UC San Diego

Sex differences in calcific aortic valve disease (CAVD) and valvular interstitial cell (VIC) mechanobiology are increasingly recognized, yet whether collagen fibril alignment, independent of matrix stiffness, contributes to these differences remains unexplored. Here, second harmonic generation imaging of human aortic valve tissue established that native collagen organization varies by layer and converges with CAVD in sex-specific manners due to changes in the fibrosa. A female-biased increase in alignment led to convergence while male alignment remained distinct, and a male-biased decrease in collagen area led to convergence while female layers remained convergent throughout health and disease. A sub-physiological stiffness collagen hydrogel platform was engineered by tuning gravitational fibrillogenesis to produce random and aligned matrices that emulate the heterogeneity of native valve ECM. Porcine VICs aligned and elongated uniformly across sex in direct response to the underlying fibril architecture, while myofibroblast activation in was not found to be significantly driven by the matrix alignment cue. Both sexes exhibited similar degrees of αSMA stress fiber formation at experimental endpoints, but male VICs showed higher-magnitude, faster-onset total αSMA expression and greater collagen matrix reorganization, while female VICs showed elevated fibronectin secretion on random matrices at early timepoints, suggesting that sex may shape the magnitude of matrix remodeling despite a shared alignment response. This work demonstrates how an alignment-tunable, sub-physiological stiffness hydrogel platform serves as a tool for isolating fibril architecture as an independent ECM cue and underscores its utility for future studies of sex-specific mechanotransduction in valvular and other collagen-remodeling tissues.