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Micro/nanostructured TiO2/ZnO coating has been shown to possess multiple functions, including antibacterial activity and bioactivity. Osteoblast-like SaOS-2 cells were employed for evaluating the in vitro osteogenic capacity of this coating and positive results were obtained. However, traditional principles of osseointegration focus only on the osteogenic differentiation alone. The effects of immunomodulation on the osteogenic activity have been largely ignored. In this study, the inflammatory responses of macrophages on the micro/nanostructured TiO2/ZnO coating were investigated. The extract media of macrophage cell line RAW264.7 cultured on the TiO2/ZnO coating were collected as indirect co-culture conditioned media. The osteogenic activity of SaOS-2 cells in the conditioned media was investigated. Adhesion, ALP activity and extracellular mineralization of cells grown in the conditioned media extracted from the micro/nanostructured TiO2/ZnO coating were found to be enhanced, compared to those grown in the conditioned media extracted from the macroporous TiO2 coating. The immune microenvironment produced by the micro/nanostructured TiO2/ZnO coating showed excellent capacity to promote osteogenesis, indicating that this coating could be a promising candidate for implant surface modification in orthopaedic and dental applications. Furthermore, this work could help us understand the interplay between the host immune system and the osteoimmunomodulatory properties of the biomaterials, and optimize the design for coating biomaterials.

Insufficient osseointegration between implants and bone, and peri-implant infection are the two most common factors contributing to implantation failure. As such, implant materials should ideally be designed with antibacterial capacity while maintaining cytocompatibility. Combining Zn, a known antibacterial agent, with micro/nanostructures on the implant surface to promote osseointegration, potentially reduces peri-implant infection. To this end, in the present study, we developed novel bifunctional micro/nanostructured titanium oxide/zinc oxide (TiO2/ZnO) coating by micro-arc oxidation (MAO) followed by hydrothermal treatment. The release rate of the Zn2+ ions was reduced by heat treatment which established a balance between antibacterial capacity and cytocompatibility. The results demonstrate a simple approach for developing a Zn-based material that balances antibacterial capacity and cytocompatibility as a promising coating material for Ti implants.

To improve the success of medical devices, implants with strong surface bioactivity are urgently required. Coatings with a macroporous structure produced by micro-arc oxidation possess advantages, such as strong adhesion to substrate and excellent resistance to wear and corrosion. Mesoporous structures contain pores with sizes of 2-50 nm, which can endow the biomaterials with the ability to enhance osteogenesis and to be loaded with diverse drugs. Thus, in this study, we aimed to evaluate the effects of both macroporous and mesoporous structures using a hierarchical macro/mesoporous structure to modify the titanium implant surface. The behaviors of SaOS-2 human osteosarcoma cells on the macro/mesoporous structure, including initial adhesion, proliferation, alkaline phosphatase (ALP) activity, and collagen secretion, were investigated. Cells that attached on the macro/mesoporous surface showed the highest cell numbers and greatest spreading area after incubation for 1, 2, and 4 h compared with the polished smooth substrate and macroporous surface in the presence of fetal bovine serum (FBS). However, in the absence of FBS, cell adhesion on the polished substrate, macroporous structure, and macro/mesoporous structure did not differ significantly. Cell proliferation on the macroporous and macro/mesoporous surfaces increased compared with that on the smooth substrate surface. Furthermore, ALP activity and collagen secretion were enhanced on the macro/mesoporous structure. Our findings provided important insights into the cellular responses to macro/mesoporous structures in the field of implant surface modification.

Zinc oxide (ZnO) has attracted a great deal of interest due to its electronic and optical properties, and has potential applications in biomedical field, while few studies have been conducted to investigate its biocompatibility. In this study, we prepared nanostructured ZnO on acid-etched pure Ti surface through a moderate hydrothermal treatment in ammonia solution (pH = 12.6). By changing Zn precursor concentration and hydrothermal duration, it was feasible to synthesis flake-like ZnO nanostructures with variable spacings. The adhesion, proliferation and alkaline phosphatase (ALP) activity of SaOS-2 cells were enhanced on the ZnO nanostructures when the Zn precursor concentration was 0.02 M and the hydrothermal duration was 4 h, compared to those on the acid-etched pure Ti without ZnO nanostructures. Narrower spacings (< 70 nm) between ZnO nanostructures are considered to contribute to this enhancement. Together, the results indicate that the superposition of ZnO nanostructures on titanium surfaces may be beneficial for the enhanced biological performance.

A dual-layer TiO2 surface with hierarchical macro and mesoporous structure was prepared by a combinational approach of micro-arc oxidation followed by evaporation-induced self-assembly of nano-crystallites. The mesoporous layer contains pores with an average size of <10 nm and consists of anatase TiO2 nanocrystallites. The dual-layer hierarchical macro/mesoporous structured TiO2 surface improves the hydrophilicity and fibronectin adsorption ability in comparison with the sole macroporous or smooth TiO2 surface. With the formation of an additional mesoporous layer on macroporous TiO2 surface, the attached number of human osteogenic sarcoma cells (SaOS-2) increases in the initial incubation of 4 h but it does not show significant difference after 24 h compared to that attached on the macroporous or smooth surfaces. Whereas, it was noticed that SaOS-2 cells have larger spread area and more stress fibers on the macro/mesoporous structured surface than those on the other surfaces. To understand the intracellular mechanism of the initial cell adhesion on the macro/mesoporous surface, the Rho/ROCK pathway was investigated to reveal the topography-induced biological functions by introducing the ROCK inhibitor Y-27632 during cell culture. In the presence of Y-27632, cells on the macroporous surface and macro/mesoporous surface both show stellate appearance, with poor assembly stress fibers and long cell membrane protrusions. Cells on the smooth surface have larger spread areas compared to the former two surfaces. And the attached cells significantly reduced but there are no differences among the three surfaces. It reveals that the ROCK inhibitor invalidates the promotion of initial cell adhesion on the macro/mesoporous structure. This study may shed light on the mechanism behind the enhancement effect of macro/mesoporous structure for initial cell adhesion.