INTRODUCTION: Targeted refinement of regenerative materials requires mechanistic understanding of cell-material interactions. The nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffold is a porous biomaterial that promotes regenerative healing of skull defects in vivo without addition of exogenous growth factors or progenitor cells, suggesting potential for clinical translation as an off-the-shelf implant. In this work, we evaluate the relationship of material stiffness, a tunable MC-GAG property, and activation of the canonical Wnt (cWnt) signaling pathway.
MATERIALS AND METHODS: Primary human bone marrow-derived mesenchymal stem cells (hMSCs) were differentiated on two MC-GAG scaffolds varying by stiffness (non-crosslinked, NX-MC, 0.3 kPa vs. conventionally crosslinked, MC, 3.9 kPa).
RESULTS: hMSCs exhibited increased expression of activated β-catenin, the major intracellular mediator, and the mechanosensitive YAP protein with near complete subcellular colocalization in stiffer MC scaffolds. Small molecule Wnt pathway inhibitors reduced activated β -catenin and YAP protein quantities and colocalization, osteogenic differentiation, and mineralization on MC, with no effects on NX-MC. Concomitantly, Wnt inhibitors increased BMP4 and phosphorylated Smad1/5 (p-Smad1/5) expression on MC, but not NX-MC. Unlike non-specific Wnt pathway downregulation, isolated canonical Wnt inhibition with β -catenin knockdown increased osteogenic gene expression and mineralization specifically on the stiffer MC. β-catenin knockdown also increased p-Smad1/5, Runx2, and BMP4 expression only on the stiffer MC material.
CONCLUSION: Our data indicates stiffness-induced activation of the Wnt and mechanotransduction pathways promotes osteogenesis in MC-GAG scaffolds. However, 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.