β-Catenin Limits Osteogenesis on Regenerative Materials in a Stiffness-Dependent Manner

Targeted refinement of regenerative materials requires mechanistic understanding of cell-material interactions. The nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffold is shown to promote skull regeneration in vivo without additive exogenous growth factors or progenitor cells, suggesting potential for clinical translation. This work evaluates modulation of MC-GAG stiffness on canonical Wnt (cWnt) signaling. Primary human bone marrow-derived mesenchymal stem cells (hMSCs) are differentiated on two MC-GAG scaffolds (noncrosslinked, NX-MC, 0.3 kPa vs conventionally crosslinked, MC, 3.9 kPa). hMSCs increase expression of activated β-catenin, the major cWnt intracellular mediator, and the mechanosensitive YAP protein with near complete subcellular colocalization on stiffer MC scaffolds. Overall Wnt pathway inhibition reduces activated β-catenin and osteogenic differentiation, while elevating BMP4 and phosphorylated Smad1/5 (p-Smad1/5) expression on MC, but not NX-MC. Unlike Wnt pathway downregulation, isolated canonical Wnt inhibition with β-catenin knockdown increases osteogenic differentiation and mineralization specifically on the stiffer MC. β-catenin knockdown also increases p-Smad1/5, Runx2, and BMP4 expression only on the stiffer MC material. Thus, while stiffness-induced activation of the Wnt and mechanotransduction pathways promotes osteogenesis on MC-GAG, activated β-catenin is a limiting agent and may serve as a useful target or readout for optimal modulation of stiffness in skeletal regenerative materials.