骨软骨脱细胞基质材料研制及修复功能动物实验研究
发布时间：2018-09-11 12:36
【摘要】：研究背景: 关节骨软骨由软骨,软骨下骨及之间的连接结构构成。人体关节活动使用量巨大,极容易在创伤、肿瘤及急慢性炎症中导致关节软骨或骨软骨损伤。软骨损伤也常会发展致滑膜、关节囊及软骨下骨,导致骨软骨损伤,骨软骨损伤通常伴随关节机械应力改变,如不治疗会进一步导致退行性关节炎发生,患者功能障碍及生活质量下降。关节软骨无血液、淋巴及神经,仅包含单一软骨细胞,细胞外基质细胞比高并且缺少局部祖细胞,关节软骨自身修复能力很差。关节软骨下骨主要对关节软骨起支撑作用,软骨下骨损伤通常采用替代物植入治疗,替代物的血管化不足将不能及时修复,大块骨缺损将明显影响其支撑作用。骨软骨之间的连接结构包含深层透明软骨、潮线、钙化软骨、粘合线及上层软骨下骨,其结构微细并复杂,骨软骨连接区损伤可能会影响其重要的生理功能。关节软骨及骨软骨损伤自身修复困难,关节骨软骨损伤治疗成为目前骨关节外科难题之一。 对软骨及骨软骨缺损修复方法较多,较有效的方法包括采用具备软骨、软骨下骨及其间的连接结构的自体及异体骨软骨移植;而仅对软骨的修复较具潜力的方法主要是组织工程技术,其中MACI(基质介导自体软骨细胞移植)技术能达到部分透明软骨修复效果,显示较好的前景。MACI对骨软骨损伤修复欠佳,而骨软骨移植物来源有限,但MACI及骨软骨技术提示带软骨、软骨下骨及其间的连接结构的骨软骨单元移植并结合种子细胞可能是骨软骨修复的潜在方案。理想的体外构建的组织工程替代结构应该模拟关节组织的自然成分及结构进而恢复其正常功能,国内外对组织工程骨软骨复合组织的构建研究,从“分层构建”的可行性初探到“一体构建”的动物实验研究取得了阶段性成果,然而目前尚存在缺损区修复组织质量缺陷、与宿主界面整合欠佳及缺乏相应力学功能等主要问题。由于骨及软骨脱细胞在技术上取得一定进展,通过粉碎软骨并采用脱细胞试剂处理的方法可以将细胞成分去除,而保留软骨细胞外基质成分。 本课题拟在此基础上,分别将含骨软骨之间连接结构的组织及软骨进行脱细胞,利用脱细胞基质材料制备含生物来源骨软骨连接结构的骨软骨支架材料。该支架包含深层透明软骨、潮线、钙化软骨层、粘合线、软骨下骨板等生理骨软骨连接结构,软骨侧构建软骨脱细胞基质支架,保留脱细胞软骨下骨,尽量模拟了骨软骨结构。通过体外接种骨髓来源间充质干细胞构建细胞-材料复合体,植入体内修复动物骨软骨缺损模型并观察软骨生成效果;并且采用骨髓来源分离扩增的内皮祖细胞接种脱细胞骨基质材料修复动物大段骨缺损,通过血管化来促进大段骨缺损的修复,为研究软骨下骨缺损修复提供新的方法。本研究将为仿生制备骨软骨复合支架材料提供依据,同时为构建更为复杂的组织工程关节创造条件。在研究内容上主要包括:(1)分别将软骨粉、软骨片及骨软骨复合组织脱细胞处理并鉴定;(2)构建含骨软骨连接结构的脱细胞骨软骨复合组织工程支架;(3)利用骨髓间充质干细胞复合含骨软骨连接结构的骨软骨支架修复羊膝关节负重区骨软骨缺损模型;(4)采用脱细胞骨基质接种骨髓来源内皮祖细胞修复兔尺骨大段骨缺损。 方法: 1.将天然人软骨在蛋白酶抑制剂保护下粉碎,采用离心分选软骨微粒,经过软骨脱细胞处理后制备软骨脱细胞微粒悬液,行组织学及生化定量分析检测。 2.制备直径为8mm含骨软骨连接结构的骨软骨组织块,其软骨侧仅保留约100μm透明软骨,经脱细胞处理后予组织学,生化定量分析检测。采用冻干法及化学交联法制备含骨软骨连接结构的脱细胞骨软骨支架,培养羊骨髓间充质干细胞,将羊骨髓间充质干细胞接种于骨软骨支架两侧,体外培养7天行组织学,组织相容性和细胞毒性检测。 3.制备羊负重区骨软骨缺损模型,分空白、植入空白支架及植入细胞支架复合物3组,3月后处死动物取标本行大体及组织学检测。 4.构建脱细胞骨支架及骨髓来源内皮祖细胞复合物,制备兔尺骨缺损模型,分空白,脱细胞骨支架及细胞支架复合物3组修复兔尺骨缺损,2,4,8周处死动物取标本行组织学检测。 结果: 1.组织学显示直径100μm以内软骨细胞微粒通过软骨脱细胞处理后无细胞碎片残留,甲苯胺蓝染色,番红O及二型胶原免疫组化染色成阳性,光镜下见软骨基质结构部分保留;生化定量结果表明DNA成分去除,保留大量细胞外基质成分; 2.