应用三层一体化高仿生支架修复关节软骨缺损的实验研究

Recently, application of biphasic monolithic scaffolds in cartilage defects repairing achieved good results. It could well resolve the problems during the tissue engineering scaffolds transplantation, such as difficulty in fixing, interface involution and slowly healed interface. And some disadvantages exist. For instance, this method could result in the downward overgrowth of tissue-engineered cartilage, leading to the structural abnormalities of cartilage and subchondral bone, which may ultimately result in abnormally stress transmission of cartilage and bone. In addition,tissue-engineered cartilages will be malnutrition in case they are too thick, which would ultimately cause the degeneration of cartilage and prevent the formation of tidemark region. Theoretically to design an intermediate layer between tissue-engineered cartilage and bone can solve the above problems. We aimed to manufacture a biomimetic three-layered scaffold with silk fibroin(100%) as a cartilage layer, silk fibroin(40%) and β-TCP(60%) as an intermediate layer, and β-TCP(100%) as a bony layer. Firstly, the scaffolds were seeded with autologous chondrocytes on the cartilage layer. And, before the scaffolds were transplanted into the defects, the bony layer were seeded with autologous BMSCs. At last, the scaffolds were embedded into articular defects by press-fitting principle in order to repair and regenerate osteochondral defects.

目前，应用双层复合支架修复关节软骨缺损取得了良好的效果，较好地解决了组织工程软骨移植时固定困难、愈合慢、界面整合不良、移植物易脱落等问题。但人们又发现，此时组织工程软骨容易向软骨下骨过度生长，导致软骨及软骨下骨结构异常，且过厚的组织工程软骨会出现局部营养不良，从而导致组织工程软骨退变。理论上，在组织工程软骨与组织工程骨之间设计一个中间过渡层（隔离带）可以解决上述问题。我们拟使用丝素蛋白及β-磷酸三钙（β-TCP）作为支架材料，通过一体化构建法制作三层一体化高仿生支架。其中软骨部分为100%的丝素蛋白（孔径150～200μm，孔隙率90%以上）、骨部分为100%的β-TCP（孔径400～500μm，孔隙率70%以上）、中间过渡层为40%的丝素蛋白和60%β-TCP（孔径30～50μm，孔隙率75%以上）。体外构建组织工程骨软骨复合组织块，最后通过压配原理嵌入动物关节软骨缺损区修复软骨缺损。

目前，应用双层支架修复关节软骨缺损取得了良好的效果，较好地解决了组织工程软骨移植时固定困难、愈合慢、界面整合不良、移植物易脱落等问题。但是人们又发现，此时组织工程软骨容易向软骨下骨过度成长，导致软骨及软骨下骨结构异常，且过厚的组织工程软骨会出现局部营养不良，从而导致组织工程软骨退变。我们应用两次冻干的方法，在双层一体化支架的软骨层与骨层支架之间设计了一条致密的“隔离带”来阻挡软骨向下过度生长。我们使用β-TCP制作骨层支架，然后在其上方应用冻干法以高浓度壳聚糖/明胶溶液为材料制作出致密的隔离带，成型后再于隔离带的上方继续应用冻干法以低浓度的壳聚糖/明胶溶液为材料制作出软骨层。通过两次冻干制作完成三层一体化支架后，使用EDC交联支架。然后，我们在支架的软骨层种植入软骨细胞，经过28天的体外培养后，我们将支架植入山羊骨软骨缺损中，在植入前，我们在骨层支架中种植入骨髓间充质干细胞（BMSCs）。除三层支架外，我们同时还会应用双层支架，自体骨软骨移植术（马赛克手术）对山羊骨软骨缺损进行处理。术后6个月，我们通过大体观察，组织学检测，生物力学测定，免疫组化分析以及生化检测等来对修复的效果进行检验。结果显示，在双层支架组中8只山羊有2只出现了软骨向下过度生长，而三层支架组中8只山羊均未出现软骨向下过度生长，且与马赛克手术相比，其修复效果与之相当。本实验的研究结果证实了三层支架可以有效地阻挡软骨向下过度生长，使得软骨修复效果更加理想化。