Effects of Zinc Ions Released from Ti-NW-Zn Surface on Osteogenesis and Angiogenesis in Vitro and in an in Vivo Zebrafish Model

Abstract

Objective: Zinc-modified titanium materials have been widely applied in oral implants, among them, our previous studies have also successfully prepared a novel acid-etched micro-structured titanium surface modified with zinc-containing nanowires (Ti-NW-Zn). However, there are very few reports concerned about the biological safeties of zinc ions released from material surface and the appropriate concentration range of released zinc ions which was more conducive to angiogenesis and bone regeneration. Danio rerio (Zebrafish) represents a powerful alternative in vivo model to study both angiogenesis and osteogenesis. Then, the aim of this study is to investigate the effects of zinc ions released from Ti-NW-Zn surfaces on angiogenesis and osteogenesis via zebrafish model, and further revealed the relationship between angiogenesis and osteogenesis via HUVECs and MC3T3-E1s in vitro models.Materials and methods: Tg (Fli‐1:EGFP)y1 zebrafish embryos were used to investigate the in vivo angiogenesis exposure to zinc ions, Wild-type Zebrafish (Danio rerio) adult fish and their embryos were used to investigate the in vivo bone regeneration and survival rate. Cell experiments including proliferation, adhesion, osteogenic differentiation and signaling pathway experiments were carried out by CCK-8, Laser scanning confocal microscope and Western blot, etc. Zinc ion release assay was detected by a Zinc assay kit.Results: The zinc ions released from Ti-NW-Zn surfaces were far lower than median lethal concentrations (LCs) of both embryos and adult fish, which was beneficial to osteogenesis and angiogenesis promotion of zebrafish model. Moreover, the appropriate concentration range of zinc ions was 1-2 ppm in vitro models, which could induce HUVECs proliferations, and the conditioned medium (CM) collected from the supernatant of 1-2 ppm zinc ions-induced HUVECs medium could obviously promote MC3T3-E1 osteoblasts behaviors via activated the MAPK/ERK signaling pathway.Conclusion: Zinc ions released from Ti-NW-Zn surfaces was at a biological safe and appropriate concentration, which could promote the angiogenesis and osteogenesis in vivo and vitro. The positive effects of the appropriate concentration of zinc ions on osteoblast behaviors might be regulated by activating the MAPK/ERK signaling pathway.