基于细胞外基质的生物仿生型凝胶研究及作用机理探讨

Hydrogel based injectable bone tissue engineering(IBTE) strategy possesses great clinical potential due to its advantages such as allowing minimally invasive procedure as so on. However, current available hydrogel system cannot precisely mimick the in vivo extracellular matrix(ECM) microenvironment during bone tissue regeneration as well as they are not suitable for load bearing segmental defect treatment. On the basis of our preliminary research work, we are the first to propose the use of osteogenic ECM (osECM), which is secreted by Mesenchymal Stem Cells under osteogenic differentiation, to construct biomimetic hydrogel system for injectable tissue engineering application. In addition, we will apply this hydrogel for segmental bone defect treatment and investigate the underlying mechanism. ① We will develop the osECM hydrogel system by systematically studying the influence of osteogenic induction condition, hypoxia culture stimulation, decellullarization technologies, fragmentation approach and so on. ② With the integrated use of scaffold implantation and guided bone regeneration membrane technology, we will explore the potential use of this hydrogel system for segmental bone defect treatment as well as tissue engineering bone graft (TEBG) in vivo, in situ construction strategy. ③ We will meticulously analyze the essential bioactive components in the osECM using high-throughput microarray and protein array technologies, and further validate the function of bioactive components and investigate their synergetic effect and interactions by RNA interference technology. The development of osECM hydrogel system will facilitate the clinical translation of IBTE and reform the current TEBG construction strategy, because of its biomimetic potential, safety, efficacy, manufacturing scalability and so on. The underlying mechanism study of osECM bioactivity will help to illuminate the understanding of the effective components of the native bone regeneration microenvironment and their subtle interaction in vivo.

基于凝胶的注射型组织工程骨因微创等优点临床前景广阔，但现有凝胶技术存在无法精确模拟适宜骨再生的细胞外基质（ECM）生理微环境（活性因子组成、释放规律等），和不适于负重的节段性骨缺损修复的问题。基于预实验，我们首次提出利用间充质干细胞成骨分化后分泌的ECM（osECM）构建仿生型凝胶的策略，用于节段性骨缺损修复，并研究作用机理：①通过研究成骨诱导、低氧刺激条件、及脱细胞、碎片化方法等，建立osECM凝胶的制备方法；②通过结合支架材料移植和引导骨再生膜技术，探索应用该凝胶修复节段性缺损和组织工程骨体内原位构建的新策略；③使用高通量基因、蛋白芯片分析osECM中关键活性因子，通过RNA干扰技术验证活性因子的功能及其间协同作用。osECM凝胶研发因天然仿生、高活性、安全、可量产等特点，可促进组织工程技术临床转化及优化现有工程化组织构建策略；其机理研究有助阐明组织修复生理微环境中活性成份及作用规律。

骨组织工程技术具有广阔的临床应用前景，但现有组织工程骨抑或效果欠佳，抑或构建过程复杂低效，且种子细胞难以获得，极大的阻碍了其临床转化。至于效果欠佳，我们可以考虑组织工程骨的仿生构建；至于工艺复杂，效率低下，我们可以考虑基于“成骨微组织”的骨组织工程策略；针对种子细胞难以获得，我们可以考虑如何提升骨组织工程中种子细胞的使用效率。而这一切，都与本课题的重点研究对象“细胞外基质”密切相关。.本课题首先优化了骨的细胞外基质的仿生制备工艺，并将其与PCL支架结合，构建了具有”纳米-微米-宏观”尺寸层级结构的Ap-Col-PCL复合支架。相比于没有修饰仿生型骨骼细胞外基质的A-PCL支架，Ap-Col-PCL复合支架具有更优的机械性能，更接近天然松质骨的硬度，更加良好的生物相容性和骨缺损修复活性，充分证实了细胞外基质在节段性骨缺损修复过程中的重要性。.此外，我们采用物理剥离与碎片化处理方式构建了 “成骨微组织”，其避免了酶消化，有效保障了细胞外基质的完整性，存在大量的成骨活性物质。同时，其没有外来材料添加，全部成分均由细胞和细胞外基质构成，避免了外来材料的残留。通过免疫蛋白印记实验，发现成骨微组织中的OCN,BMP2,COL1a和DKK1含量均显著高于消化细胞，这一现象可能也是得益于成骨微组织中存在大量的细胞外基质。.在此基础上，我们进一步构建了基于成骨微组织的大段节段性骨缺损原位修复策略。这种策略直接利用体外构建的成骨微组织，复合作为“钢筋”的支架材料，直接移植入体内缺损部位进行大段节段性骨缺损修复。相比于传统的基于消化细胞的组织工程策略，这一策略具有更高的细胞利用效率和更佳的组织修复效果。尽管需要进一步的优化，但是其将是大段节段性骨缺损更有前景的临床解决方案。.在该课题的资助下，申请人以通讯作者身份发表SCI论文5篇（IF合计36分，其中3篇IF>8），参加国内与国际会议受邀与主旨报告多次。