Biocompatibility and inflammatory response of silver tungstate, silver molybdate, and silver vanadate microcrystals

Silver tungstate (α-Ag2WO4), silver molybdate (β-Ag2MoO4), and silver vanadate (α-AgVO3) microcrystals have shown interesting antimicrobial properties. However, their biocompatibility is not yet fully understood. Cytotoxicity and the inflammatory response of silver-containing microcrystals were analyzed in THP-1 and THP-1 differentiated as macrophage-like cells, with the alamarBlue™ assay, flow cytometry, confocal microscopy, and ELISA. The present investigation also evaluated redox signaling and the production of cytokines (TNFα, IL-1β, IL-6, and IL-8) and matrix metalloproteinases (MMP-8 and -9). The results showed that α-AgVO3 (3.9 μg/mL) did not affect cell viability (p > 0.05). α-Ag2WO4 (7.81 μg/mL), β-Ag2MoO4 (15.62 μg/mL), and α-AgVO3 (15.62 μg/mL) slightly decreased cell viability (p ≤ 0.003). All silver-containing microcrystals induced the production of O2 − and this effect was mitigated by Reactive Oxygen Species (ROS) scavenger and N-acetylcysteine (NAC). TNFα, IL-6 and IL-1β were not detected in THP-1 cells, while their production was either lower (p ≤ 0.0321) or similar to the control group (p ≥ 0.1048) for macrophage-like cells. The production of IL-8 by both cellular phenotypes was similar to the control group (p ≥ 0.3570). The release of MMP-8 was not detected in any condition in THP-1 cells. Although MMP-9 was released by THP-1 cells exposed to α-AgVO3 (3.9 μg/mL), no significant difference was found with control (p = 0.7). Regarding macrophage-like cells, the release of MMP-8 and -9 decreased in the presence of all microcrystals (p ≤ 0.010). Overall, the present work shows a promising biocompatibility profile of, α-Ag2WO4, β-Ag2MoO4, and α-AgVO3 microcrystals.


Introduction
In recent years, several studies have evaluated the antimicrobial properties of medical materials functionalized with nanoparticles or antibiotics to improve their properties and prevent infections (Tran and Webster, 2013;Castro et al., 2014;Zhu et al., 2014;Castro et al., 2016a;Castro et al., 2016b;Hogan et al., 2019;Rangel et al., 2020;Verza et al., 2021). Silver has been used for centuries to treat infections and the use of silver and silver-containing particles has increased in the past few years (Politano et al., 2013). The literature shows that this metal has antimicrobial properties against a variety of microorganisms, such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans (Kim et al., 2009;Panáček et al., 2009;Martínez-Gutierrez et al., 2012;Dizaj et al., 2014;Shang et al., 2019). However, a limited number of studies have investigated its biocompatibility (Zhu et al., 2014;Rangel et al., 2020;Verza et al., 2021).
The inflammatory response is a complex, multi-step process that occurs during injury and infection (Turner et al., 2014;Tu et al., 2022). Inflammation is part of the immune response and aims to eliminate the offending agent and initiate the healing process leading to tissue and functional restoration (Tu et al., 2022). The literature reports that silver particles, especially nanoparticles, have unique chemical and physical properties responsible for their antimicrobial activity. It is already known that metallic particles can indirectly induce the production of ROS due to the presence of metallic ions (Haro Chávez et al., 2018;Assis et al., 2019). According to the literature, ROS, including the superoxide anion (O2• -), activates the NF-κB (nuclear factor kappa B) and MAPK (mitogen-activated protein kinase) pathways, which are responsible for stimulating IL-1β, TNFα and IL-6 genes (Ndengele et al., 2005;Martínez-Gutierrez et al., 2012;Murphy et al., 2016;Yu et al., 2020;Canaparo et al., 2021). Thus, the presence of ROS can activate the immune response (Akter et al., 2018) and stimulate the immune system to produce various cytokines and other inflammatory mediators (Parks et al., 2004;Abdulkhaleq et al., 2018).
Given the potential application of α-Ag 2 WO 4 , β-Ag 2 MoO 4 and α-AgVO 3 in dental materials and medical devices to prevent oral infections, it is imperative to establish their ability to mitigate any excessive inflammatory responses. Furthermore, the role of matrix metalloproteinases (MMPs), which are responsible for tissue remodeling and healing (Araújo et al., 2011), must be understood. Previous studies have shown that MMPs are strongly associated with periodontal disease, leading to the loss of periodontal attachment and bone destruction (Franco et al., 2017;Al-Majid et al., 2018). Among the 23 types of MMPs already identified, the upregulation of MMP-8 and -9 has been related to periodontitis and peri-implantitis (Franco et al., 2017;Checchi et al., 2020) and it is associated with disease progression and bone loss (Arakawa et al., 2012;Al-Majid et al., 2018). High levels of MMP-8 and -9 are found in periodontal tissues where the disease is established, possibly indicating severity and progression of the pathology (Franco et al., 2017;Al-Majid et al., 2018;Checchi et al., 2020). Additionally, MMP production can contribute to the failure of dental restorations (Hashimoto et al., 2016). Therefore, therapies aimed at controlling MMP production, while avoiding cytotoxic and genotoxic effects and reducing their levels, have the potential to effectively prevent periodontal disease and peri-implantitis.

