3.1 Effect of Zn2+ on macrophage viability

Only a few studies have employed THP-1 derived macrophages to assess the immunomodulatory function of zinc in tissue regeneration [25]. In addition, the therapeutically effective concentrations of zinc are not clear. Hence, this study explored the effect of zinc on THP-1 derived macrophages by incubating cells in culture media with different Zn2+ concentrations.

After a 24-h incubation period, the metabolic activity of the macrophages was determined. Subsequently, the half maximal inhibitory concentration (IC50) was determined to characterize the cytotoxic inhibitory potency of Zn2+ on macrophages. Results indicated that lower concentrations of Zn2+ (4.7 and 37.5 μM) did not significantly affect macrophage viability, while a gradual decline in macrophage cell viability was observed upon exposure to Zn2+  ≥ 300 μM, with an IC50 of 510 μM. Further increase of Zn2+ concentrations (3000 μM) resulted in diminished cell metabolism and severely affected cell viability (Fig. 1A).

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

Cell numbers were assessed through dsDNA content quantification after exposing THP-1 derived macrophages to different concentrations of Zn2+. The results revealed a trend which was consistent with the above-described viability measurements (Fig. 1B). A decline in dsDNA content was observed for treatments ≥ 300 μM Zn2+ (p < 0.001). Higher Zn2+ concentration from 600 μM to 1200 μM resulted in a significant reduction of the dsDNA content (p < 0.001), and above 1200 μM Zn2+ the dsDNA content of macrophages remained consistently low, showing no statistically significant difference compared to the groups at 600 μM and 1200 μM.

Cell viability was further assessed using a LIVE/DEAD staining assay (Fig. 1C). Considering the results from the CCK-8 and dsDNA assays, we excluded treatments with 1200 μM and 1500 μM Zn2+. Consistent with previous observations, LIVE/DEAD staining revealed an increase in dead cells (red) with a decrease in living cells (green) as the Zn2+ concentration increased from 300 μM (Fig. 1C). Quantification of the LIVE/DEAD results indicated a clear reduction of viable cells at a threshold concentration between 300 and 3000 μM (p < 0.001), which was in line with cell viability and dsDNA content measurements (Fig. 1D).

Together, these findings revealed a dose-dependent toxic effect of Zn2+ on THP-1 derived macrophages. Previous studies have reported various safety concentration ranges for Zn2+. For instance, Song et al. reported an IC50 of ZnCl2 at 205.1 μM in mouse Ana-1 macrophages [26], while Yoshikawa et al. reported an IC50 of ZnSO4 in rat adipocytes at ~ 1580 μM [27]. The safety concentrations of Zn2+ for human macrophages observed in the current study are in agreement with these reported concentration ranges. However, the reported concentration ranges were different for various studies, most likely due to different cell models and experimental setups. Previous work has attributed Zn2+ cytotoxicity to the formation of zinc-containing nanoparticles in cell culture media at a range between 50 to 30,000 μM [28]. However, we did not observe similar flocculent white precipitates in any Zn2+-supplemented cell culture media. Consequently, based on our cell viability results, we narrowed the Zn2+ concentration range to 0–300 μM in subsequent studies.

3.2 Effect of Zn2+ on macrophage polarization

To assess the impact of Zn2+ on macrophage polarization, THP-1 derived macrophages were exposed to Zn2+-containing media. Control groups contained M0 macrophages that were polarized toward either the M1 or M2 phenotype using LPS/IFN-γ or IL-4/IL-13, based on an established protocol [19]. Following a 48-h incubation, the specific macrophage phenotype was determined as a function of Zn2+ concentration by staining with the M1 macrophage surface marker CCR7 (red) and the M2 macrophage surface marker CD36 (green). Both control groups treated with either LPS/IFN-γ or IL-4/IL-13 exhibited CCR7 and CD36 expression, corresponding to non-exclusive marker expression for M1 and M2 human macrophages. However, cells treated with LPS/IFN-γ expressed CCR7 in a more pronounced manner, while cells treated with IL-4/IL-13 showed higher CD36 expression (Fig. 2A), consistent with a previous study from our group [19]. Quantitative analysis of the images revealed that macrophages treated with LPS/IFN-γ exhibited a significant increase in CCR7 staining (75.1% ± 6.6%) compared to cells treated with IL-4/IL-13 (46.1% ± 9.5%; p < 0.001). Conversely, macrophages treated with IL-4/IL-13 contained a higher proportion of CD36-positive cells (53.9% ± 9.5%) than cells treated with LPS/IFN-γ (24.9% ± 6.6%; p < 0.001) (Fig. 2B). Without exposure to Zn2+, macrophages tended toward an M1 phenotype. With increasing Zn2+ concentrations, the proportion of CD36-positive cells increased, indicating a tendency toward an M2 phenotype. Specifically, at Zn2+ concentrations of 37.5 μM, the population of CD36-positive cells showed a 1.5-fold increase compared to 0 μM group (p < 0.01). An additional increase of the Zn2+ concentration to 300 μM did not enhance CD36-staining.

