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

项目摘要 识别促进疾病的细胞通路或有效的前瞻性治疗依赖于 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学和生理学，以帮助预测人类疾病的进展， 潜在的治疗选择。长期目标是利用一种新的体外肺芯片（LOAC）微流感， 预测外源性物质如何导致炎症、纤维化和免疫调节性肺部疾病的快速诊断装置 在人类身上。总体目标是创造第一个完全有机的武装冲突法，在结构上由一个细胞支撑， 衍生的细胞外基质（ECM），并且包括先天免疫细胞以模拟器官水平的功能。的 拟议研究的基本原理是利用多孔硅（PSi）的独特性质，而不是以前的 探索在微流体平台的制造中彻底改变材料科学领域， 动态ECM变化。在强有力的初步数据的指导下，将通过以下方式实现总体目标： 以下三个具体目标：1）确定最佳参数和细胞机制，以消除干扰 2）确定LOAC内共培养细胞的表达水平， crete和创建自己的ECM;和3）发展多细胞肺泡结构，以激活免疫细胞， 外渗和ECM重塑使用肺动脉高压的体外模型。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）对于修饰 溶解PSi将通过扫描电子显微镜（SEM）定量PSi的溶出速率， 将通过原子力显微镜（AFM）完成表面分析，以可重复地创建柔性的、结构上 完整的膜在第二个目标下，工作假设是内皮细胞（EC）将表达并 分泌细胞来源的ECM蛋白。从头合成的ECM组分的分泌将通过 RT-PCR、旋转盘和光片显微镜、LC/MS分析和AFM。第三个目标是基于初步的 数据表明，上皮细胞（EPCs）、EC和MAC可以成功地共培养，并且在长时间内存活。 在PSi膜上进行长期培养。工作假设是在缺氧条件下，MAC将 激活并释放可溶性介质，导致EC凋亡和ECM重塑增加 将通过共聚焦IF显微镜和活细胞成像进行定量。这项研究具有创新性， 在我们看来，因为它代表了一个实质性的偏离现状，利用独特的charac- PSi是一种生物相容性和可生物降解的材料。此外，利用PSi提供了ca- 产生细胞特异性ECM的能力，其将释放包括生长因子和细胞外基质的生物化学信号。 蛋白酶拟议的研究是有意义的，因为它预计将有广泛的翻译重要性 预防和治疗多种肺部疾病。最后，治疗方面的进展 对PSi纳米材料的发展和导致体内降解的因素进行了展望。

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.