Preparation of microcrystals
Silver tungstate, silver molybdate, and silver vanadate were prepared as previously described (Fabbro et al., 2016;Foggi et al., 2017a;Oliveira et al., 2017) Aesar, Haverhill, MA, United States) or 1 × 10 −3 mol of ammonium metavanadate (NH 4 VO 3 ; 99.99% purity; Sigma-Aldrich, St. Louis, MI, United States) were diluted in 50 mL of distilled water. Temperatures of 70°C for α-Ag 2 WO 4 and β-Ag 2 MoO 4 and 10°C for α-AgVO 3 were used. After reaching the temperatures required, the solutions were mixed, instantly forming a precipitate. These precipitates were washed with distilled water to a pH of 7 and oven-dried at 60°C for 12 h. After synthesis, all microcrystals were diluted in PBS to 2 mg/mL (stock solution), and the samples were maintained in the dark and at room temperature until further use.

Physicochemical assessment and silver concentration
The structural characterization of the materials was performed at long-range, a D/Max-2500 PC diffractometer (Rigaku, Japan) with Cu Kα radiation (λ = 1.54056 Å) in the 2θ range of 10°-80°a t a scan rate of 0.01°min −1 . To analyze the morphologies, a scanning electron microscope with a field emission gun (FEG-SEM) FEI Model Inspect F50, operating at 5 kV was used. Particle count analysis was performed using ImageJ software, with a minimum Frontiers in Bioengineering and Biotechnology frontiersin.org 02 count of 100 particles. The silver content present in the microcrystals suffers oxidation during the synthesis process. To calculate the amount of oxidized silver [Ag + ] in the microcrystals structure, first, the microcrystal concentration was converted from µg/mL to µmol/mL using the following equation: silver content in each microcrystal concentration = microcrystal concentration (µmol/mL) × 10 -6 /Molecular Weight of the microcrystal. Then, the amount of silver was calculated based on the number of mols released by each microcrystal.

