【作者】 李晓；

【导师】 李伟；

【作者基本信息】 山东大学 ， 骨科学（专业学位）， 2024， 硕士

【摘要】 背景:骨关节炎(Osteoarthritis,OA)是一种病理机制复杂的退行性疾病,终末期骨关节炎常伴随疼痛和关节功能障碍,严重降低患者的生活质量。炎症反应在骨关节炎的发病过程中扮演了重要角色,其中白细胞介素1β(Interleukin-1β,IL-1β)是最关键的炎症因子之一,它可以激活软骨细胞产生炎症反应,导致软骨退变与骨赘形成。然而,目前的治疗手段如口服药物、关节腔注射和手术治疗等均存在各自的弊端和局限性。因此,我们亟需探索一些更安全有效的治疗方式。水凝胶作为一种新型药物递送系统成为当前的研究热点,借鉴天然软骨结构的水凝胶具备弹性和韧性,能够模拟软骨在关节中发挥机械支撑作用;其多孔结构类似于天然细胞外基质(Extracellular matrix,ECM),不仅为细胞提供了生存空间,还促进营养物质与生物信号的传递;同时它的高含水量能减少软骨摩擦,发挥润滑作用;基于透明质酸(Hyaluronic acid,HA)、明胶(Gelatin,Gel)与壳聚糖(Chitosan,CS)等天然物质设计的水凝胶具备生物相容性和抗炎功能;水凝胶的药物缓释特性有助于延长药物在关节内的作用时间,提高治疗效果。以上特性使水凝胶成为治疗骨关节炎的理想材料。麝香酮(Muscone,Mus)是麝香的主要活性成分,近些年的研究中发现其具有抗炎、抗氧化、抗肿瘤、抗纤维化、抗凋亡等生物活性,还具有体外促进干细胞迁移的作用。因此,我们设计了负载麝香酮的二醛壳聚糖(Dialdehyde chitosan,DACS)/明胶水凝胶(Mus@DACS/Gel)作为一种可注射的关节腔药物递送系统。水凝胶与麝香酮同步发挥抗炎作用,抑制骨关节炎进展,避免软骨损伤。同时,释放的麝香酮促进内源性干细胞的迁移和成软骨分化,从而实现对关节炎中损伤软骨的修复。目的:本研究通过DACS中醛基与明胶中氨基发生席夫碱反应的自组装原理,将麝香酮负载于水凝胶中制备Mus@DACS/Gel。通过体内、外实验研究Mus@DACS/Gel水凝胶同步抑制骨关节炎进展及促进软骨修复的能力。方法:1.采用自组装原理合成DACS/Gel水凝胶,并通过红外、扫描电镜、透射电镜证明其制备成功;测试其可注射性、自愈合性、溶胀率及流变特性;进行Mus@DACS/Gel水凝胶的体外降解实验并记录药物的缓释过程。2.通过活死染色与细胞计数试剂盒-8(Cell Counting Kit-8,CCK-8)评估Mus@DACS/Gel的细胞毒性。利用ROS荧光染色、MMP-13免疫荧光染色与实时荧光定量PCR(qPCR),证明Mus@DACS/Gel体外抑制软骨细胞炎症的能力。通过迁移实验、细胞骨架染色、阿利新蓝染色、Col2α1免疫荧光染色及qPCR分析,评估Mus@DACS/Gel的促干细胞迁移与成软骨分化性能。3.构建大鼠骨关节炎模型,通过血常规、血生化评估Mus@DACS/Gel体内毒性,利用组织病理学分析、Micro-CT扫描和免疫组化染色评估Mus@DACS/Gel的体内抗炎和促成软骨性能。结果:1.DACS与Gel混合在PBS溶液中静置,形成可经过针管注射且能断裂后自修复的淡黄色凝胶;红外测定结果中亚胺键在1650 cm-1的特征峰表明DACS/Gel成功制备;SEM和TEM可观测到DACS/Gel疏松多孔的微观结构;溶胀实验和流变测试证明了其良好的溶胀性和力学性能;体外降解和药物释放实验中,Mus@DACS/Gel可在体外条件下存在超过2 w,并依靠其降解实现麝香酮的缓释。以上结果证明Mus@DACS/Gel的成功制备。2.对于软骨细胞,活死染色与CCK-8实验显示Mus@DACS/Gel并未造成明显的细胞死亡或活性下降;ROS荧光染色、MMP-13免疫荧光染色与qPCR实验表明Mus@DACS/Gel可以下调IL-1β诱导的关节炎软骨细胞内ROS水平以及ADAMTS-5、MMP-3和MMP-13的基因表达。对于干细胞,细胞迁移实验证明麝香酮具有干细胞趋化作用;细胞骨架染色实验表明干细胞可以在Mus@DACS/Gel培养基上良好地铺展;阿利新蓝染色、Col2α1免疫荧光染色及qPCR分析显示Mus@DACS/Gel可以促进干细胞粘多糖的分泌及Col2α1、SOX-9和Acan的基因表达。上述结果说明Mus@DACS/Gel具备良好的生物相容性,在体外具有良好的抗炎和促成软骨分化能力。3.在体内实验中,Mus@DACS/Gel处理组大鼠的血常规、血生化及重要器官的H&E染色未发现病理改变;术后4w收集的膝关节Micro-CT扫描、H&E和番红O-固绿染色结果显示,与对照组相比,Mus@DACS/Gel减轻了骨关节炎中新生骨赘形成、炎性细胞浸润和软骨磨损降解;免疫组化染色显示Mus@DACS/Gel能显著促进软骨组织中Col2α1的表达,抑制MMP-13的表达。上述结果表明Mus@DACS/Gel在体内同样具备良好的生物相容性和同步抑制骨关节炎进展、保护软骨及促进软骨修复的能力。结论及意义:1.我们成功制备了负载麝香酮的关节腔药物递送系统Mus@DACS/Gel水凝胶;2.Mus@DACS/Gel在体外、体内都具有抑制骨关节炎进展及促进软骨修复的能力;3.该水凝胶制备方法简单可行,为骨关节炎的非手术治疗提供了一个有效策略。更多还原

【Abstract】 BackgroundOsteoarthritis(OA)is a degenerative disease with complex pathological mechanisms,and end-stage osteoarthritis is often accompanied by pain and joint dysfunction,which seriously reduces the quality of life of patients.Inflammation plays an important role in the pathogenesis of osteoarthritis,and interleukin-1β(IL-1β)is one of the most critical inflammatory factors,which can activate chondrocytes to produce inflammatory responses,leading to cartilage degeneration and osteophyte formation.However,current treatments such as oral medications,intra-articular injections,and surgical treatment have their own drawbacks and limitations.Therefore,there is an urgent need to explore some safer and more effective treatment modalities.As a new