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

To further elucidate the effect of Zn2+ on macrophage polarization, we assessed the secretion of pro-inflammatory cytokines IL-1β and TNF-α, as well as pro-regenerative cytokines IL-10 (data not shown as all values were below the detectable standard range) and TGF-β (Fig. 2C-E). Our results revealed that IL-1β and TNF-α were significantly elevated for M1 controls (p < 0.001). Treatments with different Zn2+ concentrations did not affect the secretion of cytokines compared to either 0 μM Zn2+ or M2 controls. To provide a comprehensive assessment of the most crucial pro- and anti-inflammatory cytokine secretion, we calculated the ratio of anti- to pro-inflammatory cytokines (TGF-β/IL-1β and TGF-β/TNF-α) (Fig. 2F, G). M1 controls exhibited the lowest ratio of anti- to pro-inflammatory cytokines compared to other groups (p < 0.05), while macrophages exposed to Zn2+ showed similar cytokine secretion levels as compared to M2 controls.

Based on the above-described results we conclude that exposure of M0 macrophages to mild Zn2+ concentrations favors macrophage polarization toward the M2 phenotype. Therefore, we developed novel Zn-containing coatings and studied their immunomodulatory effects from the perspective of potential application in peri-implant wound healing.

3.3 Coating characteristics

Chitosan is a biocompatible polymer, which has been commonly used for biomedical applications and for electrophoretic deposition (EPD) coatings. The cytocompatibility of chitosan EPD coatings for dental application was confirmed in our previous publication [21]. However, our results also indicated that chitosan alone did not sufficiently support cell adhesion. To address this, we incorporated gelatin into our coatings in the subsequent studies, as the RGD (Arg- Gly-Asp) sequences in gelatin are known to enhance cell adhesion [22].

Detailed characterization of the coatings by using scanning electron microscopy, energy-dispersive X-ray spectroscopy techniques, water contact angles, adhesion strength, Fourier-transform infrared spectroscopy and zinc release profile can be found in our previous publication [22]. The coatings remained stable, showing no morphological changes up to the maximum experimental period, i.e. > 7 days. The swelling and biodegradability of the coating were not tested in the current study. A former study reported that the swelling rate of a chitosan/gelatin scaffold was around 80% and 290% after 12 h and 24 h, respectively, in PBS [29]. Additionally, it was observed that the chitosan–gelatin matrix could maintain its microstructure for up to 8 weeks, with chitosan remaining present. These findings suggest that the physical properties of the coatings may align well with the healing process of peri-implant gingival tissue [30].

The release of Zn2+ can be regulated by the amount of zinc incorporated and the pH, as demonstrated in our previous article [22]. In the current Zn/CS/Gel coating, where the highest zinc content was incorporated, a sustained release of Zn2+ was achieved at approximately 4.62 μM under pH = 7 and 34.62 μM under pH = 5.5. Both concentrations were below the cytotoxic threshold, as confirmed by the results of this study (Fig. 1).

3.4 Effects of Zn/CS/Gel coatings on macrophage behavior

To explore the immunomodulatory effects of Zn/CS/Gel coatings, THP-1 derived macrophages were cultured on both experimental coatings and uncoated cpTi control surfaces. To ensure comparability, we established three control conditions, wherein macrophages were cultured on uncoated cpTi surfaces upon stimulation of polarization into either LPS/IFN-γ stimulated M1 or IL-4/IL-13 stimulated M2 macrophages. We assessed in vitro cell adhesion, cytocompatibility, and polarization state.

