脱细胞软骨基质三维支架的制备及特性研究

Preparation of a three-dimentional acellular cartilage matrix scaffold and its characteristics

[目的]探索脱细胞软骨基质三维多孔支架的制备及其应用于关节软骨组织工程的可行性。[方法]新鲜牛膝关节软骨粉碎后,梯度离心法获取软骨微粒,采用改进的Courtman改良法处理细胞后,再冷冻干燥,制备脱细胞软骨基质三维多孔支架。然后,采用京尼平对三维支架进行交联,再次冷冻干燥后,对支架材料进行大体、组织学染色及扫描电镜观察,分别测定支架的孔隙率、溶胀率、降解率。最后,分离培养兔骨髓基质细胞(BMSCs),采用MTT法检测BMSCs在支架材料上的生长、增殖情况,以柱形图表示。[结果]大体观察显示支架呈疏松多孔状,京尼平交联后整体呈深蓝色。组织学观察显示支架材料无软骨细胞碎片残留,HE染色、甲苯胺兰染色观察均未见软骨细胞残留。测量示支架孔隙率为90%,溶胀率为(1314±337)%,降解率2周为(13.69±7.3)%,4周为(25.99±8.9)%。MTT法显示细胞在支架上生长良好,与对照组DMEM培养液吸光度值比较,差异无统计学意义(P>0.05),提示支架无细胞毒性。扫描电镜显示支架内孔洞较明显,BMSCs通过细胞突起黏附于支架表面,黏附良好,能较好地在其上生长。[结论]经改进的Courtman改良法处理的软骨基质三维多孔支架脱细胞更彻底,保留了软骨的天然细胞外基质成分,天然交联剂京尼平交联后支架的细胞相容性好,抗降解性得到了提高,是一种适用于软骨组织工程的良好载体。

[Objective]To prepare a cartilage acellular matrix scaffold and to explore its feasibility in cartilage tissue engineering. [Methods]Microparticles about 100 μm^154 μm were prepared after calf cartilage physically shattered and experienced gradient centrifugation,and then treated by a modified Courtman＇s four-step method which was improved to produce acellular cartilage matrix.After this treatment the microparticles were made into 3% suspension which was placed into moulds.With the freeze-drying method,3-D cartilage acellular matrix （CACM） was prepared.The scaffolds were cross-linked by a neotype crosslinking agent genepin for 48h,and then placed into glycine solution server times for removing redundant genepin.The freeze-drying method was used to prepare CACM.The scaffolds were investigated by gross observation,histological staining （haematoxylin-eosin,toluidine blue） ,scanning electron microscope （SEM） observation and porosity measurement,water absorption rate and degradation rate analysis.After being cultivated for ten days,bone marrow stranal cells （BMSCs） of rabbit were seeded into the scaffold.MTT test and SEM were done to assess the growth and proliferation of BMSCs.[Results]Gross observation showed the scaffolds had a loosely porous and dark blue appearance after being cross-linked by genepin.The histological staining （haematoxylin-eosin,toluidine blue staining） showed that there were no chondrocyte fragments in the scaffold.The CACM scaffold had 90% porosity,（1314±337） % water absorption rate,and （13.69±7.3）% or （25.99±8.9） % degradation rate at 2 or 4 weeks.MTT test showed that BMSCs grew well in the 3-D CACM scaffolds of logarithmic trend,supporting that the scaffolds had no cytotoxic effect on BMSCs.SEM micrographs indicated that the scaffolds were porous and the cells covered the scaffolds firmly with cell processes.[Conclusion]The improved Courtman＇s four-step method makes a more thoroughly acellular scaffold.The 3-D CACM scaffold retains most of extracelluar matrix.After being cross-linked by genepin,the 3-D CACM scaffold has good biocompatibility and degradation rate of the scaffolds is decreased,which makes it a suitable carrier for cartilage tissue engineering.