In vitro THP-1 and macrophages-like cell culture and growth conditions
The cell line THP-1 (human monocytes from peripheral blood) was obtained from the Rio de Janeiro Cell Bank (BCRJ; cell line code 0234) and routinely cultured at 37°C in a 5% CO 2 -humidified environment in Roswell Park Memorial Institute medium (RPMI-1640; Sigma-Aldrich, St. Louis, MO, United States), supplemented with 2 mM of glutamine (LONZA, Basel, Switzerland), 10 mM of HEPES (Sigma-Aldrich, St. Louis, MO, United States), 1 mM of sodium pyruvate (Sigma-Aldrich, St. Louis, MO, United States), 4.5 g/L of glucose (Synth, Diadema, SP, Brazil), 1.5 g/L of sodium bicarbonate (Synth, Diadema, SP, Brazil), 1% of antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, MO, United States), 10% of fetal bovine serum (FBS; Gibco, Grand Island, NY, United States), and 0.09% of β-mercaptoethanol (Gibco, Grand Island, NY, United States). To obtain the macrophages-like from THP-1 cells, before each experiment, the THP-1 cells were seeded and stimulated with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, St. Louis, MO, United States) (Park et al., 2007), which was added to the cell culture medium and maintained at 37°C in a 5% CO 2 -humidified environment to achieved the macrophage phenotype. After 48 h, the supernatant was discarded and the macrophages cells were washed twice with PBS. Subsequently, a fresh medium was added and maintained overnight before the assays.

Cell viability assay
Cell viability was performed after 24 h of contact with silvercontaining microcrystals, and it was assessed by alamarBlue ™ assay. THP-1 and macrophages-like cells (1 × 10 6 /well) were seeded on 12-well plates at a final volume of 3 mL of RPMI medium with 5% FBS and maintained at 37°C in 5% CO 2 . After 16 h, the cells were washed with PBS, and the cell culture medium without FBS was added with silver-containing microcrystals (α-Ag 2 WO 4 : 7.81 μg/mL; β-Ag 2 MoO 4 : 15.62 μg/mL; α-AgVO 3 : 3.9 μg/mL and 15.62 μg/mL). The plates were maintained at 37°C in 5% CO 2 , and after 4 h and 24 h an aliquot of 100 µL of the supernatants from each well were collected and stored at −20°C until the cytokine production assay. After 24 h of contact with the microcrystals, the cells were incubated for 4 h in a fresh cell culture medium containing 10% of alamarBlue ™ reagent (Invitrogen, Carlsbad, CA, United States). Then, 200 µL of each well was transferred in quadruplicate to a black 96-well plate, and the fluorescence emission was measured (excitation: 544 nm; emission: 590 nm; Fluoroskan Ascent II, ThermoFisher Scientific, Waltham, MA, United States). Standard cell culture conditions were used as live cell control (CT) and cells incubated with 10 µL of lysis buffer solution (LB; Triton-x 100 9%; Sigma-Aldrich, St. Louis, MO, United States) were used as dead cell control. This assay was performed in quadruplicate and on three different occasions.

Production of pro-inflammatory cytokines
The IL-1β, TNFα, IL-6, and IL-8 cytokines production was assessed after THP-1 and macrophage-like cells were exposed to α-Ag 2 WO 4 (7.81 μg/mL), β-Ag 2 MoO 4 (15.62 μg/mL), and α-AgVO 3 (3.9 μg/mL and 15.62 μg/mL) microcrystals, at 4 and 24 h of exposure. The samples were obtained as described in section 2.5 and maintained at-20°C until the analysis. The Human Inflammatory Cytokine Kit (Cat. No. 551811; BD Biosciences, San Jose, CA, United States) was used according to the manufacturer's instructions. Briefly, while the samples thawed at room temperature, the lyophilized Human Inflammatory Cytokines Standards were reconstituted with 2 mL of Assay diluent, and then a serial dilution was performed from 1:2 until 1:256. The negative control (0 pg/ mL) was prepared only with Assay Diluent. Next, a mix of capture beads was prepared and 50 µL was added in each tube (standard curve and samples). Then, 50 µL of standard cytokines or samples were added to each corresponding tube, and finally 50 µL of Human Inflammatory PE Detection Reagent were added to all tubes. After 3 h of dark room incubation, 1 mL of Wash Buffer was added to all tubes and centrifuged at 200 g for 5 min. The supernatants were carefully discarded, and the pellets were resuspended in 300 µL of Wash Buffer. The samples were analyzed using a BD FACSAria ™ Fusion Flow Cytometer (BD Biosciences, San Jose, CA, United States), and all data obtained were evaluated with the FCAP Array software v3 (BD Biosciences, San Jose, CA, United States).

