Osteoblast Attachment on Bioactive Glass Air Particle Abrasion-Induced Calcium Phosphate Coating

Abstract

Air particle abrasion (APA) using bioactive glass (BG) effectively decontaminates titanium (Ti) surface biofilms and the retained glass particles on the abraded surfaces impart potent antibacterial properties against various clinically significant pathogens. The objective of this study was to investigate the effect of BG APA and simulated body fluid (SBF) immersion of sandblasted and acid-etched (SA) Ti surfaces on osteoblast cell viability. Another goal was to study the antibacterial effect against Streptococcus mutans. Square-shaped 10 mm diameter Ti substrates (n = 136) were SA by grit blasting with aluminum oxide particles, then acid-etching in an HCl-H₂SO₄ mixture. The SA substrates (n = 68) were used as non-coated controls (NC-SA). The test group (n = 68) was further subjected to APA using experimental zinc-containing BG (Zn4) and then mineralized in SBF for 14 d (Zn4-CaP). Surface roughness, contact angle, and surface free energy (SFE) were calculated on test and control surfaces. In addition, the topography and chemistry of substrate surfaces were also characterized. Osteoblastic cell viability and focal adhesion were also evaluated and compared to glass slides as an additional control. The antibacterial effect of Zn4-CaP was also assessed against S. mutans. After immersion in SBF, a mineralized zinc-containing Ca-P coating was formed on the SA substrates. The Zn4-CaP coating resulted in a significantly lower Ra surface roughness value (2.565 μm; p < 0.001), higher wettability (13.35°; p < 0.001), and higher total SFE (71.13; p < 0.001) compared to 3.695 μm, 77.19° and 40.43 for the NC-SA, respectively. APA using Zn4 can produce a zinc-containing calcium phosphate coating that demonstrates osteoblast cell viability and focal adhesion comparable to that on NC-SA or glass slides. Nevertheless, the coating had no antibacterial effect against S. mutans.

Keywords: S. mutans; biofilm; biomineralization; implant; osteoblast; peri-implantitis; titanium; zinc.

Figure 1

Typical surface profiles of the NC-SA (A), Zn4-APA (B), and Zn4-CaP (C) surfaces. The images were acquired with a 5× objective lens and utilizing a field of view multiplier of 0.5×.

Figure 2

SEM images of NC-SA (A,D,G), Zn4-APA (B,E,H), and Zn4-CaP (C,F,I). Magnifications 250× (A–C), 2500× (D–F) and (C,F,I) 5000×. Arrows in (H) show some of the attached glass particles. The window in (I) shows the same picture in 25,000× magnification, illustrating the possible HA crystals.

Figure 3

Viable S. mutans on the Zn4-CaP and NC-SA substrates.

Figure 4

Localization of focal adhesion in MC3T3-E1 cells. Focal adhesion staining of the cells on cover glass, Zn4-CaP, and NC-SA after 48 h culture (images presented here represent one culture). Hoechst 33258 was used for nucleus staining (blue), anti-vinculin was employed for focal adhesion labeling (green), and TRITC-phalloidin was utilized for visualizing the actin cytoskeleton (red). A fluorescence microscope was used to capture the images with 20× objective (with or without 4× zoom). The focal adhesion-like structures were observed at the peripheries of cells on all surfaces. On cover glasses, these structures were thin dash-like structures (arrowheads), whereas, on Zn4-CaP and NC-SA, the structures were more spread over the surface of the substrate (arrows). On cover glass and Zn4-CaP, the actin cytoskeleton of the cells is normal, but on NC-SA, actin localizes mainly in nuclei, and only a small amount is visible in the cytoplasm.

Figure 5

MTT assay results of MC3T3-E1 cells cultured on cover glass, Zn4-CaP, and NC-SA. Cell viability on Zn4-CaP and NC-SA substrates was assessed relative to cover glasses, serving as controls with their viability set to 100%. The data represent the mean (SD) pooled from three independent cell cultures.