新型梯度人工软骨的构建及其力学性质影响软骨细胞行为的机制研究

Artificial cartilage material has good prospects in the repair of cartilage defects. Polyvinyl alcohol - nano-hydroxyapatite hydrogel, one of such materials, has quite good biomechanical behavior. But it could not be fully integrated with the surrounding cartilage. Previous studies have shown that the mechanical properties of the scaffold material can affect the adhesion and proliferation ability of chondrocyte, while the mechanism remain unknown. Our previous studies have found that poly-lactic glycolic acid-Polyvinyl alcohol-nano-hydroxyapatite hydrogel has a high porosity and a controllable elastic modulus. In this study, A series of the composite hydrogel with different mechanical properties will be prepared, then co-cultured with chondrocyte to ascertain the best cell-mechanics environment for its adhesion and proliferation. We expect to explicit the mechanism of its affection of chondrocyte behaviors, and explore the mechanical - biological coupling relations and regulatory pathways between the scaffold materials - extracellular matrix and cells. Furthermore, we proposed a method for designing of a novel gradient artificial cartilage, for which the central zone meets the biomechanical requirements which can completely replace the cartilage, and surrounding zone meets the cell-mechanical requirements which can integration with chondrocytes. The purpose of our research is to construct such new kind of artificial cartilage, to study the whole biomechanical properties as well as the ability to integrate with chondrocytes, then provide basis for the bionic design of artificial cartilage material.

人工软骨材料在修复软骨缺损的多种方案中具有良好的前景，其中聚乙烯醇-纳米羟基磷灰石水凝胶生物力学性能良好，但存在不能和周围软骨完全整合的问题。前人的研究表明支架材料的力学性质可影响软骨细胞粘附、增殖，但其机制不明。我们的前期研究发现聚乳酸-羟基乙酸共聚物-聚乙烯醇-纳米羟基磷灰石水凝胶孔隙率高且弹性模量可控。本研究拟通过制备一系列不同力学性质的复合水凝胶诱导软骨细胞，确定其粘附、增殖的最佳细胞力学环境。研究其影响软骨细胞行为的机制，探索支架材料-细胞外基质-细胞间的力学耦合关系及力学-生物学调控途径。同时本课题组提出一种新型的梯度人工软骨设计方案- - 其中央区符合生物力学要求能完全取代软骨功能，周围区符合细胞力学要求能与软骨细胞整合。拟实际构建出此种新型人工软骨，研究其整体生物力学性能以及和软骨细胞的整合能力，从而为仿生设计人工软骨材料提供依据。

如何有效的修复关节软骨缺损始终是医学界尚待解决的难题之一。结合机械共混羟基磷灰石（HA）、乳化溶剂挥发法制备聚乳酸‐羟基乙酸共聚物（PLGA）微球、冻融法制备物理交联的聚乙烯醇（PVA）水凝胶的方法制备出PLGA-HA-PVA水凝胶多孔支架。对其进行表征显示所制备PLGA-HA-PVA水凝胶多孔支架内部有明显的多孔结构，孔隙由几十微米至一百多微米数量级，平均孔径随PVA含量的增加而降低，随着PVA含量的增加，水凝胶支架的孔隙率下降。将PLGA-HA-PVA水凝胶多孔支架进行体内外成软骨研究。体外实验测定细胞粘附率，不同时间点对各组材料进行HE染色、甲苯胺蓝染色、II型胶原免疫组织化学染色分析细胞的增殖情况并采用Wenstern-blot法检测糖胺聚糖和II型胶原蛋白的表达。同时， PLGA-HA-PVA与软骨细胞体外混合培养后植入裸鼠背部皮下；分别于不同时间点取出标本，分析其成软骨情况。体内外实验表明配比为30wt%PLGA，5wt%HA， 15wt%PVA的PLGA-HA-PVA水凝胶支架的水凝胶多孔支架更适合软骨修复。然后采用二步成形法成功制备出PLGA-HA-PVA支架修饰的羟基磷灰石-聚乙烯醇（HA-PVA）人工软骨，其核心为HA-PVA水凝胶（配比为5wt%HA、20wt%PVA），周缘为PLGA-HA-PVA支架（配比为30wt%PLGA、5wt%HA、15wt%PVA）。进行显微结构及生物力学检测，结果显示所制备出的新型人工软骨为一种典型的粘弹性材料，表现出和软骨类似的生物力学性质，对压缩应力的敏感性高于拉伸应力。同时，进一步探讨软骨损伤的机制研究，ELISA检测关节软骨及滑液内HIF‑1α，结果显示软骨组织与滑液内HIF‑1α水平与骨关节炎严重程度正相关，且软骨组织与滑液内HIF‑1α水平具有正相关性。分离膝关节骨关节炎患者及正常成人软骨细胞，通过不同干预后real-time PCR及Western blotting分别检测关节软骨内microRNA-30a及其靶基因Sox9 mRNA的表达，显示骨关节炎患者软骨细胞内MicroRNA-30a表达上调，其靶基因Sox9表达下调；过表达MicroRNA-30a时引起Sox9基因、糖胺聚糖及II型胶原蛋白表达下调，加入MicroRNA-30a的抑制剂能有效抑制ECM的降解。