自体骨髓间充质干细胞外基质（ECM）支架在软骨组织工程中的应用研究

发布时间：2018-09-04 09:27

【摘要】：第一部分aBMSC-dECM支架的制作及其性能研究目的探讨通过收集自体骨髓间充质干细胞外基质(autologous bone marrow mesenchymal stem cell-derived extracellular matrix, aBMSC-dECM)制备三维多孔状支架的可行性,并对支架性能进行分析。方法分离、培养2周龄新西兰大白兔骨髓间充质干细胞,原代培养4周后收集其分泌的aBMSC-dECM膜,采用交联和冻干技术制备三维多孔状支架,对支架行HE染色、扫描电镜、抗压强度测试。结果aBMSC-dECM支架呈三维多孔状海绵样结构,孔隙分布均匀且有较好的连通性,孔径大小为304.4±108.2μm；孔隙率为93.3%±4.5%；密度为28.7±1.4mg/ml;抗压强度为6.8±1.5KPa。结论通过收集aBMSC-dECM膜可用于制备组织工程支架,该支架材料具有理想的三维空间结构特征及力学特性,有望成为一种具有较高应用价值的组织工程支架。第二部分aBMSC-dECM支架在软骨再生中的应用目的探讨aBMSC-dECM支架在软骨再生中的应用可行性。方法分离、培养2周龄新西兰大白兔骨髓间充质干细胞,原代培养4周后收集其分泌的aBMSC-dECM膜,采用交联和冻干技术制备三维多孔状支架。分离培养自体软骨细胞并植入aBMSC-dECM支架,将细胞-支架复合物分别行体外和裸鼠皮下种植培养制备组织工程软骨,对照组使用atelocollagen支架。于种植后48h对细胞-支架复合物行Live-Dead染色。于体外培养1周、2周和4周后对组织工程软骨行组织学分析、Real-Time PCR和Western blotting检测。于体内植入1周、2周和3周后对组织工程软骨行大体观察、体积测量、组织学分析、生化成分分析及抗压强度测量。结果Live-Dead染色显示aBMSC-dECM支架内的软骨细胞维持较好的生物活性；与对照组相比,体外培养4周后aBMSC-dECM支架组组织工程软骨内基质含量更多、分布更均匀,特定基因和蛋白表达更高；体内植入3周,aBMSC-dECM支架组组织工程软骨的体积增长和均质性均优于对照组,具有更高软骨基质成分和抗压强度。结论aBMSC-dECM支架有利于维持细胞活性和生物学功能,能促进组织工程软骨的形成,可成为一种具有较高应用价值的组织工程软骨支架材料。第三部分aBMSC-dECM支架对自体骨髓间充质干细胞成软骨分化的作用目的探讨aBMSC-dECM支架对自体骨髓间充质干细胞成软骨分化的作用。方法分离、培养2周龄新西兰大白兔骨髓间充质干细胞,原代培养4周后收集其分泌的aBMSC-dECM膜,采用交联和冻干技术制备三维多孔状支架。将自体骨髓间充质干细胞植入aBMSC-dECM支架,细胞-支架复合物分别行体外和裸鼠皮下种植培养,对照组使用atelocollagen支架。体外培养随机分成四组：①C+组：BMSCs/atelocollagen支架复合物置于含有成软骨分化诱导因子TGF-β3的培养体系；②E+组：BMSCs/aBMSC-dECM支架复合物置于含有TGF-β3的培养体系；③C-组：BMSCs/atelocollagen支架复合物置于不含有TGF-β3的培养体系；④E-组：BMSCs/aBMSC-dECM支架复合物置于不含有TGF-β3的培养体系；分别于体外培养3d、10d和21d对各组再生组织行组织学分析、Real-Time PCR和生化成分分析。裸鼠皮下种植前,先将aBMSC-dECM支架组和atelocollagen支架组细胞-支架复合物置于不含有TGF-β3的培养体系体外培养1w；分别于体内植入后1w、2w和3w对再生组织行大体观察、体积测量、生软骨分化检测和成骨分化检测。结果体外培养过程中,与C+组和C-组相比,E+和E-组再生组织中蛋白聚糖、Ⅱ型胶原表达逐渐增多,COL2A1、ACAN及SOX9基因表达量持续升高,再生组织的总DNA含量维持在一个较高水平,GAG含量和GAG/DNA比值持续升高。体内培养初期,aBMSC-dECM支架组再生组织中蛋白聚糖、Ⅱ型胶原表达呈强阳性,但随时间延长表达逐渐减弱,成骨分化逐渐增强；而atelocollagen支架组在体外培养过程中再生组织体积逐渐缩小,基质中未见蛋白聚糖、Ⅱ型胶原表达,至后期组织大部分区域被新生骨组织替代结论aBMSC-dECM支架可促进和维持自体骨髓间充质干细胞成软骨分化,有利于组织工程软骨形成。第四部分aBMSC-dECM支架结合骨髓刺激术修复关节软骨缺损的实验研究目的探讨结合aBMSC-dECM支架植入,对骨髓刺激术(BMS)修复关节软骨缺损的作用。方法选取健康成年新西兰大白兔24只,分离、培养骨髓间充质干细胞,原代培养4w后收集其分泌的aBMSC-dECM膜,采用交联和冻干技术制备三维多孔状支架。于双侧膝关节股骨滑车部位制作全层软骨缺损模型,右侧膝关节(BMS组)行骨髓刺激术修复软骨缺损,左侧膝关节(BMS+Scaffold组)在完成骨髓刺激术后,在缺损处植入aBMSC-dECM支架。分别于术后6w,12w对两组再生软骨行大体观察、组织学检查及评分、生化成分分析。结果术后12w,与BMS组相比,BMS+Scaffold组再生软骨覆盖更完整,与周边正常软骨整合较好；BMS+Scaffold组再生软骨基质中蛋白聚糖、Ⅱ型胶原含量和分布接近正常软骨,以圆形、椭圆形透明软骨样细胞为主,纤维走行垂直于关节面,ICRS评分显著高于BMS组；BMS+Scaffold组再生软骨基质中DNA及GAG含量接近正常软骨。结论结合aBMSC-dECM支架植入可有效提高骨髓刺激术修复软骨缺损疗效,具有较高的临床应用价值。