drug delivery system,hydrogels have become a research hotspot,and the hydrogels that draw on the natural cartilage structure have elasticity and toughness,which can mimic the mechanical support role of cartilage in joints,and its porous structure is similar to that of the natural extracellular matrix(ECM),which not only provides a living space for cells,but also promotes the transmission of nutrients and biological signals,and its high water content can reduce cartilage friction and play a lubricating role.Hydrogels designed based on natural substances such as hyaluronic acid(HA),gelatin(Gel)and chitosan(CS)have biocompatible and anti-inflammatory functions,and the sustained-release properties of hydrogels can help prolong the action time of drugs in the joints and improve the therapeutic effect.The above properties make hydrogels an ideal material for the treatment of osteoarthritis.Muscone(Mus)is the main active ingredient of musk,and in recent years,it has been found that it has anti-inflammatory,antioxidant,anti-tumor,anti-fibrotic,anti-apoptosis and other biological activities,and also has the effect of promoting stem cell migration in vitro.Therefore,we designed muskone-loaded dialdehyde chitosan(DACS)/gelatin hydrogel(Mus@DACS/Gel)as an injectable articular drug delivery system.Hydrogel and muskone exert anti-inflammatory effects simultaneously,inhibit the progression of osteoarthritis,and avoid cartilage damage.At the same time,the released muskone promotes the migration and chondrogenic differentiation of endogenous stem cells,thereby achieving the repair of damaged cartilage in arthritis.ObjectiveIn this study,the preparation of Mus@DACS/Gel by loading muskone into a hydrogel was based on the principle of self-assembly of Schiff base reaction between aldehyde group in DACS and amino group in gelatin.In vivo and in vitro experiments were used to study the ability of Mus@DACS/Gel hydrogel to simultaneously inhibit the progression of osteoarthritis and promote cartilage repair.Methods1.The self-assembly principle was used to synthesize DACS/Gel hydrogels,and the preparation was proved by infrared,scanning electron microscopy and transmission electron microscopy,and its injectability,self-healing,swelling rate and rheological properties were tested,and the in vitro degradation experiments of Mus@DACS/Gel hydrogels were carried out and the sustained release process of the drugs was recorded.2.The cytotoxicity of Mus@DACS/Gel was assessed by the Cell Counting Kit-8(CCK-8).ROS fluorescence staining,MMP-13 immunofluorescence staining and real-time quantitative PCR(qPCR)were used to demonstrate the ability of Mus@DACS/Gel to inhibit chondrocyte inflammation in vitro.The pro-stem cell migration and chondrogenesis differentiation performance of Mus@DACS/Gel were evaluated by migration assay,cytoskeletal staining,alixin blue staining,Col2α1 immunofluorescence staining and qPCR analysis.3.A rat model of osteoarthritis was constructed,and the in vivo toxicity of Mus@DAC S/Gel was evaluated