Biomanufacturing Vascular Matrix-Mimetic Hydrogels from Quantified Arterial Micromechanics and Microstructure

The microstructural and micromechanical properties of the arterial extracellular matrix (ECM) are central to vascular cell behavior but are disrupted by disease and aging. Elastin and fibrillar collagens provide essential cues, yet the effects of disease-associated ECM remodeling on smooth muscle function remain poorly defined. Here, we imaged fibrillar ECM components across layers of healthy and hypertensive arteries in a large animal model and quantified their stiffness with submicron and piconewton resolution. These animal-derived data guided the fabrication of artery-mimetic hydrogels using image-derived 3D projection micro-stereolithography (µSTL) and thiol-ene clickable bioinks. The resulting hydrogels replicated fibril features down to 2 µm with emulated curvatures and tunable stiffness spanning physiological to pathological ranges (1-35 kPa). Systematic optimization of printing parameters, including light intensity, exposure time, monomer concentration, crosslinker type, optical configurations, and hydrogel swelling solutions, ensured high-fidelity structural replication. Vascular smooth muscle cells cultured on these hydrogels revealed that diseased ECM conditions promoted hypertrophy, impaired contractility, and reduced calcium responsiveness. Quantitative PCR showed reduced expression of contractile markers (MYH11, SMTN) and marked upregulation of PTK2 (focal adhesion kinase) on hypertensive-mimetic matrices, implicating focal adhesion-dependent mechanotransduction in this response. This image-guided biofabrication platform enables precise engineering of vascular microenvironments for mechanistic studies of remodeling, therapeutic testing, and modeling of complex vascular disease states. STATEMENT OF SIGNIFICANCE: Arterial extracellular matrix stiffens and reorganizes in hypertension, yet in vitro models rarely reproduce these micron-scale changes. We measured layer-specific stiffness and fibrillar architecture in arteries from normotensive and hypertensive steers, a large animal model with fattening-induced hypertension. These data were directly translated into projection micro-stereolithography of thiol-ene hydrogels. The resulting matrices, high-fidelity 3D hydrogel prints, reproduced fibrillar features down to 2 µm with emulated curvatures and tunable stiffness spanning 1-35 kPa. Vascular smooth muscle cells cultured on hypertensive-mimetic matrices exhibited hypertrophic morphology, reduced calcium responsiveness and agonist-induced contractility, altered contractile-marker expression, and increased PTK2 expression, linking disease-associated matrix remodeling to altered mechanotransduction. This measurement-to-fabrication workflow provides a physiologically informed platform for mechanistic studies of vascular remodeling and evaluation of therapeutic responses.