Ⅱ型胶原—透明质酸复合支架材料的构建及在软骨组织工程应用的初步研究
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摘要
目的
软骨组织自我修复能力差,如何修复因急性创伤或骨性关节炎等疾病导致的软骨缺损已成为骨科领域面临的一大难题。微小的损伤可能会引起软骨进一步的损伤和退化。组织工程技术成为软骨缺损修复新的手段,此技术的关键是建立由细胞和生物材料构成的三维空间复合体。本研究的目的是在体外构建一种模拟软骨组织成分的Ⅱ型胶原-透明质酸复合支架材料,探讨其作为新型软骨组织工程支架材料的可行性；并在体外分离培养骨髓间充质干细胞与Ⅱ型胶原-透明质酸复合支架材料复合培养体外构建组织工程软骨三维空间复合物,通过绿色荧光蛋白(GFP)标记示踪观察细胞在材料上的黏附、增殖和分化情况并检测软骨细胞特异性X型胶原的表达,初步评价骨髓间充质干细胞和Ⅱ型胶原-透明质酸复合支架材料构建的复合物作为软骨移植物的可行性。
方法
用本研究所以往的方法,提取Ⅱ型胶原溶液。利用电泳、紫外最大吸收峰的方法来测定提取胶原的纯度。经聚乙二醇将胶原原液浓缩,从而控制胶原冻干后的孔径大小。采用部分匀浆法和冷冻干燥法制备Ⅱ型胶原-透明质酸复合材料,并采用碳化二亚胺／N-羟基琥珀酰亚胺法对Ⅱ型胶原-透明质酸复合海绵进行交联改性,制备出Ⅱ型胶原-透明质酸复合支架材料(CⅡ-HA)。同时以常规制备的Ⅰ型胶原-透明质酸复合支架材料(CⅠ-HA)作为对照组。通过激光共聚焦显微镜、扫描电镜、红外光谱分析仪、拉力测试机等手段对材料的结构和理化性能进行分析。
采用全骨髓培养法分离培养骨髓间充质干细胞(Bone marrow stem cells, BMSCs)。通过成脂成骨诱导对分离培养的BMSCs进行鉴定。将BMSCs种植到CⅡ-HA材料上,XTT法测定细胞的增殖情况,并利用免疫荧光法检测细胞的X型胶原表达情况。采用携带有绿色荧光蛋白基因(Green fluorescent protein, GFP)的慢病毒对BMSCs进行基因转染,通过GFP对BMSCs进行标记,用荧光显微镜观察细胞在材料上的生长情况。
结果
Ⅱ型胶原的电泳图显示为α和β条带,没有杂蛋白的条带出现；Ⅱ型胶原在233.8nm处有紫外最大吸收峰。Ⅱ型胶原-透明质酸复合支架材料具有良好的三维孔洞结构,孔洞均匀且相互交通,平均孔径为92.63±32.51μm,符合软骨组织生长的合适孔径大小,而对照组CⅠ-HA材料则为202.16±76.75μm,两者具有统计学差异。CⅡ-HA材料孔隙率和平衡含水率为94.90±6.37%和97.13±0.32%。CⅡ-HA材料具有良好的力学性能,交联后力学性能达到79.67±11.7KPa,与常规制备的CⅠ-HA材料的17.38±1.8KPa相比,具有明显的统计学差异。此外,红外光谱图显示,Ⅱ型胶原与透明质酸复合交联后,形成新的小峰(1402cm-1),说明形成新的化学键。
骨髓间充质干细胞(BMSCs)分离培养后,通过成脂成骨诱导鉴定干细胞特性发现,成脂诱导21天后,有脂滴分布；成骨诱导21天后形成钙结节,细胞呈成骨细胞样结构。传代培养的BMSCs种植到CⅡ-HA支架材料上,X型胶原免疫荧光呈阳性。慢病毒能够成功将GFP基因转染到BMSCs中,转染后的BMSCs能够正常增殖。GFP示踪的BMSCs种植到CⅡ-HA支架材料后,细胞生长状况良好。
结论
制备的Ⅱ型胶原-透明质酸支架材料经交联后,具有良好的三维孔洞结构,材料孔径大小适合,孔洞分布均匀且相互交通。力学性能优异,孔隙率及平衡含水率均符合组织工程软骨支架材料的要求。故本研究制备的Ⅱ型胶原-透明质酸支架材料有望成为软骨组织工程的合适的仿生支架材料。
骨髓间充质干细胞能够在CⅡ-HA材料上良好的增殖,且部分骨髓间充质干细胞已向软骨细胞方向分化,GFP示踪的骨髓间充质干细胞能够在材料上生长状况良好。本研究制备的细胞-材料三维复合物基本符合组织工程化移植物要求的条件,能够进一步的进行体内的实验研究。
Objection
Articular cartilage defects repair is a difficult medical problem in orthopeadic area due to the limited capability for repair and self-regeneration. A minor injury of particular cartilage may lead to progressive damage and degeneration. Tissue engineering can potentially be used for cartilage repair. Establishment of a 3D system consisting of biomaterials and cells plays a vital role in tissue engineering. In this study, we prepared a collagen typeⅡ-hyaluronic acid copolymer porous scaffold(CⅡ-HA) which imitated the ingredients and ratios of natural cartilage matrix. Bone marrow mesenchymal stem cells(BMSCs) were obtained and cultured in vitro, and then seeded in the CⅡ-HA matrices. In order to investigate the feasibility of the BMSC/CⅡ-HA as tissue engineering cartilage graft, the viability and differentiation of BMSCs were detected. The expression of chondrocyte specific collagen type X in the cells were also detected.
