Development of Zinc-Containing Chitosan/Gelatin Coatings with Immunomodulatory Effect for Soft Tissue Sealing around Dental Implants

Abstract

Background: Soft tissue integration (STI) around dental implant abutments is a prerequisite to prevent bacterial invasion and achieve successful dental implant rehabilitation. However, peri-implant STI is a major challenge after dental abutment placement due to alterations in the immune microenvironment upon surgical dental implant installation.

Methods: Based on known immunomodulatory effects of zinc, we herein deposited zinc/chitosan/gelatin (Zn/CS/Gel) coatings onto titanium substrates to study their effect on macrophages. First, we exposed macrophages to cell culture media containing different zinc ion (Zn²⁺) concentrations. Next, we explored the immunomodulatory effect of Zn/CS/Gel coatings prepared via facile electrophoretic deposition (EPD).

Results: We found that Zn²⁺ effectively altered the secretome by reducing the secretion of pro-inflammatory and enhancing pro-regenerative cytokine secretion, particularly at a Zn²⁺ supplementation of approximately 37.5 μM. Zn/CS/Gel coatings released Zn²⁺ in a concentration range which effectively stimulated pro-regenerative macrophage polarization as demonstrated by M2 macrophage polarization. Additionally, the impact of these Zn²⁺-exposed macrophages on gingival fibroblasts incubated in conditioned medium showed stimulated adhesion, proliferation, and collagen secretion.

Conclusion: Our promising results suggest that controlled release of Zn²⁺ from Zn/CS/Gel coatings could be applied to immunomodulate peri-implant STI, and to enhance dental implant survival.

Keywords: Chitosan; Gelatin; Macrophage; Soft tissue integration; Zinc ion.

Fig. 1

Zn²⁺ effects on macrophage viability. A THP-1 derived macrophage viability in percentage (%) after 24-h exposure to Zn²⁺ containing media. B dsDNA content of cells after 24-h incubation with different concentrations of Zn²⁺. C Fluorescent images of living (green) and dead cells (red) after incubation with Zn²⁺ containing media for 24 h; scale bar = 100 μm. D Quantification of living and dead cells depicted in total cell percentage (%). These findings revealed a dose-dependent toxic effect of Zn.²⁺ on THP-1 derived macrophages. **p < 0.01, ***p < 0.001

Fig. 2

Phenotypic characterization and secretome analysis of macrophages upon 48-h culture in Zn²⁺ containing media. A Immunostaining images of macrophages with M1 macrophage marker CCR7 (red) and M2 macrophage marker CD36 (green); scale bar = 50 μm. B Quantification of CCR7 and CD36 positively stained cells from fluorescent microscopy, depicted as total cell population percentage (%). C Secretion of pro-inflammatory cytokines IL-1β, D TNF-α; and E anti-inflammatory cytokine TGF-β. F Ratio of TGF-β and IL-1β, and G TGF-β and TNF-α. Results indicate that exposure of M0 macrophages to mild Zn.²⁺ concentrations favors macrophage polarization toward the M2 phenotype. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Effects of Zn/CS/Gel coatings on macrophage adhesion. A Schematic illustration of cell seeding and digital photographs of the substrates; B dsDNA content of macrophages after culture on different surfaces for 1 and 3 days; C SEM images of macrophages on different surfaces after 3 days of culture; scale bar = 20 μm. Results indicate that Zn.²⁺ stimulated macrophage adhesion to this polymer-based surface. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Effects of coatings on THP-1 derived macrophages viability. A LIVE/DEAD cell staining after culturing macrophages for 24 h; scale bar = 100 μm. B Quantification of the percentage of live cells, depicted as total cell population percentage (%); C macrophage viability assessed using the CCK-8 assay after 24 h of cell culture (cell metabolism in cpTi-M0 was regarded as 100% indicated by red dashed line). Results indicate that Zn.²⁺-containing coating enhances cellular metabolic activity of macrophages. *p < 0.05, **p < 0.01

Fig. 5

Effects of Zn/CS/Gel coatings on macrophage polarization and secretome upon culture of 3 days. A Immunostaining images of macrophages with the M1 macrophage marker CCR7 (magenta) and the M2 macrophage marker CD36 (green); scale bar = 20 μm. B Quantification of M1 and M2 macrophage population, depicted as total cell population percentage (%). C IL-1β, D IL-6; E TNF-α, and anti-inflammation cytokines F TGF-β. Ratio of (G) TGF-β/IL-1β, H TGF-β/IL-6; I TGF-β/ TNF-α. These findings demonstrate that Zn/CS/Gel coatings modulate the inflammatory response by downregulating the pro-inflammatory cytokine secretion and upregulating anti-inflammatory cytokine secretion. *p < 0.05. a: significance level = 0.05; A: significance level = 0.01

Fig. 6

Indirect effects of Zn/CS/Gel coatings on hGF viability. A LIVE/DEAD cell staining after 24-h incubation; scale bar = 100 μm. B hGFs morphology upon culture in CM after cytoskeleton staining; upper image scale bar = 20 μm, lower image scale bar = 10 μm. C hGF viability after incubation with CM for 1, 3 and 7 days. Results showed that CM-M1 compromised fibroblast growth. *p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 7

Indirect effects of Zn/CS/Gel coatings on hGF behavior. A Immunofluorescence staining of vinculin upon 1-day culture; scale bar = 50 μm. B Quantitative vinculin staining; C Immunofluorescence staining of Col I upon 7-day culture; scale bar = 50 μm. D Quantitative Col I staining. Results showed that CM-Zn/CS/Ge could create a pro-regenerative microenvironment beneficial for fibroblast adhesion and collagen deposition. *p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 8

Indirect effects of Zn/CS/Gel coatings on hGF mitochondrial activity. A Representative immunofluorescence images and skeleton of mitochondria per fibroblast upon 1-day culture; immunofluorescence image scale bar = 20 μm, skeleton image scale bar = 10 μm; B Quantitative analysis of mitochondria number per cell. C Quantitative analysis of mitochondrial area per cell. D Quantitative analysis of mitochondria branch number per cell. Results indicate that CM-Zn/CS/Gel elevates mitochondrial activity in fibroblasts. a: significance level = 0.05; A: significance level = 0.01