摘要
目的探讨制备兔耳廓天然软骨细胞外基质材料的新方法,制备适用于组织工程应用的天然生物支架材料。方法①采用多步骤去污剂—酶处理技术抽提兔耳廓软骨细胞制备天然软骨细胞外基质(Nature Extracellular Matrix,NECM),对NECM行HE染色、澳新蓝染色、维多利亚蓝染色、VanGieson染色并进行组织学及扫描电镜观察;②以灰家兔NECM为支架材料和新西兰大白兔耳廓软骨细胞为种子细胞进行体外培养,倒置显微镜下观察其亲水性和对细胞的吸附力,并通过四氮唑盐比色法(MTTAssay)观察NECM对软骨细胞的增殖作用。结果通过HE染色、蛋白多糖、胶原及弹力纤维特染、扫描电镜观察分析证实经过多步骤去污剂—酶法抽提软骨细胞制备的天然生物支架材料里,不含细胞及细胞器成分,仅含有不溶解的胶原、弹性蛋白和蛋白多糖,亦即NECM;体外培养实验发现,兔耳廓软骨细胞易进入脱细胞异体软骨基质结构裸露的空穴,且生长旺盛,组织学检查证实兔耳廓软骨NECM上裸露的骨陷窝已部分被软骨细胞占据。MTTAssay检测证实NECM对软骨细胞增殖有促进作用。结论本实验制备的NECM脱细胞彻底、细胞外基质成分保留较完整,并具有促进细胞粘附、增殖和分化生长的作用,是一种较为理想的天然生物支架材料。
Objective To develop a novel manufacturing method of nature cartilaginous extracellular matrix scaffold materials using rabbit auricle cartilage and prepare a kind of scaffolds for tissue engineering. Methods ① Apply multistep detergent and enzymatic extraction process to extract cells of auricle cartilage of rabbit to get nature extracellular matrix （NECM） and the obtained products were stained with HE, victoria blue, alcian blue, and Van Gieson, then observed by optical microscope. Unstained NECM and cartilages of rabbits was observed by scanning electron microscope. ② With NECM of grey rabbit as scaffold, we cultivated the auricle cartilage cells of New Zealand white rabbits in vitro. The hydrophilia and absorption of the cells were observed with inverted microscope. The cell proliferation on NECM was detected by MTT assay. Result By using HE staining and proteoglycan-, collagen-, and elastic fibrin-specific staining and scanning electron microscopy, we demonstrated that the multistep detergent and enzymatic extraction process could extract cartilage cells, leaving behind a nature extracellular matrix （NECM） scaffold, which was composed of indissoluble collagen, elastic fibrin and proteoglycan. The cultured auricle chondrocytes of rabbits in vitro were easy to enter cartilage lacuna of allogenic NECM and grew well. The evidence of histology confirmed that the cartilage lacunas of allogenic NECM of rabbit auricle cartilage were partly occupied by cartilage cells. MTr assay results showed NECM could promote the proliferation of cartilage cells. Conclusion A complete cell-free NECM can be manufactured by above technique, keeping intact extracellular matrix with the potential for promoting cell attachment, proliferation, growth and differentiation. Altogether, the results suggest that it is an ideal nature extracellular matrix scaffold materials.
出处
《中华耳科学杂志》
CSCD
2005年第3期194-199,共6页
Chinese Journal of Otology
基金
国家杰出青年科学基金(39925035)
国家自然科学基金(39670781
30070809)资助