组织学显示含部分透明软骨的骨软骨组织块脱细胞处理后无细胞碎片残留,软骨侧甲苯胺蓝染色,番红O及二型胶原免疫组化染色成阳性,骨软骨连接结构功能基本保留;生化定量结果表明DNA成分去除,保留大量细胞外基质成分;含骨软骨连接结构的脱细胞骨软骨支架经组织学检测骨软骨支架连接完好,羊骨髓间充质干细胞在支架上生长良好,软骨侧番红O及二型胶原免疫染色阳性,电镜及组织学显示细胞生长良好,有细胞外基质分泌; 3.修复羊负重区骨软骨缺损模型实验结果显示细胞支架复合修复组骨软骨有较好修复,空白支架组软骨下骨基本修复、软骨侧无明显修复,空白对照组未见明显修复,缺损边缘软骨退变; 4.骨髓来源干细胞能通过体外分离扩增,接种骨髓来源的内皮祖细胞的脱细胞骨基质支架体内修复大段骨缺损后,修复组织内微血管密度较空白支架组高(p0.05),并能对兔尺骨大段骨缺损进行一定程度的修复。结论: 粉碎的软骨微粒、厚度100μm左右软骨片及仅含100μm厚透明软骨的骨软骨组织通过脱细胞处理,均可去除组织内细胞成分,保留大部分细胞外基质结构功能;通过冻干及化学交联可制备含骨软骨连接结构脱细胞骨软骨支架,该支架软骨侧为软骨脱细胞多孔支架,软骨下骨侧为脱细胞骨,骨软骨连接结构保留,该支架具备良好细胞相容性,无明显细胞毒性;含骨软骨连接结构的脱细胞骨软骨支架接种种子细胞能较好的修复羊负重区骨软骨缺损;脱细胞骨支架及骨髓来源内皮祖细胞复合物,对兔尺骨缺损修复及促进微血管生成具备一定作用。
[Abstract]:Research background:
Articular cartilage is made up of cartilage, subchondral bone and connective structures. The use of large amounts of human joint activities is extremely easy to cause articular cartilage or osteochondral injury in trauma, tumor and acute or chronic inflammation. Cartilage injury often develops into synovium, articular capsule and subchondral bone, resulting in osteochondral injury, often accompanied by osteochondral injury. The articular cartilage has no blood, lymph and nerve, only a single chondrocyte, a high ratio of extracellular stromal cells and a lack of local progenitor cells. The articular cartilage has a poor ability to repair itself. Subchondral bone injuries are usually treated with substitutes implanted. Inadequate vascularization of substitutes will not be able to be repaired in time, and large bone defects will significantly affect the supporting effect. Bone-cartilage junctions include deep hyaline cartilage, tidal line, calcified cartilage, adhesive line and upper subchondral bone with fine structure. It is difficult for articular cartilage and osteochondral injury to repair itself. The treatment of articular cartilage injury has become one of the difficult problems in osteoarthroplasty.
