Engineering Dynamic 3D-Bioprinted Models of Pulmonary Vascular Disease

项目摘要/摘要 肺动脉高压(PAH)是一种进行性和不治之症，其特征是 肺血压、肺动脉重塑，并最终发展为右心 心力衰竭。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 而不是治愈疾病。现有的细胞培养技术是发现新细胞的重要障碍 PAH的治疗靶点，因为这些系统不能充分复制人类的关键方面 生理学，具体地说，肺血管的复杂三维结构和时间依赖性 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， 迫切需要开发新的工具和技术，使我们能够研究糖尿病的发病机制 PAH随着时间的推移。我们建议开发一种新的基于聚乙二醇基的光调谐材料 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 患者来源成纤维细胞周围按需局部微环境(即僵硬)的特性 使用聚焦光封装在3D打印的血管模型中，目标是模拟PAH 体外致病机制。这里实施的先进生物材料平台将为 由多种细胞类型组成的日益复杂的生物模型在流下培养 赞助商实验室的发展揭示了对人类减少的新的机械学见解 疾病。这个项目将把申请者和由生物工程师组成的不同的指导团队聚集在一起。 和临床医生-专门研究心血管和肺部疾病的科学家，以进一步开发肺 劳动力。团契培训将包括通过肺动脉高压的临床经验 突破性倡议、在研究会议上的演讲和帮助专业人员的盛大回合 在Kurt Stenmark博士的心肺研究实验室学习开发和动手培训 理解多环芳烃所必需的实验技术。完成后的模型将提供一个平台 测试和验证治疗肺动脉高压的方法，促进该领域的转化性研究。我们 提出两个具体目标来论证这一方法的可行性。目标一：设计一个动态的3D- 弹性模数可控的生物印刷细胞培养平台。目的二：调查 维度和材料模数对患者来源细胞成纤维细胞激活的影响。AIM I的成功 将通过流变学特性和细胞活性分析进行测量。AIM II将测量成纤维细胞 通过免疫组织化学和简明的qRT-PCR阵列激活以比较表型变化 在3D生物打印血管中培养的健康患者来源的细胞中，模拟病理的 细胞外基质，生长在2D水凝胶基质上的健康细胞，以及新鲜分离的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.