【作者】 周红梅；

【导师】 李德富； 刘其松；

【作者基本信息】 四川大学 ， 生物工程（专业学位）， 2022， 硕士

【摘要】 作为一类高致残率及高发病率的慢性疾病,骨软骨缺损的发生率逐年增长并出现年轻化的趋势,致使80%的骨软骨缺损患者行动受阻,25%的患者无法进行日常活动,患者面临着难以缓解的疼痛及高额医疗费用。由于软骨缺乏血管、淋巴及神经组织,骨软骨的自我修复能力极为有限且缓慢。人工关节不仅在性能上无法与生物组织匹配,随之而来的术后并发症也是亟待解决的关键问题。具备优良生物活性的生物材料可在诱导自身内源性细胞增殖并修复缺损组织后降解,这在一定程度上规避了传统治疗手段潜在的风险。透明软骨、钙化软骨、软骨下骨的生化组成与结构的不同决定了每一层细胞外微环境的差异,而胞外微环境与细胞代谢和增殖等功能活动密切相关,单一的组成无法针对性地为细胞提供最佳微环境。多层支架可分别为软骨细胞和成骨细胞提供适宜的生长和增殖条件,研究表明多层支架在骨软骨修复中的质量优于单层或双层支架。因此,一类分别针对骨软骨生化成分与结构且可诱导自体组织修复的多层支架具有极大的应用价值。本课题中,以天然骨软骨的主要成分:胶原、透明质酸钠和纳米羟基磷灰石为原材料,制备模拟天然骨软骨的成分与结构的连续性胶原基异质多层支架并对其进行结构及性能研究。本论文具体开展了以下研究工作:(1)以胶原、透明质酸钠、纳米羟基磷灰石为原料制备单层支架。分别根据透明软骨、钙化软骨及软骨下骨的基质组成调整各层支架中各成分的质量比:透明软骨层中胶原:透明质酸钠=1:1、钙化软骨层中胶原:透明质酸钠:纳米羟基磷灰石=1:1:1、软骨下骨层中胶原:透明质酸钠:纳米羟基磷灰石=4:1:5。选用不同量的EDC/NHS调节胶原的氨基与透明质酸钠的羧基之间的交联度,分别得到低交联度、中交联度与高交联度的透明软骨层、钙化软骨层及软骨下骨层支架。(2)对单层支架进行结构表征。结果表明,胶原与透明质酸钠通过酰胺键、酯键交联形成支架网络结构,纳米羟基磷灰石附着于支架网络表面。EDC/NHS的增加会降低孔径及孔隙率,提高交联度。交联度较低的支架出现方向性孔,透明软骨层与钙化软骨层的孔更为致密,软骨下骨层的孔相对疏松,支架的孔隙率均高于85%,利于营养物质运输及细胞增殖。(3)利用溶剂浇筑法及冷冻干燥技术依次将单层支架复合为异质多层支架并对支架进行了结构表征及性能测试。结果表明,异质多层支架的孔之间高度互通且分布均匀,各层之间界面连续。交联作用及纳米羟基磷灰石极大地降低了支架的溶胀性能并提高了机械性能,支架降解速率变缓、降解率降低。此外,支架在具有优良的生物相容性的同时可促进创面凝血,利于骨软骨组织修复。支架无细胞毒性,且透明软骨层显著表现出促进软骨细胞增殖的作用,相对地,软骨下骨层更利于成骨细胞的增殖。综上所述,本课题制备的胶原基异质多层支架具备针对性诱导内源性细胞增殖的作用,可应用于骨软骨修复领域。更多还原

【Abstract】 Osteochondral defects is a chronic diseases with high disability and morbidity,the incidence of osteochondral defects increases year by year and it shows a trend of getting younger.80% of patients with osteochondral defects are blocked in movement,25% of patients are even unable to carry out daily activities,the patients are faced with pain that is difficult to relieve and high medical costs.Lacking of blood vessels,lymph,nerves in cartilage,the self-healing of osteochondral is extremely limited and slow.Artificial joints are not only unable to match biological tissue in terms of performance,but the accompanying postoperative complications are also a crucial problem to be solved urgently.Biomaterials with excellent biological activitives could degrade after inducing the proliferation of endogenous repair cells and repairing the defect,which avoids the potential risks of traditional treatments to a certain extent The differences in biochemical composition and structure of hyaline cartilage,calcified cartilage and subchondral bone determine the differences in the extracellular microenvironment of each layer which is closely correlated to the functional activities of cell metabolism and proliferation.Scaffold with changeless component cannot provide the optimal microenvironment for osteochondral.In comparison,multilayer scaffold offfers specific growth and proliferation conditions for chondrocytes and osteoblasts respectively.Research has shown that the quality of multilayer scaffold in osteochondral defects repair is superior to the single or double scaffold.Therefore,multilayer scaffold which mimicing biochemical composition and structure of osteochondral is well worthy of practice.In this project,collagen,sodium hyaluronate and nano-hydroxyapatite,the main components of natural osteochondral,were used as raw materials to prepare continuous collagen-based heterogeneous multilayer scaffolds which mimicng the composition and structure of natural osteochondral,and its structure and performance were systematically studied.The following research works were carried out in this paper:(1)The single layer scaffolds were prepared by collagen,sodium hyaluronate and nano-hydroxyapatite.According to the natural hyaline cartilage,calcified cartilage and subchondral bone matrix,the mass ratio of each component in each layer scaffold was adjusted respectively: the mass ratio of collagen to sodium hyaluronate in hyaline cartilage layer was 1:1,the mass ratio of collagen to sodium hyaluronate to nano-hydroxyapatite in calcified cartilage layer was 1:1:1,the mass ratio of collagen to sodium hyaluronate to nano-hydroxyapatite in subchondral bone layer was 4:1:5.Different amount of EDC/NHS were choosen to regulate the crosslinking degree between the amino of collagen and the carboxyl of sodium hyaluronate,resulting in low,medium and high crosslinking degree of hyaline cartilage layer,calcified cartilage layer and subchondral bone layer.(2)The single layer scaffolds were characterized.The results showed that the scaffolds were crosslinked by amide and ester bonds,nano-hydroxyapatite was attached to the internal surface of scaffold.The increase of EDC/NHS reduced the pore size and porosity,while improving the cross-linking degree.The scaffolds with low cross-linking degree show directional pores,and the hyaline cartilage layer and calcified cartilage layer scaffolds were dense,while the subchondral bone layer scaffold was relatively loose.The porosity of scaffolds were all above 85%,which is beneficial to nutrient transportation and cell proliferation.(3)The single layer scaffolds were successively combined to heterogeneous multilayer scaffolds by solvent casting method and freeze-drying,and the characterization and properties of heterogeneous multilayer scaffolds were carried out.The results showed that the pores of the scaffolds were highly interconnected and evenly distributed,and the continuous interfaces between the scaffolds were found.Crosslinking and nano-hydroxyapatite greatly reduced the swelling ratio and mechanical properties of scaffolds.Compared to collagen scaffold,the degradation rate of crosslinked scaffolds slowed down,while the remianing weight of crosslinked scaffolds increased.Additionally,scaffolds with excellent biocompatibility also promoted blood coagulation,which is conducive to osteochondral tissue repair.The hyaline cartilage layer significantly promotes the proliferation of chondrocytes,while the subchondral bone layer is relatively more conducive to the proliferation of osteoblasts.In conclusion,the collagen-based heterogeneous multilayer scaffolds prepared in this paper specifically induce the proliferation of endogenous repair cells,which can be applied in osteochondral repair.更多还原