基于关节软骨多元耦合仿生的分层制备及其在多参数调控动态环境中有效构建的协同作用机制

The repairing idea about the articular cartilage defects has changed gradually from the "Cartilage-bone" to the "bone-bone" integrated interface, due to the gradient gradualness and complex physiological characteristics of hierarchical structure of articular cartilage. Therefore, the efficient constructing and integrating all physical layers of articular cartilage has become the key problem to be solved urgently. This study will give a multivariate coupling bionics and preparation of all physical layers of articular cartilage and create the dynamic bioreactor systems (PE-DPB and PE-RWVB) for constructing all physical layers. The dynamic pressure module with the function of periodic pressure and mass transfer enhancement will be designed and created. And the module will be optimized, based on its influence on the flow distribution, shear force, dynamic pressure changes and cytokine transfer efficiency according to the simulation and experiment analysis. The effective scale and appropriate additive amount of derived bone powder in the C/GP/Co/Bp gradient hydrogel scaffolds will be defined. Meanwhile, the composite proportion of gradient hydrogel, changes of surface and internal structure and the relationship between the release efficiency of factors such as TGF-β2 and the dynamic environment in PE-DPB will also be analyzed. On this basis, these parameters such as dynamic pressure pulse electromagnetic field of PE-DPB, gradient hydrogel scaffolds, hypoxia and low pH environment will be integrated. The synergy for the construction of engineered hyaline cartilage (including calcified cartilage zone) caused by these parameters will be considered and studied synthetically.

关节软骨因其分层结构的梯度渐进性和复杂生理特性，其损伤的修复思路逐渐从“软骨-骨”向“骨-骨”的整合界面转移。如何能体外有效构建并整合其各生理分层，是亟待解决的关键问题。本项目拟多元耦合仿生制备关节软骨各生理分层和用于各分层动态构建的生物反应器系统(PE-DPB和PE-RWVB)。设计、优化和制备起周期性加压和强化传质作用的动压器组件，从模拟和实验两方面综合分析该动压器对软骨培养槽的流场分布、剪切力和动压变化及细胞因子传递效率的影响。界定C/GP/Co/Bp梯度水凝胶支架中衍生骨粉的有效尺度和适宜添加量。分析梯度水凝胶的复合比例、表面和内部结构变化及所处PE-DPB动态环境与TGF-β2等因子释放效率的关系。在此基础上，通过试验设计探究PE-DPB的动压脉冲电磁场、梯度水凝胶支架、低氧和低pH环境等参数整合作用对工程化透明软骨(含软骨钙化层)组织有效构建的协同作用机制。

关节软骨因其分层结构的梯度渐进性和复杂生理特性，其损伤的修复思路逐渐从“软骨-骨”向“骨-骨”的整合界面转移。如何能体外有效构建并整合其各生理分层，是亟待解决的关键问题。本项目基于多元耦合理念通过迭代分层法制备了Cs/GP/Co(Gel)/Bp(nHAP)复合梯度支架，采用系列脱细胞、脱蛋白、脱钙、脱脂等处理猪源股骨构建了具有良好生物相容性和骨诱导能力的衍生支架；通过设计、优化和制备起周期性电磁刺激的电磁场及起周期性加压和强化传质作用的动压器组件，制备了用于各分层动态构建的生物反应器系统，PE-DPB和PE-RWVB；并通过模拟和实验综合分析了动态流场和动压器等对骨/软骨培养槽的流场分布、剪切力和动压变化及细胞因子传递效率的影响。研究表明，PE-RWVB为60rpm，24h/d培养细胞14 d后，ADSCs扩增为静态的1.56±0.04倍；动态环境结合EMF(频率为75Hz，电磁强度为1mT)探究了对ADSCs的刺激作用，发现EMF处理后的细胞分别扩增2.29±0.06倍(DMEM)和1.59±0.05倍(OM)；当Co(Gel):Cs为7:3时的梯度支架具有良好的孔隙率(86.07±2.06%)、吸水率(1130.05±15.79%)和压缩模量(0.46±0.05MPa)；Cs3/GP/Co(Gel)7/nHAP仿生梯度支架上ADSCs粘附良好，能分泌更多细胞外基质；PE-DPB动态条件为60rpm、24h/d处理细胞14d后，和静态相比，细胞能扩增1.1±0.07倍；动态环境促进Col II扩增1.19±0.05倍，糖胺聚糖扩增1.12±0.02倍；结合EMF(频率为75Hz, 电磁强度为2mT)处理细胞，细胞扩增1.47±0.05倍(DMEM)，Col II基因表达扩增7.14±0.33倍，Col X基因表达缩减1.68±0.06倍；通过系列试验完成了PE-DPB三维动压脉冲电磁场、梯度水凝胶支架、低氧和低pH等对工程化透明软骨组织有效构建的协同作用机制探究。将分别构建的透明软骨/软骨下骨组织进行复合构建，并使用此复合构建组织成功完成了动物关节软骨损伤的修复。