There are many methods for repairing cartilage and osteochondral defects, including autogenous and allograft cartilage transplantation with cartilage, subchondral bone and its connective structure, while tissue engineering is the most promising method for cartilage repair only, in which MACI (matrix-mediated autologous chondrocyte transplantation) technique can achieve partial repair. MacI is not good for the repair of osteochondral injury, but the source of osteochondral grafts is limited. However, MACI and osteochondral techniques suggest that the transplantation of osteochondral units with cartilage, subchondral bone and its connective structures and the combination of seeding cells may be a potential option for osteochondral repair. The constructed tissue engineering substitution structure should simulate the natural composition and structure of joint tissue to restore its normal function. The construction of tissue engineering osteochondral composite tissue has been studied at home and abroad. From the feasibility of "layered construction" to the animal experimental study of "integrated construction", the results have been achieved at different stages. Due to the advances in bone and cartilage acellular technology, chondrocyte components can be removed by comminuting cartilage and using acellular reagents, while extracellular matrix components can be retained.
On this basis, the tissue and cartilage containing the connective structure between osteochondria were acellularized, and the osteochondral scaffolds containing biological osteochondral connective structure were prepared using acellular matrix materials. The scaffolds include deep hyaline cartilage, tidal line, calcified cartilage layer, adhesive line, subchondral bone plate and other physiological osteochondral connective materials. The bone marrow-derived mesenchymal stem cells (BMSCs) were inoculated in vitro to construct the cell-material complex and implanted in vivo to repair the animal osteochondral defect model and observe the effect of cartilage formation. Endothelial progenitor cells (EPCs) were seeded with acellular bone matrix (ABM) to repair large bone defects in animals, and vascularization was used to promote the repair of large bone defects. This study will provide a new method for the study of subchondral bone defect repair. Conditions include: (1) acellular treatment and identification of cartilage powder, cartilage slices and osteochondral composite tissue; (2) construction of acellular osteochondral composite tissue engineering scaffold with osteochondral junction; (3) repair of sheep knee joint negative by bone marrow mesenchymal stem cells and osteochondral composite scaffold with osteochondral junction structure. (4) Bone marrow-derived endothelial progenitor cells (EPCs) were seeded with acellular bone matrix to repair large ulnar bone defects in rabbits.
Method:
1. The natural human cartilage was crushed under the protection of protease inhibitor. The cartilage particles were centrifuged and separated. After the cartilage was acellular treated, the cartilage acellular particles suspension was prepared for histological and biochemical quantitative analysis.
2. The osteochondral tissue block with osteochondral junction structure of 8 mm in diameter was prepared, and only about 100 micron hyaline cartilage was retained on the cartilage side. After acellular treatment, histological and biochemical quantitative analysis were carried out. The acellular osteochondral scaffold containing osteochondral junction structure was prepared by freeze-drying method and chemical cross-linking method. Bone marrow mesenchymal stem cells were seeded on both sides of osteochondral scaffolds and cultured in vitro for 7 days for histological, histocompatibility and cytotoxicity tests.
3. Prepare sheep osteochondral defect model in load-bearing area, and divide into three groups: blank, blank and cellular scaffold composite. Samples were sacrificed 3 months later for gross and histological examination.
4. Acellular bone scaffold and bone marrow-derived endothelial progenitor cell complex were constructed to prepare rabbit ulna defect model. The rabbit ulna defect was repaired by three groups: blank group, acellular bone scaffold group and cell scaffold complex group. The rabbits were sacrificed at 2,4,8 weeks for histological examination.
Result:
1. Histological examination showed that chondrocyte particles with diameter less than 100 micron were free of cell debris after acellular treatment, toluidine blue staining, Safranine O and collagen II immunohistochemical staining were positive, and the structure of cartilage matrix was partly preserved under light microscope.
