Modification of the surface of titanium with multifunctional chimeric peptides to prevent biofilm formation via inhibition of initial colonizers

Abstract

Background: Prevention of bacterial colonization remains a major challenge in the field of oral implant devices. Chimeric peptides with binding, antimicrobial, and osteogenesis motifs may provide a promising alternative for the inhibition of biofilm formation on titanium (Ti) surfaces.

Methods: In this study, chimeric peptides were designed by connecting an antimicrobial sequence from human β-defensin-3 with a Ti-binding sequence and arginine-glycine-aspartic acid using a glycine-glycine-glycine linker. Binding to the Ti substrate and antimicrobial properties against streptococci were evaluated. Significant improvement in reduction of bacterial colonization onto the Ti surface was observed, with or without the presence of saliva or serum. The MC3T3-E1 cells grew well on the modified Ti surfaces compared with the control group.

Results: The data showed that the three peptide functional motifs maintained their respective functions, and that the antibiofilm mechanism of the chimeric peptide was via suppression of sspA and sspB gene expression.

Conclusion: These results indicated that the endogenous peptide fragments engineered on the Ti surface could provide an environmentally friendly approach for improving the biocompatibility of oral implants.

Keywords: Ti-binding peptide-1; arginine-glycine-aspartic acid-containing peptides; human β-defensin-3; modification of titanium surface; multifunctional chimeric peptide.

Figure 1

(A–C) Molecular characteristics of the peptides (TBP-1-RGDS-hBD3-1, TBP-1-RGDS-hBD3-2, and TBP-1-RGDS-hBD3-3) showing amphipathic properties (top: hydrophilic; bottom: hydrophobic).

Figure 2

(A and C) Circular dichroism spectra of TBP-1-RGDS-hBD3-1, TBP-1-RGDS-hBD3-2, and TBP-1-RGDS-hBD3-3 dissolved in PBS at 37°C. (B and D) Raman spectra of TBP-1-RGDS-hBD3-1, TBP-1-RGDS-hBD3-2, and TBP-1-RGDS-hBD3-3. (E) Zeta potential results of TBP-1-RGDS-hBD3-1, TBP-1-RGDS-hBD3-2, and TBP-1-RGDS-hBD3-3. Notes: Data are shown as the mean ± SEM; n=3. *P<0.01 compared with the TBP-1-RGDS-hBD3-2 groups.

Figure 3

XPS wide-scan spectra of Ti surfaces: Ti treated with PBS (control), and Ti treated with TBP-1-RGDS-hBD3-1, TBP-1-RGDS-hBD3-2, or TBP-1-RGDS-hBD3-3 dissolved in PBS. Elemental composition of Ti disc surfaces with or without chimeric peptide treatment, as determined via XPS. Abbreviations: XPS, X-ray photoelectron spectroscopy; Ti, titanium.

Figure 4

Antibacterial effects of TBP-1-RGDS-hBD3-1, TBP-1-RGDS-hBD3-2, and TBP-1-RGDS-hBD3-3 against single-species (Streptococcus oralis, Streptococcus gordonii, or Streptococcus sanguinis) biofilms. Biofilms treated with the three peptides (1/2 MIC) were incubated for (A) 24 hours or (B) 72 hours. Data are shown as the mean ± SEM; n=3. *P<0.01 compared with the control groups. Abbreviations: MIC, minimal inhibitory concentration; SEM, standard error of the mean.

Figure 5

Two-dimensional CLSM images of blank Ti and Ti surfaces treated with TBP-1-RGDS-hBD3-3 (1/2 MIC), TBP-1-RGDS-hBD3-3, and serum, or TBP-1-RGDS-hBD3-3 and saliva, against Streptococcus oralis (Aa and Ad), Streptococcus gordonii (Ae and Ah), and Streptococcus sanguinis (Ai and Al). Overlay images show dead cells (red) and living cells (green), scale bar =100 µm. Antibacterial rates (R) against S. oralis, S. gordonii, and S. sanguinis adhered on the surface of Ti treated with TBP-1-RGDS-hBD3-3 (1/2 MIC) for 24 (B) or 72 hours (C). Data are shown as the mean ± SD; n=3. Abbreviations: CLSM, confocal laser scanning microscopy; Ti, titanium; MIC, minimal inhibitory concentration.

Figure 6

Two-and three-dimensional CLSM renderings of blank Ti, Ti treated with TBP-1-RGDS-hBD3-3, with TBP-1-RGDS-hBD3-3 and serum, or with TBP-1-RGDS-hBD3-3 and saliva, incubated with Streptococcus oralis (Aa and Ad), Streptococcus gordonii (Ae and Ah), and Streptococcus sanguinis (Ai and Al) biofilms for 36 hours. Overlay images show dead cells (red) and living cells (green) stained with AO/EB and TBP-1-RGDS-hBD3-3 (blue) stained with AMC. Scale bar =100 µm. (B) SEM images of S. gordonii biofilms that were treated with TBP-1-RGDS-hBD3-3 at 320 µg/mL for 24 hours. (C) TEM micrographs of the inner structures of S. gordonii that were treated with TBP-1-RGDS-hBD3-3 at 320 µg/mL for 12 hours. Black arrows: disruption of the cell membrane and release of cellular contents. (D) Gene expression of sspA and sspB in S. gordonii treated with TBP-1-RGDS-hBD3-3 or left untreated. S. gordonii planktonic cells (1×10⁸ CFU/mL) and cells that were treated overnight with TBP-1-RGDS-hBD3-3 for 24 hours were harvested for qRT-PCR. *P<0.05. Abbreviations: CLSM, confocal laser scanning microscopy; Ti, titanium; AMC, 7-amino-4-methylcoumarin; SEM, scanning electron microscopy; TEM, transmission electron microscopy; RQ, relative quantification.

Figure 7

(A) Proliferation of MC3T3-E1 cells cultured on surfaces of blank Ti (control) and Ti plates treated with TBP-1-RGDS-hBD3-3 for 1–7 days. (B) Live/dead assay of MC3T3-E1 cells stained with AO/EB (green: live; red: dead) after being cultured for 1–7 days on Ti plates that were either untreated (control) or treated with TBP-1-RGDS-hBD3-3 at 640 µg/mL. Abbreviation: Ti, titanium.