Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech, Georgia Institute of Technology, 315 Ferst Drive NW, Atlanta, GA 30332-0363, USA.
Acta Biomater. 2011 Jun;7(6):2740-50. doi: 10.1016/j.actbio.2011.02.030. Epub 2011 Feb 23.
The Wnt signaling pathway inhibitor Dickkopf-2 (Dkk2) regulates osteoblast differentiation on microstructured titanium (Ti) surfaces, suggesting involvement of Wnt signaling in this process. To test this, human osteoblast-like MG63 cells were cultured on tissue culture polystyrene or Ti (smooth PT (Ra=0.2 μm), sand-blasted and acid-etched SLA (Ra=3.22 μm), modSLA (hydrophilic SLA)). Expression of Wnt pathway receptors, activators and inhibitors was measured by qPCR. Non-canonical pathway ligands, receptors and intracellular signaling molecules, as well as bone morphogenetic proteins BMP2 and BMP4, were upregulated on SLA and modSLA, whereas canonical pathway members were downregulated. To confirm that non-canonical signaling was involved, cells were cultured daily with exogenous Wnt3a (canonical pathway) or Wnt5a (non-canonical pathway). Alternatively, cells were cultured with antibodies to Wnt3a or Wnt5a to validate that Wnt proteins secreted by the cells were mediating cell responses to the surface. Wnt5a, but not Wnt3a, increased MG63 cell differentiation and BMP2 and BMP4 proteins, suggesting Wnt5a promotes osteogenic differentiation through production of BMPs. Effects of exogenous and endogenous Wnt5a were synergistic with surface microstructure, suggesting the response also depends on cell maturation state. These results indicate a major role for the non-canonical, calcium-dependent Wnt pathway in differentiation of osteoblasts on microstructured titanium surfaces during implant osseointegration.
Wnt 信号通路抑制剂 Dickkopf-2（Dkk2）调节成骨细胞在微结构钛（Ti）表面的分化，提示 Wnt 信号通路在此过程中起作用。为了验证这一点，将人成骨样 MG63 细胞培养在组织培养聚苯乙烯或 Ti（光滑 PT（Ra=0.2μm）、喷砂酸蚀 SLA（Ra=3.22μm）、modSLA（亲水 SLA））上。通过 qPCR 测量 Wnt 通路受体、激活剂和抑制剂的表达。非经典通路配体、受体和细胞内信号分子以及骨形态发生蛋白 BMP2 和 BMP4 在 SLA 和 modSLA 上上调，而经典通路成员下调。为了证实非经典信号通路的参与，每天用外源性 Wnt3a（经典通路）或 Wnt5a（非经典通路）培养细胞。或者，用 Wnt3a 或 Wnt5a 的抗体培养细胞，以验证细胞分泌的 Wnt 蛋白介导细胞对表面的反应。Wnt5a 而非 Wnt3a 增加了 MG63 细胞分化和 BMP2 和 BMP4 蛋白，表明 Wnt5a 通过产生 BMPs 促进成骨分化。外源性和内源性 Wnt5a 的作用与表面微观结构协同，表明该反应还取决于细胞成熟状态。这些结果表明，在植入物骨整合过程中，非经典、钙依赖性 Wnt 通路在成骨细胞在微结构钛表面的分化中起主要作用。
