Decontamination methods to restore the biocompatibility of contaminated titanium surfaces

Abstract

Purpose: The reaction of cells to a titanium implant depends on the surface characteristics of the implant which are affected by decontamination. The aim of this study was to evaluate the cytocompatibility of titanium disks treated with various decontamination methods, using salivary bacterial contamination with dental pellicle formation as an in vitro model.

Methods: Sand-blasted and acid-etched (SA) titanium disks were used. Three control groups (pristine SA disks [SA group]; salivary pellicle-coated SA disks [pellicle group]; and biofilm-coated, untreated SA disks [NT group]) were not subjected to any decontamination treatments. Decontamination of the biofilm-coated disks was performed by 14 methods, including ultrasonic instruments, rotating instruments, an air-powder abrasive system, a laser, and chemical agents. MG63 cells were cultured in the presence of the treated disks. Cell proliferation assays were performed on days 2 and 5 of cell culture, and cell morphology was analyzed by immunofluorescence and scanning electron microscopy (SEM). A vascular endothelial growth factor (VEGF) assay was performed on day 5 of culture.

Results: The cell proliferation assay revealed that all decontaminated disks, except for the 2 groups treated using a plastic tip, showed significantly less cell proliferation than the SA group. The immunofluorescence and SEM analyses revealed that most groups showed comparable cell density, with the exception of the NT group, in which the cell density was lower and bacterial residue was observed. Furthermore, the cells grown with tetracycline-treated titanium disks showed significantly lower VEGF production than those in the SA group.

Conclusions: None of the decontamination methods resulted in cytocompatibility similar to that of pristine SA titanium. However, many methods caused improvement in the biocompatibility of the titanium disks in comparison with the biofilm-coated, untreated titanium disks. This suggests that decontamination is indispensable for the treatment of peri-implantitis, even if the original biocompatibility cannot be restored.

Keywords: Biocompatible materials; Decontamination; Dental implants; Peri-implantitis.

Figure 1. EDS of the treated titanium disks. (A) Chemical composition (weight percent) of the prepared disks. (B) Chemical composition (atomic percent) of the prepared disks.

EM: EMS metal, EP: EMS plastic, SM: Satelec metal, SP: Satelec plastic, ST: Satelec titanium, iB: iBrush, Ti: Tigran brush, GB: GingiBrush, Pf: Perioflow, CA: citric acid, Tc: tetracycline, NT: no treatment, SA: sand-blasted and acid-etched, EDS: energy-dispersive X-ray spectroscopy, EDTA: ethylenediaminetetraacetic acid.

Figure 2. Cell proliferation analysis. The Cell Counting Kit-8 assay was performed on days 2 and 5. (A) Cell proliferation on day 2. Cell viability in the Ti, GB, and laser groups was significantly higher than in the SA group. (B) Cell proliferation on day 5. Cell viability in 14 groups (EM, SM, ST, iB, Ti, GB, Pf, laser, H₂O₂, CA, EDTA, Tc, NT, and pellicle) was significantly lower than in the SA group.

EM: EMS metal, EP: EMS plastic, SM: Satelec metal, SP: Satelec plastic, ST: Satelec titanium, iB: iBrush, Ti: Tigran brush, GB: GingiBrush, Pf: Perioflow, CA: citric acid, Tc: tetracycline, NT: no treatment, SA: sand-blasted and acid-etched, OD: optical density, EDTA: ethylenediaminetetraacetic acid. ^a)Significantly different from the SA group (P<0.05); ^b)Significantly different from the NT group (P<0.05).

Figure 3. Morphology of the cells under immunofluorescence microscopy. The F-actin in cells was stained by rhodamine-phalloidin. Nuclei were stained with DAPI.

EM: EMS metal, EP: EMS plastic, SM: Satelec metal, SP: Satelec plastic, ST: Satelec titanium, iB: iBrush, Ti: Tigran brush, GB: GingiBrush, Pf: Perioflow, CA: citric acid, Tc: tetracycline, NT: no treatment, SA: sand-blasted and acid-etched, DAPI: 4′,6-diamidino-2-phenylindole, EDTA: ethylenediaminetetraacetic acid.

Figure 4. Morphology of the cells under scanning electron microscopy. The scale bar in the figures indicates 40 μm and 20 μm.

EM: EMS metal, EP: EMS plastic, SM: Satelec metal, SP: Satelec plastic, ST: Satelec titanium, iB: iBrush, Ti: Tigran brush, GB: GingiBrush, Pf: Perioflow, CA: citric acid, Tc: tetracycline, NT: no treatment, SA: sand-blasted and acid-etched, EDTA: ethylenediaminetetraacetic acid.

Figure 5. VEGF assay at day 5. The VEGF level in the Tc and NT groups was significantly different from that in the SA group.

VEGF: vascular endothelial growth factor, EM: EMS metal, EP: EMS plastic, SM: Satelec metal, SP: Satelec plastic, ST: Satelec titanium, iB: iBrush, Ti: Tigran brush, GB: GingiBrush, Pf: Perioflow, CA: citric acid, Tc: tetracycline, NT: no treatment, SA: sand-blasted and acid-etched, EDTA: ethylenediaminetetraacetic acid. ^a)Significant difference (P<0.05).