摘要
目的探讨软骨基质来源定向结构支架复合成软骨诱导BMSCs体内构建组织工程软骨的可行性。方法利用温度梯度热诱导相分离技术制备牛膝关节软骨基质来源定向结构支架并检测其力学性能;同时采用传统冷冻干燥方法制备普通多孔状非定向结构支架。分离、培养2月龄新西兰大白兔BMSCs并成软骨诱导分化,按5×105个/块分别接种于两种支架,扫描电镜和MTT法观测细胞在支架上增殖情况。体外培养1周后,将两种细胞-支架复合物植入4周龄裸鼠皮下,分别在2、4周后取材对构建的组织工程软骨进行组织学、生物化学及生物力学检测。结果定向结构支架压缩弹性模量显著高于非定向结构支架(t=201.099,P=0.000),体外培养3～9 d时定向结构支架上细胞增殖亦高于非定向结构支架(P<0.05)。裸鼠体内培养4周后构建的定向及非定向组织工程软骨番红O染色、甲苯胺蓝染色及Ⅱ型胶原免疫组织化学染色均呈阳性,Ⅰ型胶原免疫组织化学染色呈阴性;在定向组织工程软骨中可见大量规则平行排列的粗大胶原纤维。培养2、4周,两组新生软骨总DNA、总糖胺聚糖及总胶原含量比较,差异均无统计学意义(P>0.05);定向组织工程软骨压缩弹性模量均高于非定向组织工程软骨(P<0.05);但两组上述指标均显著低于正常膝关节软骨(P<0.05)。结论软骨基质来源定向结构支架复合BMSCs后在体内成功构建定向组织工程软骨,并有效提高了新生软骨的机械力学性能,可进一步用于软骨组织工程研究。
Objective To investigate the feasibility of fabricating an oriented scaffold combined with chondrogenic- induced bone marrow mesenchymal stem cells （BMSCs） for enhancement of the biomechanical property of tissue engineered cartilage in vivo. Methods Temperature gradient-guided thermal-induced phase separation was used to fabricate an oriented cartilage extracellular matrix-derived scaffold composed of microtubules arranged in parallel in vertical section. No-oriented scaffold was fabricated by simple freeze-drying. Mechanical property of oriented and non-oriented scaffold was determined by measurement of compressive modulus. Oriented and non-oriented scaffolds were seeded with chondrogenic-induced BMSCs, which were obtained from the New Zealand white rabbits. Proliferation, morphological characteristics, and the distribution of the cells on the scaffolds were analyzed by MTT assay and scanning electron microscope. Then cell-scaffold composites were implanted subcutaneously in the dorsa of nude mice. At 2 and 4 weeks after implantation, the samples were harvested for evaluating biochemical, histological, and biomechanical properties. Results The compressive modulus of oriented scaffold was significantly higher than that of non-oriented scaffold （t=-201.099, P=O.O00）. The cell proliferation on the oriented scaffold was significantly higher than that on the non-oriented scaffold from 3 to 9 days （P 〈 0.05）. At 4 weeks, collagen type II immunohistochemical staining, safranin O staining, and toluidine blue staining showed positive results in all samples, but negative for collagen type I. There were numerous parallel giant bundles of densely packed collagen fibers with chondrocyte- like cells on the oriented-structure constructs. Total DNA, glycosaminoglycan （GAG）, and collagen contents increased withtime, and no significant difference was found between 2 groups （P 〉 0.05）. The compressive modulus of the oriented tissue engineered cartilage was significantly higher than that of the non-oriented tissue engineered cartilage at 2 and 4 weeks after implantation （P 〈 0.05）. Total DNA, GAG, collagen contents, and compressive modulus in the 2 tissue engineered cartilages were significantly lower than those in normal cartilage （P 〈 0.05）. Conclusion Oriented extracellular matrix-derived scaffold can enhance the biomechanical property of tissue engineered Cartilage and thus it represents a promising approach to cartilage tissue engineering.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2013年第5期513-519,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家高技术研究发展计划(863)资助项目(2012AA020502)
国家自然科学基金资助项目(31070860)~~
关键词
软骨组织工程
定向结构支架
细胞外基质
BMSCS
生物力学
Cartilage tissue engineering Oriented scaffold Extracellular matrix Bone marrowmesenchymal stem cells Biomechanics
