E2 - Modeling Pulmonary Hypertension with 3D-Printed Vascular Scaffolds: A Platform for Mechanobiology and Personalized Therapy

Introduction: : Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by elevated pulmonary artery pressure, leading to right-sided heart failure and eventually death. Current diagnostic approaches often identify PAH only in advanced stages, limiting the effectiveness of available treatments. Recent studies suggest that abnormal mechanotransduction, influenced by changes in the extracellular matrix (ECM) stiffness, plays a crucial role in endothelial dysfunction at the early stages of PAH. This research aims to develop a 3D-printed vascular model to better understand the cellular responses to mechanical stress and ECM stiffness in PAH. By creating healthy and diseased vascular models, this study will investigate early-stage vascular remodeling and provide insights into the disease's progression, ultimately contributing to the development of early diagnostic tools and personalized therapeutic strategies for PAH.

Materials and

Methods: : A 3D-printed vascular model was created using Gelatin Methacryloyl (GelMA) and Polyethylene Glycol Diacrylate (PEGDA) hydrogel scaffolds to mimic the mechanical properties of both healthy and PAH-affected arteries. The scaffolds were fabricated with varying stiffness and wall thickness to replicate diseased vascular conditions, using a BIONOVA X light-based bioprinter. A photomask-based approach controlled the exposure time and light intensity to generate stiffness gradients mimicking healthy (1-5 kPa) and PAH-affected (10-15 kPa) arteries. Human-derived smooth muscle cells (SMCs) were seeded, and cellular responses were analyzed through live/dead staining, immunofluorescence for collagen and elastin ECM deposition, and Yap/Taz nuclear translocation. Gene expression of contractile and synthetic markers were quantified using RT-qPCR. SMC contractility and calcium activity were analyzed using fluorescence indicator dyes, and response metrics such as peak amplitude and oscillation frequency. Cell proliferation was assessed using PicoGreen DNA and nuclei density quantification across healthy and diseased scaffold regions.

Results, Conclusions, and Discussions:: The 3D-printed GelMA.PEGDA scaffolds (4:1 ratio) supported optimal adhesion, viability, and maintenance of a contractile phenotype in SMCs. Spatial stiffness gradients enabled precise fabrication of local matrix mechanics resulting in diseased modeling that accurately replicated PAH-associated mechanical properties (10–15 kPa). Additionally, SMCs aligned along micropatterned grooves and elongated in stiffer regions, recapitulating the mechanical heterogeneity of PAH. Enhanced nuclear localization of Yap/Taz indicated activation of PAH-associated mechanotransduction pathways. ECM staining revealed increased deposition of collagen I and elastin, consistent with PAH fibrotic remodeling. PicoGreen quantification showed increased DNA content in stiffened regions, suggesting mechanoresponsive PASMC proliferation. RT-qPCR analysis demonstrated a shift in SMC phenotype, with upregulation of synthetic markers and downregulation of contractile markers in cells cultured on the diseased scaffolds. Elevated intracellular calcium transients were observed in high-stiffness regions, consistent with the expected promotion of a more contractile phenotype in response to increased matrix stiffness. Although dedifferentiation under softer conditions typically reduces calcium signaling and contractility, the mechanical cues provided by stiffer substrates appear to partially restore contractile features and calcium dynamics via mechanotransduction pathways. Collectively, these results confirm that our model offers distinct advantages over conventional 2D and in vivo models by replicating key aspects of early PAH, including matrix remodeling, phenotype switching, and mechanoresponsive signaling. With future integration of endothelial co-culture and perfusion, this system holds strong potential for translational applications in drug screening, biomarker discovery, and patient-specific therapeutic strategies.

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