COA:细胞外基质来源的双层支架的研制及其修复犬膝关节负重区骨软骨缺损的实验研究

背景与目的 骨软骨损伤常见且修复困难，本研究探讨采用软骨细胞外基质（CECM)与脱细胞骨基质（ACBM)为材料制作新型组织工程骨软骨双层支架的可行性,并检测其联合骨髓基质干细胞（bone marrow mesenchymal stem cells，BMSCs）修复犬膝关节负重区骨软骨联合缺损的疗效。 方法 ⑴新型组织工程骨软骨双层支架的骨部分以犬松质骨制备的ACBM为原料，软骨部分以人CECM为材料：将天然软骨粉碎成微丝并脱细胞处理后配成30 g/L的乳状悬液,将ACBM浸于盛CECM悬液的模具中，采用冷冻冻干法制备CECM/ACBM双层支架并交联。进行组织学、扫描电镜、Micro-CT观察,测定支架孔隙率，采用MTT法分析支架浸提液毒性。⑵ 将成软骨诱导的BMSCs种植到双相支架上体外构建组织工程骨软骨复合体，并以此复合体修复犬膝关节股骨髁负重区骨软骨缺损，分为细胞-双相支架组(实验组)和单纯支架组（对照组），分别在3和6个月时取材，根据大体、组织学、生物力学、生物化学、Micro-CT等检测结果进行半定量或定量评估。 结果 支架评估：组织学显示双层支架去细胞彻底,无细胞碎片残留；CECM部分番红O及Ⅱ型胶原免疫荧光染色呈阳性。扫描电镜及Micro-CT观察显示支架内孔洞相互贯通呈海绵状，CECM部分孔径(155±34）μm，孔隙率为91.3%±2%；ACBM部分具有天然骨的孔径和空隙率，骨软骨部分结合良好。MTT法显示，不同浓度支架浸提液与对照培养液吸光度值比较，差异无统计学意义（P>0.05）。在修复犬膝关节骨软骨缺损的实验中，结果显示两组以纤维软骨或透明软骨对缺损有不同的修复，大体以及组织学评分表明实验组明显优于对照组，生物力学测试表明6月实验组修复软骨的刚度为正常膝关节软骨的70.1%，与生物化学分析结果一致；软骨下骨的刚度达到正常膝关节的74.96%。Micro-CT结果表明实验组与对照组软骨下骨重建不具备明显差异。结论 CECM /ACBM骨软骨双层支架保留了骨、软骨的细胞外基质成分，具备良好的孔径和孔隙率，骨-软骨两层间结合良好，无毒，生物相容性良好，可作为支架载体用于组织工程骨软骨复合体的构建。骨软骨双相支架复合成软骨诱导的BMSCs能成功修复犬膝关节负重区的骨软骨缺损，

COA:Evaluation of an ECM-derived acellular matrix biphasic scaffold/cell construct in repair of large articular high-load-bearing osteochondral defects in a canine model

Background osteochondral lesion repair is challenging in orthopedic surgery. The objective of this study was to develop an extracellular matrix-derived, integrated biphasic scaffold and to investigate the regeneration potential of the scaffold loaded with chondrogenically induced bone marrow-derived mesenchymal stem cells (BMSCs) in the repair of large, high-load-bearing osteochondral defects in a canine model. Methods The biphasic scaffolds were fabricated by combining a decellularization procedure with a simple freeze-drying technique and characterized by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). osteochondral constructs were tissue-engineered in vitro using chondrogenically induced BMSCs and biphasic scaffold, and assessed by SEM for cell attachment. Osteochondral defects (4.2-mm diameter ×6-mm deep) were created in femoral condyles. The defects were treated with constructs of a biphasic scaffold with chondrogenically induced BMSCs or cell-free scaffolds (control group). The repaired defects were evaluated for gross morphology and by histological, biochemical, biomechanical and micro-CT analysis at 3 and 6 months post-implantation. Results Most of the defects of the experimental group showed good osteochondral repair, and macroscopic and histologic scores were superior to those of control group. Micro-CT analysis of the subchondral bone showed mature trabecular bone regularly formed at both 3 and 6 months, with no significant difference between the experimental and control groups. Quantification of glycosaminoglycan of the repaired cartilage and biomechanical evaluation of the cartilage and subchondral bone at 6 months gave slightly lower values than those for normal osteochondral tissue. Conclusion The extracellular matrix-derived, integrated biphasic scaffold has high potential in repair of large, high-load-bearing osteochondral defects.