Division of Plastic and Reconstructive Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, 90095, USA.
Research Service, Greater Los Angeles VA Healthcare System, Los Angeles, CA, 90073, USA.
Adv Healthc Mater. 2021 Dec;10(23):e2101467. doi: 10.1002/adhm.202101467. Epub 2021 Oct 8.
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.
目标再生材料的精细化需要对细胞-材料相互作用有机械理解。研究表明，纳米颗粒矿化胶原糖胺聚糖（MC-GAG）支架在体内促进颅骨再生，无需添加外源性生长因子或祖细胞，这表明其具有临床转化的潜力。这项工作评估了 MC-GAG 刚度对经典 Wnt（cWnt）信号的调制。原代人骨髓间充质干细胞（hMSCs）在两种 MC-GAG 支架（未交联的 NX-MC，0.3 kPa 与传统交联的 MC，3.9 kPa）上分化。hMSCs 增加了激活的β-连环蛋白（cWnt 的主要细胞内介质）和机械敏感的 YAP 蛋白的表达，这些蛋白在较硬的 MC 支架上几乎完全亚细胞共定位。总体 Wnt 通路抑制降低了激活的β-连环蛋白和成骨分化，同时提高了 MC 上的 BMP4 和磷酸化 Smad1/5（p-Smad1/5）的表达，但 NX-MC 上没有。与 Wnt 通路下调不同，用β-连环蛋白敲低单独抑制经典 Wnt 会特异性增加较硬的 MC 上的成骨分化和矿化。β-连环蛋白敲低还仅在较硬的 MC 材料上增加 p-Smad1/5、Runx2 和 BMP4 的表达。因此，虽然刚度诱导的 Wnt 和机械转导途径的激活促进了 MC-GAG 上的成骨作用，但激活的β-连环蛋白是一个限制因素，它可能作为优化骨骼再生材料刚度调制的有用靶点或读出信号。
