3D-bioprinted, phototunable hydrogel models for studying adventitial fibroblast activation in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculature, characterized by elevated pulmonary blood pressure, remodeling of the pulmonary arteries, and ultimately right ventricular failure. Therapeutic interventions for PAH are limited in part by the lack of in vitro screening platforms that accurately reproduce dynamic arterial wall mechanical properties. Here we present a 3D-bioprinted model of the pulmonary arterial adventitia comprised of a phototunable poly(ethylene glycol) alpha methacrylate (PEG-αMA)-based hydrogel and primary human pulmonary artery adventitia fibroblasts (HPAAFs). This unique biomaterial emulates PAH pathogenesis in vitro through a two-step polymerization reaction. First, PEG-αMA macromer was crosslinked off-stoichiometry by 3D bioprinting an acidic bioink solution into a basic gelatin support bath initiating a base-catalyzed thiol-ene reaction with synthetic and biodegradable crosslinkers. Then, matrix stiffening was induced by photoinitiated homopolymerization of unreacted αMA end groups. A design of experiments approach produced a hydrogel platform that exhibited an initial elastic modulus (E) within the range of healthy pulmonary arterial tissue (E = 4.7 ± 0.09 kPa) that was stiffened to the pathologic range of hypertensive tissue (E = 12.8 ± 0.47 kPa) and supported cellular proliferation over time. A higher percentage of HPAAFs cultured in stiffened hydrogels expressed the fibrotic marker alpha-smooth muscle actin than cells in soft hydrogels (88 ± 2% versus 65 ± 4%). Likewise, a greater percentage of HPAAFs were positive for the proliferation marker 5-ethynyl-2'-deoxyuridine (EdU) in stiffened models (66 ± 6%) compared to soft (39 ± 6%). These results demonstrate that 3D-bioprinted, phototunable models of pulmonary artery adventitia are a tool that enable investigation of fibrotic pathogenesis in vitro.

肺动脉高压（Pulmonary arterial hypertension, PAH）是一种进行性肺血管疾病，其特征为肺动脉压力升高、肺动脉重构，最终引发右心室衰竭。目前肺动脉高压的治疗干预手段有限，部分原因在于缺乏能够精准再现动脉管壁动态力学特性的体外筛选平台。在此我们提出一种3D生物打印的肺动脉外膜模型，该模型由光可调聚乙二醇α甲基丙烯酸酯（poly(ethylene glycol) alpha methacrylate, PEG-αMA）基水凝胶以及原代人肺动脉外膜成纤维细胞（primary human pulmonary artery adventitia fibroblasts, HPAAFs）构成。这种独特的生物材料通过两步聚合反应在体外模拟肺动脉高压的发病机制：首先，将酸性生物墨水溶液通过3D生物打印至碱性明胶支撑浴中，使PEG-αMA大分子单体以非化学计量比交联，引发碱催化的巯基-烯反应，结合合成且可生物降解的交联剂；随后，通过未反应αMA末端基团的光引发均聚反应诱导基质硬化。通过实验设计方法构建的水凝胶平台，初始弹性模量（E）处于健康肺动脉组织的范围（E=4.7±0.09 kPa），可被硬化至高血压组织的病理范围（E=12.8±0.47 kPa），且能够长期支持细胞增殖。与软质水凝胶中的细胞相比，在硬化水凝胶中培养的HPAAFs表达纤维化标志物α-平滑肌肌动蛋白的比例更高（88±2% 与 65±4%）。同样，与软质模型（39±6%）相比，硬化模型中表达增殖标志物5-乙炔基-2'-脱氧尿苷（5-ethynyl-2'-deoxyuridine, EdU）的HPAAFs比例更高（66±6%）。上述结果表明，3D生物打印的光可调肺动脉外膜模型是一种能够在体外研究纤维化发病机制的工具。