[Abstract]:The first part is about the fabrication and performance of aBMSC-dECM scaffold.
Objective To investigate the feasibility of fabricating three-dimensional porous scaffolds by collecting autologous bone marrow mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM) and analyze the performance of the scaffolds.
Methods Bone marrow mesenchymal stem cells (BMSCs) from 2-week-old New Zealand white rabbits were isolated and cultured. The aBMSC-dECM membrane secreted by BMSCs was collected after 4 weeks of primary culture. Three-dimensional porous scaffolds were prepared by cross-linking and freeze-drying techniques. The scaffolds were stained with HE, scanning electron microscopy and compressive strength was tested.
Results The aBMSC-dECM scaffold was a three-dimensional porous sponge-like structure with uniform pore size of 304.4 (+ 108.2) micron, porosity of 93.3 (+ 4.5%), density of 28.7 (+ 1.4 mg/ml) and compressive strength of 6.8 (+ 1.5 KPa).
Conclusion The aBMSC-dECM membrane can be used to fabricate tissue engineering scaffolds. The scaffolds have ideal three-dimensional structure and mechanical properties, and are expected to be a tissue engineering scaffold with high application value.
The second part is the application of aBMSC-dECM scaffold in cartilage regeneration.
Objective to investigate the feasibility of aBMSC-dECM scaffold in cartilage regeneration.
Methods Bone marrow mesenchymal stem cells (BMSCs) from 2-week-old New Zealand white rabbits were isolated and cultured. The aBMSC-dECM membrane secreted by BMSCs was collected after 4 weeks of primary culture. Three-dimensional porous scaffolds were prepared by cross-linking and freeze-drying techniques. Tissue-engineered cartilage was prepared and the control group was treated with atelocollagen scaffold.Cell-scaffold complex was stained with Live-Dead 48 hours after implantation.Tissue-engineered cartilage was cultured in vitro for 1 week,2 weeks and 4 weeks.Histological analysis,Real-Time PCR and Western blotting were used to detect tissue-engineered cartilage.Gross appearance of tissue-engineered cartilage was observed after implantation for 1 week,2 weeks and 3 weeks. Observation, volume measurement, histological analysis, biochemical composition analysis and compressive strength measurement.
