The results showed that, regardless of the treatment used, there was no difference in the values of cell proliferation in the different times of analysis. Similarly, cell viability values did not differ, especially after 24 h and 72 h. Corroborating the results of this study, Baltriukiene et al. [17] used grade 2 titanium disks with different surface treatments and demonstrated that the modified titanium surface did not affect human gingival fibroblast viability. Interestingly, some studies show that smooth surfaces increase the proliferation and adhesion of fibroblasts [18, 19], which raises the question of whether the recommended acid treatment of titanium surfaces indeed has an advantage in terms of promoting greater biological sealing. In addition, increased surface roughness promotes bacterial colonization and increased retention [20, 21], contributing to increased biofilm formation.

In the present study, human gingival fibroblasts were used in a similar strategy as per other reports in the literature [10, 15, 22,23,24,25]. In spite of in vivo studies representing the ideal environment to investigate tissue responses, in vitro studies facilitate the investigation of a specific and isolated factor of the tissue response [26]. In addition to being widely used in cell biocompatibility studies, considering the various topographic surface features, fibroblasts are responsible for the biosynthesis of collagen and other extracellular matrix molecules.

To establish the amount of protein secreted by fibroblasts, we chose type I collagen, as it is the most abundant protein in the extracellular matrix of gingival connective tissue, representing almost 90% of the total organic matrix in mature bone. The results revealed differences between groups only at 48 h of culture, where the lowest titers were observed when surfaces were treated with acid solutions. At 24 h and 72 h, no significant difference was observed regarding collagen secretion, though the highest quantification occurred at 72 h for the surfaces treated for 60 min. These results are in agreement with the findings by Ramaglia et al. [9] and Velasco-Ortega et al. [24], in which they suggested that roughened surfaces may improve the biological behavior of fibroblasts as well as the process of perimplant soft tissue healing and osseointegration compared to machined surfaces.