Influence of Bioinspired Lithium-Doped Titanium Implants on Gingival Fibroblast Bioactivity and Biofilm Adhesion

Abstract

Soft tissue integration (STI) at the transmucosal level around dental implants is crucial for the long-term success of dental implants. Surface modification of titanium dental implants could be an effective way to enhance peri-implant STI. The present study aimed to investigate the effect of bioinspired lithium (Li)-doped Ti surface on the behaviour of human gingival fibroblasts (HGFs) and oral biofilm in vitro. HGFs were cultured on various Ti surfaces-Li-doped Ti (Li_Ti), NaOH_Ti and micro-rough Ti (Control_Ti)-and were evaluated for viability, adhesion, extracellular matrix protein expression and cytokine secretion. Furthermore, single species bacteria (Staphylococcus aureus) and multi-species oral biofilms from saliva were cultured on each surface and assessed for viability and metabolic activity. The results show that both Li_Ti and NaOH_Ti significantly increased the proliferation of HGFs compared to the control. Fibroblast growth factor-2 (FGF-2) mRNA levels were significantly increased on Li_Ti and NaOH_Ti at day 7. Moreover, Li_Ti upregulated COL-I and fibronectin gene expression compared to the NaOH_Ti. A significant decrease in bacterial metabolic activity was detected for both the Li_Ti and NaOH_Ti surfaces. Together, these results suggest that bioinspired Li-doped Ti promotes HGF bioactivity while suppressing bacterial adhesion and growth. This is of clinical importance regarding STI improvement during the maintenance phase of the dental implant treatment.

Keywords: biofilm; gingival fibroblasts; implants; nanostructure; soft-tissue integration; surface modification; titanium.

Figure 1

Top view SEM images showing the surface of Ti substrates. (a) Lithium-incorporated Ti (Li_Ti), (b) alkaline-treated Ti (without Li) (NaOH_Ti) and (c) mechanically micro-machined Ti (Control_Ti). All scale bars represent 1 um.

Figure 2

Viability and proliferation of human gingival fibroblasts. (a) Confocal microscopy images of Live-Dead staining over Li_Ti, NaOH_Ti and Control_Ti substrates at day 1, (b) analysis of PicoGreen assay for DNA content. * p < 0.05.

Figure 3

Human gingival fibroblasts’ attachment. (a–f) Nuclei (blue) and actin F (red) staining using confocal microscopy images of HGFs at 4 and 24 h, (g–i) SEM images of the HGF over Ti groups, ×1000 magnification.

Figure 4

Cell morphology analysis at 4 and 24 h culture. (a) Nuclei counts, (b) cell length, and (c) length-to-width ratio (Aspect Ratio). Three-dimensional confocal microscopy images in Figure 3 were used for the analysis, * p < 0.05.

Figure 5

Human gingival fibroblasts characterisation 7 days after culture. (a–c) Confocal microscopy images of HGF at day 7 showing nuclei (blue) and Actin F (red) staining, (d) PicoGreen assay for DNA content, (e–i) HGF gene expression showing the fold change of COL-I, COL-III, CXCL8, fibronectin and IL1β. Dotted lines refers to the reference value * p < 0.05; # p < 0.01.

Figure 6

Multiplex ELISA quantification of conditioned media concentrations of FGF-2 (a), MMP-8 (b), MMP-1 (c), PDGF-BB (d) and VEGF (e) from HGF cultures with Ti substrates. * p < 0.05.

Figure 7

Metabolic activity (a,c), and live (green)/dead (red) staining (b,d) of single species (S. aureus) and multispecies (saliva) biofilm on three Ti substrates (Li_Ti, NaOH_Ti and Control_Ti) after 1 and 3 days of culture. * p < 0.05 by two-way ANOVA with Tukey’s multiple comparison test.