透明质酸基多功能水凝胶的构建及其髓核/纤维环一体化再生修复研究

Lumbar degenerative disease caused by the imbalance of extracellular matrix synthesis and degradation is a clinical common disease. Current, the existing clinical treatments can not effectively prevent or reverse the degeneration of the lumbar intervertebral disc, and eventually, the intervertebral fusion has to be performed, which results in restricted lumbar spinal motion and degeneration of adjacent segment. Therefore, in view of the pathogenesis and clinical treatment requirements for lumbar disc degeneration, this project aim to design new multifunctional hyaluronic acid based hydrogels to regenerative repair the nucleus pulposus/annulus fibrosus simultaneously. Through reconstruct the physiological structure and biomechanical function of the lumbar intervertebral disc to prevent and reverse its degeneration. Hyaluronic acid hydrogel system with good biocompatible, injectable, interface bonding and good mechanical properties will be synthesized by "click chemistry". The relationship between the structure and properties of hydrogels will be studied, and the effects of hydrogels on cell activity and behavior will be investigated. Through the Schiff-base reaction, the final integrated regeneration and repair of nucleus pulposus/nucleus fibrous will be achieved. And then we elucidate its mechanism of effective plugging and resistance to the transmission of nucleus pulposus. Through in vitro degenerative model and animal experiments, we explore the biomechanical and molecular mechanisms of hydrogel reverse the degeneration of nucleus pulposus and promote the repair of annulus fibrosus. All these work will provide experimental foundation and theoretical basis to design and develop novel nucleus pulpous/annulus brosus integrated material with regenerative and restored function, and is expected to achieve a new breakthrough of clinical treatment methods of lumbar intervertebral degeneration.

腰椎间盘退行性疾病是临床常见疾病，其产生的根本原因是髓核细胞外基质合成与降解失衡。现有治疗方式均不能有效阻止或逆转其进程，最终不得不采取椎间融合术，导致患者腰椎运动受限及临近节段退变等。因此，本项目从腰椎间盘退变机制和临床治疗需求出发，以新型透明质酸基多功能水凝胶重建髓核力学及生物学微环境，阻止和逆转退变进程，实现腰椎间盘一体化再生修复。采用“点击化学”构建具有生物相容、可注射、界面结合和良好力学性能的透明质酸基水凝胶体系，研究水凝胶分子结构与性能之间的关系，并探讨其对细胞活性及行为的影响规律；通过席夫碱反应实现髓核/纤维环一体化再生修复设计，阐明其对纤维环有效封堵及抵抗髓核传递压力的作用机理；通过体外椎间盘退变模型研究和动物体内实验，揭示水凝胶逆转髓核退变、促进纤维环修复的生物力学及分子生物学机制，为开发新型腰椎间盘退变再生修复材料提供实验基础和理论依据，实现临床治疗方法的新突破。

腰椎间盘（IVD）退变是临床常见疾病，现有治疗方式不能有效阻止或逆转IVD退变。水凝胶能为IVD修复提供接近天然组织的微环境，是一种理想的修复材料，但是材料性能单一、对退变机制了解不足使水凝胶设计难以满足修复IVD的需求。.在本项目研究中，我们通过苯硼酸酯、“点击化学”、自由基聚合和席夫碱等高效反应，结合乳液法、冷冻成胶等方法对水凝胶功能进行设计，获得了可注射、组织粘附、高强度、药物递送等功能各异的凝胶材料，实现了良好的IVD修复效果。此外，本项目结合离体器官模型、基因测序等方法展示了对IVD退变机制的认识。.在髓核（NP）修复方面，我们设计了一系列可注射水凝胶：通过苯硼酸酯和Diels-Alder反应构建的透明质酸水凝胶具多功能性；进一步复合硫酸软骨素，水凝胶获得优异抗炎性能，可抑制NP退变；利用苯硼酸酯反应和光引发自由基聚合，制备了负载EGCG的水凝胶，成功保护髓核细胞免受氧化应激和炎症损伤；同类型反应结合乳液法，制备了负载活性因子的微凝胶组装体，其可募集并促进BMSCs向NP分化，抑制食蟹猴IVD退变；借助酶交联构建的纤维蛋白-透明质酸水凝胶可调节退变人髓核细胞的表型。.在纤维环（AF）修复方面，我们构建了一系列力学性能优异、生物活性突出的修复材料：基于Diels-Alder反应和自由基聚合制备的水凝胶在离体器官中实现了与宿主AF良好结合；利用冷冻成胶、席夫碱反应制备的负载外泌体的丝素冷冻凝胶以及粘附性水凝胶，可封堵AF缺损并抑制退变；复合活性因子和功能性细胞的胶原-聚氨酯支架表现出良好的AF再生潜能。.在机理研究方面，我们通过RNA深度测序获得腰椎间盘突出型退变的标志性microRNA，并建立一次性冲击载荷离体器官模型，探究急性创伤型IVD退变机制，阐明了创伤造成的早期IVD退变的转录组变化和基因调控网络。.本项目的研究阐明了水凝胶的功能设计对IVD修复的影响机制，初步揭示IVD退变的部分机制。以上研究成果为开发新型髓核/纤维环一体化再生修复材料提供实验基础和理论依据。