MMPs signaling
To evaluate the production of MMP-8 and -9, THP-1 and macrophage-like cells were seeded in 25-cm 2 flasks at a concentration of 5 × 10 5 cells/flask in RPMI culture medium containing 5% FBS and 5% CO 2 at 37°C. After 16 h, the cells were washed with PBS, and fresh culture medium, without FBS, containing α-Ag 2 WO 4 (7.81 μg/mL), β-Ag 2 MoO 4 (15.62 μg/mL), and α-AgVO 3 (3.9 μg/mL and 15.62 μg/mL) microcrystals were added to the correspondent treatment flask. The cells were maintained at 37°C in 5% CO 2 for 24 h. Negative control cells (CT) were maintained under standard cell culture conditions, and the positive control of MMP production was assessed with cells incubated with 1 μg/mL of lipopolysaccharide from Escherichia coli (LPS; Sigma-Aldrich, St. Louis, MO, United States). Subsequently, the supernatants were collected and stored at −20°C until analysis. This assay was performed in duplicate on two independent occasions. Before the ELISA assay, the amount of total protein in each sample was measured with the Bradford protein assay (Bradford, 1976) (Sigma-Aldrich, St Louis, MO, United States) using bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, United States) as the standard. Spectrophotometric measurements were performed at 595 nm (EZ Read 400 Microplate Reader; BioChrom, Cambourne, CAM, United Kingdom). The MMPs (−8 and −9) production was detected with the MMP-8 Human ELISA Kit (ab100609, Abcam, Cambridge, CBE, United Kingdom) and MMP-9 SimpleStep ELISA Kit (ab246539; Abcam, Cambridge, CBE, United Kingdom), according to the manufacturer's instructions. The OD was immediately read at 600 nm using a microplate reader (EZ Read 400 Microplate Reader; BioChrom, Cambourne, CAM, United Kingdom). The final data were normalized by the amount of protein per sample. This assay was performed in triplicate in a single occasion.

Statistical analysis
All data obtained were analyzed for normality (Shapiro-Wilk's test) and homoscedasticity (Levene test). The statistical analysis of cell viability and O 2 − production was performed with one-way ANOVA, followed by Tukey's post hoc on the IBM SPSS Statistics software (version 23). For cytokine and MMP production, a 95% confidence interval (CI) was defined to compare the results among groups. A significance level of 5% was adopted.

Microcrystals' characterization and silver concentration
The XRD and FEG-SEM analyses are shown in Figure 1. For the α-Ag 2 WO 4 sample, the orthorhombic phase was obtained, with a Pn2n space group (PDF 34-61) ( Figure 1A). This phase has a complex structure, formed by several clusters of [AgO x ] (x = 2, 4, 6, and 7) and distorted octahedral clusters of [WO 6 ] . Its morphology is composed of hexagonal micro rods ( Figure 1B) of average length and width of 0.95 ± 0.35 and 0.15 ± 0.06 µm, respectively. The β-Ag 2 MoO 4 phase was also obtained, with cubic structure and Fd-3m space group (PDF 8-473) ( Figure 1C). This structure has a lower complexity in terms of constituent clusters, being formed by distorted octahedral and tetrahedral clusters of [AgO 6 ] and [MoO 4 ], respectively . Its morphology does not have a polyhedral microstructure, known as bean-like morphology ( Figure 1D). These particles have a high degree of aggregation, coalescing in many cases. The average length and width obtained for this sample was 3.80 ± 0.80 and 1.40 ± 0.31 µm, respectively. For α-AgVO 3 , it is observed that the pure phase is obtained, without any additional peak, referring to the monoclinic phase with C2/c space group (PDF 89-4396) ( Figure 1E). This phase is formed by distorted octahedral clusters of [AgO 6 ] and distorted clusters of [VO 4 ] (Silva et al., 2019). Its morphology is homogeneous, with the shape of 4-Frontiers in Bioengineering and Biotechnology frontiersin.org sided micro rods ( Figure 1F). Its average length and width are 9.17 ± 4.98 and 0.52 ± 0.18 µm, respectively, showing high sample size dispersibility. The results obtained for the three samples are in agreement with those published in previous works (Oliveira et al., 2017;Assis et al., 2021;Teodoro et al., 2022).