Method
Type II collagen solutions were obtained by the method of our previously shown, and identified by SDS-PAGE electrophoresis and the maximum absorption wavelength. To control the pore size of materials, the initial collagen solutions were condensed with polyethylene glycol (PEG) at 4℃. The collagen typeⅡ-hyaluronic acid compound were prepared by partial homogenaization and then freeze-dried. CⅡ-HA compound were crosslinked with EDC/NHS. Collagen type I-hyaluronic acid matrics were prepared routinely as control group. The characteristics of the prepared scaffolds were analyzed by laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM), FT-IR analyzer, tensile tests and so on.
Bone marrow stem cells were obtained and purified by adhesion culture in vitro. The morphology of BMSCs was observed under the phase contrast microscope Some of BMSCs were induced into adipotic cells or osteoblast cells in special supplement medium. Afterwards, specific markers of differentiation of both adipocytic and osteogenic cells were detected. BMSCs were seeded into the CⅡ-HA matrices, the proliferation of BMSCs was monitored via XTT test, and the expression of collagen type X was detected by immunofluorescence. The GFP labeled BMSCs were planted into crosslinked CⅡ-HA, the cells viability was observed by LSCM.
Result
The result of SDS-PAGE electrophoresis shows typical a andβbands of the type II collagen, which indicated that the preparation collagen is pure and free of contaminating proteins. The peak of absorption maximum wavelength was 233.8nm as the same as the collagen should be. Collagen scaffolds had the interconnected porous structure, and the pore were well-distributed. The pore size of CⅡ-HA was 92.63±32.51μm which was more suitable for the growth of cartilage tissue, compared with the control group(CⅠ-HA) that was 202.16±76.75μm, and there was significant difference between two groups. Average porosity and water capacity of CⅡ-HA scaffolds reached to 93.39% and 97.78%, respectively. The mechanical properties of cross-linked CⅡ-HA increased significantly, reached to 79.67±11 .7KPa, While CⅠ-HA was 17.38±1.8KPa. Moreover, the infrared spectrum showed collagen type II and hyaluronic acid formed a new small peak (1402cm-1) after cross-linking that indicated there was a bond formation.
BMSCs were isolated and cultured in vitro. BMSCs were cultured in osteogenic or adipogenesis medium have been shown that cells we isolated could be differentiated down the osteogenic or adipogenic lineage, as evidenced by calcium deposition or droplet after 21 days. BMSCs were seeded in CⅡ-HA scaffold for 5 days, the expression of collagen type X was positive. The green fluorescent protein (GFP) gene can successfully be transducted into BMSCs by lentivirus. The BMSCs showed in normal condition after the transduction. GFP labeled BMSCs were seeded into CⅡ-HA scaffold, the cells were in normal condition and the expression of collagen type X in these cells was positive.
Conclusion
The CⅡ-HA has good three-dimensional pore structure. The pore size of CⅡ-HA matrices was suitable for cell growth, Besides, the pores were interconnective and well-distributed. CⅡ-HA scaffolds had suitable mechanical properties, porosity, and water capacity for cartilage tissue engineering. This type II collagen-hyaluronic acid scaffold suppose to be an ideal biomaterial for cartilage tissue engineering.
Our results indicated that CⅡ-HA scaffold may create an appropriate environment for the proliferation of BMSCs to chondrocyte differentiation. This CⅡ-HA/BMSCs graft may be suitable for implanting into full-thickness articular cartilage defect in vivo study.
