3D生物打印负载转化生长因子β3的软骨复合支架  

Cartilage composite scaffold loaded with transforming growth factor beta 3 using three-dimensional bioprinting

作　　者：杨振 李浩 付力伟[1,2] 高仓健 姜双鹏 王福鑫 苑志国 孙志强 查康康 田广招 曹福洋 眭翔 刘舒云[2] 郭全义[2] Yang Zhen;Li Hao;Fu Liwei;Gao Cangjian;Jiang Shuangpeng;Wang Fuxin;Yuan Zhiguo;Sun Zhiqiang;Zha Kangkang;TianGuangzhao;Cao Fuyang;Sui Xiang;Liu Shuyun;Guo Quanyi(Medical College of Nankai University,Tianjin 300071,China;Institute of Orthopedics,the First Medical Center,Chinese PLA General Hospital,Beijing Key Laboratory of Regenerative Medicine in Orthopedics,Key Laboratory of Musculoskeletal Trauma&War Injuries,PLA,Beijing 100853,China)

机构地区：[1]南开大学医学院,天津市300071 [2]解放军总医院第一医学中心骨科研究所,骨科再生医学北京市重点实验室,全军骨科战创伤重点实验室,北京市100853

出　　处：《中国组织工程研究》2021年第34期5445-5452,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家重点研发计划课题(2019YFA0110600),项目负责人:郭全义;国家自然科学基金(81772319),项目负责人:郭全义。

摘　　要：背景:通过募集内源性干细胞原位再生软骨损伤的治疗策略,是未来软骨组织工程研究的新方向。目的:构建既能募集干细胞又能促进其黏附和增殖,且有利于新生组织成熟的组织工程软骨复合支架。方法:将脱细胞软骨细胞外基质(extracellular matrix,ECM)与甲基丙烯酸酯化明胶(methacrylated gelatin,GelMA)混合配制光敏性生物墨水,利用3D生物打印技术分别制备单纯聚己内酯[poly(Ɛ-caprolactone),PCL]支架、PCL/GelMA/ECM支架。将转化生长因子β3(transforming growth factorβ3,TGF-β3)负载于生物墨水中制备PCL/GelMA/ECM/TGF-β3支架,检测其缓释性能。从形态学、组织学、生物化学、生物力学等角度评价PCL/GelMA/ECM支架的物理化学性质。利用CCK-8法检测PCL/GelMA/ECM支架的细胞毒性。将脂肪间充质干细胞接种于PCL/GelMA/ECM支架上,1,4,7 d后,共聚焦显微镜下观察细胞活性,扫描电镜观察细胞黏附。将PCL/GelMA/ECM支架植入SD大鼠皮下,组织学观察炎性细胞浸润与支架降解。利用Transwell小室实验检测PCL/GelMA/ECM支架、PCL/GelMA/ECM/TGF-β3支架对脂肪间充质干细胞迁移的影响,以单独培养的细胞为阴性对照。结果与结论:①PCL/GelMA/ECM支架呈现三维立体多孔网状结构,无细胞成分,含有Ⅱ型胶原和糖胺聚糖等软骨特异性成分,支架弹性模量为(14.24±2.44)MPa;②PCL/GelMA/ECM支架无明显的细胞毒性;③脂肪间充质干细胞紧密贴附于PCL/GelMA/ECM支架上,细胞活性良好,可分泌细胞外基质;④PCL/GelMA/ECM支架植入大鼠皮下1周后有轻度急性炎症反应,3周后炎性反应减轻,并可见逐步支架降解;⑤PCL/GelMA/ECM/TGF-β3支架具有良好的缓释性能,可持续释放TGF-β3达60 d;⑥相对于阴性对照组,PCL/GelMA/ECM支架、PCL/GelMA/ECM/TGF-β3支架均可促进脂肪间充质干细胞的迁移,其中以PCL/GelMA/ECM/TGF-β3支架促迁移作用更显著;⑦结果表明,3D打印PCL/GelMA/ECM/TGF-β3支架可促BACKGROUND:The therapeutic strategy of in situ regeneration of cartilage injury by recruiting endogenous stem cells is a new research direction of cartilage tissue engineering in the future.OBJECTIVE:To construct a tissue engineering cartilage composite scaffold that can not only recruit stem cells,promote cell adhesion and proliferation,but also be beneficial to the maturation of neo-tissue.METHODS:Acellular cartilage extracellular matrix(ECM)and methacrylate gelatin(GelMA)were mixed to prepare photosensitive bio-ink,and threedimensional bioprinting technology was used to prepare polycaprolactone(PCL)scaffolds and PCL/GelMA/ECM scaffolds.Transforming growth factorβ3(TGF-β3)was loaded into bio-ink to prepare PCL/GelMA/ECM/TGF-β3 scaffold,and its sustained release performance was tested.The physical and chemical properties of PCL/GelMA/ECM scaffolds were evaluated from the point of view of morphology,histology,biochemistry and biomechanics.CCK-8 assay was used to detect the cytotoxicity of PCL/GelMA/ECM scaffolds.After adipose-derived mesenchymal stem cells were seeded on PCL/GelMA/ECM scaffold for 1,4 and 7 days,the cell viability was observed by confocal microscope and the cell adhesion was observed by scanning electron microscope.PCL/GelMA/ECM scaffolds were implanted subcutaneously in SD rats,and the infiltration of inflammatory cells and the degradation of scaffolds were observed histologically.The effects of PCL/GelMA/ECM scaffold and PCL/GelMA/ECM/TGF-β3 scaffold on the migration of adipose-derived mesenchymal stem cells were detected by Transwell chamber test,and the cells cultured alone were used as negative control.RESULTS AND CONCLUSION:(1)The PCL/GelMA/ECM scaffolds had a three-dimensional porous reticular structure,without cellular components,contained cartilage-specific components such as type II collagen and glycosaminoglycan and its elastic modulus was(14.24±2.44)MPa.(2)PCL/GelMA/ECM scaffolds showed no obvious cytotoxicity.(3)Adipose-derived mesenchymal stem cells were adhered closely to the

关 键 词：骨 材料 支架 转化生长因子Β3 3D生物打印 软骨损伤 脂肪间充质干细胞 招募 迁移 

分 类 号：R459.9[医药卫生—治疗学] R318.08[医药卫生—临床医学]