Cell viability
The cell viability was evaluated by alamarBlue ™ assay ( Figure 2).

Intracellular O 2 − quantification and imaging
The generation of O 2 − by the cells after the contact with silvercontaining microcrystals was evaluated with fluorescence emission using a DHE probe. The THP-1 cells exposed to silver-containing microcrystals increased the production of O 2 − . As expected, when cells were incubated with microcrystals and the NAC scavenger was added (+NAC), there was a drop in the O 2 − production ( Figure 3A).  The highest decrease regarding O 2 − production was observed when THP-1 cells were exposed to α-Ag 2 WO 4 (7.81 μg/mL) + NAC and α-AgVO 3 (3.9 μg/mL) + NAC, but yet they were similar to control group (CT; p > 0.170) ( Figure 3A).
The CLSM images confirmed the data obtained with the intracellular fluorescence emission quantification. The THP-1 ( Figure 4)

Production of pro-inflammatory cytokines
The flow cytometry analysis showed that THP-1 cells produced only IL-8 at both 4 and 24 h ( Figures 6A, B, respectively). For this cell line, only α-Ag 2 WO 4 (7.81 μg/mL) was able to increase IL-8 production after 4 h of contact (p = 0.0136), when compared to the control group (CT). However, after 24 h of contact, there were no significant differences (p ≥ 0.7161) in the production of IL-8 between all experimental groups and the control group. The other cytokines evaluated (IL-1β, TNFα, and IL-6) were not detected in this cell line at any conditions (data not shown).  For macrophage-like cells, all cytokines were detected (Figure 7). The production of TNFα, IL-6 and IL-1β was lower than CT after 4 h of contact with all silver-containing microcrystals (p ≤ 0.0321; Figures 7A, C, E). In contrast, when compared to control, no significant changes in the production of IL-8 were observed after 4 h of exposure to all experimental microcrystal (p ≥ 0.1789; Figure 7G). After 24 h of exposure to α-AgVO 3 (15.62 μg/mL), macrophage-like cells showed a reduction in TNFα production (p = 0.0035) ( Figure 7B). At this time, no significant changes in IL-6 production were observed, regardless the experimental microcrystals (p ≥ 0.1549; Figure 7D). Similar results were observed for IL-1β and IL-8, except for α-AgVO 3 (15.62 μg/mL) and β-Ag 2 MoO 4 (15.62 μg/mL), where there was an increased production of IL-1β (p = 0.0006) and IL-8 (p = 0.0039), respectively, after 24 h of exposure ( Figures 7F, H).

