含钙化层仿生组织工程骨软骨支架的制备

发布时间：2019-07-02 10:29

【摘要】：背景及目的: 人体关节活动量巨大,极容易在创伤、肿瘤及急慢性炎症中导致关节骨软骨损伤。骨软骨损伤通常伴随关节机械应力改变,如不及时治疗会进一步导致退行性骨关节炎发生,大多软骨损伤也会发展至滑膜、关节囊及软骨下骨,导致更严重的骨软骨损伤,患肢功能障碍及生活质量下降。关节骨软骨由软骨,软骨下骨及之间的钙化层构成。关节软骨基本无血液、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,导致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,软骨下骨替代物的血管化不足将导致其不能及时修复,大块软骨下骨缺损将明显影响其支撑作用。骨软骨之间的钙化层包含深层透明软骨、潮线、钙化软骨、粘合线及上层软骨下骨,其结构微细并复杂,难于体外构建,但骨软骨连接区具备重要的生理功能。目前临床对软骨的修复较好方法主要是MACI技术,能达到部分透明软骨修复效果;对骨软骨损伤修复的主要方法包括自体及异体骨软骨移植,其具备软骨、软骨下骨及其间的钙化层,能取得一定临床效果。但是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、软骨下骨及其间的钙化层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结构进而恢复其正常功能,目前的骨软骨组织工程构建方法难以模拟包含骨软骨钙化层在内的骨软骨组织。由于骨及软骨脱细胞在技术上取得一定进展,通过粉碎软骨基质的方法可以将组织细胞成分去除,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钙化层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钙化层的骨软骨支架材料,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钙化层,骨软骨上方交联多孔软骨脱细胞基质支架并体外接种骨髓来源间充质干细胞构建细胞-材料复合体,观察细胞生物学改变。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。本研究主要内容包括:(1)分别将软骨粉、软骨片及骨软骨复合组织脱细胞处理,鉴定脱细胞程度并检验脱细胞对骨软骨钙化层功能的影响;(2)构建含骨软骨钙化层的脱细胞骨软骨复合组织工程支架,鉴定骨软骨支架并体外检测支架生物相容性。方法: 1.将天然羊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,检测其组织学,生化定量分析及理化性能检测。 2.制备直径为8mm含骨软骨钙化层的骨软骨组织块,其软骨侧仅保留约100um透明软骨,经脱细胞处理后予组织学,生物化学及理化性能检测。 3.采用冻干法及化学交联法制备含骨软骨钙化层的脱细胞骨软骨支架,予组织学、生物化学及理化性能检测,同时将扩增培养的羊骨髓间充质干细胞接种于含钙化层骨软骨支架两侧,体外培养7天行组织相容性和细胞毒性检测。结果: 1.组织学显示直径100um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,骨软骨钙化层功能基本保留;生化定量结果表明DNA成分去除,保留大量细胞外基质成分; 3.含骨软骨钙化层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,电镜及组织学显示细胞生长良好,有细胞外基质分泌。结论: 1.直径100um内软骨组织可以通过脱细胞处理去除细胞结构并保留大量细胞外基质成分;2.仅含100um厚透明软骨的骨软骨组织通过脱细胞处理,可去除组织内细胞成分,并保留骨软骨间钙化层;3.通过冻干及化学交联可制备含骨软骨钙化层脱细胞骨软骨支架,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,骨软骨钙化层保留,该支架具备良好细胞相容性,无明显细胞毒性。
[Abstract]:Background and purpose: The joint activity of the human body is huge, and the joint bone cartilage can be easily caused in the wound, the tumor and the acute and chronic inflammation Injury. The damage of the bone cartilage is usually accompanied by a change in the mechanical stress of the joint, such as the non-timely treatment can further lead to the occurrence of degenerative osteoarthritis, and most of the cartilage damage can also be developed to the synovium, the joint capsule and the subchondral bone, resulting in more severe bone-cartilage damage, limb dysfunction and quality of life. The articular cartilage is composed of the cartilage, the subchondral bone and the calcified layer between the subchondral bone and the cartilage. The articular cartilage is basically free of blood, lymph and nerves, only contains a single cartilage cell, the extracellular matrix cell ratio is high and the local progenitor cells are absent, which leads to the self-healing capacity of the articular cartilage itself. The articular cartilage subchondral bone is mainly supported by the articular cartilage, and the hypovascularisation of the subchondral bone substitute will cause it to not be repaired in time, and the subchondral bone defect will significantly affect its support The calcified layer between the bone and cartilage comprises the deep transparent cartilage, the tidal line, the calcified cartilage, the adhesive line and the upper layer of the subchondral bone, the structure is fine and complex, it is difficult to construct in vitro, but the joint area of the bone cartilage has an important physiological function. The method for repairing the cartilage is mainly based on the MACI technique, which can achieve the effect of partial transparent cartilage repair, and the main method for repairing the bone cartilage injury comprises the autograft and the allogenic bone cartilage graft, which is provided with the cartilage, the subchondral bone and the calcified layer between the cartilage and the subchondral bone, The clinical effect is fixed. However, the repair of the bone cartilage damage is not good, but the source of the bone cartilage graft is limited, but the MACI and the bone cartilage technique suggest that the bone cartilage unit with the cartilage, the subchondral bone and the calcified layer between the cartilage, the subchondral bone and the calcified layer between the cartilage and the subchondral bone may be a bone cartilage repair. The alternative structure of the ideal in vitro construction of the tissue engineering should mimic the natural structure of the joint tissue and restore its normal function, and the present construction method of the bone cartilage tissue engineering is difficult to simulate the calcified layer containing the bone cartilage. The bone and cartilage tissue. Due to the