摘要
背景：体外单层诱导骨髓问充质干细胞转化为软骨细胞后，但仍存在难以长期扩增培养，容易出现去极化的现象。目的：观察体外诱导骨髓间充质干细胞出现软骨细胞表型后，在自制复合壳聚糖，胶原三维多孔支架上构建组织工程化软骨的特点。设计、时间及地点：以细胞为对象的单一样本观察，于2006—12／2007—09在首都医科大学细胞与遗传实验室完成。材料：1岁龄骨骼成熟的健康绵羊。雌雄不拘，体质量20～30kg。方法：采用密度梯度离心法分离羊骨髓间充质干细胞，体外培养至第3代时，实验组以特定诱导液培养2周，以未经诱导培养的细胞为对照，两组均接种于自制的壳聚糖／胶原三维多孔支架中。主要观察指标：在相差显微镜和扫描电镜下观察细胞形态、增殖和功能改变情况，Ⅱ型胶原免疫组织化学染色鉴定形成软骨组织的特征。结果：壳聚糖/胶原接种细胞悬液后，细胞迅速扩散到支架孔隙内，分布均匀，不易脱落，有较好的亲水性和黏附性。实验组细胞在三维立体诱导培养环境中，一直成球状聚集生长，生长旺盛，2周时肉眼即可隐约看见支架孔隙内的细胞团块，4周时则更加明显，甚至向支架表面外向生长。组织学检查发现诱导分化后的细胞分泌大量的细胞外基质，Ⅱ型胶原免疫组织化学染色阳性说明分泌的细胞外基质中含有软骨组织特有的Ⅱ型胶原，形成类似于正常软骨组织的组织工程化软骨。结论：在壳聚糖肢原三维立体诱导培养环境中，骨髓间充质干细胞能够很好的转化为软骨细胞，保持表型不变，继续增殖、代谢以及分泌细胞外基质，形成软骨组织。
BACKGROUND： Although bone marrow mesenchymal stem cells （MSCs） can be induced to chondrocytes in vitro, it is still difficult to culture them and prone to depolarization.
OBJECTIVE： To investigate the feasibility of constructing tissue engineering cartilage by induced MSCs seeded into chitosan/collagen three-dimensional porous scaffolds in vitro. DESIGN, TIME AND SETTING： A single sample observation based on cells was carded out in the laboratory of Cell and Genetics, Capital University of Medical Sciences （Beijing, China） from December 2006 to September 2007.
MATERIALS： Healthy sheep of one year old and either gender, weighing 20-30 kg and representing skeletal maturation, were adopted in this study.
METHODS： MSCs were isolated by density centrifugation with Percoll solution from bone marrow aspirated from sheep iliac crest. MSCs at the third passage were induced for two weeks and seeded into chitosardcollagen porous scaffold. While the cells cultured without the induction liquid were taken as controls.
MAIN OUTCOME MEASURES： The morphology, proliferation and function of MSCs were observed with contrast phase microscope and scanning electron microscope. Collagen Ⅱ immunohistochemistry stain was applied to identify the cartilages.
RESULTS： MSCs diffused into the pores of chitosan/collagen scaffold rapidly and attached well, manifestating well distribution and good hydrophilicity. MSCs of the experimental group grew to glomeration in the three-dimensional scaffolds, and cell aggregates were seen at the second weeks and grew out of the scaffolds at the fourth week. Histological stain showed that, MSCs secreted a great amount of extracellular matrix, which was positive for type Ⅱ collagen by immunohistochemistry stain and identical with the tissue engineering cartilage of normal tissues. CONCLUSION： Chitosan/collagen three-dimensional complex scaffold is very good for cartilage tissue engineering. MSCs can be induced into chondrocytes and seeded into the scaffold to construct tissue engineering cartilage successfully, meanwhile keeping phenotype of chondrocytes, proliferating and secreting extracellular matrix.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第23期4406-4409,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
