负载SDF-1组织工程气管支架的构建及其原位诱导上皮再生的实验研究

Tracheal stenosis is one of the most common complications associated with congenital heart disease. Currently, tissue engineered trachea (TET) has highlighted the plight of limited clinical therapy for long segment congenital tracheal stenosis. Nevertheless, the poor regeneration of airway epithelium post transplantation seriously hampers further development of TET. In our pilot research, tracheal cartilage rings had already been successfully constructed with assistance from 3D printing technology. But it`s frustrating to find that restenosis of reconstructed tracheal lumens tends to appear in very early stage after operation due to unsatisfied airway epithelium regeneration. Taking into account tracheal epithelial progenitor/stem cells are hard to be isolated and cultured in vitro, biomimetic tracheal scaffold which could facilitate the in-situ regeneration of airway epithelium is urgently needed for improving the prognosis after TET transplantation. It has been established by some recent researches that ideal airway epithelium regeneration could be achieved by recruitment of epithelial progenitor/stem cells, which might be conducted by stromal cell-derived factor-1(SDF-1) via SDF-1/CXCR4 signaling pathway. The particular underlying mechanism of recruitment still remains obscure and scarce related applications in area of TET have been reported hitherto. Therefore, our project will thoroughly explore the molecular mechanism of epithelial progenitor/stem cells recruitment and its potential application in constructing novel bistratal TET scaffold. Briefly, the SDF-1 encapsulated tube-like scaffold will be fabricated through electrospinning and taken as the inner part of scaffold. Then cartilaginous ring constructed by 3D printing would be used as outer part to assemble intact scaffold. To systematically evaluate the potential of promoting airway epithelium regeneration at various levels, tracheal defects of rabbit model will be restored through transplantation of the novel scaffold eventually. This project will pave new road for patients with congenital heart disease and severe tracheal stenosis.

气管狭窄在先天性心脏病人群中较为常见，然而临床治疗手段有限。组织工程气管带来希望，但气管上皮化是制约其进一步发展的主要因素之一。我们前期结合3D打印技术已成功构建气管软骨层，体内移植实验亦发现因缺乏气管上皮早期即出现管腔狭窄。由于气管上皮干细胞分离获取困难，若能制备出可直接诱导上皮原位再生的组织工程气管支架，将有望提高气管体内移植效果。近期研究表明基质细胞衍生因子-1(SDF-1)可通过SDF-1/CXCR4信号轴募集气管上皮干细胞完成上皮自我修复，但在组织工程气管中研究甚少，募集机制亦不明。故本项目在前期基础上，拟利用静电纺丝制备负载SDF-1组织工程气管支架，探讨支架对气管上皮干细胞的募集作用及调控机制，进而优化支架的募集潜能，再结合前期的3D打印软骨环支架组合成双层气管支架，并移植修复大动物气管缺损以评价该支架原位诱导气管上皮再生的能力，为先天性心脏病伴气管狭窄患者提供新的治疗策略。

功能上皮化在维持组织工程气管 (TET) 的长期管腔通畅中起关键作用。由于自体上皮迁移缓慢，移植的TET的自发上皮化过程迟缓。本课题拟采用负载SDF-1气管支架以达到促进气管上皮迁移。体外实验证实了3ng/ml的SDF-1促进上皮细胞迁移效果最好。但是进一步实验中发现PCL支架打印的过程中需要溶解于有机溶剂，而SDF-1在此过程中大量失活。因此，考虑移植前在 TET 上接种气管基底细胞 (TBCs) 可能有利于加速上皮形成，但体外 TBCs 的快速扩增仍然相对困难。在这项研究中，我们提出了一种有前景的扩增策略，使 TBCs 在体外快速增殖。 TBCs从兔自体气管黏膜中分离出来，并与来源于3T3-J2细胞的外泌体共培养。在与外泌体成分共培养后，TBCs 可以在体外大力增殖并保持其多向分化的潜能。与单独培养的TBCs在30天左右才能扩增至2代形成鲜明对比，而且大多数单培养的细胞进入凋亡晚期。另一方面，采用3D打印技术设计制造了一种具有良好力学性能和生物相容性的仿生管状双层支架。然后分别在支架外层植入自体软骨细胞，在内层植入TBCs，体外构建负载两种细胞的TET。然后在体内对TET进行预血管化，并进行带蒂移植以修复受体兔的长节段缺损。结果发现，接种在双层支架上的软骨细胞和 TBCs 发育良好。术后2周，TET管腔表面几乎完全覆盖纤毛上皮，相比之下，没有预接种TBCs的TET上皮化在术后近2个月完成。总之，TBCs 与 3D 打印双层支架的有希望的扩展策略促进了移植 TET 的快速上皮化过程，这可能对临床和转化医学具有重要意义