To investigate the adhesion of THP-1 derived macrophages on different surfaces, the amount of dsDNA of adhered cells was quantified. For cells cultured on cpTi, the adhesion of M1 macrophage controls was significantly lower than the other two controls, particularly after 3 days of culture (p < 0.05). Macrophages in the Zn/CS/Gel group exhibited superior adhesion compared to all other groups, either at Day 1 or Day 3. Specifically, compared to macrophages cultured on cpTi surfaces without extra stimulation, cells cultured on coatings (w/- Zn) showed distinct adhesion characteristics. The CS/Gel coating reduced macrophage adhesion, while Zn/CS/Gel significantly enhanced macrophage adhesion (p < 0.01) (Fig. 3A), suggesting that Zn2+ stimulated macrophage adhesion to this polymer-based surface. The absence of a similar phenomenon in the previous study involving Zn2+ treatment might be attributed to the initiation of macrophage stimulation after their differentiation from THP-1 monocytes in tissue culture plates. When we cultured macrophages on disks, we directly added the THP-1 monocyte suspension to the wells containing different substrates. Moreover, prior studies have reported that macrophage adhesion to titanium surfaces is mainly dependent on chemical properties rather than surface roughness, which aligns with our observations [31, 32]. Additionally, the observed improvement in macrophage adhesion on Zn2+-containing coatings compared to zinc-free surfaces is consistent with findings from previous studies [33, 34].

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

SEM images revealed different macrophage morphologies for the various experimental groups. Macrophages adhered abundantly on both coating types, especially on Zn/CS/Gel coatings (Fig. 3B). Previous studies reported that anti-inflammatory cytokines like TGF-β regulate the expression of cell adhesion molecules, such as integrins, and hence affect the adhesion of macrophages [35, 36]. Additionally, TGF-β can also control intracellular signaling pathways, such as the SMAD signaling pathway, thereby influencing gene expression related to macrophage adhesion and the reorganization of the cellular cytoskeleton [37, 38].

To determine coating cytocompatibility, THP-1 derived macrophages were seeded onto various surfaces. Observations from the microscopy images revealed that the majority of macrophages were alive (green fluorescent staining), suggesting cytocompatibility of all surfaces for THP-1 derived macrophages, except for an increased number of dead cells (red fluorescent staining) upon treatment with LPS/IFN-γ. Compared to cells on pure titanium, macrophages on the coatings tended to cluster, which corresponds to our SEM observations (Fig. 4A). Quantification of the percentage of live cells confirmed our observations, indicating slightly lower viability of macrophages for cpTi-M1 controls compared to other groups. This decrease may be attributed to the inflammatory status induced by pro-inflammatory factors, leading to reduced cell viability [39].

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

Furthermore, cell metabolism was measured through a CCK-8 assay after culturing THP-1 derived macrophages for 24 h. Interestingly, cells on Zn/CS/Gel coatings exhibited the highest metabolic activity compared to the cells in other groups (p < 0.01) (Fig. 4B). Considering that THP-1 derived macrophages do not proliferate once activated, we hypothesize that the Zn2+-containing coating may enhance cellular metabolic activity. Combining these results with our previous study where THP-1 derived macrophages were incubated with Zn2+ containing media, we speculate that the increased metabolism observed Zn/CS/Gel coatings is likely due to enhanced cell adhesion to this surface.

To ascertain the polarization of macrophages cultured for 3 days on different surfaces, immunofluorescent staining was employed to label CCR7 (magenta, M1 macrophage marker) and CD36 (green, M2 macrophage marker) in THP-1 derived macrophages. Macrophages treated with LPS/IFN-γ as control exhibited substantial CCR7 expression with limited CD36 expression, while the other two controls displayed both CCR7 and CD36 expression. Macrophages cultured on both types of coating similarly exhibited both CCR7 and CD36 expression (Fig. 5A).

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

Quantitative analysis of macrophage polarization (Fig. 5B) indicated less CCR7 staining for macrophages on Zn/CS/Gel coatings compared to M1 control macrophages (p < 0.05). Moreover, macrophages cultured on Zn/CS/Gel coatings exhibited comparable CD36 expression to M2 control macrophages on cpTi surfaces. Additionally, macrophage morphology correlated with the earlier SEM observations indicating that M1 macrophages were smallest, while macrophages cultured on Zn/CS/Gel coatings showed enhanced spreading (Figs. 3B and 5A).

Our results revealed a higher amount of M2 macrophages on Zn/CS/Gel coatings, which aligns with findings from previous studies [33, 34]. While various surfaces induced macrophage polarization to different extents, none of the groups in these previously performed studies distinctly directed THP-1 derived macrophages into the M1 or M2 phenotypes. Herein, on the contrary, we employed M1 and M2 controls stimulated according to an established protocol on titanium surfaces. Previous studies have emphasized that material-activated macrophage phenotypes may not precisely mirror conventionally cytokine-activated states due to the intricate nature of macrophages [19]. A decrease in M1 phenotype characteristics does not necessarily correlate with a simultaneous increase in M2 phenotype characteristics and vice versa. A key factor of macrophage-mediated immunomodulatory functions in wound healing involves cytokine secretion. Therefore, for assessment of immunomodulatory response of macrophages on biomaterials, measurement of cytokine secretion levels and quantification of M1 and M2 morphological phenotypes are essential.

