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胶原-透明质酸钠-纤维蛋白胶复合去抗原松质骨
支架的实验研究
硕士研究生
:穆继宏
     
:胡蕴玉 教授
第四军医大学西京医院骨科,西安 710032
                                     
 
关节软骨的损伤和病变在临床上较常见,以受力最大的膝关节和髋关节为多。软骨的自身修复能力有限,且关节软骨的损伤往往伴发软骨下骨损伤,同时,骨坏死到后期会导致关节面塌陷而破坏关节软骨,故无论长骨干骺端的病变原发于骨还是软骨,后期均会引起骨软骨的破坏及缺损。目前治疗骨软骨缺损的方法很多,组织工程方法是研究的热点。本研究设计构建了一种以纤维蛋白胶为粘合剂的新型骨软骨一体化支架材料,并对其结构特性、生物相容性及其修复软骨缺损的能力进行探索研究。研究结果证明该支架材料能满足细胞的黏附、生长和增殖,对兔关节骨软骨缺损具有良好的修复作用。
1. 胶原-透明质酸钠-纤维蛋白胶复合去抗原牛松质骨支架的制备及结构特征
目的:探索胶原-透明质酸钠-纤维蛋白胶多孔支架复合去抗原牛松质骨的骨软骨一体化支架的制备方法及其结构特征。方法:氯仿/甲醇脱脂,过氧化氢脱蛋白,稀盐酸部分脱钙制备去抗原牛松质骨;酸溶法制备鼠尾胶原;将胶原、透明质酸钠及纤维蛋白胶混匀,利用纤维蛋白胶的粘合性与去抗原牛松质骨复合,冻干后经碳化二亚胺交联24h,成为骨软骨一体化的支架材料。通过大体观察和扫描电镜观察了解材料的孔径和交通情况。结果:支架材料大体呈白色,骨相与软骨相分界明显,结合紧密,吸水性较好;经交联和复合的一体化骨软骨支架材料具有良好的三维多孔结构,骨相孔径约300500µm,软骨相孔径约80120µm,孔隙较均匀。结论:胶原-透明质酸钠-纤维蛋白胶多孔支架复合去抗原牛松质骨一体化支架的大体形态、物理性状和空间结构可以满足细胞黏附生长及体内修复缺损的要求。
2. 胶原-透明质酸钠-纤维蛋白胶复合去抗原牛松质骨支架材料的生物相容性研究
目的:探索胶原-透明质酸钠-纤维蛋白胶多孔支架复合去抗原牛松质骨的骨软骨一体化支架的生物相容性。方法:分离、增殖仔兔骨髓基质细胞(BMSCs),取第3BMSCs接种至支架材料复合培养, 135710d后,行MTT检测并绘制细胞生长曲线;取第1d7d标本,行扫描电镜观察。结果:扫描电镜观察可见1d7d的支架材料的骨相及软骨相均有细胞黏附,复合7d的材料上黏附的细胞明显较复合1d广泛,且细胞周围有基质分泌。MTT比色实验,OD值随时间增长,值10d到最大,显示细胞在支架上的生长良好,生长曲线呈对数生长趋势。结论:胶原-透明质酸钠-纤维蛋白胶多孔支架复合去抗原牛松质骨的一体化材料具有良好的生物相容性, 可用于修复骨软骨联合缺损的组织工程支架材料的研究。
3.应用胶原-透明质酸钠-纤维蛋白胶复合去抗原牛松质骨支架修复兔关节骨软骨缺损的研究
 目的:应用胶原-透明质酸钠-纤维蛋白胶多孔支架复合去抗原牛松质骨的骨软骨一体化支架修复兔关节骨软骨缺损,观察其体内的修复作用。方法:12只大耳白兔,于后膝关节股骨髁部非负重区制造约5.0mm的缺损,随机选取6只兔的右后膝缺损旷置处理,作为对照组;其余缺损均填充胶原-透明质酸钠-纤维蛋白胶复合去抗原牛松质骨支架,作为实验组。在12周和24周后取材,大体观察和组织学染色观察缺损修复的情况。结果:大体观察12周和24周后实验组的缺损组织,可见其被支架材料填充,表面光滑,而对照组突出于关节面,结构不规则。组织学观察显示,12周的修复体边界基本与正常组织整合,材料大部分降解,软骨层有透明软骨样组织形成;24周实验组新生的软骨组织更接近于正常软骨,骨小梁结构完整,松质骨支架已基本被降解。结论:胶原-透明质酸钠-纤维蛋白胶多孔支架复合去抗原牛松质骨的一体化材料修复兔关节骨软骨缺损具有较好的修复效果,基本恢复关节软骨的组织结构,有望成为修复骨软骨联合缺损的组织工程支架材料。
 
