具有自主诱导特性的可降解材料及骨髓基质干细胞在其构建的三维支架上区域诱导分化的研究

The general methods to fulfil the aim of inducing cell differentiation on scaffold in the joint regeneration research are mainly focus on using growth factors directly or sustain releasing. However, these growth factors administration methods result in side effects on the cells which don't need the diffusing factors without any control. It's necessary to control the factors function in a constrained spatial area to ensure the tissue's correct differentiation direction. Until now, no research is focus on this. In this proposal, we are going to research the basic principal towards adhesion and enrichment of growth factors on material surface based on the specific interactions between the factor and their affinity peptide on surface. The anti-protein nonspecific adhesion on material surface, bioactivity maintaining mechanism of the adhesive factors, and the adhesive factor inductive mechanism proposed by that through a key signal transduction pathway activation to mediate the gene expression related to cell differentiation, will be discussed. The basic design principal and the inductive mechanism of the self-inductive biomaterial will be interpreted. This will be useful to enrich the growth factors in the low concentration medium or tissue environments on material surface to induce the cells on it differentiating to the right direction avoiding the side effects of the diffusing factors. Combining with the additive materials fabrication method, we are going to make the porous scaffold which has the zonal inductive property. On that scaffold, the zonal induction function of the growth factors will be demonstrated and the mechanism will be interpreted by inducing the bone mesenchymal cells differentiate to chondrocytes in the specific spatial area of the scaffold, in vitro, in site and ectopically. Through this research, the basic design principal of the joint regeneration will be established and the main key problem of deficiency of the idea scaffold in the joint regeneration research field will be solved either.

采用生长因子直接添加或缓释的方法是当前关节再生研究解决在同一支架上软骨与骨诱导分化的主要方法。该方法难以避免因子的扩散对不需要该因子的细胞潜在的负面影响，需要限定生长因子的效应空间来诱导特定组织的准确分化，目前缺乏相关的研究。 本项目研究材料分子结构上生长因子的特异性结合位点对因子吸附与富集的基础原理，通过结合材料表面抗蛋白非特异性吸附，生长因子空间活性构象维持机制的探索，结合富集的因子对细胞分化相关信号通路刺激并介导关键基因的表达这一诱导分化机制的探索，阐明自主诱导材料的设计原理及诱导机理。实现因子在低浓度无效应环境中，通过材料的富集调控并诱导的细胞分化，避开因子扩散的影响。采用三维打印技术构建基于该材料的骨软骨诱导分化区域控制三维支架，并进行体外诱导分化、体内原位修复及异位再生研究，阐明支架材料的区域诱导分化控制机制。明确特定区域内准确诱导细胞分化调控的关节再生三维支架的设计原理。

生长因子的使用是体内外进行组织再生诱导的重要技术手段，限制生长因子扩散导致的对周边其他细胞额外的作用将对组织的再生构建具有重要的作用。天然细胞外基质对生长因子具有锚定功能，在一定程度上限定了其空间扩散，并对粘附在基质上细胞的生物学功能进行调控。本研究通过在材料表面结合特定生长因子亲和肽，模拟细胞外基质的作用，限定生长因子的空间效应，实现在生物材料表面特异性结合的生长因子与细胞膜表面受体直接相互作用，调控细胞功能。研究以以TGF-β为模型分子，筛选了三种亲和肽分子，并对其进行叠氮化修饰；同时合成出了炔基含量范围为0.18- 1.02mmol/g 的可降解聚氨酯材料；优选Cu2+/抗坏血栓钠催化体系在55℃时，叠氮亲和肽与炔基的点击最高接枝率超过90%；材料表面亲和肽能够与TGF-β实现特异性结合，亲和肽减少了TGF-β从材料表面的脱吸附，同时使生长因子在材料表面保持更松散的天然结构，杂蛋白存在不影响该特异性结合；合成的材料通过3D打印制备出了多孔支架，该支架同样具有表面富集多肽的效果；细胞实验表明，通过亲和肽系留在材料表面的TGF-β能够通过与细胞膜表面受体相结合从而促进细胞表达Acan、Sox9以及Col2等特异性软骨基因；受限于3D打印设备在打印精度上的限制，系留TGF-β的层区诱导实验还在进行之中，但系留在材料表面的TGF-β成功抑制了早期大鼠肌肉植入时的炎症反应并促进大鼠肌细胞的早期增殖，且其生物学效应与其表面系留浓度正相关。相关研究表明通过表面亲和肽的特异性结合，生长因子能够在材料表面实现其生物学功能，从而大大降低生长因子扩散导致的非特异性诱导结果。该研究结果对未来设计适于组织精确结构再生的多孔支架材料具有重要的意义。