基于细胞外基质的结构特征仿生构建软骨基质及其促进软骨缺损修复的研究

Based on the requirements of cartilage repair and the structure and function of extracellular matrix(ECM), we conceived a unique designing approach of how to biomimetically integrate TGF-β1 growth factor on tissue engineering scaffold able to sequester and deliver in the sustained release for cartilage repair. The main researches include preparing collagen matrix integrated with TGF-β1-loaded PLGA micro-nanoparticles, conjugating heparin both within PLGA particles and on the collagen matrix to double stable the TGF-β1. The research focus is on the bioactive stability and release dynamics of the scaffold in vitro, and the effect of sizes and dosage of particles integrated with collagen matrix on proliferation of seeded chondrocytes in vitro and enhancement of cartilage healing in vivo. The biomimetic matrices are not only used for cartilage tissue engineering, but also for tissue regeneration therapy. The research will promote the progress of tissue engineering, regenerative medicine and drug delivery technology, and lay a foundation for the clinical application of tissue engineering scaffolds...Joint pain and osteoarthritis due to cartilage damage are the commonest forms of impairment in middle and older people. Cartilage undergoes limited spontaneous repair owing to lack of the blood supply.The interest in cartilage repair is growing worldwide, with particular focus on tissue engineering and cell-based therapies. It appears likely that controlled release of stimulatory growth factors as molecular signals will be necessary. Cells receive numerous signals from their ECM. Within a biomechanical context provided by this elastic milieu, cells adhere by receptor-mediated interactions with ECM components, mediated by specialized adhesion receptors such as integrins and others. These receptors transmit stress from the ECM, through the membrane to the cytoskeleton within the cell in a dynamic and concerted manner. In addition to adhesion proteins, the ECM also sequesters and presents a number of morphoregulatory molecules including growth factors, which control processes of cell division, differentiation, and multicellular morphogenesis. The growth factors bind ECM components such as heparan sulfate, and reside there until released by enzymatic processes or dissociation. These characteristics of the ECM guide the design of the matrices for cartilage repair in this study.

针对软骨缺损难以自行修复和现有临床治疗方案的缺陷与不足，以及细胞外基质的结构、组成和功能，本项目提出了独特的研究思路以仿生构建软骨修复基质，即在胶原基质中引入转化生长因子TGF-β1作为信号分子，使其处于俘获状态及在应用时持续释放。主要研究内容包括：将TGF-β1经聚乳酸-乙醇酸包埋后复合到胶原基质上，制成仿生软骨基质或支架；在包埋的粒子内部键合肝素成分，以稳定生长因子的活性；并在胶原基质上引入肝素点位，以捕捉从粒子内部扩散出的生长因子，达到活性双重稳定的效果。重点研究生长因子的体外活性稳定性和释放动力学，以及不同粒径范围的粒子和剂量对促进软骨细胞增殖和软骨缺损修复的影响。期望该仿生软骨基质既可用于软骨组织工程研究，又可在软骨缺损组织再生治疗上发挥作用。本项目研究将推动组织工程、再生医学和药物控释技术的发展，为组织工程支架材料的临床应用研究打下基础。

对聚乳酸羟基乙酸（PLGA）进行改性处理，使其接枝氨基以利于肝素的结合，进而加强对细胞生长因子的吸附能力。通过溶剂挥发法，以转化生长因子TGF-beta1为缓释药物制备了PLGA粒子，将其与胶原基质进一步复合可以得到包埋生长因子的软骨基质或支架。理化性能检测验证了胶原基质、粒子和软骨基质作为生长因子控制释放载体的可行性。.以顺丁烯二酸酐和PLGA为原料制得改性聚乳酸羟基乙酸(MPLGA)；再以MPLGA和乙二胺为原料进一步改性制备乙二胺改性聚乳酸羟基乙酸(EMPLGA)，并通过对PLGA接枝氨基的方法以接枝肝素进而增强PLGA对生长因子的吸附作用，同时利用接枝乙二胺克服聚乳酸羟基乙酸及其降解产物的酸性问题。利用茚三酮反应、FT-IR和DSC对PLGA、MPLGA和EMPLGA进行全面的比较和表征。粒子的稳定性实验主要考察4℃低温实验条件下粒子外观形貌、载药量、突释率、包封率、体外释放行为来评价。实验结果表明：缓释粒子样品经过4℃低温留样实验后粒子的外观形貌、平均粒径、载药量、突释率、包封率、粒径分布、体外释放行为也没有发生明显的变化。缓释粒子的活性检测实验表明能够在一定程度上保持其生物活性，可在体外可释出具有生物活性的生长因子，并可以达到有效生理浓度以促进细胞的分裂与增殖。.采用酶解法从牛跟腱中提取胶原，通过醋酸溶解制成均匀的胶原凝胶，经冷冻干燥制成胶原海绵。运用碳化二亚胺和羟基琥珀酰亚胺（EDC/NHS）交联法对胶原海绵改性加工以接枝肝素，制备胶原基质，提高吸附生长因子的能力。将缓释粒子与胶原基质进一步复合以仿生构建包埋活性粒子的软骨基质或支架，并进一步研究体外释放情况。该软骨基质具有细胞外基质的特征，期望在将来的实际应用时具有细胞外基质的功能。实验结果表明软骨基质能够有效地缓解药物释放过程中的突释问题，并使突释率由19.8%降至4％。且体外释放药物浓度比较稳定，药效缓释时间更加长。同时，胶原软骨基质的研究为将来的体内软骨修复研究或组织工程支架材料的研制提供实验依据。