODS: BMSCs of rabbits were primarily cultured and subcultured in vitro, and then divided into two groups according to the difference of induction factors: experimental group receiving transforming growth factor (TGF)-β1 and insulin-like growth factor (IGF)-Ⅰ; blank control group. After three weeks, the two groups were detected by methyl thiazolyl tetrazolium (MTT) assay, measurement of glycosaminoglycan (GAG) and immunohistochemistry. Chondrocytes in the experimental group were incubated in type Ⅰ and Ⅱ collagen scaffolds, and then divided into four groups: group 1: chondrocytes and type Ⅱ collagen scaffolds co-cultured statically; group 2: chondrocytes and type Ⅱ collagen scaffolds co-cultured under the mimic microgravity environment; group 3: chondrocytes and type Ⅰ collagen scaffolds co-cultured statically; group 4: chondrocytes and type Ⅰ collagen scaffolds co-cultured under the mimic microgravity environment. One week later, hematoxylin-eosin staining and toluidine blue staining were performed.
RESULTS AND CONCLUSION: The results of the MTT assay (absorbance value) and the GAG content in experimental group were higher than in blank control group. Immunohistochemical detection of collagen Ⅱ was positive in experimental group. Results from hematoxylin-eosin staining and toluidine blue staining have demonstrated that composited cell number in type Ⅱ collagen scaffolds was evidently more than that of typeⅠ collagen. Composited cell number under the mimic microgravity environment was evidently more than that of static culture in the same scaffold. Above-described results have confirmed that the mimic microgravity environment is conducive to adhesion and proliferation of high-density cells, and contributes to signal transmission among cells to provide suitable microenvironment for maintaining cell growth and metabolism. Collagen type Ⅱ scaffold can be used as a more satisfying scaffold material for chondrocytes than collagen typeⅠ.

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