脱细胞软骨基质三维支架材料修复兔关节骨软骨缺损的实验研究
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摘要
关节软骨缺乏直接的血液供应、淋巴循环和神经支配,代谢能力低,自身难以修复,而目前临床常用的修复方法均有一定局限性,脱细胞基质以其良好的生物相容性、细胞吸附性及良好的亲水性,成为组织工程支架选择的又一亮点。与人工材料相比,天然材料的优点是生物相容性好、结构成分与ECM相似,适合种子细胞的生长、增殖及分化,与其他天然仿生材料相比,由于软骨细胞ECM生化成分与结构复杂,脱细胞基质支架材料可以更加接近软骨组织的复杂天然结构及生物学特性。本研究通过制备脱细胞软骨基质三维支架修复兔关节骨软骨缺损。
第一部分脱细胞软骨基质三维支架的制备及特性研究
目的:探索脱细胞软骨基质三维多孔支架的制备及其特性研究。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。结果:大体观察示支架呈疏松多孔状,京尼平交联后整体呈深蓝色。组织学观察示支架材料无软骨细胞碎片残留,苏木精-伊红(HE)染色,甲苯胺兰染色观察均未见软骨细胞残留。扫描电镜显示支架内孔洞较明显。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)%。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材料机械强度和抗降解性得到了提高。
第二部分脱细胞软骨基质三维支架的生物相容性研究
目的:运用兔骨髓基质细胞(BMSCs)接种于支架上评价支架的生物相容性。方法:原代培养兔骨髓基质细胞(BMSCs),传至第二代后以2.0×106个/ml的细胞浓度接种于支架上,采用MTT法检测BMSCs在支架材料上后1、3、5、7、9d的生长、增殖情况,并描绘出柱形图。1w后扫描电镜观察兔骨髓基质细胞于支架上的黏附情况。结果: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1w后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:天然交联剂京尼平交联后支架的细胞生物相容性好,细胞在材料上能很好的黏附和生长增殖,可作为骨软骨组织工程的良好载体。
第三部分脱细胞软骨基质三维支架复合bBMP后修复兔关节骨软骨缺损
目的:运用制备好的脱细胞软骨基质三维支架修复兔关节骨软骨缺损。方法:用盐酸胍溶液溶解bBMP,将脱细胞软骨基质支架浸泡于此溶液中,包于透析膜内,蒸馏水透析4d后冷冻干燥,环氧乙烷消毒备用。扫描电镜观察复合bBMP后支架的结构以及BMSCs接种9d后在支架上的生长状况。取日本大耳兔12只,在其股骨髁部造成一直径4mm深达骨髓腔的缺损,其中18个膝盖植入复合有bBMP的脱细胞软骨基质支架,作为实验组,剩余的6个膝盖旷置为空白对照组。术后12w,24w取材,大体观察及HE、甲苯胺兰组织学观察。结果:扫描电镜观察可见9d后复合体材料表面有大量细胞黏附生长,并连接成片状覆盖于支架表面,细胞生长旺盛,广泛分布于支架上。12w,实验组缺损处可见白色组织大部分覆盖于缺损表面,与周围正常组织界限不明显,组织学观察为新生软骨,能见到透明软骨结构及软骨下骨结构,支架基本降解;对照组缺损明显,以纤维样组织填充于缺损部位。24w,实验组缺损处可见白色组织基本完全覆盖于缺损表面,与周围正常组织界限基本消失,组织学观察为新生透明软骨,结构层次分明,并与软骨下骨连接紧密;对照组为纤维样组织修复,缺损仍然明显。结论:在体内关节腔和骨髓腔不同的微环境诱导下,复合bBMP的脱细胞软骨基质支架材料能对兔关节骨软骨缺损达到较好的修复效果,为骨软骨组织工程修复提供了又一方法。
实验结果表明,脱细胞软骨基质三维支架材料改进的Courtman改良法处理后脱细胞更彻底,保留了天然软骨的细胞外基质成分,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,复合bBMP后的支架材料对兔关节骨软骨缺损修复效果良好,是一种适用于骨软骨组织工程的良好载体。
Articular cartilage is hard to repair because it lacks a direct blood supply,lymph circulation ,innervation and has a low metabolism.Presnt clinical common repair method has certain limitation.Acullular matrix which has good biocompatibility and hydrophilicity becomes another choice for tissue engineering scaffold.Compared with artificial material,natural materials have an advantage of good biocompatibility,similarity to structure composition of ECM and suitability for cell’s growth,proliferation and differentiation.Compared to other natural materials,a acellular matrix scaffold can get more closer to cartilage’s complex natural structure and biological characteristics.In this study we prepared a cartilage acellular matrix scaffold and repaired the articular osteochondral defect.
