Objective: To manufacture a poly (lactic-co-glycolic acid) (PLGA) scaffold by low temperature deposition three-dimensional (3D) 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 3D 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 cell-scaffold 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 3D 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 3D structure. Histological and immunohistochemical qualitative analysis demonstrated that DACECM was completely decellularized, retaining the glycosaminoglycans and collagen typeⅡ. Biomechanical examination showed that the compression modulus of DACECM oriented scaffold was significantly lower than those of the other two scaffolds ( P<0.05). There was no significant difference between PLGA scaffold and PLGA/DACECM oriented scaffold ( P>0.05). Subcutaneously embedded HE staining of the three scaffolds showed that the immunological rejections of DACECM and PLGA/DACECM oriented scaffolds were significantly weaker than that of the PLGA scaffold. Scanning electron microscope observation of the cell-scaffold complex showed that chondrocytes did not obviously adhere to PLGA scaffold, and a large number of chondrocytes adhered and grew on PLGA/DACECM oriented scaffold and DACECM oriented scaffold. CCK-8 assay showed that with the extension of culture time, the number of cells cultured in the three kinds of scaffold extracts and the control group increased. There was no significant difference in the absorbance ( A) value between the groups at each time point ( P>0.05).
Conclusion: The PLGA/DACECM oriented scaffolds have no cytotoxicity, have excellent physicochemical properties, and may become a promising scaffold material of tissue engineered cartilage.
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，各组细胞数量均呈递增趋势，各时间点组间吸光度（ A）值比较差异均无统计学意义（ P>0.05）。.
结论: PLGA/DACECM 取向支架具有无细胞毒性、优良的理化性能，有望成为一种组织工程软骨支架材料。.
Keywords: Cartilage tissue engineering; decellularized articular cartilage extracellular matrix; physicochemical property; poly (lactic-co-glycolic acid); scaffold material; three-dimensional printing technology.