Department of Biomedical Engineering, Virginia Commonwealth University, 800 E. Leigh St, Room 1071, Richmond, VA 23219, United States.
Department of Biomedical Engineering and Nursing, Duquesne University, 600 Forbes Ave, Pittsburg, Pennsylvania 15282, United States.
Acta Biomater. 2019 Dec;100:223-234. doi: 10.1016/j.actbio.2019.10.009. Epub 2019 Oct 5.
Decellularized tissues offer a unique tool for developing regenerative biomaterials or in vitro platforms for the study of cell-extracellular matrix (ECM) interactions. One main challenge associated with decellularized lung tissue is that ECM components can be stripped away or altered by the detergents used to remove cellular debris. Without characterizing the composition of lung decellularized ECM (dECM) and the cellular response caused by the altered composition, it is difficult to utilize dECM for regeneration and specifically, engineering the complexities of the alveolar-capillary barrier. This study takes steps towards uncovering if dECM must be enhanced with lost ECM proteins to achieve proper epithelial barrier formation. To achieve this, the epithelial barrier function was assessed on dECM coatings with and without the systematic addition of several key basement membrane proteins. After comparing barrier function on collagen I, fibronectin, laminin, and dECM in varying combinations as an in vitro coating, the alveolar epithelium exhibited superior barrier function when dECM was supplemented with laminin as evidenced by trans-epithelial electrical resistance (TEER) and permeability assays. Increased barrier resistance with laminin addition was associated with upregulation of Claudin-18, E-cadherin, and junction adhesion molecule (JAM)-A, and stabilization of zonula occludens (ZO)-1 at junction complexes. The Epac/Rap1 pathway was observed to play a role in the ECM-mediated barrier function determined by protein expression and Epac inhibition. These findings revealed potential ECM coatings and molecular therapeutic targets for improved regeneration with decellularized scaffolds. STATEMENT OF SIGNIFICANCE: Efforts to produce a transplantable organ-scale biomaterial for lung regeneration has not been entirely successful to date, due to incomplete cell-cell junction formation, ultimately leading to severe edema in vivo. To fully understand the process of alveolar junction formation on ECM-derived biomaterials, this research has characterized and tailored decellularized ECM (dECM) to mitigate reductions in barrier strength or cell attachment caused by abnormal ECM compositions or detergent damage to dECM. These results indicate that laminin-driven Epac signaling plays a vital role in the stabilization of the alveolar barrier. Addition of laminin or Epac agonists during alveolar regeneration can reduce epithelial permeability within bioengineered lungs.
去细胞化组织为开发再生生物材料或用于研究细胞-细胞外基质（ECM）相互作用的体外平台提供了独特的工具。与去细胞化肺组织相关的一个主要挑战是，用于去除细胞碎片的去污剂可能会去除或改变 ECM 成分。如果不去表征去细胞化肺 ECM（dECM）的组成以及改变组成所引起的细胞反应，就很难将 dECM 用于再生，特别是用于工程化肺泡-毛细血管屏障的复杂性。本研究旨在探讨 dECM 是否必须增强失去的 ECM 蛋白以实现适当的上皮屏障形成。为此，在有系统地添加几种关键基底膜蛋白的情况下，评估 dECM 涂层的上皮屏障功能。在比较不同组合的胶原 I、纤连蛋白、层粘连蛋白和 dECM 作为体外涂层的屏障功能后，肺泡上皮在补充层粘连蛋白的情况下表现出更好的屏障功能，这一点可以通过跨上皮电阻（TEER）和通透性测定来证明。用层粘连蛋白增加屏障阻力与 Claudin-18、E-钙粘蛋白和连接黏附分子（JAM）-A 的上调以及紧密连接复合物中 zonula occludens（ZO）-1 的稳定有关。观察到 Epac/Rap1 途径在 ECM 介导的屏障功能中发挥作用，这是由蛋白质表达和 Epac 抑制决定的。这些发现揭示了潜在的 ECM 涂层和分子治疗靶点，可用于改善去细胞化支架的再生。意义声明：迄今为止，为肺再生生产可移植器官规模的生物材料的努力尚未完全成功，这是由于细胞-细胞连接形成不完全，最终导致体内严重水肿。为了充分了解 ECM 衍生生物材料上肺泡连接形成的过程，本研究对去细胞化 ECM（dECM）进行了表征和定制，以减轻由于 ECM 组成异常或去污剂对 dECM 的损伤导致的屏障强度降低或细胞附着减少。这些结果表明，层粘连蛋白驱动的 Epac 信号在肺泡屏障的稳定中起着至关重要的作用。在肺泡再生过程中添加层粘连蛋白或 Epac 激动剂可以降低生物工程肺中的上皮通透性。
