摘要
目的基于低温沉积3D打印技术构建新型组织工程半月板支架,评价该支架理化性质及生物相容性。方法取新鲜猪膝关节半月板,采用改良物理化学联合方法脱细胞处理,获得脱细胞半月板基质匀浆;经大体观察、HE及DAPI染色观察脱细胞效果,甲苯胺蓝、番红O及天狼猩红染色评估黏多糖和胶原保留情况。然后制备脱细胞半月板基质生物墨水,通过低温沉积3D打印技术制备新型组织工程半月板支架。扫描电镜观察微观结构;与脂肪来源干细胞共培养后,采用细胞计数试剂盒8(cell counting kit 8,CCK-8)检测支架细胞相容性,死/活细胞染色和细胞骨架染色观察细胞活性和形态;植入大鼠皮下后组织学染色评估支架炎症细胞浸润与降解情况。结果脱细胞处理后半月板基质匀浆呈透明凝胶状,DAPI和组织学染色示免疫原性的核酸去除,同时黏多糖及胶原成分保留。采用低温沉积3D打印技术成功构建新型组织工程半月板支架,扫描电镜示支架呈分级大孔-微孔的微观结构;CCK-8检测示支架具有良好细胞相容性;死/活细胞染色示支架可有效维持细胞活性(>90%);细胞骨架染色示支架有利于细胞黏附和铺展;支架植入大鼠皮下1周后有轻度炎症反应,3周后未见明显炎症反应,并可见支架逐步降解。结论基于低温沉积3D打印技术构建的新型组织工程半月板支架具有分级大孔-微孔的微观结构和良好细胞相容性,有利于细胞黏附和生长,为下一步体内研究奠定基础。
Objective To investigate the construction of a novel tissue engineered meniscus scaffold based on low temperature deposition three-dimenisonal(3D)printing technology and evaluate its biocompatibility.Methods The fresh pig meniscus was decellularized by improved physicochemical method to obtain decellularized meniscus matrix homogenate.Gross observation,HE staining,and DAPI staining were used to observe the decellularization effect.Toluidine blue staining,safranin O staining,and sirius red staining were used to evaluate the retention of mucopolysaccharide and collagen.Then,the decellularized meniscus matrix bioink was prepared,and the new tissue engineered meniscus scaffold was prepared by low temperature deposition 3D printing technology.Scanning electron microscopy was used to observe the microstructure.After co-culture with adipose-derived stem cells,the cell compatibility of the scaffolds was observed by cell counting kit 8(CCK-8),and the cell activity and morphology were observed by dead/live cell staining and cytoskeleton staining.The inflammatory cell infiltration and degradation of the scaffolds were evaluated by subcutaneous experiment in rats.Results The decellularized meniscus matrix homogenate appeared as a transparent gel.DAPI and histological staining showed that the immunogenic nucleic acids were effectively removed and the active components of mucopolysaccharide and collagen were remained.The new tissue engineered meniscus scaffolds was constructed by low temperature deposition 3D printing technology and it had macroporous-microporous microstructures under scanning electron microscopy.CCK-8 test showed that the scaffolds had good cell compatibility.Dead/live cell staining showed that the scaffold could effectively maintain cell viability(>90%).Cytoskeleton staining showed that the scaffolds were benefit for cell adhesion and spreading.After 1 week of subcutaneous implantation of the scaffolds in rats,there was a mild inflammatory response,but no significant inflammatory response was observed after 3 weeks,and the scaffolds gradually degraded.Conclusion The novel tissue engineered meniscus scaffold constructed by low temperature deposition 3D printing technology has a graded macroporous-microporous microstructure and good cytocompatibility,which is conducive to cell adhesion and growth,laying the foundation for the in vivo research of tissue engineered meniscus scaffolds in the next step.
作者
陈明学
吴江
殷瀚
眭翔
刘舒云
郭全义
CHEN Mingxue;WU Jiang;YIN Han;SUI Xiang;LIU Shuyun;GUO Quanyi(Department of Orthopaedics,Beijing Jishuitan Hospital Affiliated to Capital Medical University,Beijing,100035,P.R.China;Institute of Orthopedics,the First Medical Center of the Chinese PLA General Hospital,Beijing Key Laboratory of Orthopaedic Regenerative Medicine,Key Laboratory of Orthopaedic War Trauma,Beijing,100853,P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2024年第6期748-754,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金青年科学基金项目(82202784)
北京市医院管理中心青年人才培养“青苗”计划(QML20230404)
北京积水潭医院科研优才计划(JSTYC202305)。
关键词
组织工程支架
低温沉积3D打印技术
半月板
脱细胞基质
Tissue engineered scaffold
low temperature deposition three-dimensional printing technology
meniscus
decellularized matrix
