In the present study, three-layer glass-ceramic coatings of different chemical compositions were synthesized and coated on the Ti6Al4V substrate using the sol–gel method. Hydroxyapatite, hydroxyapatite–wollastonite, and wollastonite–anorthite were coated on the Ti6Al4V substrate, respectively. X-ray diffraction results showed that crystallinity of the main phase in the first (hydroxyapatite (94 wt%)), second (wollastonite (88 wt%)), and third (anorthite (80 wt%)) layers were about 70%, 61%, and 55%, respectively. Scanning electron microscopy analysis showed that when the applied coatings were heat-treated at 800 °C, the morphology of the first layer was changed from spherical to needle-like morphology. The values of microhardness for the first, second, and third layers of the coating were measured as 400, 503.3, and 750.4 Hv, respectively. The mean values of the surface roughness for the mentioned layers, which was analyzed using atomic force microscopy (AFM), were 20, 54, and 63 nm, respectively. The results showed that the coated samples interacted with SBF medium, resulting in the increment of pH and the formation of hydroxyapatite on the coated substrate. MTT assay indicated that the coating process and the coating layers had no adverse effect on the viability of the human osteoblastic SaOS2 osteosarcoma cell line. Cell attachment results showed that the SaOS2 cell line was well attached and expanded on each layer of the coating.

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