 
关键词:骨软骨缺损胶原透明质酸钠纤维蛋白胶去抗原松质骨
 


 
Studies on a scaffold material composed of collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer and
antigen-extracted cancellous bone
 
Candidate for master: Mu Jihong
Supervisor: Hu Yunyu
Department of orthopaedics,xijing hospital, Fourth Military Medical University,
Xi’an 710032, China
 
Damage to the articular cartilage is a common clinical occurrence, which mostly occurs in knee and hip joints due to the great stress they are exposed to. However, the self-repair capacity of cartilage is limited, and the articular cartilage damage is often associated with injuries of subchondral bone. Meanwhilethe pathological changes in long bone metaphyses will lead to destruction of cartilage and bone defects, no matter where these changes occur. There are many treatment options for the osteochondral defects, and tissue engineering is a focus of research. In this study we designed and constructed a new type of osteochondral scaffold and explored its morphology and biocompatibility. The results showed that the scaffold material could meet the demand for adhesion, growth and proliferation of the BMSCs. This material was used in repairing rabbit articular cartilage defect with satisfactory results.
1. Preparation and structural characteristics of collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone
ObjectiveTo explore the preparation and structural characteristics of collagen-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone. MethodsWe prepared the rat tail collagen and antigen-extracted bovine cancellous bone by ourselves. The osteochondral scaffold was constructed of collagen-sodium hyaluronate-fibrin glue tri-copolymer scaffold and antigen-extracted bovine cancellous bone with adhesion of the two parts by fibrin glue.Then it was freeze-dried and cross-linked by carbodiimide for 24h. Pore size and internal pores of the osteochondral scaffold were observed grossly and by SEM. ResultsThe osteochondral scaffold after crosslinking and adhesion had a porous structure. The bone scaffold was bonding with the cartilage scaffold solidly. Pore size of bone scaffold is about 300~500μm and pore size of cartilage is about 80~120μm. ConclusionThe osteochondral scaffold of the collagen-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone has proper structural characteristicswhich can  meet the structural requirements of the cell adhesion ,growth and repair defects in vivo.
2. The study on the biocompatibility of collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone.
ObjectiveTo explore the biocompatibility of collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone. MethodsRabbit bone marrow stromal cells were isolated and amplified before they were seeded into the scaffold at the third passage. Proliferation of the cells on the scaffolds was examined using MTT method and the growth curve was drawn. The scaffolds with cells were dehydrated and observed by SEM after 1d and 7d. Results:The examination under SEM indicated that the BMSCs adhered and spread well on the scaffold after 1d and 7d, and the extracellular matrices were also seen around the BMSCs after 7d. ConclusionThe osteochondral scaffold of the collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone has a good biocompatibility , which make it a useful scaffold in the osteochondral tissue engineering.
3. The study on repairing osteochondral defects in rabbit by collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone.
ObjectiveTo explore the repair of osteochondral defect by acollagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone in vivo. Methods12Rabbits were used as experiment animals. After creating defects 5mm in diameter, the defects in six knees of the rabbits were filled up with nothing at all, and other eighteen defects were filled up with collagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone. After 12 weeks and 24 weeks respectively,all animals were killed ,then the newly formed tissues were observed by gross obversation and histology. Results:After 12 weeks and 24 weeks,tissue defects in the experimental group were filled with scaffold material and had smooth surface,while the control group in the articular surface showed irregular structure. The scaffolds after 12 weeks were absorbed and the defects were repaired with hyaline cartilage.After 24 weeks,the new tissue was similar to normal cartilage and the cancellous bone has basically been degraded. ConclusioncollagenⅠ-sodium hyaluronate-fibrin glue tri-copolymer scaffold bonding with antigen-extracted bovine cancellous bone has a good reparative capacity for the osteochondral defects in rabbits and can be expected to be used for an osteochondral tissue engineered scaffold.
 
Key wordsOsteochondral defectcollagenSodium hyaluronateFibrin glueAntigen-extracted cancellous bone
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