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.