摘要
目的采用低温沉积3D打印技术制备聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA]支架,复合脱细胞软骨细胞外基质(decellularized articular cartilage extracellular matrix,DACECM)制备PLGA/DACECM组织工程软骨支架,探讨其理化特性。方法利用低温沉积3D打印技术制备PLGA支架。采用改良式物理、化学脱细胞方法制备DACECM混悬液。利用冷冻干燥和物理化学法交联技术制备DACECM取向支架,同法将DACECM混悬液与PLGA支架复合制备PLGA/DACECM取向支架。通过大体观察、扫描电镜观察3种支架宏观、微观结构,组织学及免疫组织化学染色定性分析DACECM取向支架成分,生物力学试验检测3种支架压缩模量。于SD大鼠皮下包埋3种支架,HE染色观察免疫排斥反应。分离培养新西兰大白兔软骨细胞,制备3种细胞-支架复合物,扫描电镜观察细胞在支架上的生长黏附情况。取鼠L-929成纤维细胞分别于3种支架浸提液进行培养,以DMEM培养液培养细胞作为对照,细胞计数试剂盒8(cell counting kit 8,CCK-8)法检测细胞增殖情况。结果大体观察和扫描电镜观察示,PLGA支架表面较光滑,可见大孔;DACECM取向支架表面粗糙,为疏松多孔相互连通的三维立体结构;PLGA/DACECM取向支架表面粗糙,大孔与小孔相互连通,具有垂直三维立体结构。组织学及免疫组织化学定性分析显示,DACECM脱细胞完全,保留了软骨基质的糖胺聚糖和Ⅱ型胶原蛋白成分。生物力学检测示,DACECM取向支架压缩模量显著低于其余2种支架(P<0.05),PLGA支架和PLGA/DACECM取向支架间差异无统计学意义(P>0.05)。SD大鼠皮下包埋实验示,DACECM取向支架和PLGA/DACECM取向支架的免疫排斥反应明显低于PLGA支架。细胞-支架复合物扫描电镜观察示,软骨细胞在PLGA支架上未见明显黏附,大量软骨细胞在PLGA/DACECM取向支架和DACECM取向支架表面黏附、生长。CCK-8检测示,随培养时间延长,各组细
Objective To manufacture a poly(lactic-co-glycolic acid)(PLGA) scaffold by low temperature deposition three-dimensional(3 D) printing technology, prepare a PLGA/decellularized articular cartilage extracellular matrix(DACECM) cartilage tissue engineered scaffold by combining DACECM, and further investigate its physicochemical properties. Methods PLGA scaffolds were prepared by low temperature deposition 3 D printing technology, and DACECM suspensions was prepared by modified physical and chemical decellularization methods.DACECM oriented scaffolds were prepared by using freeze-drying and physicochemical cross-linking techniques.PLGA/DACECM oriented scaffolds were prepared by combining DACECM slurry with PLGA scaffolds. The macroscopic and microscopic structures of the three kinds of scaffolds were observed by general observation and scanning electron microscope. The chemical composition of DACECM oriented scaffold was analyzed by histological and immunohistochemical stainings. The compression modulus of the three kinds of scaffolds were measured by biomechanical test.Three kinds of scaffolds were embedded subcutaneously in Sprague Dawley rats, and HE staining was used to observe immune response. The chondrocytes of New Zealand white rabbits were isolated and cultured, and the three kinds of cellscaffold complexes were prepared. The growth adhesion of the cells on the scaffolds was observed by scanning electron microscope. Three kinds of scaffold extracts were cultured with L-929 cells, the cells were cultured in DMEM culture medium as control group, and cell counting kit 8(CCK-8) was used to detect cell proliferation. Results General observation and scanning electron microscope showed that the PLGA scaffold had a smooth surface and large pores;the surface of the DACECM oriented scaffold was rough, which was a 3 D structure with loose pores and interconnected;and the PLGA/DACECM oriented scaffold had a rough surface, and the large hole and the small hole were connected to each other to construct a vertical 3 D stru
作者
张彬
沈师
鲜海
代永静
郭维民
李旭
张学亮
王振勇
李浩江
彭礼庆
罗旭江
刘舒云
鲁晓波
郭全义
ZHANG Bin;SHEN Shi;XIAN Hai;DAI Yongjing;GUO Weimin;LI Xu;ZHANG Xueliang;WANG Zhenyong;LI Haojiang;PENG Liqing;LUO Xujiang;LIU Shuyun;LU Xiaobo;GUO Quanyi(Department of Bone and Joint Surgery,Affiliated Hospital of Southwest Medical University,Luzhou Sichuan,646000,P.R.China;Institute of Orthopedics,Chinese PLA General Hospital,Beijing Key Lab of Regenerative Medicine in Orthopedics,Key Lab of Musculoskeletal Trauma & War Injuries of Chinese PLA,Beijing,100853,P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2019年第8期1011-1018,共8页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家重点研发计划资助项目(2018YFC1105902)
国家自然科学基金面上项目(81772319)~~
关键词
软骨组织工程
支架材料
3D打印技术
聚乳酸-羟基乙酸共聚物
脱细胞软骨细胞外基质
理化特性
Cartilage tissue engineering
scaffold material
three-dimensional printing technology
poly (lacticco- glycolic acid)
decellularized articular cartilage extracellular matrix
physicochemical property
