Inhibition of the Wnt Pathway Reduces Activation of the YAP/TAZ Mechanotransduction Pathways and Osteogenic Differentiation in Nanoparticulate Mineralized Collagen Glycosaminoglycan Materials

Abstract

PURPOSE: Developing surgically practical regenerative materials requires in-depth understanding of cell-material interaction mechanisms. We previously identified a novel nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) material—inspired by the extracellular matrix—with the ability to induce calvarial bone healing without added exogenous growth factors or progenitor cells, suggesting that this material may support the development of off-the-shelf implants for skull defects. We have demonstrated that modulating the material’s stiffness activates the mechanotransduction pathway via YAP and TAZ, thus improving osteogenic differentiation. Given the strong relationship between the mechanotransduction pathways and Wnt activation, this work seeks to determine the necessity of the Wnt pathways for the activity of MC-GAG in relationship to differences in stiffness. METHODS: MC-GAG scaffolds were prepared with lyophilization. Crosslinking was performed with 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide at a molar ratio of 5:2:1 EDC:NHS:COOH; COOH represents the amount of collagen in the scaffold. Noncrosslinked (NX-MC) and crosslinked (MC) GAG scaffolds were cultured with primary bone-marrow-derived human mesenchymal stem cells. Osteogenic differentiation was assessed with a combination of quantitative real-time reverse transcriptase polymerase chain reaction for osteogenic differentiation markers, western blot and confocal microscopy for activation and subcellular localization of intracellular mediators, and alizarin red staining and micro-computed tomography for mineralization. Wnt pathways were inhibited using small molecular inhibitors IWR1 (specifically targeting the canonical Wnt/b-catenin pathway) and IWP2 (targeting both canonical and non-canonical Wnt pathways). Analyses of variance and Tukey post hoc testing were performed with a P < 0.05 significance threshold. RESULTS: Inhibiting the Wnt pathways with IWR1 and IWP2 resulted in decreased mineralization of primary human mesenchymal stem cells on MC scaffolds, whereas NX-MC scaffolds were unaffected. Both inhibitors downregulated, but did not eliminate, gene expression of bone markers, including alkaline phosphatase, collagen 1, and bone sialoprotein II primarily affecting MC over NX-MC. However, a reciprocal increase in BMP4 expression was found in the presence of both inhibitors. On western blot analysis, both inhibitors reduced protein expression of active b-catenin, YAP, and TAZ on MC materials, whereas the less stiff NX-MC material demonstrated no differences in expression. Despite the reduction in bone markers and mineralization, a reciprocal increase in Runx2 and Smad1/5 phosphorylation was noted in the presence of IWR1 and IWP2 specifically on the MC scaffolds, but not on NX-MC. On confocal microscopy, untreated materials exhibited colocalization of YAP and active b-catenin in the cytosolic compartment on NX-MC and in both cytosolic and nuclear compartments on MC. While IWR1 and IWP2 did not affect YAP and active b-catenin staining on NX-MC scaffolds, they reduced nuclear localization of active b-catenin on MC scaffolds, with IWP2 demonstrating more of an effect than IWR1. CONCLUSIONS: Mechanistic understanding of regenerative materials is required for safe clinical translation. We have shown that stiffness of MC-GAG, a promising skull regenerative material, can improve osteogenic capabilities via YAP/TAZ-mediated mechanotransduction. In this work, we showed that the osteogenic properties, imparted by the stiffness of MC-GAG, function through the Wnt signaling pathways, such that inhibition downregulates YAP/TAZ expression, mineralization, and expression of bone markers.