2. Histology showed that there was no residual cell fragments, toluidine blue staining on cartilage side, Safranine O and collagen II immunohistochemical staining were positive, and the structure and function of osteochondral junction were basically preserved after acellular treatment of osteochondral tissue with hyaline cartilage. The acellular osteochondral scaffold with cartilage junction structure was well-connected by histological examination. The goat bone marrow mesenchymal stem cells grew well on the scaffold. Safranine O and collagen II immunostaining were positive on the cartilage side. Electron microscopy and histology showed that the cells grew well and secreted extracellular matrix.
3. The experimental results of repairing sheep's osteochondral defects in the load-bearing area showed that the osteochondral defects were repaired well in the cell scaffold group, and the subchondral bones were basically repaired in the blank scaffold group, and the cartilage side was not repaired obviously in the blank control group.
4. Bone marrow-derived stem cells can be isolated and amplified in vitro, and the acellular bone matrix scaffolds inoculated with bone marrow-derived endothelial progenitor cells can repair large bone defects in vivo. The microvessel density in the repaired tissues is higher than that in the blank scaffolds group (p0.05), and can repair large bone defects of rabbit ulna to a certain extent.
By acellular treatment, the chondrocartilage with a thickness of about 100 microns, and the osteochondral tissue with only 100 microns of hyaline cartilage can remove the intracellular components and retain most of the structure and function of extracellular matrix. The scaffolds have good cell compatibility and no obvious cytotoxicity. The seeding cells of acellular osteochondral scaffolds with osteochondral junction can repair the bone and cartilage defects of sheep in the load-bearing area. Endothelial progenitor cell complex can play an important role in repairing ulnar defects and promoting angiogenesis in rabbits.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
本文编号：2236708
[Abstract]:Research background:
Articular cartilage is made up of cartilage, subchondral bone and connective structures. The use of large amounts of human joint activities is extremely easy to cause articular cartilage or osteochondral injury in trauma, tumor and acute or chronic inflammation. Cartilage injury often develops into synovium, articular capsule and subchondral bone, resulting in osteochondral injury, often accompanied by osteochondral injury. The articular cartilage has no blood, lymph and nerve, only a single chondrocyte, a high ratio of extracellular stromal cells and a lack of local progenitor cells. The articular cartilage has a poor ability to repair itself. Subchondral bone injuries are usually treated with substitutes implanted. Inadequate vascularization of substitutes will not be able to be repaired in time, and large bone defects will significantly affect the supporting effect. Bone-cartilage junctions include deep hyaline cartilage, tidal line, calcified cartilage, adhesive line and upper subchondral bone with fine structure. It is difficult for articular cartilage and osteochondral injury to repair itself. The treatment of articular cartilage injury has become one of the difficult problems in osteoarthroplasty.
There are many methods for repairing cartilage and osteochondral defects, including autogenous and allograft cartilage transplantation with cartilage, subchondral bone and its connective structure, while tissue engineering is the most promising method for cartilage repair only, in which MACI (matrix-mediated autologous chondrocyte transplantation) technique can achieve partial repair. MacI is not good for the repair of osteochondral injury, but the source of osteochondral grafts is limited. However, MACI and osteochondral techniques suggest that the transplantation of osteochondral units with cartilage, subchondral bone and its connective structures and the combination of seeding cells may be a potential option for osteochondral repair. The constructed tissue engineering substitution structure should simulate the natural composition and structure of joint tissue to restore its normal function. The construction of tissue engineering osteochondral composite tissue has been studied at home and abroad. From the feasibility of "layered construction" to the animal experimental study of "integrated construction", the results have been achieved at different stages. Due to the advances in bone and cartilage acellular technology, chondrocyte components can be removed by comminuting cartilage and using acellular reagents, while extracellular matrix components can be retained.
