Peptide coatings enhance keratinocyte attachment towards improving the peri-implant mucosal seal

Abstract

There is a critical need for preventing peri-implantitis as its prevalence has increased and dental implants lack features to prevent it. Research strategies to prevent peri-implantitis have focused on modifying dental implants to incorporate different antimicrobial agents. An alternative strategy consists of barring the expansion of the biofilm subgingivally by forming a long-lasting permucosal seal between the soft tissue and the implant surface. Here, we innovatively biofunctionalized titanium with bioinspired peptide coatings to strengthen biological interactions between epithelial cells and the titanium surface. We selected laminin 332- and ameloblastin-derived peptides (Lam, Ambn). Laminin 332 participates in the formation of hemidesmosomes by keratinocytes and promotes epithelial attachment around teeth; and ameloblastin, an enamel derived protein, is involved in tissue regeneration events following disruption of the periodontium. Lam, Ambn or combinations of both peptides were covalently immobilized on titanium discs. Successful immobilization of the peptides was confirmed by contact angle goniometry, X-ray photoelectron spectroscopy and fluorescent labelling of the peptides. Additionally, we confirmed the mechanical and thermochemical stability of the peptides on Ti substrates. Proliferation and hemidesmosome formation of human keratinocytes (TERT-2/OKF-6) were assessed by immunofluorescence labelling. The peptide-coated surfaces increased cell proliferation for up to 48 h in culture compared to control surfaces. Most importantly, formation of hemidesmosomes by keratinocytes was significantly increased on surfaces coated with Ambn + Lam peptides compared to control (p < 0.01) and monopeptide coatings (p < 0.005). Together, these results support the Ambn + Lam multipeptide coating as a promising candidate for inducing a permucosal seal around dental implants.

Figure 1

Water contact angles for all control and peptide-coated titanium surfaces before challenges and after mechanical (ultrasonication in water for 2h) and thermochemical (8 days in PBS, 37°C, pH=7.4) stability challenges. Error bars represent Standard Deviations.

Figure 2

Visualization of fluorescently-labeled peptide coatings and control surfaces. A. Before challenge; B. After ultrasonication for 2h in water; and C. After ultrasonication for 2h in water and 8 days of immersion in PBS at 37°C, pH=7.4. Immobilized fluorescently-labeled peptides were Lam-TAMRA (red), Ambn-FAM (green) and GL13K-FAM (green).

Figure 3

Proliferation of keratinocytes on all control and biofunctional peptide-coated Ti surfaces. A. Number of keratinocyte cells (TERT-2/OKF-6) after 48h in culture. Error bars represent Standard Deviations. B. Keratinocyte proliferation over time for all tested surfaces in culture.

Figure 4

HDs formation by keratinocytes (TERT-2/OKF-6) on all control and biofunctional peptide-coated Ti surfaces. A. After 24h in culture and B. After 48h in culture. Error bars represent Standard Deviations.