PartⅠ. Preparation of a three-dimentional acellular cartilage matrix scaffold and its characteristics.
Objective: To prepare a cartilage acellular matrix scaffold and to evaluate its characteristics. Methods: Calf cartilage microparticles were prepared after being physically shattered and gradient centrifugation, and then treated by a modified Courtman’s four-step method which is improved to produce the acellular cartilage matrix.3-D cartilage acellular matrix were prepared with the freeze-drying method.The scaffolds were cross-linked by a neotype crosslinking agent Genepin for 48h,and then placede into glycine solution server times for removing redundant Genepin.The freeze-drying method was used to prepare the CACM.The scaffolds were investigated with gross observation,histological staining(hematoxylin-eosin,toluidine blue),Scan electronic microscope(SEM) and porosity measurement,water absorption rate and degradation rate analysis. Results: Gross observation showed the scaffolds were loose porous and dark blue cross-linked by Genepin.The histological staining(haematoxylin- eosin, toluidine blue staining)showed that there were no chondrocyte fragments in the scaffolds.SEM showed that the scaffolds were porous.The CACM scaffold had 90% porosity,(1314±337)% water absorption rate and (13.69±7.3)%, (25.99±8.9)% degradation rate(2w,4w). Conclusion: The Courtman’s four-step method which is improved makes acellular effects more thoroughly and a retention of the natural cartilage extracellular matrix components.The neotype crosslinking agent Genepin makes scaffolds’mechanical strength and resistance to degradation be enhanced.
PartⅡ.Evaluation of Biocompatibility of acellular cartilage matrix scaffolds
Objective: Evaluate the biocompatibility of acellular cartilage matrix scoffolds by use of bone marrow stromal cells seeded onto scoffolds. Methods: After being cultivated for ten days,BMSCs of rabbit were seeded onto the scaffold with a cell density of 2.0×106/ml. MTT test and SEM were done to assess the growth and proliferation of BMSCs. Results: MTT test showed that BMSCs grew well in the 3-D CACM scaffolds of logarithmic trend, Absorbance compared with the control group,the difference was not statistically significant(P>0.05) ,supporting that the scaffolds had no cytotoxic effect on BMSCs.SEM micrographs indicated that cells covered the scaffolds firmly with cell processes. Conclusion: After being cross-linked by Genepin,the 3-D CACM scaffold has good biocompatibility and can be a good choice for osteochondral tissue engineering.
PartⅢ.Repair of the articular osteochondral defects in rabbits with the bBMP-scaffold complexes
Objective: To repair the articular osteochondral defects in rabbits with the bBMP-scaffold complexes. Methods: After dissolving 40mg bBMP in 4ml,4M guanidine hydrochloride,put the scaffolds into the solution.Put the scaffolds into a dialysisi-membrane and dialyze to distilled water for 4 days,and then use the freeze-drying method to prepare the bBMP-scaffold complexes.Seed BMSCs onto complexes at a concentration of 2.0×106/ml.9d later,SEM was used to assess the growth of BMSCs. The Articular osteochondral defects of rabbits with 4mm in diameter and reaching medullary cavity were made in the femoral condyles.18 bBMP-scaffold complexes were implanted into articular osteochondral defects of rabbits,6 articular osteochondral defects without treatment were the control group.At 12th and 24th week, Reparative effect of the defects was investigated by gross observation and histological staining (hematoxylin-eosim,toluidine blue). Results: SEM showed that BMSCs grew very well onto bBMP-scaffold complexes and the structure of the scaffold was the same as before. At 12th week,the defects with bBMP-scaffold complexes were filled with hyaline tissue. Histological staining indicated that the defects were mostly repaired with chondrogenesis and subchondral osteogenesis regeneration.The scaffolds were almost degraded.The defects were still existed in control group filled with fibrous tissue.At 24th week, defects with bBMP-scaffold complexes were completely repaired with chondrogenesis and subchondral osteogenesis fully regenerated indicated by histological staining.The control group were filled with fibrous tissue at the surface of the defects and still had deep defects. Conclusion: We found that BMSCs could differentiated into bone and cartilage induced by vivo different environment.The bBMP-scaffold complexes shows a good effect in repairing the articular osteochondral defects in rabbits.
