脂肪间充质干细胞复合壳聚糖-胶原-硫酸软骨素多孔支架体外成软骨能力的实验研究

The study of in vitro chondrogenesis of adipose tissue-derived stromal cells loading on chitosan-collagen-chondroitin sulfate scaffold

目的制备壳聚糖．胶原．硫酸软骨素三维支架，并与大鼠脂肪间充质干细胞复合培养，探讨其作为软骨组织工程支架的可行性。方法冷冻干燥法制备壳聚糖-胶原-硫酸软骨素复合多孔海绵支架材料，采用体积法和称重法测定支架的孔隙率和吸水性，扫描电镜观察支架材料的形态结构。分离培养大鼠脂肪间充质干细胞，流式细胞学检测大鼠脂肪间充质干细胞的表面标志CD29、CD34、CD44、CD45，将传至3代的细胞，以2×10^6／ml的密度接种于自制的壳聚糖-胶原-硫酸软骨素三维支架上。实验组为含TGF-β1的培养基，对照组无TGF-β1培养3周后，通过免疫组织化学，RT-PCR及westenblot方法对诱导后的细胞进行鉴定。结果制备的壳聚糖-胶原-硫酸软骨素三维支架具有合适的三维多孔结构，孔隙率为（92．23±1．68）％，孔径为100—130μm。复合培养3周后，Ⅱ型胶原免疫组织化学染色成阳性，RT-PCR结果表明有蛋白聚糖和Ⅱ型胶原mRNA的表达，westenblot检测出Ⅱ型胶原蛋白的表达。结论壳聚糖．胶原．硫酸软骨素复合支架材料可为脂肪间充质干细胞生长分化及组织形成提供一个良好的环境，在软骨组织工程的支架材料领域有较广泛的应用前景。

Objective The aim of the study was to produce a novel porous chitosan-collagen-chondroitin sulfate scaffold loading with rat adipose tissue-derived stromal ceils and to evaluate this tricopolymer as a new biomimetic biodegradable polymer scaffold for application of the articular cartilage tissue engineering. Methods The porous chitosan-collagen-chondroitin sulfate tricopolymer scaffold were fabricated by the freeze-drying technique, The porosity and water content were valuated by measuring the mass and volume of the scaffold. The microstructure of the scaffold was observed via scanning electron microscopy. The rat adipose tissue-derived stromal cells were isolated and cultured with standard culture medium. The CD29, CD34 and CD44 CD45 expressions of the third passage cells were detected by flow cytometry. ADSCs from the three passage were seeded into the porous chitosancollagen-chondroitin sulfate tricopolymer scaffold with 2 × 10^6/ml and then they were cultured in the condition respectively in the TGF-β1 group and the control group for three weeks. The differentiation of the ADSCs into the chondrocytes was identified by the histological, immunohistochemical staining RT-PCR and western blot methods Results The porous structure of the scaffold facilitated the penetration and attachment of the seeded cells. The porosity was 92.23 ± 1.68% and the pore size was 100 - 130 μm. Immunohistochemical staining of the neocartilage with antl-type Ⅱ collagen showed the presence of type Ⅱ collagen in the ECM of tissue engineered cartilage. RT-PCR showed that Col2al and Agc mRNA were expressed in theexperimental group, but not in the control group. Western-blot detection confirmed positive collagenⅡ expression in experimental group. Conclusion These results show that the chitosan-collagen-chondroitin sulfate copolymer matrices can provide an appropriateenvironment for the generation of cartilage-like tissues and have a potential application in the cartilage tissue engineering scaffold field.