fact that bone and cartilage decells have made some progress in the art, the tissue cell components can be removed by the method of crushing the cartilage matrix, while the chondrocytes are retained. The subject is to take the tissue and the cartilage of the calcified layer between the bone-containing cartilage and the cartilage to decell, and use the acellular matrix material to prepare the bone cartilage support material containing the biological origin bone cartilage calcification layer, which comprises the deep transparent cartilage, the tide line and the calcified cartilage. a cell-material complex is constructed by a layer, an adhesive line, a cartilage subchondral bone plate, a physiological bone cartilage calcification layer, a cross-linked porous cartilage acellular matrix scaffold above the bone cartilage, and the bone marrow-derived mesenchymal stem cells are inoculated in vitro to construct a cell-material complex, Biological changes. The study will provide the basis for biomimetic preparation of the bone-cartilage composite scaffold material, and at the same time to construct a more complex tissue engineering closure The main contents of this study are as follows: (1) the cartilage powder, the cartilage sheet and the bone cartilage composite tissue are respectively decell-treated, the degree of decell is identified, and the acellular-to-bone cartilage is tested. the effect of the function of the calcified layer; (2) the scaffold of the acellular bone cartilage composite tissue engineering scaffold containing the bone cartilage calcification layer is constructed, the bone cartilage scaffold is identified and the in vitro test is carried out, test support The method comprises the following steps of:1, the natural sheep cartilage is crushed under the protection of a protease inhibitor, 2. Preparation of a bone-cartilage tissue mass with a diameter of 8 mm with a bone-cartilage-calcified layer, whose cartilage side only retains about 100 um of the transparent cartilage, and the cartilage side only retains about 100 um of the transparent cartilage, and the cartilage side only retains about 100 um of the transparent cartilage, and the cartilage side only retains about 100 um of the transparent cartilage, and The histological, biochemical and physical and chemical properties of the bone-containing cartilage-containing layer were prepared by freeze-drying and chemical cross-linking. The bone marrow of the cultured sheep was also detected by the methods of histology, biochemistry and physical and chemical properties. Mesenchymal stem cells are seeded on both sides of the calcified layer of bone cartilage support in vitro The histocompatibility and cytotoxicity of 7 day lines were tested. The results were as follows:1. There were no cell debris left, toluidine blue staining, Panyu O and type 2 after the treatment of the chondrocyte with the diameter of 100 um. The immunohistochemical staining of the collagen was positive, and the structure of the cartilage matrix was retained under the light microscope. The results showed that the DNA component was removed and a large amount of extracellular matrix components were retained. Cartilage-side methamidon-blue staining, Panyu-O and 2-type collagen immunohistochemical staining were positive, and the function of the bone-cartilage calcification layer was basic. the biochemical quantitative results indicate that the DNA component is removed and a large amount of the extracellular matrix component is retained; and the acellular bone cartilage scaffold containing the bone cartilage calcification layer is histologically detected, the bone cartilage scaffold is connected in good condition, and the bone marrow mesenchymal stem cells of the sheep in a stand The growth of the cells was good, and the electron microscope and the histology showed that the cells were well-grown and the extracellular matrix was secreted. in that 00 um, the cartilage tissue can remove the cell structure by decell treatment and retain a large amount of extracellular matrix component;2. only 100 um thick transparent soft tissue the bone cartilage tissue of the bone can remove the cell components in the tissue by decell treatment, and the calcification layer between the bone cartilage can be retained; and 3. the bone-containing cartilage-containing calcified layer acellular bone cartilage scaffold can be prepared by freeze-drying and chemical cross-linking, wherein the cartilage side of the scaffold is a cartilage acellular porous scaffold, a cartilage,
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R314

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