Figure 3. Adhesion to nascent proteins controls hMSC mechanosensing in degradable hydrogels.

a Representative images (scale bar 200 μm, inset 20 μm) of nascent protein deposition in MMP-degradable NorHA hydrogels after 6 days treatment without (control, note same image as in Fig. 2b), with monoclonal antibodies against integrin alpha 2 (anti α2, 20 μg/mL) or human fibronectin (HFN7.1, 5 μg/mL) or with soluble RGD (sol RGD, 0.5 mM). b Quantification of cell aspect ratio and accumulated nascent protein thickness deposited by hMSCs encapsulated in degradable NorHA hydrogels (n = 133 cells (control), n = 155 cells (anti α2), and n = 152 cells (HFN7.1), n = 124 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in a). c Representative images and d quantification of nuclear/cytoplasmic (nuc/cyto) YAP/TAZ ratios of hMSCs encapsulated in degradable NorHA hydrogels, cultured for 6 days in adipogenic-osteogenic media (scale bar 50 μm), quantifications: n = 60 cells (control), n = 51 cells (anti α2), and n = 40 cells (HFN7.1), n = 39 cells (sol RGD) from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, * p ≤ 0.05, one-way ANOVA with Bonferroni post hoc, red dots indicate measurements for magnified images in c). e Immunostaining for fatty-acid binding protein (FABP, adipogenic marker) and osteocalcin (Oc, osteogenic marker) after 14 days in adipogenic-osteogenic media (scale bar 50 μm). f Quantification of positively stained cells (percentage,%) towards osteogenesis (Oc positive) and adipogenesis (FABP positive) after 14 days, n = 6 samples from 2 biologically independent experiments), mean ± SD, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, two-way ANOVA with Bonferroni post hoc).