基于高度取向超细纤维的肌腱组织微环境的综合仿生构建研究

Poor tendon repair is often a clinical challenge due to the lack of ideal tendon graft. Electrospun aligned fibers, resembling the ultrastructure of tendon, have been a promising materials to promote tendon regeneration. However, the aligned fibers alone are not capable enough to induce tendon regeneration. Based on different growth factors and mechanical stimulation are also required during the process of tendon regeneration and our previous studies, this project is designed to induce tendon regeneration by electrospun highly aligned ultrafine fibers through mimicking the tendon specific environment (structure and composition, growth factors and mechanics) and evaluate its efficacy of tendon regeneration. It will firstly develop a stable jet based coaxial electrospinning technique to enable readily fabricating aligned ultrafine core-shell structured composite fibers loading with TGF-β3 and BMP-12, respectively. Those governing processing variables that affect controllable fabrication will be explored. And the controlled release behavior, release kinetics and biological function of TGF-β3 and BMP-12 from nanofibers will be investigated. Then the promoting tenogenesis effect and working mechanism of thus made biomimetic composite scaffolds under mechanical stimulation will be determined by cell study. Finally we will evaluate its efficacy of tendon regeneration in a rabbit Achilles tendon repair model. A successful implementation of the proposed project would not only benefit the development of innovative technology for the generation of high performance biomimetic fibers with great potential of practical value, but also enable to provide insightful scientific evidence for fundamentally understanding, at the cellular and molecular levels, of the action mechanisms of these biomimetic scaffolds, prior to clinical applications of them in future.

临床上因缺乏合适的移植物而导致修复肌腱损伤困难。电纺取向纤维可模拟肌腱组织的微结构已成为诱导肌腱再生的新型材料，但单一的结构仿生诱导肌腱再生的效果有限。基于肌腱再生中还需要不同的生长因子及力学刺激的支持和我们的前期研究结果，本课题提出基于高度取向超细纤维的肌腱组织微环境的综合仿生构建的研究思路并探究其在促进肌腱再生时的作用功效，将研究：1）基于稳定射流同轴电纺丝方法的TGF-β3和BMP-12负载技术，探讨形成壳-芯纤维的可控制备参数，并在体外研究TGF-β3和BMP-12的顺序缓释行为、缓释动力学及生物活性；2）在细胞水平探讨仿生纤维支架在结合力学刺激时的促干细胞成肌分化作用和机制；3）评估该载双因子壳芯取向超细纤维对兔跟腱缺损修复的功效。本项目将为发展具有实用价值的新型仿生功能纤维提供新材料，为从细胞和分子水平理解仿生材料对干细胞的成肌诱导作用机制和今后的临床应用提供科学依据。

在肌腱组织工程中，制备能仿生天然肌腱组织中细胞外基质微环境仿生构建肌腱组织有重要意义。本项目通过稳定射流同轴电纺丝（SJCES）法制备了经肌腱ECM主要成分I型胶原（COL1）和硫酸软骨素（CS）修饰的PLLA壳芯超细取向纤维支架。同时负载小分子生物活性物质精氨酸（L-Arg）和透明质酸（HA）后，COL1-CS（壳）/PLLA（芯）纤维能够顺序释放L-Arg和HA，且具有良好的细胞生物相容性。对hMSCs在PLLA及COL1-CS（壳）/PLLA（芯）纤维支架上的生长进行形貌观察、细胞增殖及成肌腱分化检测，结果表明，COL1-CS（壳）/PLLA（芯）取向纤维能够促进hMSCs的铺展与增殖，基因水平肌腱相关基因SCX, TN-C及COL1呈高表达，标志性蛋白（TNMD）也表现为高阳性表达率，证明该仿生纤维支架具有优良的成肌腱诱导能力。进一步在引入力学刺激条件下培养，发现力学信号可以协同生物信号（TGFβ3和 BMP12）诱导hMSCs肌腱分化。而且，我们发现TGF-β信号相关分子TGFβ2, TGFβ3, TGFβRII, Smad3在COL1-CS（壳）/PLLA（芯）纤维支架上高表达，说明COL1-CS（壳）/PLLA（芯）纤维诱导成肌腱分化是通过TGFβ信号通路进行。体内动物实验证明此材料具有良好的体内生物相容性，并能够促进肌腱损伤的再生修复。上述研究结果为探索和发展新型、实用、高性能的仿生纤维材料应用于肌腱（及其他结构特异性组织）的仿生构建提供了新方法、新材料、和新科学依据。