Surface characteristics on commercial dental implants differentially activate macrophages in vitro and in vivo

Abstract

Objectives: Biomaterial implantation provokes an inflammatory response that controls integrative fate. M2 macrophages regulate the response to implants by resolving the inflammatory phase and recruiting progenitor cells to aid healing. We have previously shown that modified titanium (Ti) disks directly induce M2 macrophage polarization. The aim of this study was to examine macrophage response to commercially available Ti or Ti alloy implants with comparable roughness and varying hydrophilicity.

Material and methods: Eleven commercially available Ti (A-F) or Ti alloy (G-K) dental implants were examined in this study. Surface topography, chemistry, and hydrophilicity were characterized for each implant. To compare the immune response in vitro, human monocyte-derived macrophages were seeded on implants and secreted pro- and anti-inflammatory proteins measured. To evaluate the inflammatory response in vivo, mice were subcutaneously instrumented with clinical implants, and implant adherent macrophage populations were characterized by flow cytometry.

Results: Macrophages on hydrophobic Implant C produced the highest level of pro-inflammatory proteins in vitro. In contrast, hydrophilic Implant E produced the second-highest pro-inflammatory response. Implants F and K, both hydrophilics, produced the highest anti-inflammatory protein secretions. Likewise, pro-inflammatory CD80hi macrophages predominated in vivo on implants C and E, and M2 CD206 + macrophages predominated on implants F and K.

Conclusions: These findings show that hydrophilicity alone is insufficient to predict the anti-inflammatory effect on macrophage polarization and that other properties-surface composition or topography-determine immune modulation. This in vivo model may be a useful screening method to compare the immunomodulatory response to clinical implants of disparate geometry or size.

Keywords: hydrophilicity; inflammation; macrophages; surface chemistry.

Figure 1:

Comparison on implant surface characteristics. Qualitative SEM analysis at 500x from the different titanium and titanium alloy implants.

Figure 2:

XPS analysis of titanium and titanium alloy implants showing atomic percentages of oxygen, carbon, titanium, zirconium, and aluminum on implant surface layer.

Figure 3:

Pro-inflammatory protein secretion from primary macrophages cultured on implants for 48 hours. p<0.05: a vs. implant A, b vs. implant B, c vs. implant C, d vs. implant D, e vs. implant E, f vs. implant F, g vs. implant G, h vs. implant H, i vs. implant I, j vs. implant J.

Figure 4:

Anti-inflammatory protein secretion from primary macrophages cultured on implants for 48 hours. p<0.05: a vs. implant A, b vs. implant B, c vs. implant C, d vs. implant D, e vs. implant E, f vs. implant F, g vs. implant G, h vs. implant H, i vs. implant I, j vs. implant J.

Figure 5:

C57Bl/6 mice were instrumented with titanium or titanium alloy implants. Implants were retrieved after 3 days post-implantation and immunophenotyping characterization from adherent cells was performed by flow cytometry to identify total macrophages (A, CD45+CD68+CD11b+), pro-inflammatory macrophages (B, CD45+CD68+CD11b+CD80+), and anti-inflammatory macrophages (C, CD45+CD68+CD11b+CD206+). p<0.05: p<0.05: a vs. implant A, b vs. implant B, c vs. implant C, d vs. implant D, e vs. implant E, f vs. implant F, g vs. implant G, h vs. implant H, i vs. implant I, j vs. implant J.