异位模拟关节软骨环境构建同种异体组织工程化软骨的研究

Previous study showed that in ectopic cartilage engineering by using allogenous chondrocytes seeded in collagen hydrogel with diffusion chamber technique, immunological tolerance has been achieved during the process of cartilage reconstruction. Successive application of the engineered ectopic cartilage in cartilage repair demonstrated the advantage over engineered cartilage in vitro, indicating the superiority of the strategy in cartilage restoration. However, the engineered ectopic cartilage is far from natural cartilage in some aspects such as biomechanical property, resulting in decreased function of newly formed cartilage compared to native cartilage. This may be attributed to the difference between ectopic site and in situ, predominantly the difference between scaffolds and extracellular matrix (ECM), lack of growth factors facilitating and maintaining cartilage growth, and lack of mechanical load at ectopic site. For the reasons, this study is intended to make improvement by way of simulating the in situ environment of cartilage. Firstly, based upon cartilage ECM, collagen/ hyaluronic acid / chondroitin sulfate compounded hydrogel is to be prepared, with the requirement of improving both biomechanical and biological property. Second, to create the biomimetic environment, it is designed encapsulating growth factors in microcapsules as drug delivery to controlled release growth factors at ectopic site. Third, in vivo dynamic site such as subcutaneous knee, muscle is to be chosen as implantation site to simulate mechanical load. This study is endeavored to create a simulated environment for engineering ectopic cartilage to enhance the function of the artificial cartilage, to eventually provide excellent resources for cartilage repair. It may provide reference for the clinical restoration of cartilage defect.

前期采用胶原凝胶复合异体细胞及扩散盒技术于宿主皮下构建异位组织工程化软骨，在软骨重建过程中可获得一定免疫适应，随后用于软骨修复相比于直接原位修复效果更好，证实该方法用于同种异体软骨修复极具优势。但是，现存的问题是异位构建的软骨与正常软骨相比还有一定差距，如生物力学性能较差。究其原因在于原位与异位的差异，主要体现在细胞外骨架（ECM）与支架材料的差别、在异位缺乏软骨正常功能维持所需的生长因子和力学刺激等，因此本课题就此进行改进：1）支架材料设计以软骨ECM为模板，选取胶原/透明质酸/硫酸软骨素三相复合水凝胶，要求保证其生物相容性的同时提高机械性能；2）采用微载体技术，将生长因子缓释于异位环境，为异位软骨形成创造类似关节软骨的局部环境；3）选择体内其他动态部位，膝部皮下、肌肉，提供体内力学刺激，以增强组织工程化软骨的功能性。该研究对于异体软骨组织工程的临床应用，实现软骨缺损的修复极具参考价值。

本课题以软骨细胞外基质为模板，选取其主要成分胶原/透明质酸/硫酸软骨素作为组织工程支架材料组分，制备三相复合水凝胶，确保支架材料的结构和成分更接近软骨基质。随后，采用扩散盒技术将复合软骨细胞的三相水凝胶接种于具有免疫性的动物背部皮下、膝部皮下及腿部肌肉等动态部位提供体内力学刺激，同时利用微载体技术将转化生长因子TGF-β1微囊注射于异位环境，为异位软骨形成创造类似关节软骨的局部环境，所获得的具有免疫适应性的工程化组织能更有效地修复关节软骨缺损。本课题将软骨组织工程支架材料的仿生化设计与异位构建相结合，获得了更具功能性的同种异体组织工程化软骨，更有利于关节软骨缺损的修复和再生，为异体软骨组织的临床应用以及软骨缺损的功能性修复提供了重要的参考价值。