基于iPS细胞的种植体周角化粘膜组织工程化重建研究

An adequate band of periimplant keratinized mucosa is crucial for biological seal around implants and the long-term success of implant therapy. Three-dimensional bioengineered soft tissue graft (BSTG) has become a research focus in recent years for oral soft tissue reconstruction. However, a BSTG based on the four-dimensional theory with optimal angiogenesis, re-epithelization, re-keratinization, and sealing effects has not been reported. In the previous studies, we found dual delivery of bFGF and KGF can improve angiogenesis during wound healing and we fabricated coaxial electrospinning materials which proved to be a new strategy to construct scaffolds with spaciotemporal release potential of multiple bioactive factors. Our previous studies focusing on induced pluripotent stem cells(iPSCs) also showed that iPSCs were promising seeding cells in soft tissue engineering. Therefore, in the view of spaciotemporal release of bFGF and KGF for the first time, a new BSTG seeded with iPSCs will be constructed. The biological characteristics of the BSTG will be evaluated in vitro by release kinetics of growth factors, identification of angiogenetic markers and keratinization markers. Animals models without periimplant keratinized mucosa will be established and grafted with the BSTG to evaluate its augmentation efficiency. Laser speckle imaging will be used to quantify its angiogenetic potential. Furthermore, the essential genes and signaling pathways regarding bFGF-KGF-induced angiogenesis, re-epitheliulization, re-keratinization will be studied. The results of this program will complement the present theory of implantation, and will be of significance in improving dental implant success.

良好的种植体周角化粘膜质量对于种植体生物封闭及远期效果至关重要。组织工程构建的三维结构粘膜已成为口腔软组织重建热点，四维模式下生物模拟粘膜时序性愈合从而提升移植物成血管、成角化上皮、促种植体生物封闭的研究仍未见报道。本课题组前期研究发现，bFGF-KGF联合使用能协同促进血管化；前期制备的同轴静电纺丝壳-核支架可实现多因子时序性缓释；而课题组围绕诱导性多能干细胞(iPSCs)的相关研究揭示了其用于口腔软组织重建的潜能。基于此，本课题首次以同轴电纺时序性缓释双因子为切入点，拟构建一类基于iPSCs的组织工程角化粘膜，通过生长因子释放动力学、结合血管化角化标志物鉴定等手段评价其体外生物学性能；构建犬种植体周粘膜缺损模型、采用激光散斑成像等技术评价材料促进种植体周软组织扩增的效果；并探究双因子介导下粘膜愈合的分子转导机制。研究结果对进一步完善种植学理论，提高牙种植体成功率具有重要意义。

良好的种植体周角化粘膜质量对于种植体生物封闭及远期效果至关重要。组织工程构建的三维结构粘膜已成为口腔软组织重建热点。组织工程化角化粘膜移植物的构建涉及种子细胞、支架材料以及生长因子。在种子细胞的选择上，iPSC诱导形成的iPSC-MSC细胞具有成角化和成血管潜能，可以作为构建种植体周围角化粘膜的理想种子细胞来源。在支架材料的设计上，材料结构对于细胞的生物学行为、宿主免疫反应有重要影响，课题组构建了随机组、取向组和网格组三种结构的支架材料。通过体外、体内试验评估了材料的理化性能、细胞毒性、对软组织再生的促进效果、对宿主免疫反应的调控作用，得出取向组单轴纳米纤维电纺材料相比其他形貌组有更佳的促进皮肤软组织创口愈合效果，同时具有调控周围免疫微环境的能力。课题组进一步通过高通量单细胞测序、蛋白组测序探究了取向组材料调控周围免疫微环境的机制，材料对于免疫微环境的调控涉及对固有免疫和适应性免疫过程的影响，取向组相比对照组在术后修复期（术后7天）有更低的固有免疫细胞数量（巨噬细胞等）、更高的效应T细胞数量，提示材料促进局部免疫进程向适应性免疫推进。免疫细胞之间、免疫细胞-组织细胞之间涉及复杂的相互作用信号网，组织修复由多种信号共同影响。课题组在取向型单轴纳米纤维电纺膜作为支架材料、iPSC-MSC细胞作为种子细胞的基础上，进一步负载不同生长因子（KGF组、bFGF组、KGF+bFGF组）。体外构建具有三种不同生长因子的组织工程化软组织移植物，并将其运用于小鼠模型，bFGF组实现了更快的表皮覆盖、促进新生血管形成和角化的效果。.取向型单轴纳米纤维电纺膜作为支架材料、iPSC-MSC细胞作为种子细胞、bFGF作为生长因子，共同构建的组织工程化移植物可能有潜力应用于软组织修复、为临床使用的生物材料的设计和选择提供参考。