Modification of Titanium-based implant materials by Zn-doped calcium phosphate coatings improves corrosion resistance, biomineralization, antibacterial efficacy and immunocompatibility

Implant-induced chronic inflammation and aseptic implant failure represent serious clinical challenges in orthopedics, dentistry, and reconstructive surgery. Our study aimed to develop an implant material that minimizes acute and chronic inflammation while providing long-term integration properties. We systematically investigated the innate immune responses modulated by the addition of Zn to CaP coatings, using whole transcriptome profiling through RNA sequencing (RNA-Seq). Overall design: The coatings were produced on Ti64 alloy (ELI grade) substrates by the microarc oxidation method (MAO). The treatment was performed in aqueous electrolytes containing C10H12CaN2Na2O8, (NH4)3PO4, KH2PO4 and C4H6O4Zn as a Zn component. The electrolyte was agitated by a magnetic stirrer. The coatings were produced by applying a unipolar waveform with a constant peak current density of 5 A dm-2, 50% duty cycle, and 1 kHz frequency. The duty cycle is defined as the ratio of the anodic on-time per cycle to the total time of the cycle. The electrical parameters were chosen in such a way that layer thicknesses between 10 and 20 µm were achieved. Ti64_CaP_Zn25, Ti64_CaP_Zn50, Ti64_CaP_Zn75, and Ti64_CaP_Zn100 were prepared by adding zinc acetate as a zinc carrier in the doping solutions to CaP, with concentrations of 25 g/L, 50 g/L, 75 g/L, and 100 g/L, respectively. Buffy coats obtained from German Red Cross Blood Service Baden-Württemberg – Hessen were diluted at a ratio of 1:1 with Ca2 +- and Mg2 +-free phosphate-buffer saline (PBS) (Biochrom, Berlin, Germany). CD14+ monocytes were isolated by 2 sequential gradient centrifugations followed by the soring with CD14 Microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Monocytes were cultivated for 6 days on the cell culture plastic or sample disks at a concentration of 2×106 cells/mL in macrophage serum-free medium X-Vivo 10 (Lonza) supplemented with 5 ng/mL M-CSF (Peprotech, Hamburg, Germany) and cytokines as follows: For M0, no cytokines were added; for M1, 100 ng/mL of IFN? (Peprotech, Hamburg, Germany) was added; and for M2, 10 ng/mL of IL-4 was added. Cells were harvested for RNA isolation and RNA-seq.