by blood routine and blood biochemistry,and the anti-inflammatory and cartilage properties of Mus@DACS/Gel were evaluated by histopathological analysis,Micro-CT scan and immunohistochemical staining.Results1.DACS and Gel were mixed with PBS solution to form a pale yellow gel that could be injected by needle tube and could be broken and self-repaired,the characteristic peak of the imine bond at 1650 cm-1 in the infrared measurement results showed that DACS/Gel was successfully prepared,the loose and porous microstructure swelling experiment and rheological test of DACS/Gel could be observed by SEM and TEM,and the swelling and mechanical properties of DACS/Gel were observed Mus@DACS by SEM and TEM.Gel can be present in vitro for more than 2 W and relies on its degradation for sustained release of muskone.The above results prove the successful preparation of Mus@DACS/Gel.2.For chondrocytes,live-dead staining and CCK-8 assay showed that Mus@DACS/Gel did not cause significant cell death or decreased activity,and ROS fluorescence staining,MMP-13 immunofluorescence staining and qPCR assay showed that Mus@DACS/Gel could down-regulate the IL-1β-induced ROS level in arthritis chondrocytes and the gene expression of ADAMTS-5,MMP-3 and MMP-13.For stem cells,cell migration assays showed that muskone had a stem cell chemotaxis,cytoskeletal staining experiments showed that stem cells could spread well on Mus@DDACS/Gel medium,and alixin blue staining,Col2α1 immunofluorescence staining and qPCR analysis showed that Mus@DACS/Gel could promote the secretion of mucopolysaccharides and the gene expression of Col2α1,SOX-9 and Acan in stem cells.The above results indicate that Mus@DACS/Gel has good biocompatibility and good anti-inflammatory and cartilage differentiation ability in vitro.3.In vivo experiments,no pathological changes were found in the blood routine,blood biochemistry and H&E staining of vital organs in the Mus@DACS/Gel treatment group,and the results of micro-CT scanning,H&E and safberry O-solid green staining of the knee joint collected at 4 w after surgery showed that compared with the control group,Mus@DACS/Gel alleviated the formation of neoosteophytes,inflammatory cell infiltration and cartilage wear and degradation in osteoarthritis,and immunohistochemical staining showed Mus@DACS/Gel significantly promoted the expression of Col2al and inhibited the expression of MMP-13 in cartilage tissue.These results indicate that Mus@DACS/Gel also has good biocompatibility in vivo and the ability to simultaneously inhibit the progression of osteoarthritis,protect cartilage and promote cartilage repair.Conclusions1.We successfully prepared Mus@DACS/Gel hydrogel,a drug delivery system loaded with muscone into the articular cavity.2.Mus@DACS/Gel has the ability to inhibit the progression of osteoarthritis and promote cartilage repair both in vitro and in vivo.3.The hydrogel preparation method is simple and feasible,providing an effective strategy for the non-surgical treatment of osteoarthritis.更多还原