To further validate macrophage polarization on different surfaces, we assessed the secretion of representative pro- and anti-inflammatory cytokines and using M1 and M2 macrophage controls. Results revealed that M1 control macrophages exhibited the highest secretion of IL-1β and IL-6, while this secretion of inflammatory mediators significantly decreased for M2 control macrophages, compared to the other two controls (p < 0.05) (Fig. 5C, D). Macrophages cultured on either type of coating displayed reduced secretion of inflammatory mediators, particularly those cultured on Zn/CS/Gel coatings (Fig. 5C-E). Similar to our former measurements, the concentration of IL-10 was below the limit of detection. TGF-β secretion was highest for macrophages cultured on Zn/CS/Gel coatings (p < 0.05) (Fig. 5F). The ratio of anti- to pro-inflammatory cytokines (TGF-β/IL-1β, TGF-β/IL-6, and TGF-β/TNF-α) showed to be highest for macrophages cultured on Zn/CS/Gel coatings (Fig. 5G-I).

These findings demonstrate that Zn/CS/Gel coatings effectively modulate the inflammatory response by downregulating the secretion of pro-inflammatory cytokines and upregulating the secretion of anti-inflammatory cytokines. The ability of zinc-based materials to facilitate the M1-to-M2 macrophage transition and promote wound healing has been well documented [17]. Hereby, we provided a new strategy to load and release Zn2+ through a simple and cost-effective coating deposition technique. In addition, effective and timely modulation of M1 and M2 macrophage polarization, along with the induction of cytokine secretion, is crucial to improve the outcome of wound healing. Yu et al. demonstrated that zinc-coated scaffolds increased M1 polarization within the initial 6 h, followed by a subsequent increase in M2 response after 24 h [40]. Bai et al. reported that zinc-doped porous microcrystalline bioactive glass facilitates an M1-to-M2 transition within 3–7 days [41]. Additionally, Lu et al. have shown that macrophages cultured on calcium phosphate cement incorporated with zinc silicate undergo M1-to-M2 conversion by day 3 [42]. These findings align with our observations after culturing macrophages on Zn/CS/Gel coatings for 3 days. Hence, we postulate that Zn/CS/Gel coatings exert immunomodulatory capacity by favoring macrophage polarization toward the M2 type.

3.5 Indirect effects of Zn/CS/Gel coatings on fibroblast behavior via macrophage-conditioned media

Since the immune environment strongly determines tissue integration, we subsequently investigated the implication of the macrophage-derived immune microenvironment on cellular behavior of soft tissue cells. Hereby, we used hGFs as model cells, since they are dominant within peri-implant soft tissue, functioning as important cellular components for the formation of a soft tissue seal [7]. Before studying the effects of differential immune stimulation on fibroblast cellular function, we verified fibroblast viability under CM stimulation obtained from macrophage cultures. After 24-h incubation, LIVE/DEAD staining indicated that cell viability was not compromised under any of the simulated immune environments (Fig. 6A). Fibroblast cell morphology showed a typical elongated spindle shape without apparent differences (Fig. 6B). Results of CCK-8 viability assays showed that fibroblasts generally grew well, as reflected by continuous proliferation up to 7 days; lowest cell viability was observed for fibroblasts cultured in CM-M1 (p < 0.05) (Fig. 6C).

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

To establish a robust peri-implant soft tissue seal, one of the most vital prerequisites is to achieve effective cell adhesion for soft tissue cells. Fibroblasts adhere to implant surfaces by developing focal adhesions, which are structures connecting the cytoskeleton, cell adhesive receptors, and ECM to an implant surface [43]. Vinculin is a cytoskeleton-related protein, which is a representative component of focal adhesions. Immunofluorescent staining showed that the vinculin expression increased in the group in which hGFs were cultured in CM-Zn/CS/Gel (p < 0.05) (Fig. 7A, B). Previously, Rout et al. found that exposure to TGF-β enhanced vinculin expression in fibroblasts to improve adhesion apparatus formation, while the direct influence of TGF-β on vinculin expression has not been extensively studied [44]. In our study, more TGF-β secretion was detected for macrophages cultured on Zn/CS/Gel, from which CM is likely to induce higher vinculin expression in fibroblasts. Moreover, previous studies have also shown that the secretion of pro-inflammatory cytokines by M1 macrophages, such as TNF-α, IL-1β, and IL-6, compromised the expression and activity of focal adhesion proteins, including vinculin [45, 46].