This experiment indicate that the Courtman’s four-step method which is improved makes acellular effects more thoroughly.The 3-D CACM scaffold reserves 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 make it a suitable carrier for osteochondral tissue engineering.After being combined with bBMP, the bBMP-scaffold complexes repair osteochondral defects better.
第一部分脱细胞软骨基质三维支架的制备及特性研究
目的:探索脱细胞软骨基质三维多孔支架的制备及其特性研究。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。结果:大体观察示支架呈疏松多孔状,京尼平交联后整体呈深蓝色。组织学观察示支架材料无软骨细胞碎片残留,苏木精-伊红(HE)染色,甲苯胺兰染色观察均未见软骨细胞残留。扫描电镜显示支架内孔洞较明显。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)%。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材料机械强度和抗降解性得到了提高。
第二部分脱细胞软骨基质三维支架的生物相容性研究
目的:运用兔骨髓基质细胞(BMSCs)接种于支架上评价支架的生物相容性。方法:原代培养兔骨髓基质细胞(BMSCs),传至第二代后以2.0×106个/ml的细胞浓度接种于支架上,采用MTT法检测BMSCs在支架材料上后1、3、5、7、9d的生长、增殖情况,并描绘出柱形图。1w后扫描电镜观察兔骨髓基质细胞于支架上的黏附情况。结果: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1w后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:天然交联剂京尼平交联后支架的细胞生物相容性好,细胞在材料上能很好的黏附和生长增殖,可作为骨软骨组织工程的良好载体。
第三部分脱细胞软骨基质三维支架复合bBMP后修复兔关节骨软骨缺损
目的:运用制备好的脱细胞软骨基质三维支架修复兔关节骨软骨缺损。方法:用盐酸胍溶液溶解bBMP,将脱细胞软骨基质支架浸泡于此溶液中,包于透析膜内,蒸馏水透析4d后冷冻干燥,环氧乙烷消毒备用。扫描电镜观察复合bBMP后支架的结构以及BMSCs接种9d后在支架上的生长状况。取日本大耳兔12只,在其股骨髁部造成一直径4mm深达骨髓腔的缺损,其中18个膝盖植入复合有bBMP的脱细胞软骨基质支架,作为实验组,剩余的6个膝盖旷置为空白对照组。术后12w,24w取材,大体观察及HE、甲苯胺兰组织学观察。结果:扫描电镜观察可见9d后复合体材料表面有大量细胞黏附生长,并连接成片状覆盖于支架表面,细胞生长旺盛,广泛分布于支架上。12w,实验组缺损处可见白色组织大部分覆盖于缺损表面,与周围正常组织界限不明显,组织学观察为新生软骨,能见到透明软骨结构及软骨下骨结构,支架基本降解;对照组缺损明显,以纤维样组织填充于缺损部位。24w,实验组缺损处可见白色组织基本完全覆盖于缺损表面,与周围正常组织界限基本消失,组织学观察为新生透明软骨,结构层次分明,并与软骨下骨连接紧密;对照组为纤维样组织修复,缺损仍然明显。结论:在体内关节腔和骨髓腔不同的微环境诱导下,复合bBMP的脱细胞软骨基质支架材料能对兔关节骨软骨缺损达到较好的修复效果,为骨软骨组织工程修复提供了又一方法。
实验结果表明,脱细胞软骨基质三维支架材料改进的Courtman改良法处理后脱细胞更彻底,保留了天然软骨的细胞外基质成分,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,复合bBMP后的支架材料对兔关节骨软骨缺损修复效果良好,是一种适用于骨软骨组织工程的良好载体。
Articular cartilage is hard to repair because it lacks a direct blood supply,lymph circulation ,innervation and has a low metabolism.Presnt clinical common repair method has certain limitation.Acullular matrix which has good biocompatibility and hydrophilicity becomes another choice for tissue engineering scaffold.Compared with artificial material,natural materials have an advantage of good biocompatibility,similarity to structure composition of ECM and suitability for cell’s growth,proliferation and differentiation.Compared to other natural materials,a acellular matrix scaffold can get more closer to cartilage’s complex natural structure and biological characteristics.In this study we prepared a cartilage acellular matrix scaffold and repaired the articular osteochondral defect.
PartⅠ. Preparation of a three-dimentional acellular cartilage matrix scaffold and its characteristics.
