Figure 4.

Stiffness activates the canonical Wnt signaling pathway on crosslinked MC-GAG. A) Representative confocal microscopic images of primary hMSCs cultured on NX-MC (top row) or MC (middle row) for 3 d stained with anti-non-p-β-catenin and Dapi. Negative control (Control) using secondary antibody only and Dapi on cells cultured on MC shown on bottom row. Scale bar indicates 10 µm. Yellow arrows indicate cells with weak non-p-β-catenin staining and sparing of the nuclei. White arrows indicate cells with strong non-p-β-catenin staining including nuclear staining. B) Quantification of non-p-β-catenin of primary hMSCs cultured on NX-MC or MC for 3 or 7 d. Western blot of primary hMSCs cultured on NX-MC or MC for 0, 3, 7, or 14 d in osteogenic differentiation medium for C) non-p-β-catenin, phosphorylated Akt (p-Akt), and total Akt. Densitometric quantification of western blot analyses demonstrating relative protein amounts of D) non-p-β-catenin to actin and E) p-Akt to total Akt. QPCR of primary hMSCs cultured on NX-MC or MC for 3 or 7 d in osteogenic differentiation medium for the F) WNT3A, G) WNT4, H) WNT5A, I) WNT5B, and J) WNT11 (n = 3). Bars represent means, errors bars represent SE. Significant posthoc comparisons following ANOVA indicated with p-values.