Behavior of Human Osteoblast Cells Cultured on Titanium Discs in Relation to Surface Roughness and Presence of Melatonin

Abstract

The aim of this work was to observe the behavior of osteoblast cells cultured in vitro on titanium discs in relation to disc surface roughness and the addition of melatonin to the culture medium. MG63 osteoblast cells were cultivated on 120 Grade 5 Ti divided into three groups: Group E, treated with dual acid etch; Group EP, treated with dual acid etch and calcium phosphate; and Group M, machined. Surface roughness was examined under a laser scanning confocal microscope (CLSM) and scanning electron microscopy (SEM). The proliferation and morphology of cells were determined under fluorescence microscopy and SEM. Messenger ribonucleic acid (mRNA) of different genes related to osteoblastic differentiation was quantified by means of real-time quantitative polymerase chain reaction (RT-PCR) assay. The greatest surface roughness was found in Group EP (Ra 0.354 µm), followed by Group E (Ra 0.266 µm), and Group M (Ra 0.131 µm), with statistically significant differences between the groups (p < 0.001). In the presence of melatonin a trend to a higher cell proliferation was observed in all groups although significant differences were only found in Group M (p = 0.0079). Among the genes studied, a significant increase in phosphate-regulating neutral endopeptidase, X-linked (PHEX) expression was observed in cells cultured on EP discs. The addition of melatonin increased osteoblast cell proliferation and differentiation, and may favor the osseointegration of dental implants.

Keywords: PHEX; dental implants; differentiation; mRNA; melatonin; osteoblasts; proliferation; roughness; titanium.

Figure 1

Microphotographs taken with an optical microscope (35×). (A) Image of a Group M disc; (B) Image of a Group E disc.

Figure 2

Microphotographs captured by scanning electron microscopy (SEM) (2000×). (A) Image of a Group M disc showing the lines produced by machining; (B) Image of a disc in Group E showing characteristic microtexture of pits and hollows.

Figure 3

Box plot of roughness (Ra) in different study groups (mean ± SD).

Figure 4

Microphotograph of a Group E disc showing the blue fluorescence of cell nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) and the green fluorescence of cell cytoplasm components stained with eosin.

Figure 5

Cell density (mean and SD) of cells grown on discs of the three experimental groups after 72 h of cell culture without or with melatonin added to the cell culture media. Statistically significant differences between groups are shown (*, p < 0.05; **, p < 0.01.).

Figure 6

Scanning electron micrograph of MG63 cells cultured on titanium disc surfaces (350×). (A) Group M disc showing osteoblast adhered over the surface following the concentric machining lines; (B) Group E disc where cells were distributed randomly over the whole surface.

Figure 7

Scanning electron microphotographs of MG63 cells cultured of titanium disc surfaces (2000×): (A) Group M disc cells of flat morphology could be seen on the machined discs in close contact with the surface with cytoplasmic prolongations and filopodia; (B) Group E cells were more rounded or cuboid, more regular and with shorter prolongations.

Figure 8

Quantification of the relative expression of the indicated genes after one week of cell culture without melatonin of the indicated genes. The mean Fc and SD value are represented for the different genes, taking the average expression of cells grown on Group M discs as control.

Figure 9

Quantification of the relative expression of the indicated genes after one week of cell culture with melatonin added to the culture media. The mean Fc and SD values are represented for the different genes, taking the average expression of cells grown on Group M discs as control.

Figure 10

Quantification of the relative expression of the indicated genes after five weeks of cell culture without melatonin of the indicated genes. The mean Fc and SD value are represented for the different genes, taking the average expression of cells grown on Group M discs as control.

Figure 11

Quantification of the relative expression of the indicated genes after five weeks of cell culture with melatonin added to culture media. The mean Fc and SD values are represented for the different genes, taking the average expression of cells grown on Group M discs as control.