In an effort to examine the effect of the microstructural changes of the Ti6Al4V alloy, two heat treatments were carried out below (Ti6Al4V800) and above (Ti6Al4V1050) its β-phase transformation temperature. After each treatment, globular and lamellar microstructures were obtained. Saos-2 pre-osteoblast human osteosarcoma cells were seeded onto Ti6Al4V alloy disks and immersed in cell culture for 7 days. Electrochemical assays in situ were performed using OCP and EIS measurements. Impedance data show a passive behavior for the three Ti6Al4V alloys; additionally, enhanced impedance values were recorded for Ti6Al4V800 and Ti6Al4V1050 alloys. This passive behavior in culture medium is mostly due to the formation of TiO2 during their sterilization. Biocompatibility and cell adhesion were characterized using the SEM technique; Ti6Al4V as received and Ti6Al4V800 alloys exhibited polygonal and elongated morphology, whereas Ti6Al4V1050 alloy displayed a spherical morphology. Ti and O elements were identified by EDX analysis due to the TiO2 and signals of C, N and O, related to the formation of organic compounds from extracellular matrix. These results suggest that cell adhesion is more likely to occur on TiO2 formed in discrete α-phase regions (hcp) depending on its microstructure (grains).

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Two heat treatments were carried out below (Ti6Al4V800) and above (Ti6Al4V1050) Ti6Al4V beta-phase transformation temperature (980 °C), with the purpose of studying the effect of microstructure on the adhesion and proliferation of fibroblast cells, as well as their electrochemical behavior. These alloys were seeded with 10,000 L929 fibroblast cells and immersed for 7 days in the cell culture at 37 °C, pH 7.40, 5% CO2 and 100% relative humidity. Cell adhesion was characterized by Scanning Electron Microscopy (SEM) and Electrochemical Impedance Spectroscopy (EIS) techniques. Polygonal and elongated cell morphology was observed independent of Ti6Al4V microstructure. Besides, C, O, P, S, Na and Cl signals were detected by Energy Dispersive X-Ray Spectroscopy (EDX), associated with the synthesis of organic compounds excreted by the cells, including protein adsorption from the medium. In certain areas on Ti6Al4V and Ti6Al4V800 alloys, cells were agglomerated (island type), likely related to the globular microstructure; meanwhile, larger cellular coverage is shown for Ti6Al4V1050 alloy, forming more than one layer on the surface, where only Ca was recorded. Impedance diagrams showed a similar passive behavior for the different Ti6Al4V alloys, mainly due to TiO2 overlaying the contribution of the organic compounds excreted by fibroblast cells.

Two heat treatments were carried out at below (Ti6Al4V800) and above (Ti6Al4V1050) the beta-phase transformation temperature (TTRANSUS = 980 °C), to study the effect of microstructural changes on osseointegration. The alloys were implanted in the femurs of hind legs of Wistar rats for 15, 30, and 60 days. Histology of the femur sections obtained for the first 15 days showed inflammatory tissue surrounding the implants and tissue contraction, which prevented osseointegration in early stages. After 30 days, trabecular bone, reduction of inflammatory tissue around the implants, and osseointegration were observed in Ti6Al4V as received and Ti6Al4V1050 alloys, while osseointegration was detected for the three alloys after 60 days. These results were supported through morphometric studies based on the analysis of Bone Implant Contact (BIC), where there was a larger bone contact after 60 days for the Ti6Al4V1050 alloy; indicating that microstructural features of the Ti6Al4V alloys influence their osseointegration, with the lamellar microstructure (Ti6Al4V1050), being the most responsive.