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

Another important function of fibroblasts in their participation to peri-implant STI is collagen secretion, since collagen fibers are the major components of the tissue matrix around dental implants [47]. Mature tissue matrix can provide a stable scaffold for tissue adhesion, supporting implant-tissue integration. Therefore, we investigated the effect of the immune microenvironment on collagen synthesis of hGFs after culturing cells in CM for 7 days. Immunofluorescent staining indicated that fibroblasts in CM-Zn/CS/Gel showed highest levels of Col I expression (p < 0.001) (Fig. 7C, D). An earlier study has reported that TGF-β stimulates Col I expression via the SMAD signal transduction pathway [48]. Regarding to our early observation that macrophages cultured on Zn/CS/Gel exhibit higher TGF-β secretion, we speculate that the CM-Zn/CS/Ge might create a pro-regenerative microenvironment beneficial for collagen deposition.

Cellular behavior is highly associated with mitochondrial activity, which can be assessed through mitochondrial number and structure. Previous studies demonstrated that increased mitochondrial activity is correlated to improved cellular metabolism and particular functions such as collagen synthesis [47, 49]. To characterize mitochondrial activity for the various experimental groups, we stained mitochondria in living fibroblasts via MitoTracker (Fig. 8A). Fibroblasts cultured in Zn/CS/Gel conditioned medium showed an increased number of mitochondria (p < 0.05) (Fig. 8B, C). The mitochondria branch number in hGFs cultured in conditioned medium Zn/CS/Gel was almost 3 times higher than those from CM-M1 and CM-M0 controls as well as CM-CS/Gel (p < 0.01) (Fig. 8D).

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

As the power source for cellular energy production, mitochondria are of vital importance for maintaining cellular growth and function such as homeostasis and differentiation [49]. Both mitochondrial number and area are critical parameters in mitochondrial biology, as these parameters reflect their function and dynamics. Mitochondria are double-membraned organelles with electron transport chain (ETC) complexes on the inner mitochondrial membrane, responsible for oxidative phosphorylation – the process generating adenosine triphosphate (ATP), the primary energy source for cellular activities. Mitochondrial branching can influence mitochondria distribution and their efficiency in ATP production. A previous study indicated that cells with more branched mitochondria tend to exhibit superior metabolic flexibility, facilitating the adaptation to fluctuating energy demands. The inner mitochondrial membrane is impermeable to most ions, creating an electrochemical gradient or potential across the membrane which is termed as mitochondrial membrane potential (MMP). A former study reported that TGF-β exposure increased the ATP content in podocytes [50]. Their results indicated that TGF-β increases mitochondrial MMP and oxygen consumption rates (OCR), resulting in enhanced reactive oxygen species (ROS) generation via the mammalian target of rapamycin (mTOR) pathway, which might contribute to wound healing. Based on the aforementioned findings, we postulate that the elevated mitochondrial activity in cells treated by CM-Zn/CS/Gel is likely attributed to the higher concentration of TGF-β.

The immunomodulatory impact of zinc in tissue regeneration has gained considerable attention, particularly due to its potential in influencing macrophage polarization states [17]. However, the application of Zn2+-containing materials to modulate the immune microenvironment in peri-implant tissue, particularly for STI remains unclear. Consequently, our study aimed to address this gap by incorporating Zn2+ into a dental abutment coating and investigating their direct effects on THP-1 derived macrophages and indirect effects on fibroblast behavior.

Compared with previous studies, we adopted a straightforward and cost-effective approach to study the effect of Zn2+ on macrophage responses, directly relevant to peri-implant wound healing. The results from ion release kinetics revealed that our Zn/CS/Gel coating exhibits pH-responsive behavior, with increased and expedited release of Zn2+ observed under acidic conditions. In combination with the findings that demonstrate the ability of the Zn2+-containing coating to stimulate pro-regenerative M2 macrophage polarization, we conclude that these coatings hold strong promise to reduce inflammation and enhance tissue repair around dental implant abutments.

In addition, the healing of tissues around different dental implant components involves complex processes that include various cell types, such as blood cells, immune cells, mesenchymal stem cell and endothelial cells. Previous studies have introduced numerous zinc-based dental materials owing to their osteoimmunological properties [33,