Objective: To prepare a cartilage acellular matrix scaffold and to evaluate its characteristics. Methods: Calf cartilage microparticles were prepared after being physically shattered and gradient centrifugation, and then treated by a modified Courtman’s four-step method which is improved to produce the acellular cartilage matrix.3-D cartilage acellular matrix were prepared with the freeze-drying method.The scaffolds were cross-linked by a neotype crosslinking agent Genepin for 48h,and then placede into glycine solution server times for removing redundant Genepin.The freeze-drying method was used to prepare the CACM.The scaffolds were investigated with gross observation,histological staining(hematoxylin-eosin,toluidine blue),Scan electronic microscope(SEM) and porosity measurement,water absorption rate and degradation rate analysis. Results: Gross observation showed the scaffolds were loose porous and dark blue cross-linked by Genepin.The histological staining(haematoxylin- eosin, toluidine blue staining)showed that there were no chondrocyte fragments in the scaffolds.SEM showed that the scaffolds were porous.The CACM scaffold had 90% porosity,(1314±337)% water absorption rate and (13.69±7.3)%, (25.99±8.9)% degradation rate(2w,4w). Conclusion: The Courtman’s four-step method which is improved makes acellular effects more thoroughly and a retention of the natural cartilage extracellular matrix components.The neotype crosslinking agent Genepin makes scaffolds’mechanical strength and resistance to degradation be enhanced.
PartⅡ.Evaluation of Biocompatibility of acellular cartilage matrix scaffolds
Objective: Evaluate the biocompatibility of acellular cartilage matrix scoffolds by use of bone marrow stromal cells seeded onto scoffolds. Methods: After being cultivated for ten days,BMSCs of rabbit were seeded onto the scaffold with a cell density of 2.0×106/ml. MTT test and SEM were done to assess the growth and proliferation of BMSCs. Results: MTT test showed that BMSCs grew well in the 3-D CACM scaffolds of logarithmic trend, Absorbance compared with the control group,the difference was not statistically significant(P>0.05) ,supporting that the scaffolds had no cytotoxic effect on BMSCs.SEM micrographs indicated that cells covered the scaffolds firmly with cell processes. Conclusion: After being cross-linked by Genepin,the 3-D CACM scaffold has good biocompatibility and can be a good choice for osteochondral tissue engineering.
PartⅢ.Repair of the articular osteochondral defects in rabbits with the bBMP-scaffold complexes
Objective: To repair the articular osteochondral defects in rabbits with the bBMP-scaffold complexes. Methods: After dissolving 40mg bBMP in 4ml,4M guanidine hydrochloride,put the scaffolds into the solution.Put the scaffolds into a dialysisi-membrane and dialyze to distilled water for 4 days,and then use the freeze-drying method to prepare the bBMP-scaffold complexes.Seed BMSCs onto complexes at a concentration of 2.0×106/ml.9d later,SEM was used to assess the growth of BMSCs. The Articular osteochondral defects of rabbits with 4mm in diameter and reaching medullary cavity were made in the femoral condyles.18 bBMP-scaffold complexes were implanted into articular osteochondral defects of rabbits,6 articular osteochondral defects without treatment were the control group.At 12th and 24th week, Reparative effect of the defects was investigated by gross observation and histological staining (hematoxylin-eosim,toluidine blue). Results: SEM showed that BMSCs grew very well onto bBMP-scaffold complexes and the structure of the scaffold was the same as before. At 12th week,the defects with bBMP-scaffold complexes were filled with hyaline tissue. Histological staining indicated that the defects were mostly repaired with chondrogenesis and subchondral osteogenesis regeneration.The scaffolds were almost degraded.The defects were still existed in control group filled with fibrous tissue.At 24th week, defects with bBMP-scaffold complexes were completely repaired with chondrogenesis and subchondral osteogenesis fully regenerated indicated by histological staining.The control group were filled with fibrous tissue at the surface of the defects and still had deep defects. Conclusion: We found that BMSCs could differentiated into bone and cartilage induced by vivo different environment.The bBMP-scaffold complexes shows a good effect in repairing the articular osteochondral defects in rabbits.
This experiment indicate that the Courtman’s four-step method which is improved makes acellular effects more thoroughly.The 3-D CACM scaffold reserves 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 make it a suitable carrier for osteochondral tissue engineering.After being combined with bBMP, the bBMP-scaffold complexes repair osteochondral defects better.
引文
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