软骨组织自我修复能力差,如何修复因急性创伤或骨性关节炎等疾病导致的软骨缺损已成为骨科领域面临的一大难题。微小的损伤可能会引起软骨进一步的损伤和退化。组织工程技术成为软骨缺损修复新的手段,此技术的关键是建立由细胞和生物材料构成的三维空间复合体。本研究的目的是在体外构建一种模拟软骨组织成分的Ⅱ型胶原-透明质酸复合支架材料,探讨其作为新型软骨组织工程支架材料的可行性；并在体外分离培养骨髓间充质干细胞与Ⅱ型胶原-透明质酸复合支架材料复合培养体外构建组织工程软骨三维空间复合物,通过绿色荧光蛋白(GFP)标记示踪观察细胞在材料上的黏附、增殖和分化情况并检测软骨细胞特异性X型胶原的表达,初步评价骨髓间充质干细胞和Ⅱ型胶原-透明质酸复合支架材料构建的复合物作为软骨移植物的可行性。
方法
用本研究所以往的方法,提取Ⅱ型胶原溶液。利用电泳、紫外最大吸收峰的方法来测定提取胶原的纯度。经聚乙二醇将胶原原液浓缩,从而控制胶原冻干后的孔径大小。采用部分匀浆法和冷冻干燥法制备Ⅱ型胶原-透明质酸复合材料,并采用碳化二亚胺／N-羟基琥珀酰亚胺法对Ⅱ型胶原-透明质酸复合海绵进行交联改性,制备出Ⅱ型胶原-透明质酸复合支架材料(CⅡ-HA)。同时以常规制备的Ⅰ型胶原-透明质酸复合支架材料(CⅠ-HA)作为对照组。通过激光共聚焦显微镜、扫描电镜、红外光谱分析仪、拉力测试机等手段对材料的结构和理化性能进行分析。
采用全骨髓培养法分离培养骨髓间充质干细胞(Bone marrow stem cells, BMSCs)。通过成脂成骨诱导对分离培养的BMSCs进行鉴定。将BMSCs种植到CⅡ-HA材料上,XTT法测定细胞的增殖情况,并利用免疫荧光法检测细胞的X型胶原表达情况。采用携带有绿色荧光蛋白基因(Green fluorescent protein, GFP)的慢病毒对BMSCs进行基因转染,通过GFP对BMSCs进行标记,用荧光显微镜观察细胞在材料上的生长情况。
结果
Ⅱ型胶原的电泳图显示为α和β条带,没有杂蛋白的条带出现；Ⅱ型胶原在233.8nm处有紫外最大吸收峰。Ⅱ型胶原-透明质酸复合支架材料具有良好的三维孔洞结构,孔洞均匀且相互交通,平均孔径为92.63±32.51μm,符合软骨组织生长的合适孔径大小,而对照组CⅠ-HA材料则为202.16±76.75μm,两者具有统计学差异。CⅡ-HA材料孔隙率和平衡含水率为94.90±6.37%和97.13±0.32%。CⅡ-HA材料具有良好的力学性能,交联后力学性能达到79.67±11.7KPa,与常规制备的CⅠ-HA材料的17.38±1.8KPa相比,具有明显的统计学差异。此外,红外光谱图显示,Ⅱ型胶原与透明质酸复合交联后,形成新的小峰(1402cm-1),说明形成新的化学键。
骨髓间充质干细胞(BMSCs)分离培养后,通过成脂成骨诱导鉴定干细胞特性发现,成脂诱导21天后,有脂滴分布；成骨诱导21天后形成钙结节,细胞呈成骨细胞样结构。传代培养的BMSCs种植到CⅡ-HA支架材料上,X型胶原免疫荧光呈阳性。慢病毒能够成功将GFP基因转染到BMSCs中,转染后的BMSCs能够正常增殖。GFP示踪的BMSCs种植到CⅡ-HA支架材料后,细胞生长状况良好。
结论
制备的Ⅱ型胶原-透明质酸支架材料经交联后,具有良好的三维孔洞结构,材料孔径大小适合,孔洞分布均匀且相互交通。力学性能优异,孔隙率及平衡含水率均符合组织工程软骨支架材料的要求。故本研究制备的Ⅱ型胶原-透明质酸支架材料有望成为软骨组织工程的合适的仿生支架材料。
骨髓间充质干细胞能够在CⅡ-HA材料上良好的增殖,且部分骨髓间充质干细胞已向软骨细胞方向分化,GFP示踪的骨髓间充质干细胞能够在材料上生长状况良好。本研究制备的细胞-材料三维复合物基本符合组织工程化移植物要求的条件,能够进一步的进行体内的实验研究。
Objection
Articular cartilage defects repair is a difficult medical problem in orthopeadic area due to the limited capability for repair and self-regeneration. A minor injury of particular cartilage may lead to progressive damage and degeneration. Tissue engineering can potentially be used for cartilage repair. Establishment of a 3D system consisting of biomaterials and cells plays a vital role in tissue engineering. In this study, we prepared a collagen typeⅡ-hyaluronic acid copolymer porous scaffold(CⅡ-HA) which imitated the ingredients and ratios of natural cartilage matrix. Bone marrow mesenchymal stem cells(BMSCs) were obtained and cultured in vitro, and then seeded in the CⅡ-HA matrices. In order to investigate the feasibility of the BMSC/CⅡ-HA as tissue engineering cartilage graft, the viability and differentiation of BMSCs were detected. The expression of chondrocyte specific collagen type X in the cells were also detected.
