Engineering Dynamic 3D-Bioprinted Models of Pulmonary Vascular Disease

项目总结/摘要 肺动脉高压（PAH）是一种进行性和不可治愈的疾病，其特征是肺动脉高压升高， 肺动脉血压，肺动脉重塑，并最终发展的权利， 心室衰竭不幸的是，临床上唯一可用的治疗方法可以缓解症状，但确实如此 而不是治愈疾病。现有的细胞培养技术代表了发现新的 PAH的治疗靶点，因为这些系统不能充分再现人类的关键方面， 生理学，特别是肺血管系统的复杂3D结构和时间依赖性 在疾病进展过程中发生的细胞外基质（ECM）机械特性的变化。因此，我们认为， 迫切需要开发新的工具和技术，使我们能够研究 PAH随时间推移。我们提出开发一类新的光可调聚乙二醇（PEG）基 水凝胶生物材料和生物制造技术，使研究人员能够控制机械 局部微环境的特性（即，3）按需围绕患者来源的成纤维细胞 使用聚焦光封装在3D打印血管模型中，目的是模拟PAH 体外致病性。这里实施的先进生物材料平台将为 包括多种细胞类型的日益复杂的生物模型， 在申办者的实验室的发展，揭示了新的机械见解，减少人类 疾病该项目将汇集申请人和由生物工程师组成的多元化指导团队 以及专门研究心血管和肺部疾病的临床科学家， 劳动力奖学金培训将包括通过肺动脉高压的临床经验 突破计划，在研究会议和大型圆桌会议上的演讲，以帮助专业人士 Kurt Stenmark博士的心血管肺研究实验室进行实践培训， 了解PAH所必需的实验技术。完成的模型将提供一个平台， 测试和验证肺动脉高压疗法，推进该领域的转化研究。我们 提出了两个具体目标，以证明这一办法的可行性。目标一：设计一个动态的3D- 具有可控弹性模量的生物打印的细胞培养平台。目的二：研究 使用患者来源的细胞激活成纤维细胞的维度和材料模量。Aim I的成功 将通过流变学表征和细胞活力测定来测量。Aim II将测量成纤维细胞 通过免疫组织化学和简洁的qRT-PCR阵列比较表型变化 在3D生物打印血管模拟物中生长的健康患者来源的细胞中， ECM、在2D水凝胶基质上生长的健康细胞和新鲜分离的PAH患者细胞。

PROJECT SUMMARY/ABSTRACT Pulmonary arterial hypertension (PAH) is a progressive and incurable disease, characterized by elevated pulmonary blood pressure, remodeling of the pulmonary arteries, and ultimately the development of right ventricular failure. Unfortunately, the only clinically available therapeutic treatments mitigate symptoms but do not cure the disease. Existing cell culture techniques represent a significant barrier to discovering new therapeutic targets for PAH because these systems do not adequately reproduce key aspects of human physiology, specifically, the complex 3D structure of the pulmonary vasculature and the time-dependent changes in extracellular matrix (ECM) mechanical properties that occur during disease progression. Therefore, an urgent need remains to develop new tools and technologies that enable us to study the pathogenesis of PAH over time. We propose to develop a new class of phototunable poly(ethylene glycol) (PEG)-based hydrogel biomaterials and biomanufacturing techniques that allow investigators to control the mechanical properties of the local microenvironment (i.e., stiffen) on-demand around patient-derived fibroblasts encapsulated within 3D-printed vascular models using focused light, with the goal of emulating PAH pathogenesis in vitro. The advanced biomaterial platform implemented here will provide the foundation for biological models of increasing complexity comprising multiple cell types that are cultured under flow under development in the sponsor's laboratory that reveal novel mechanistic insights into reduction of human disease. This project will bring together the applicant and a diverse mentoring team made up of bioengineers and clinician-scientists specializing in cardiovascular and pulmonary diseases to further develop the pulmonary workforce. Fellowship training will include clinical experiences through the Pulmonary Hypertension Breakthrough Initiative, presentations at research conferences and grand rounds to aid professional development, and hands-on training in Dr. Kurt Stenmark's Cardiovascular Pulmonary Research Lab to learn experimental techniques essential to understanding PAH. The completed model will provide a platform for testing and validating therapies for pulmonary hypertension, advancing translational research in this field. We propose two specific aims to demonstrate the feasibility of this approach. AIM I: Engineer a dynamic 3D- bioprinted cell culture platform with controllable modulus of elasticity. AIM II: Investigate the influence of dimensionality and material modulus on fibroblast activation using patient-derived cells. The success of Aim I will be measured through rheological characterization and cell viability assays. Aim II will measure fibroblast activation through immunohistochemistry and a concise qRT-PCR array to compare phenotypic changes among healthy patient-derived cells grown in 3D-bioprinted blood vessel mimics that emulate pathological ECM, healthy cells grown on 2D hydrogel substrates, and freshly isolated PAH patient cells.