基于3D多孔支架材料PGS/PLLA共培养体系构建组织工程化颞下颌关节盘的实验研究

In clinic, up to 70% of the temporomandibular joint (TMJ) diseases is involved in pathological changes of the articular disc. Since disc cells cannot self-repair after damage and current treatment cannot effectively promote the recovery of the diseased disc eventually leading to progressive destruction of joint tissue and occurrence of osteoarthritis, it is crucial to promote and sustain the long-term regeneration of the TMJ disc cells. Scaffoldless self-assembling technique is a method of constructing in vitro tissue based on the exogenous biochemical and biomechanical stimulation to repair the damaged articular disc. Its bottleneck is the construct contraction at present, which leads to the decrease of cell viability and matrix formation in the central zone of the construct. Our previous study has found that high expression of alpha smooth muscle protein and changes of cytoskeleton integrity are involved in the construct contraction. In our preliminary experiments, we observed that poly(glycerol sebacate) / poly L lactic acid (PGS/PLLA) scaffold could improve cell adhesion and matrix strength. So we speculate that application of porous scaffold with good adhesion and strong support to build the co-culture system of BMSCs and disc cells with the help of the nutritional function of BMSCs may be an effective strategy to solve the problem of construct contraction. In this study, ice crystal method and freeze-drying technology will be employed to fabricate a controllable porous three-dimensional PGS/PLLA scaffold and assembly an engineering construct composed of BMSCs and disc cells so as to observe their biological and biomechanical behaviors from tissue, cell and molecule levels, and to obtain optimized conditions for long-term growth and forming cartilage-like structure of the construct. Our ultimate goal is to reveal the in vitro assembly rule of the TMJ disc replacement and lay down basis for its in vivo transplantation.

临床高达70%的颞下颌关节疾病与关节盘的病变有关。由于关节盘细胞损伤后难以自我修复，最终导致关节盘组织破坏，故促进关节盘细胞再生十分重要。无支架自组装技术是基于外源性生化与生物力刺激，通过体外构建组织修复受损关节盘的方法。但无支架培养的瓶颈问题是基体收缩。课题组前期研究发现，α平滑肌蛋白高表达和细胞骨架完整性改变与基体收缩有关，预实验中观察到聚癸二酸丙三醇酯/左旋聚乳酸（PGS/PLLA）支架能改善细胞粘附和组装基体的强度。推测利用粘附性好支持力强的多孔支架，借助BMSCs的营养作用构建与关节盘细胞的共培养体系，可能是解决基体收缩的有效机制。课题将采用冰晶冷冻干燥技术制备三维PGS/PLLA多孔可控支架，组装BMSCs和关节盘细胞基体，从组织-细胞-分子三个层次观察其生物学和力学行为，探索出基体长期生长和成类软骨组织的最佳条件，为揭示关节盘置换体的体外组装规律和进行体内移植奠定基础。

TMJ关节盘是颞下颌关节疾病临床诊治的关键。关节盘细胞损伤后难以自我修复，故研究关节盘细胞与组织的再生在TMJ组织修复和类器官研究中至关重要。本课题在探索克服前期研究自组装基体收缩机制的基础上，设计和制备了3D-PGS/PLLA多孔支架材料并行性能表征，利用孔径控制、氧等离子和聚多巴胺改性改善支架性能，并与山羊BMSCs和TMJ关节盘细胞的共培养体系构建组装基体。研究结果发现：2µmol/L的细胞松弛素B、0.1～1nM的星孢菌素可干预山羊TMJ关节盘细胞增殖和细胞骨架代谢，调控细胞形态，影响关节盘自组装基体的构建；2%、4%、8%单轴拉伸刺激会影响TMJ关节盘细胞和细胞骨架蛋白的形态；山羊原生TMJ关节盘主要生化成分中水含量为77.04%，胶原、PGs、GAGs含量分别为3.01μg/ml、0.93μg/ml和1.73μg/ml；在4%O2和8%O2培养条件下，山羊TMJ关节盘细胞表现出较21%常O2更好的活力，葡萄糖转运体（GLUT-1）和AMP依赖蛋白激酶（AMPKα1）的表达依赖于葡萄糖浓度；血清剥夺会诱导细胞发生凋亡和明显的自噬现象；联合应用10ng/ml TGF-β1、500ng/ml BMP-2可对山羊BMSCs进行良好的干性维持和向纤维软骨样分化诱导，经诱导的自组装基体同时具有良好的抗压特性，证实共培养体系是实现关节盘细胞有效增殖的最佳途径；利用静电纺丝技术、冷冻结晶法、盐析法及3D打印均可制备PGS与PLLA复合多孔支架材料，优化质量比为2.5:1～3:1，不同孔径的PGS支架材料对山羊TMJ关节盘细胞增殖有一定影响，采用孔径控制、氧等离子处理和接枝聚多巴胺和胶原，可显著增强PGS/PLLA 3D多孔支架支架材料的强度和细胞粘附性。以上研究结果为揭示TMJ关节盘体外组装规律和进行体内移植奠定了必要的基础。