In Vitro Molecular Study of Titanium-Niobium Alloy Biocompatibility

Abstract

Titanium dental implants have common clinical applications due to their biocompatibility, biophysical and biochemical characteristics. Although current titanium is thought to be safe and beneficial for patients, there are several indications that it may release toxic metal ions or metal nanoparticles from its alloys into the surrounding environment, which could lead to clinically relevant complications including toxic reactions as well as immune dysfunctions. Hence, an adequate selection and testing of medical biomaterial with outstanding properties are warranted. This study was designed to explore the biocompatibility of smooth titanium-niobium alloy (S_TiNb) versus smooth titanium commercially pure (S_TiCp)-a reference in implantology. All experiments were performed in vitro using human osteoblast-like SaOs-2 and monocyte THP-1 cell lines as models. Cell adhesion and growth morphology were determined by scanning electron microscopy, while cell viability was evaluated using WST-1 assay. Because niobate anions or niobium nanoparticles can be released from implants during biomaterial-cell interaction, potential immunotoxicity of potassium niobate (KNbO₃₎ salt was evaluated by examining both metabolic activity and transcriptomic profiling of treated THP-1 monocytes. The main findings of this study are that S_TiCp and S_TiNb discs do not show an impact on the proliferation and viability of SaOs-2 cells compared to polystyrene surfaces, whereas a significant decrease in THP-1 cells' viability and metabolic activity was observed in the presence of S_TiNb discs compared to the control group. However, no significant changes were found neither at the metabolic activity nor at the transcriptomic level of THP-1 monocytes exposed to KNbO₃ salt, suggesting that niobium has no effect on the immune system. Overall, these data imply a possible toxicity of S_TiNb discs toward THP-1 cells, which may not be directly related to niobium but perhaps to the manufacturing process of titanium-niobium alloy. Thus, this limitation must be overcome to make titanium alloy an excellent material for medical applications.

Keywords: biocompatible materials; cytotoxicity; dental implant; immunotoxicity; monocyte; niobium; osteoblast; potassium niobate; titanium; transcriptome.

Figure 1

SEM images of: (A) mirror polished (S_TiNb) and (B) rough (R_TiCp) discs. Bars indicate the scale (similar magnification).

Figure 2

Grown (A) and metabolic activity (B) of SaOs-2 cells onto to S_TiCp and S_TiNb discs compared to CTRL polystyrene surface. Single-classification ANOVA. The number and the letter displayed onto the bars indicate, respectively, sampling replicates and statistical group. Groups not sharing the same letter are different on the 95% level (Tukey-Kramer method). Absorbance at 450 nm is expressed in mA; ‘CTRL’, ‘TiNb’ (S_TiNb) and ‘TiCp’ (S_TiCp) groups: growth on polystyrene, smooth TiNb and smooth TiCp alloys, respectively, for 24, 48, 72 and 96 h.

Figure 3

Grown (A) and metabolic activity (B) of THP-1 cells onto S_TiNb discs compared to CTRL polystyrene surface. The number and the letter displayed onto the bars indicate, respectively, sampling replicates and statistical group. Groups not sharing the same letter are different on the 95% level (Tukey–Kramer method). Absorbance at 450 nm is expressed in mA; ‘CTRL’ and ‘TiNb’ (S_TiNb) groups: growth on polystyrene and smooth TiNb alloy, respectively, for 24, 48, 72 and 96 h.

Figure 4

SEM image of SaOs-2 bone cells seeded on the surface of S_TiCp (mirror polished) disc at initial magnification of 300× (A) and 1000× (B) after a 72-h-incubation period.

Figure 5

SEM image of SaOs-2 bone cells seeded on the surface of S_TiNb (mirror polished) disc at initial magnification of 300× (A) and 1000× (B) after a 72-h incubation period.

Figure 6

Metabolic activity of THP-1 cells exposed to various concentrations of KNbO₃ determined by WST-1 assay.

Figure 7

Network of gene-gene interactions in control and exposed SaOs-2 cells to 25 µg/mL of KNbO₃ salt as analyzed by String database v11.5. Sixty-eight retrieved genes were plotted as they were recognized in String DB©. The line color of network edges indicates the type of gene interaction evidence by: (i) database in light blue line, (ii) experimental in purple line, (iii) neighborhood in green line, (iv) fusion in red line (evidence), (v) co-occurrence in blue line, (vi) text mining in yellow line, (vii) black line (co-expression evidence) and, finally, (viii) protein homology in violet.