In vivo 3 dimensional cartilage tissue engineering by adipose derived stem cells

由于人体脂肪干细胞的获取相对困难，本项目将脂肪干细胞的种属从人改为GFP转基因小鼠。因此，我们首次从GFP转基因小鼠中分离到了类似的人脂肪干细胞（adipose derived stem cells，ASC），并将其在对照培养基中增殖后，在诱导培养基中采用微团培养技术诱导ASC向软骨细胞分化2周。随后，ASCs成功分化为直径为1至2 mm的球形软骨。该培养技术也适用于在无支架条件下再生直径为8 ~ 10 mm的较大尺寸软骨。当这些结构在体内植入到血管丰富的空间，如筋膜下或肌内区域，它们被发现在宏观上和组织学上保持形状和大小。接下来，我们研究了人工多肽（PuraMatrix）是否<TM>可以作为合适的支架用于使用ASCs的软骨再生。将<TM>含有3 × 10^6个ASC的1% PuraMatrix^在软骨形成培养基中孵育，并观察长达8周。孵育2周后，这些混合物显示出软骨形成基质的产生，这通过阿尔新蓝染色证实。此外，在孵育8周后，表型仍然相同。

The species of adipose derived stem cells was changed from human to GFP transgenic mice in this project because human specimen was relatively difficult to obtain. Accordingly, we firstly identified that similar population of human adipose derived stem cells (ASCs) can be isolated from GFP transgenic mice.After the proliferation of ASC in control medium, they were induced into chondrogenic differentiation in the induction medium using micromass culture technique for 2 week. Subsequently, ASCs were successfully differentiated into spheroid-shape cartilage of 1 to 2mm in diameter. This culture technique was also applied to regenerate relatively big size of cartilage which is 8 to 10mm in diameter without any scaffolds. When these structures were implanted in vivo into the vascular-rich space such as subfascia or intramuscular region, they were found to keep in shape and size both macroscopically and histologically.Next, we investigated whether artificial polypeptide (PuraMatrix^<TM>) could be suitable scaffold for chondrogenic regeneration using ASCs. 1% PuraMatrix^<TM> with 3x10^6 ASCs were incubated in the chondrogenic media and observed up to 8 weeks. After 2 weeks of incubation, these mixtures exhibited the production of the chondrogenic matrices which was confirmed by Alcian blue staining. In addition, the phenotype was still identical after 8 weeks of incubation.