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

Zhou, Q.; Ren, X.; Oberoi, M. K.; Caprini, R. M.; Dewey, M. J.; Kolliopoulos, V.; Yamaguchi, D. T.; Harley, B. A. C.; Lee, J. C.

doi:10.1101/2021.06.15.447161

bioRxiv · 10.1101/2021.06.15.447161

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

Zhou, Q.·Ren, X.·Oberoi, M. K.·Caprini, R. M.·Dewey, M. J.·Kolliopoulos, V.·Yamaguchi, D. T.·Harley, B. A. C.·Lee, J. C.

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Abstract

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 calvaria defects in vivo without addition of exogenous growth factors or progenitor cells, suggesting its potential as an off-the-shelf implant for reconstructing skull defects. In this work, we evaluate the relationship between material stiffness, a tunable MC-GAG property, and activation of the canonical Wnt (cWnt) signaling pathway. 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). hMSCs exhibited increased expression of activated {beta}-catenin, the major cWnt intracellular mediator, and the mechanosensitive YAP protein with near complete subcellular colocalization in stiffer MC scaffolds. Small molecule Wnt pathway inhibitors reduced activated {beta}-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 {beta}-catenin knockdown increased osteogenic gene expression and mineralization specifically on the stiffer MC. {beta}-catenin knockdown also increased p-Smad1/5, Runx2, and BMP4 expression only on the stiffer MC material. Our data indicates stiffness-induced activation of the Wnt and mechanotransduction pathways promotes osteogenesis in MC-GAG scaffolds. However, activated {beta}-catenin is a limiting agent and may serve as a useful target or readout for optimal modulation of stiffness in skeletal regenerative materials. One Sentence Summary{beta}-Catenin limits stiffness-induced osteogenenic differentiation on nanoparticulate mineralized collagen glycosaminoglycan materials

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Zhou, Q., Ren, X., Oberoi, M. K., Caprini, R. M., Dewey, M. J., Kolliopoulos, V., Yamaguchi, D. T., Harley, B. A. C., Lee, J. C.. 2021-06-16. Beta-Catenin Limits Osteogenesis on Regenerative Materials in a Stiffness-Dependent Manner. https://doi.org/10.1101/2021.06.15.447161

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