Results Live-Dead staining showed that the chondrocytes in the aBMSC-dECM scaffold maintained good biological activity; compared with the control group, the aBMSC-dECM scaffold group had more tissue-engineered cartilage matrix content, more uniform distribution, higher specific gene and protein expression after 4 weeks of culture in vitro; after 3 weeks of implantation, the aBMSC-dECM scaffold group had higher tissue-engineered cartilage content, more uniform distribution, and higher specific gene and protein expression. The volume growth and homogeneity were better than those of the control group, with higher cartilage matrix composition and compressive strength.
Conclusion The aBMSC-dECM scaffold can maintain cell viability and biological function, promote the formation of tissue-engineered cartilage, and can be used as a tissue-engineered cartilage scaffold material with high application value.
The third part is the effect of aBMSC-dECM scaffold on chondrogenic differentiation of autologous bone marrow mesenchymal stem cells.
Objective to investigate the effect of aBMSC-dECM scaffold on chondrogenic differentiation of autologous bone marrow mesenchymal stem cells.
Methods Bone marrow mesenchymal stem cells (BMSCs) from 2-week-old New Zealand white rabbits were isolated and cultured. The aBMSC-dECM membrane secreted by BMSCs was collected after 4 weeks of primary culture. Three-dimensional porous scaffolds were prepared by cross-linking and freeze-drying techniques. The control group was cultured with atelocollagen scaffolds and randomly divided into four groups: (1) C + group: BMSCs / atelocollagen scaffold complex was placed in the culture system containing TGF - beta 3; E + group: BMSCs / aBMSC - dECM scaffold complex was placed in the culture system containing TGF - beta 3; and (3) C - group: BMSCs / atelocollagen scaffold complex; Group E: BMSCs/aBMSC-dECM scaffold complex was placed in a culture system without TGF-beta 3. Histological analysis, Real-Time PCR and biochemical analysis of regenerated tissues were performed on 3, 10 and 21 days after culture in vitro, respectively. Gross observation, volume measurement, chondrogenic differentiation and osteogenic differentiation were performed at 1, 2 and 3 weeks after implantation.
Results The expression of proteoglycan and type II collagen in E + and E - regenerated tissues increased gradually, the expression of COL2A1, ACAN and SOX9 genes increased continuously, the total DNA content of regenerated tissues maintained at a higher level, the GAG content and the ratio of GAG to DNA increased continuously. The expression of proteoglycan and type II collagen was strongly positive in regenerated tissues, but gradually weakened with time, and the osteogenic differentiation was gradually strengthened; while the volume of regenerated tissues in atelocollagen scaffold group was gradually reduced, no proteoglycan was found in matrix, and the expression of type II collagen was found in most areas of the late tissue, which was then regenerated by bone tissue. Replace
Conclusion The aBMSC-dECM scaffold can promote and maintain the chondrogenic differentiation of autologous bone marrow mesenchymal stem cells and is beneficial to the formation of tissue-engineered cartilage.
The fourth part is aBMSC-dECM scaffold combined with bone marrow stimulation to repair articular cartilage defects.
Objective to investigate the effect of bone marrow stimulation (BMS) combined with aBMSC-dECM stent implantation on repairing articular cartilage defects.
Methods Twenty-four healthy adult New Zealand white rabbits were selected to isolate and culture bone marrow mesenchymal stem cells. The aBMSC-dECM membrane secreted by bone marrow mesenchymal stem cells was collected after primary culture for 4 weeks. Three-dimensional porous scaffolds were prepared by cross-linking and freeze-drying techniques. After bone marrow stimulation, aBMSC-dECM scaffolds were implanted into the left knee joint (BMS+Scaffold group). The regenerated cartilage of the two groups were observed, histologically examined and scored, and biochemical compositions were analyzed 6 and 12 weeks after operation.
Results At 12 weeks after operation, the regenerated cartilage in BMS+Scaffold group was more complete and integrated with the surrounding normal cartilage than that in BMS+Scaffold group, and the contents and distribution of proteoglycan and collagen type II in the regenerated cartilage matrix in BMS+Scaffold group were close to that of normal cartilage, mainly round and oval hyaline chondrocytes with the fibers running perpendicular to the articular surface. The levels of DNA and GAG in BMS+Scaffold group were higher than those in BMS group.
Conclusion The combination of aBMSC-dECM stent implantation can effectively improve the effect of bone marrow stimulation in repairing cartilage defects and has high clinical value.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

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