中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (12): 2127-2131.doi: 10.3969/j.issn.1673-8225.2010.12.010

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

壳聚糖-丝素支架与真皮间充质干细胞的体外复合培养

张文元，杨亚冬，房国坚   

  1. 浙江省医学科学院生物工程所，浙江省杭州市  310013
  • 出版日期:2010-03-19 发布日期:2010-03-19
  • 作者简介:张文元★，男，1961年生，浙江省温岭市人，汉族，2000年浙江省医学科学院毕业，硕士，研究员，主要从事干细胞组织工程研究。 zhangwy61@163.com
  • 基金资助:

    浙江省自然科学基金(Y2090404)，皮肤干细胞复合壳聚糖-丝素共混膜构建组织工程皮肤及动物模型修复研究。浙江省科技计划项目(2008C33025)，骨髓干细胞/壳聚糖-丝蛋白的组织工程软骨及模型治疗。浙江省医药卫生科学研究基金(2007A002)，真皮间充质干细胞对大鼠皮肤缺损创面的促愈合作用研究。浙江省医药卫生科学研究基金(2009A003)，皮肤干细胞复合壳聚糖-丝素共混膜制作组织工程皮肤及模型治疗。

Co-culture of chitosan-silk fibroin scaffold and dermal mesenchymal stem cells in vitro

Zhang Wen-yuan, Yang Ya-dong, Fang Guo-jian   

  1. Institute of Medical Bioengineering, Zhejiang Academy of Medical Sciences, Hangzhou  310013, Zhejiang Province, China
  • Online:2010-03-19 Published:2010-03-19
  • About author:Zhang Wen-yuan★, Master, Researcher, Institute of Medical Bioengineering, Zhejiang Academy of Medical Sciences, Hangzhou 310013, Zhejiang Province, China zhangwy61@163.com
  • Supported by:

    the Natural Science Foundation of Zhejiang Province, No. Y2090404*; Science and Technology Planning Project of Zhejiang Province, No. 2008C33025*; Foundation for Medical and Health Research of Zhejiang Province, No. 2007A002*; 2009A003*

PDF

435

被引次数

6

摘要：

背景：壳聚糖与丝素有各自的优点和不足，将两者共混，可取长补短，改善性能。进行壳聚糖-丝素支架与真皮间充质干细胞体外复合培养的研究，可进一步为工程化组织构建打下基础。
目的：将壳聚糖-丝素支架与大鼠真皮间充质干细胞体外复合培养，探讨壳聚糖-丝素支架对真皮间充质干细胞吸附、生长及增殖的影响。
方法： ①壳聚糖和丝素以质量比为 3︰7充分搅拌混匀，冻干制作壳聚糖-丝素海绵状支架，并测其孔径、孔隙率等指标。②将该支架与大鼠真皮间充质干细胞复合进行体外培养，倒置相差显微镜观察细胞与支架复合生长、基质形成以及细胞与支架结合的情况。分别于培养6，12，24 h消化支架上的细胞，计算细胞吸附率。于培养2，4，6，8，10 d后，用MTT法检测细胞增殖率情况。以无支架的细胞培养作为对照组。
结果与结论： ①倒置相差显微镜及扫描电镜下观察发现该支架有分布较均匀且细密的小孔，孔与孔之间相互连通，孔径大小较均匀，孔径在100~150 μm之间，孔隙率为90.3%。②24 h内，真皮间充质干细胞对支架的黏附率随着时间的增加而逐渐增高，24 h时的黏附率为93%，表明真皮间充质干细胞可良好地黏附于壳聚糖-丝素支架上。③MTT法检测细胞增殖结果显示，于培养各检测时间点，实验组与对照组比较差异均无显著性意义。于培养早期(第2，4天)，对照组增殖细胞数略高于实验组；于培养第10天，实验组的增殖细胞数略高于对照组。倒置相差显微镜观察细胞于支架上的形态和附着良好，并有较旺盛的细胞基质分泌。表明真皮间充质干细胞在该支架上能良好地生长、增殖。提示壳聚糖-丝素支架是真皮间充质干细胞体外培养的良好支架材料。

关键词: 壳聚糖-丝素支架, 真皮间充质干细胞, 吸附率, 增殖率, MTT法　　

Abstract:

BACKGROUND: Chitosan and silk fibroin have its own merits and drawbacks, the co-culture of these two materials can improve performance. Studies with regard to co-culture of chitosan-silk fibroin scaffold and dermal mesenchymal stem cells (DMSCs) in vitro can provide a foundation for further engineering tissue construction. 
OBJECTIVE: To co-culture of chitosan-silk fibroin scaffold and rat DMSCs in vitro, and to explore the effects of chitosan-silk fibroin scaffold on the adherence, growth, and proliferation of DMSCs.
METHODS:  ① Chitosan was mixed with silk fibroin at ratio of 3:7, and then the mixture was prepared for chitosan-silk fibroin scaffold after freeze drying. The pore size and porosity of scaffold was measured. ② The scaffold was co-cultured with rat DMSCs, the cell growth, matrix formation, as well as the combination of cells and scaffold was observed under an inverted phase contrast microscope. In addition, the cell adherence rate was calculated at 6, 12, and 24 hours after digestion. The cell proliferation was detected by MTT at 2, 4, 6, 8, and 10 days after co-culture. The normal cultured cells without scaffold were served as controls. 
RESULTS AND CONCLUSION:  ① The uniformity distributed pores with sizes of 100-150 μm could be seen under microscopes, whose average porosity was 90.3%. ② The cell adherence rate exhibited a time increasing trend within 24 hours, which reached to 93% after 24 hours. It suggested that DMSCs adhered well on chitosan-silk fibroin scaffold. ③MTT results demonstrated that there had no significant difference between the experimental and control groups at each culture detection points. However, the proliferation of the control group was greater than that of the experimental group at days 2 and 4 after culture, which was conversely at 10 days after culture. Under an inverted phase contrast microscope, DMSCs grew well and secreted rich cell matrix on chitosan-silk fibroin scaffold. All results suggested that chitosan-silk fibroin scaffold is suitable for DMSCs culture in vitro.

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