Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb The Wnt/β-catenin signaling pathway is regulated by titanium with nanotopography to induce osteoblast differentiation Rodrigo P.F. Abuna, Fabiola S. Oliveira, Helena B. Lopes, Gileade P. Freitas, Roger R. Fernandes, Adalberto L. Rosa, Marcio M. Beloti ⁎ Bone Research Lab, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil ARTICLE INFO Keywords: CRISPR Nanotopography Osteoblast Titanium Wnt ABSTRACT Wnt/β-catenin signal transduction is involved in the homeostatic control of bone mass. It is well established that a titanium surface with nanotopography (Ti-Nano) favors osteoblast differentiation by modulating different signaling pathways. However, few studies have investigated the participation of the Wnt/β-catenin pathway in the osteogenic effect of nanoscale topographies. In this study, we aimed to determine whether the Wnt/β-catenin signaling pathway is involved in the elevated osteogenic potential of Ti-Nano. MC3T3-E1 cells were cultured on Ti-Nano and machined Ti (Ti-Control) for evaluation of the expression of Wnt/β-catenin signaling pathway- related genes. Based on the results to real-time PCR, the Wnt receptor Fzd4 was selected and silenced by CRISPRi. The resulting cells were cultured on both Ti surfaces, and several events involved in osteoblast differentiation were evaluated. The results revealed that Fzd4 gene silencing, corresponding to negative modulation of Wnt/β- catenin, inhibits expression of the osteoblast phenotype. It is worthy of note that this inhibitory effect on os- teoblast differentiation was more pronounced in cells grown on Ti-Nano compared with those grown on Ti- Control. By disrupting Fzd4 gene expression, we have shown that the elevated osteogenic potential of Ti-Nano is due to activation of the Wnt/β-catenin signaling pathway, which reveals a new mechanism to explain osteoblast differentiation induced by nanotopography. Such an understanding of the intracellular machinery involved in surface guiding of osteoblast fate may contribute to the development of smart biomaterials to modulate the process of implant osseointegration. 1. Introduction The nanotopography surface of titanium (Ti) implants enhances the osteoblast-substrate interaction, producing modifications in protein adsorption and focal cell adhesion that improve the rate of osseointe- gration. Nanoscale surfaces are common in the extracellular matrix microenvironment; they generate mechanical effects that influence processes such as cell migration and polarization [1]. Synthetically, nanoscale surfaces are created not only to regulate initial cell interac- tions with the substrate but also to modify the intracellular signaling pathways and, consequently, influence the phenotype and biological responses to implants [2,3]. In recent years, cellular mechanisms that are triggered by nanotopography have been unveiled; nonetheless, the Wnt signaling pathway that is responsible for tissue and organ struc- tural organization, including that of bone, remains incompletely un- derstood in this context. WNTs are lipid-modified glycoproteins that activate cell surface receptor-mediated signal transduction pathways, which are essential during embryonic development. WNT transduction effects are pleio- tropic, thus regulating a plethora of cellular activities such as mitogenic stimulation, cell fate commitment, proliferation, and differentiation, in addition to homeostasis in adult tissues. WNT proteins act through Frizzled (FZD) receptors, which transduce the signal through either the canonical β-catenin pathway or the non-canonical pathway [4]. Cano- nical signaling is referred to as the Wnt/β-catenin pathway and consists of a heterotrimeric complex composed of WNT ligands, low-density li- poprotein receptor-related protein transmembrane coreceptors (LRP), and signaling transmembrane receptors FZD [5–7]. This pathway is initiated when a ligand binds to the FZD and recruits the coreceptor LRP5/6 in an oligomeric complex called signalosome [8]. Then, a complex cascade involving casein kinase 1, axin, the phosphoprotein Disheveled, and glycogen synthase kinase 3 leads to cytosolic stabili- zation of β-catenin and its subsequent translocation to the nucleus, where it dislocates the Groucho co-repressor and interacts with the T- cell transcription factor/lymphoid enhancer (TCF/LEF) to mediate the effects of the Wnt pathway on gene transcription [9,10]. Both gain- and https://doi.org/10.1016/j.colsurfb.2019.110513 Received 19 June 2019; Received in revised form 31 August 2019; Accepted 17 September 2019 ⁎ Corresponding author at: School of Dentistry of Ribeirão Preto, University of São Paulo, Av. do Café s/n, 14040-904, Ribeirão Preto, SP, Brazil. E-mail address: mmbeloti@usp.br (M.M. Beloti). Colloids and Surfaces B: Biointerfaces 184 (2019) 110513 Available online 20 September 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved. T