Utilizing biodegradable porous silicon membranes as a novel design for lung-on-achip microfluidic devices to investigate extracellular matrix interactions.

项目摘要 确定促进疾病的细胞通路或哪些预期疗法有效依赖于 合适的哺乳动物模型。因此，迫切需要先进的人体模型系统 可以准确地再现人体解剖和生理学，以帮助预测人类疾病的进展和AS- Sess潜在的治疗选择。长期目标是利用一种新型的体外芯片肺(LOAC)微流感-- 预测外源物质如何导致炎症性、纤维化和免疫调节性肺部疾病的IDIC设备 在人类身上。总体目标是创建第一个完全有机的LOAC，它在结构上由细胞支撑- 衍生的细胞外基质(ECM)，并包括先天免疫细胞来模拟器官水平的功能。这个 拟议研究的基本原理是利用多孔硅(PSI)的独特性质，而不是以前 探索在微流控平台的制造中给材料科学领域带来革命性的变化 动态ECM变化。在强劲的前期数据的指引下，总体目标将通过追求实现 以下三个具体目标：1)确定溶解超薄的最佳参数和细胞机制 长期培养过程中的多孔硅；2)确定LOAC内共培养的细胞在多大程度上符合 并创建自己的ECM；以及3)开发多细胞肺泡结构，以激活免疫细胞 利用体外肺动脉高压模型进行细胞外渗和细胞外基质重建。在第一个目标下， 基于初步数据的工作假设是，人类巨噬细胞(Mac)是修改 并溶解PSI。PSI的溶出度将通过扫描电子显微镜(SEM)和 表面分析将由原子力显微镜(AFM)完成，以在结构上可重复地创建灵活的 完整的膜。在第二个目标下，工作假设是内皮细胞(ECs)将表达和 分泌细胞来源的细胞外基质蛋白。从头合成的细胞外基质成分的分泌将通过 RT-PCR、转盘和光片显微镜、LC/MS分析和AFM。第三个目标是基于初步的 有数据表明，上皮细胞、内皮细胞和巨噬细胞可以成功地共培养，并在长期培养中存活。 PSI膜上的长期培养。工作假说是在低氧条件下，Mac将 激活并释放可溶性介质，导致内皮细胞凋亡和增加ECM重塑 这将通过共聚焦IF显微镜和活细胞成像进行量化。这项拟议的研究具有创新性， 在我们看来，因为它代表着利用独特的个性与现状的实质性偏离-- PSI是一种生物相容和可生物降解的材料。此外，利用PSI还提供了 能够产生细胞特异性的细胞外基质，释放包括生长因子和细胞外的生化信号 蛋白酶。这项拟议的研究意义重大，因为预计它将具有广泛的翻译重要性 在预防和治疗广泛的肺部疾病方面。最后，治疗方面的进展 PSI纳米材料的发展和导致体内降解的因素是值得期待的。

Project Summary Identifying the cellular pathways that promote disease or which prospective therapies are effective relies upon appropriate mammalian models. Therefore, there is an urgent need for advanced human model systems that can accurately reproduce human anatomy and physiology to help predict human disease progression and as- sess potential treatment options. The long-term goal is to utilize a novel in vitro lung-on-a-chip (LOAC) microflu- idic device to predict how xenobiotics lead to inflammatory, fibrotic and immunomodulatory pulmonary diseases in humans. The overall objective is to create the first fully organic LOAC that is structurally supported by a cell- derived extracellular matrix (ECM) and includes innate immune cells to simulate organ-level functionality. The rationale for the proposed research is to employ the unique properties of porous silicon (PSi) not previously explored to revolutionize the field of material science in the fabrication of microfluidic platforms that incorporates dynamic ECM changes. Guided by strong preliminary data, the overall objective will be accomplished by pursing the following three specific aims: 1) Identify the optimal parameters and cellular mechanisms to dissolve ultrathin porous silicon during long-term culture; 2) Determine the extent to which co-cultured cells within the LOAC se- crete and create their own ECM; and 3) Develop a multicellular alveolar structure to activate immune cells leading to extravasation and ECM remodeling using an in vitro model of pulmonary hypertension. Under the first aim, the working hypothesis based on preliminary data is that human macrophages (MACs) are essential to modify and dissolve PSi. Dissolution rates of PSi will be quantified through scanning electron microscopy (SEM) and surface analysis will be completed by atomic force microscopy (AFM) to reproducibly create flexible, structurally intact membranes. Under the second aim, the working hypothesis is that endothelial cells (ECs) will express and secrete cell-derived ECM proteins. Secretion of de novo synthesized ECM components will be quantified through RT-PCR, spinning disk and light sheet microscopy, LC/MS analysis and AFM. The third aim based on preliminary data indicate epithelial cells (EPCs), ECs and MACs can be successfully co-cultured and are viable during long- term culture on PSi membranes. The working hypothesis is in the presence of hypoxic conditions, MACs will become activated and release soluble mediators leading to apoptosis of ECs and increased ECM remodeling that will be quantified through confocal IF microscopy and live cell imaging. The proposed research is innovative, in our opinion, because it represents a substantive departure from the status quo by utilizing the unique charac- teristics of PSi, which is a biocompatible and biodegradable material. In addition, utilizing PSi provides the ca- pability to create a cell specific ECM that will release biochemical cues including growth factors and extracellular proteinases. The proposed research is significant because it is expected to have broad translational importance in the prevention and treatment of a wide range of pulmonary diseases. Finally, therapeutic advancements in the development of PSi nanomaterials and factors that lead to degradation in vivo are expected.