Fibrosa-like hydrogels preferentially induced production of osteogenic microcalcification by VICs. Red = NIRF Osteosense 680 imaging agent (nano- and microcalcifications), green = TUNEL apoptosis assay, blue = Hoechst nuclear stain: (a) VICs isolated from non-diseased human AV did not undergo calcification when cultured in NM on/in either 2D tissue culture polystyrene (TCPS), 3D fibrosa-like (F-like) hydrogels, or 3D spongiosa-like (S-like) hydrogels (representative images); (b,c) Importantly, under OM treatment this naïve VIC population developed significant production of microcalcification only in 3D F-like hydrogels that mimic the biomechanics of the disease-prone layer of the native tissue. OM treatment did not induce significant microcalcification on 2D TCPS or in 3D S-like hydrogels. (d) Apoptosis was negligible and unchanged across all conditions, confirming that apoptotic cell death was not the source of preferential calcification in OM-treated F-like hydrogels. Mean + SEM; *** p < 0.001; n = 3 samples per condition (3 images per sample); scale bar = 50 μm.