Method
Type II collagen solutions were obtained by the method of our previously shown, and identified by SDS-PAGE electrophoresis and the maximum absorption wavelength. To control the pore size of materials, the initial collagen solutions were condensed with polyethylene glycol (PEG) at 4℃. The collagen typeⅡ-hyaluronic acid compound were prepared by partial homogenaization and then freeze-dried. CⅡ-HA compound were crosslinked with EDC/NHS. Collagen type I-hyaluronic acid matrics were prepared routinely as control group. The characteristics of the prepared scaffolds were analyzed by laser scanning confocal microscopy (LSCM), scanning electron microscopy (SEM), FT-IR analyzer, tensile tests and so on.
Bone marrow stem cells were obtained and purified by adhesion culture in vitro. The morphology of BMSCs was observed under the phase contrast microscope Some of BMSCs were induced into adipotic cells or osteoblast cells in special supplement medium. Afterwards, specific markers of differentiation of both adipocytic and osteogenic cells were detected. BMSCs were seeded into the CⅡ-HA matrices, the proliferation of BMSCs was monitored via XTT test, and the expression of collagen type X was detected by immunofluorescence. The GFP labeled BMSCs were planted into crosslinked CⅡ-HA, the cells viability was observed by LSCM.
Result
The result of SDS-PAGE electrophoresis shows typical a andβbands of the type II collagen, which indicated that the preparation collagen is pure and free of contaminating proteins. The peak of absorption maximum wavelength was 233.8nm as the same as the collagen should be. Collagen scaffolds had the interconnected porous structure, and the pore were well-distributed. The pore size of CⅡ-HA was 92.63±32.51μm which was more suitable for the growth of cartilage tissue, compared with the control group(CⅠ-HA) that was 202.16±76.75μm, and there was significant difference between two groups. Average porosity and water capacity of CⅡ-HA scaffolds reached to 93.39% and 97.78%, respectively. The mechanical properties of cross-linked CⅡ-HA increased significantly, reached to 79.67±11 .7KPa, While CⅠ-HA was 17.38±1.8KPa. Moreover, the infrared spectrum showed collagen type II and hyaluronic acid formed a new small peak (1402cm-1) after cross-linking that indicated there was a bond formation.
BMSCs were isolated and cultured in vitro. BMSCs were cultured in osteogenic or adipogenesis medium have been shown that cells we isolated could be differentiated down the osteogenic or adipogenic lineage, as evidenced by calcium deposition or droplet after 21 days. BMSCs were seeded in CⅡ-HA scaffold for 5 days, the expression of collagen type X was positive. The green fluorescent protein (GFP) gene can successfully be transducted into BMSCs by lentivirus. The BMSCs showed in normal condition after the transduction. GFP labeled BMSCs were seeded into CⅡ-HA scaffold, the cells were in normal condition and the expression of collagen type X in these cells was positive.
Conclusion
The CⅡ-HA has good three-dimensional pore structure. The pore size of CⅡ-HA matrices was suitable for cell growth, Besides, the pores were interconnective and well-distributed. CⅡ-HA scaffolds had suitable mechanical properties, porosity, and water capacity for cartilage tissue engineering. This type II collagen-hyaluronic acid scaffold suppose to be an ideal biomaterial for cartilage tissue engineering.
Our results indicated that CⅡ-HA scaffold may create an appropriate environment for the proliferation of BMSCs to chondrocyte differentiation. This CⅡ-HA/BMSCs graft may be suitable for implanting into full-thickness articular cartilage defect in vivo study.
引文
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