On this basis, the tissue and cartilage containing the connective structure between osteochondria were acellularized, and the osteochondral scaffolds containing biological osteochondral connective structure were prepared using acellular matrix materials. The scaffolds include deep hyaline cartilage, tidal line, calcified cartilage layer, adhesive line, subchondral bone plate and other physiological osteochondral connective materials. The bone marrow-derived mesenchymal stem cells (BMSCs) were inoculated in vitro to construct the cell-material complex and implanted in vivo to repair the animal osteochondral defect model and observe the effect of cartilage formation. Endothelial progenitor cells (EPCs) were seeded with acellular bone matrix (ABM) to repair large bone defects in animals, and vascularization was used to promote the repair of large bone defects. This study will provide a new method for the study of subchondral bone defect repair. Conditions include: (1) acellular treatment and identification of cartilage powder, cartilage slices and osteochondral composite tissue; (2) construction of acellular osteochondral composite tissue engineering scaffold with osteochondral junction; (3) repair of sheep knee joint negative by bone marrow mesenchymal stem cells and osteochondral composite scaffold with osteochondral junction structure. (4) Bone marrow-derived endothelial progenitor cells (EPCs) were seeded with acellular bone matrix to repair large ulnar bone defects in rabbits.
Method:
1. The natural human cartilage was crushed under the protection of protease inhibitor. The cartilage particles were centrifuged and separated. After the cartilage was acellular treated, the cartilage acellular particles suspension was prepared for histological and biochemical quantitative analysis.
2. The osteochondral tissue block with osteochondral junction structure of 8 mm in diameter was prepared, and only about 100 micron hyaline cartilage was retained on the cartilage side. After acellular treatment, histological and biochemical quantitative analysis were carried out. The acellular osteochondral scaffold containing osteochondral junction structure was prepared by freeze-drying method and chemical cross-linking method. Bone marrow mesenchymal stem cells were seeded on both sides of osteochondral scaffolds and cultured in vitro for 7 days for histological, histocompatibility and cytotoxicity tests.
3. Prepare sheep osteochondral defect model in load-bearing area, and divide into three groups: blank, blank and cellular scaffold composite. Samples were sacrificed 3 months later for gross and histological examination.
4. Acellular bone scaffold and bone marrow-derived endothelial progenitor cell complex were constructed to prepare rabbit ulna defect model. The rabbit ulna defect was repaired by three groups: blank group, acellular bone scaffold group and cell scaffold complex group. The rabbits were sacrificed at 2,4,8 weeks for histological examination.
Result:
1. Histological examination showed that chondrocyte particles with diameter less than 100 micron were free of cell debris after acellular treatment, toluidine blue staining, Safranine O and collagen II immunohistochemical staining were positive, and the structure of cartilage matrix was partly preserved under light microscope.
2. Histology showed that there was no residual cell fragments, toluidine blue staining on cartilage side, Safranine O and collagen II immunohistochemical staining were positive, and the structure and function of osteochondral junction were basically preserved after acellular treatment of osteochondral tissue with hyaline cartilage. The acellular osteochondral scaffold with cartilage junction structure was well-connected by histological examination. The goat bone marrow mesenchymal stem cells grew well on the scaffold. Safranine O and collagen II immunostaining were positive on the cartilage side. Electron microscopy and histology showed that the cells grew well and secreted extracellular matrix.
3. The experimental results of repairing sheep's osteochondral defects in the load-bearing area showed that the osteochondral defects were repaired well in the cell scaffold group, and the subchondral bones were basically repaired in the blank scaffold group, and the cartilage side was not repaired obviously in the blank control group.
4. Bone marrow-derived stem cells can be isolated and amplified in vitro, and the acellular bone matrix scaffolds inoculated with bone marrow-derived endothelial progenitor cells can repair large bone defects in vivo. The microvessel density in the repaired tissues is higher than that in the blank scaffolds group (p0.05), and can repair large bone defects of rabbit ulna to a certain extent.
By acellular treatment, the chondrocartilage with a thickness of about 100 microns, and the osteochondral tissue with only 100 microns of hyaline cartilage can remove the intracellular components and retain most of the structure and function of extracellular matrix. The scaffolds have good cell compatibility and no obvious cytotoxicity. The seeding cells of acellular osteochondral scaffolds with osteochondral junction can repair the bone and cartilage defects of sheep in the load-bearing area. Endothelial progenitor cell complex can play an important role in repairing ulnar defects and promoting angiogenesis in rabbits.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329
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