Engineered Hydrogels as Valve Tissue Mimetics That Recapitulate Sexual Dimorphisms Observed in Calcification and Osteopontin Activity

Abstract

Objective: Recent clinical reports indicate that patients with calcific aortic valve stenosis (AVS) have sexually dimorphic disease phenotypes, where female patients have reduced valve calcification compared to males. Yet, the cellular mechanisms contributing to this dimorphism remain unknown. Our results reveal that diseased valve leaflets from a female tend to have reduced and smaller-scale microcalcifications compared to males. Additionally, osteopontin (OPN) expression, a pro-fibrotic but anti-calcific bone sialoprotein, is elevated in human female valve tissue sections. We hypothesize that OPN may play a role in reducing calcification in female aortic valves. Methods: To this end, we use a hydrogel system with cellularly-degradable crosslinkers and adhesive peptides to culture VICs isolated from male and female pigs in 3D. We further introduced a secondary interpenetrating network of collagen type I and studied differences in the cellular response to osteogenic stimuli. Results: The hydrogel matrix supported VIC survival, matrix remodeling, and recapitulated the mineralization differences that are observed in human diseased valve tissue. Specifically, female valve tissue and female VICs cultured in pro-calcifying hydrogels have more punctate calcification (< 3000 μm2) relative to male samples (> 3000 μm2) (Figure 1). Further, OPN localization shifted from the hydrogel extracellular matrix to the nucleus in female cells in response to exogenously delivered calcification stimuli. Conclusions: OPN localization corresponded to sites of VIC-mediated matrix calcification, suggesting OPN may have a role in suppressing mineralization growth in AVS progression. This 3D hydrogel culture system should prove useful to test additional hypotheses related to sex-specific AVS calcification and potentially guide the development of sex-specific treatments. Figure 1. Von Kossa stained histological sections display reduced calcification of female samples (left) relative to male (right), in both tissue sections of human diseased aortic valve (top) and within in vitro 3D hydrogel valve mimetics (bottom). Scale bar = 100 μm