shows the surface morphology of a Porous sample obtained by immersion treatment in acid solution. The acid etching produces a homogeneous surface characterized by microcavities surrounded by tapered summits. This pattern of roughness produces a homogeneous surface without preferential roughness orientation.
shows that the immersion of the Porous surface in a solution containing fluoride ions did not change the microcavity morphology, and the sharp edges persisted. At higher magnification, the presence of flatter areas and smaller micropeaks may be noted although these surfaces remain tapered. This change may be associated with the high reactivity of fluorine ions and the chemical susceptibility of titanium oxide to these ions, which may produce a coalescence of peaks. These results are consistent with those of Ellingsen and Lyngstadaas [9
] and Johansson et al. [8
], which showed that titanium surfaces treated with fluoride present smoother microtopographies and lower Ra
values than acid-treated surfaces without fluoride. demonstrates the presence of microcavities, summits, and conglomerates on their edges, most likely due to the corrosion process and consequent decrease in surface roughness for the surface subjected to immersion in solution containing fluoride.
Images obtained by atomic force microscopy () show that both the Porous and PorousNano surfaces exhibit microcavities surrounded by summits. Like the high-resolution SEM images, the AFM images indicate that summits and microcavities of the PorousNano sample surface have smoother edges although they remain tapered. These sharp edges seem to assist or facilitate the adsorption of FN and cells.
As measured based on the images obtained through AFM, the roughness of the PorousNano surface sample was lower than that of the Porous surface, demonstrating that treatment with fluoride reduced the summit height, most likely due to the reaction of titanium oxide with fluoride ions. This ultrastructural aspect of the summits contributes to the more homogeneous roughness pattern of the PorousNano surface, in addition to the presence of smoother areas and larger microcavities.
The presence of only one crystalline phase of titanium was revealed by X-ray diffraction of the Porous and PorousNano samples. It is likely that the immersion in a solution containing fluoride ions adds only a small amount of this element to the titanium surface and that this trace amount of fluoride cannot be detected by the XRD technique for the analysis of thin films (grazing incidence technique).
Approximately 80% of the FN allowed to interact with Porous and PorousNano surfaces was adsorbed (2.473
AU). This result demonstrates that the chemical treatment with acids (Porous) and chemical treatment with acids followed by immersion in solution containing fluoride ions (PorousNano) did not affect the incorporation of biomolecule; that is, the presence of the fluoride ion did not influence the protein adsorption. Dos Santos et al. [15
] observed that FN adsorption to anodized titanium samples was 68%. It can be concluded that titanium surfaces have an affinity for fibronectin and that differences in the percentage of incorporation in different studies most likely are due to the conditions under which the FN was reacted with the surfaces (pH used, for example) and/or the various treatments performed on them.
Cell counting in a hematimetric chamber is a sensitive and accurate technique for the evaluation of cell adhesion to titanium surfaces. In this study, the PorousNano surface showed a stronger association with osteoblastic cells (2,3 × 105
cells/mL) than the Porous surface (7,9 × 104
cells/mL) after one hour of interaction. Because 106
cells/mL were taken to interact with surfaces, approximately 8% adhered to the Porous sample, while 23% were associated with the PorousNano sample. These indices suggest that the surface subjected to chemical treatment followed by immersion in a solution containing fluoride ions favors the adhesion of most cells during the initial interaction period. As mentioned earlier, the association indices of the Porous and PorousNano surfaces without fibronectin were considered null for evaluations of the influence of protein on cell behavior. Thus, the number of cells associated with the PorousNano with FN surface increased 57.4% compared with the same surface without the biological variable. For the Porous with FN surface, the increase in cell adhesion was 44.7% compared to the same area without the protein. These indices show that the protein variable is responsible for the significant increase in the number of cells attached to the surfaces, confirming the results of Ku et al. [16
], who also reported an increase in the adhesion rate of cells to surfaces treated with recombinant fibronectin. They showed that, for TiO2
, cell adhesion was initiated after 3 hours and had significantly lower cell numbers for all measurement points compared with FN. The present work showed the same results.
The cell-surface interaction index of PorousNano with FN was approximately 28% higher than that of the Porous with FN surface. This study demonstrates that, among the four types of surfaces examined, the PorousNano with fibronectin coating most favors the adsorption and adhesion of osteoblastic cells during the tested interaction period. In addition, the study provides strong evidence that FN incorporation into titanium surfaces is much more relevant for biocompatibility and the consequent acceleration of the osseointegration process than surface treatment with acid and/or immersion in solution containing fluoride ions.
A total of 1.8 × 106
cpm) were allowed to interact with Porous, Porous-FN, PorousNano, and PorousNano-FN titanium surfaces for three hours. After one hour of interaction, 92% of the cells were associated with the PorousNano-FN surface, and 90% of cells were associated with the Porous-FN surface, while 70% and 64% of cells were associated with the PorousNano and Porous surfaces (without FN), respectively. These results confirm that the protein coating accelerated the adsorption of cells during the initial interaction period (adaptation period). This can be explained by the fact that when fibronectin is allowed to interact with titanium samples under ideal conditions of pH such that its cryptic sites are exposed, the fibronectin signals to osteoblasts to activate the cell cycle and initiate the secretion of ECM proteins.
The Porous-FN and PorousNano-FN surfaces showed similar behavior during the three-hour interaction, both during the initial adherence of cells (approximately 90% for both surfaces) and in their proliferation. The cell number increased by 14% for the sample PorousNano-FN and 12% for Porous-FN in the first 3 hours of interaction (). Ku et al. [16
] also demonstrated that the biomimetization of titanium surfaces with fibronectin increased the adhesion, proliferation, and differentiation rates of cells.
In samples without FN, this study showed that within one-to-three hours of interaction, the number of cells attached to the PorousNano surface increased by 32%, while the number of cells attached to the Porous surface increased by 15%. This difference shows that the surface that received acid treatment followed by immersion in a solution containing fluoride ions (Nano) showed accelerated cell division and proliferation compared to the Porous surface. shows that the PorousNano surface without fibronectin coating exhibited the greatest increase in cpm as a function of time over 3 hours. Ellingsen and Lyngstadaas [9
] and Johansson et al. [8
] showed that fluoride-treated surfaces have a greater capacity to react with biological tissues and nuclear phosphate crystals in vitro
, in addition to offering greater osseointegration resistance in vivo
. Although previous studies have used different methodologies for fluoride treatment, their results also suggest that the presence of fluoride ions on titanium surfaces facilitates various osseointegration processes. Analysis of the experimental cell adhesion and proliferation data presented in showed that the cell behavior was similar in all samples containing fibronectin. The results of this study show that the FN is critical to the biocompatibility of surfaces of titanium implants, but when this protein is not present, treatment with acids and fluorides seems to favor more tissue integration than treatment with acid only (i.e., no fluoride).