Biological Effects of New Titanium Surface Coatings Based on Ionic Liquids and HMGB1: A Cellular and Molecular Characterization in Lewis Rats

Abstract

High Mobility Group Box 1 (HMGB1) is a redox-sensitive molecule that plays dual roles in tissue healing and inflammation. We previously demonstrated that HMGB1 is stable when anchored by a well-characterized imidazolium-based ionic liquid (IonL), which serves as a delivery vehicle for exogenous HMGB1 to the site of injury and prevents denaturation from surface adherence. However, HMGB1 exists in different isoforms [fully reduced HMGB1 (FR), a recombinant version of FR resistant to oxidation (3S), disulfide HMGB1 (DS), and inactive sulfonyl HMGB1(SO)] that have distinct biological functions in health and disease. Thus, the goal of this study was to evaluate the effects of different recombinant HMGB1 isoforms on the host response using a rat subcutaneous implantation model. A total of 12 male Lewis rats (12-15 weeks) were implanted with titanium discs containing different treatments (n = 3/time point; Ti, Ti-IonL, Ti-IonL-DS, Ti-IonL-FR, and Ti-IonL-3S) and assessed at 2 and 14 days. Histological (H&E and Goldner trichrome staining), immunohistochemistry, and molecular analyses (qPCR) of surrounding implant tissues were employed for analysis of inflammatory cells, HMGB1 receptors, and healing markers. Ti-IonL-DS samples resulted in the thickest capsule formation, increased pro-inflammatory, and decreased anti-inflammatory cells, while Ti-IonL-3S samples demonstrated suitable tissue healing similar to uncoated Ti discs, as well as an upregulation of anti-inflammatory cells at 14 days compared to all other treatments. Thus, results from this study demonstrated that Ti-IonL-3S are safe alternatives for Ti biomaterials. Future studies are necessary to investigate the healing potential of Ti-IonL-3S in osseointegration scenarios.

Keywords: immunomodulation; implant coating; inflammation; subcutaneous.

Figure 1.

Capsule thickness of subcutaneous tissue representing a healing panel of uncoated (Ti) and coated [ionic liquid (Ti-IonL), disulfide HMGB1(Ti-IonL-DS), fully reduced HMGB1 (Ti-IonL-FR), and non-oxidizable HMGB1 (Ti-IonL-3S)] at 2 and 14 days post-implant placement. Scale bar = 50 μm and 200 μm, staining: GT (A). Quantitative results of capsule thickness over time are presented as mean ± SD for each parameter at implantation sites during 2 and 14 days (B). The asterisk symbol indicates statistical significance between implant treatments within the same time point (n = 3; p < 0.05).

Figure 2.

Histological evaluation of subcutaneous implantation sites representing a healing panel of uncoated (Ti) and coated [ionic liquid (Ti-IonL), disulfide HMGB1(Ti-IonL-DS), fully reduced HMGB1 (Ti-IonL-FR), and non-oxidizable HMGB1 (Ti-IonL-3S)] at 2 and 14 days post-implant placement. Scale bar = 20 μm, staining: H&E, original magnification 40× (A). Histomorphometry connective tissue parameters (blood vessels, inflammatory cells, and fibers + fibroblasts) over time (B). Quantitative results are presented as mean ± SD for each parameter at implantation sites during 2 and 14 days. The asterisk symbol indicates statistical significance between implant treatments within the same time point (n = 3; p < 0.05).

Figure 3.

Immunohistochemistry (IHC) of macrophage surface markers (CD86 and CD163) of subcutaneous tissue surrounding Ti and coated [ionic liquid (Ti-IonL), disulfide HMGB1(Ti-IonL-DS), fully reduced HMGB1 (Ti-IonL-FR), and non-oxidizable HMGB1 (Ti-IonL-3S)] discs at 2 days post-implant placement (A). Dark cells: positive labeling. Scale bar: 20 μm, original magnification 40×, counterstaining Mayer’s hematoxylin, chromogen DAB. Quantification of markers identified by immunohistochemical analysis is shown as means ± SD for area density (%) at the implant site (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001) between treatments (n = 3) (B). Quantitative results are presented as mean ± SD for each parameter at implant sites after 2 and 14 days.

Figure 4.

Immunohistochemistry (IHC) of HMGB1 and CXCR4 in subcutaneous tissue surrounding Ti and coated [ionic liquid (Ti-IonL), disulfide HMGB1(Ti-IonL-DS), fully reduced HMGB1 (Ti-IonL-FR), and non-oxidizable HMGB1 (Ti-IonL-3S)] discs at 2 days post-implant placement (A). Dark cells: positive labeling. Scale bar: 20 μm, original magnification 40×, counterstaining Mayer’s hematoxylin, chromogen DAB. Quantification of markers identified by immunohistochemical analysis is shown as means ±SD for area density (%) at the implant site (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001) between implant treatments (n = 3) (B). Quantitative results are presented as mean ± SD for each parameter at implantation sites during 2 and 14 days.

Figure 5.

Fold change of inflammatory and wound healing gene expression in Ti and coated [ionic liquid (Ti-IonL), disulfide HMGB1(Ti-IonL-DS), fully reduced HMGB1 (Ti-IonL-FR), and non-oxidizable HMGB1 (Ti-IonL-3S)] discs at 2 and 14 days post-implant placement, relative to a non-surgery control. Quantification of expression is shown as means ±SD for area density (%) at the implant site (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001) between treatments (n = 3). Quantitative results are presented as mean ± SD for each parameter at implantation sites during 2 and 14 days.