This study was approved by the Research Ethics Committee of the São Leopoldo Mandic Research Institute, Campinas/SP (protocol No. 59866216.6.0000.5374). Titanium disks were commercially pure, grade 4 (n = 108), measuring 6 mm in diameter by 2 mm in thickness, provided by the company Conexão Sistemas de Próteses (Arujá, São Paulo).

For the treatment of titanium disk surfaces, sulfuric, nitric, and hydrochloric acid solutions were used for 20 min (n = 36) and 60 min (n = 36). As a control, disks with no surface treatment (n = 36, machined surface only) were used. The concentration of each acid in the solutions is not described because the company did not wish to disclose it. Roughness was measured using a contact profilometer (Mitutoyo, Surftest model SJ200, Brazil, Suzano). Four linear measurements were performed on each sample according to DIN ISO 1302 standard, and the arithmetic mean of the absolute values for each disk (Ra) was calculated. The average roughness (Ra) obtained was 0.1 to 0.15 μm, for 20 min of acid treatment, and 0.5 to 0.7 μm, for 60 min of acid treatment. Figure 1 shows the morphological characteristics of the surfaces obtained.

The disks were sterilized in ethylene oxide (Acecil, Campinas, São Paulo) and used for the subsequent experiments. Three different human fibroblast cell lines were obtained from the Cell Bank of the Cell Culture Laboratory (São Leopoldo Mandic Research Institute, Campinas) to control for phenotypic and genotypic variations. These cells were previously isolated by primary culture of human gingiva from three patients [12].

The cells were cultured on 96-well plates (Corning, NY, USA) at an initial concentration of 110 cells/mm². For the cell proliferation assay, the fibroblasts were detached using 0.05% trypsin and counted on a hemocytometer to calculate proliferation indices at 24 h, 48 h, and 72 h. Cell viability assay was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, Sigma), at the same time periods. The optical density was read at 570–650 nm on a plate reader (Epoch, Bio-Tek, Winooski, VT), and data were expressed as absorbance.