Production of MMP-8 and -9
The release of MMPs by THP-1 and macrophage-like cells, after 24 h of exposure to silver-containing microcrystals, was measured by the ELISA. It was not possible to detect the production of MMP-8 by THP-1 cells, even under standard cell culture conditions or in the presence of LPS (data not shown). The release of MMP-9 was not detected when these
In Figure 9 we have a summary of the main findings of this work.
It is already known that metal particles can indirectly induce ROS production due to the presence of metal ions (Haro Chávez et al., 2018;Assis et al., 2019). This oxidative stress can be
Frontiers in Bioengineering and Biotechnology frontiersin.org responsible for the cytotoxicity of metal particles, considering that an increase in ROS generation can lead to cell damage and even cell death (Hashimoto et al., 2016;Canaparo et al., 2021;Liu et al., 2021). In this work, silver-containing microcrystals induced the production of superoxide (O 2 − ) by THP-1 and macrophage-like cells. Interestingly, the production of superoxide by THP-1 cells when incubated with the silver-containing microcrystals was higher than that of H 2 O 2 control. This is probably due to rapidly degradation of H 2 O 2 , limiting superoxide production by the cells. In contrast, the silver-containing microcrystals may promote a more sustained superoxide production. This is because, based on their mechanism of action, when in an aqueous environment, these silver-containing microcrystals degrade into complex clusters that interact with water and oxygen molecules, leading to the decomposition of these molecules into ROS (Foggi et al., 2017a;Foggi et al., 2017b;Oliveira et al., 2017;Foggi et al., 2020). When the NAC ROS scavenger was added to the cells, together with the microcrystals, the O 2 − signaling was reversed. This was already expected because NAC is a ROS scavenger. In a previous study, Brzicova et al. (2019) also observed an increase in superoxide production after THP-1 cells were maintained in contact with silver nanoparticles for 24 h. However, no significant differences were observed among concentrations and times of exposition (Brzicova et al., 2019).
According to the literature, ROS, including anion superoxide (O 2 • -), activate the NF-κB (nuclear factor kappa B) and MAPK (mitogen-activated protein kinase) pathways, which stimulates the expression of genes responsible for IL-1β, TNFα and IL-6 production (Ndengele et al., 2005;Martínez-Gutierrez et al., 2012;Murphy et al., 2016;Yu et al., 2020;Canaparo et al., 2021). This may occur by oxidative stress, which is induced when the antioxidant ability of the cells is overcome by ROS generation (Park et al., 2011;Yu et al., 2020;Canaparo et al., 2021). In the present study, despite the high production of O 2 − by THP-1 cells, there was no detection of IL-1β, TNFα, and IL-6. Only IL-8 was detected, but it was not significantly different from the control group, except for THP-1 cells in contact with α-Ag 2 WO 4 (7.81 μg/mL) for 4h, where an increase in IL-8 production was observed. Furthermore, the exposure of macrophages-like to silver-containing microcrystals resulted in a decreased or similar production of the IL-1β, TNFα, IL-6, and IL-8 pro-inflammatory cytokines after 4 h. This decrease or similar production was maintained after 24 h for all cytokines evaluated, except for the increased production of IL-1β and IL-8, when macrophage-like cells were exposed to α-AgVO 3 (15.62 μg/ mL) and β-Ag 2 MoO 4 (15.62 μg/mL), respectively. Previous findings have reported macrophage inflammatory responses caused by silver nanoparticles (Martínez-Gutierrez et al., 2012;Murphy et al., 2016). This may be attributed to the higher amount of ROS generated by silver nanoparticles due to their relatively large surface area (Park et al., 2011) compared to microcrystals. Another explanation is that nanoparticles can penetrate cell membranes and form clusters inside cell cytoplasm, inducing the inflammatory process (Martínez-Gutierrez et al., 2012), which does not occur with microcrystals due to their larger size. The findings reported here showed that even with the high production of O 2 − , this was easily reversed in the presence of a ROS scavenger, indicating that O 2 − production by these particles may be self-limited and, consequently, less capable of inducing significant inflammatory responses. Thus, the low cytotoxicity of α-Ag 2 WO 4 (7.81 μg/mL) and α-AgVO 3 (3.9 μg/mL) could be explained by the reversible REDOX signaling by O 2 − , which is considered an important property of both microcrystals. Additionally, literature reports suggest
Frontiers in Bioengineering and Biotechnology frontiersin.org that, among the ROS produced by cells, the O 2 − pathway is less harmful (Schieber and Chandel, 2014).
The present investigation also reveled that when THP-1 and macrophage-like cells were stimulated with silver-containing microcrystals, the production of MMP-8 and MMP-9 decreased. Considering that TNFα is a physiological inducer of MMP-9 (Heidinger et al., 2006), the reduced amount of TNFα produced in the presence of silver-containing microcrystals may explain the decrease in MMP-9 production by the cells. Previous studies have demonstrated that MMPs play a role in pathological and healing processes in the oral environment, particularly in relation to periodontal disease, leading to the loss of periodontal attachment and bone destruction (Franco et al., 2017;Al-Majid et al., 2018;Zhang et al., 2018). Among the 23 types of MMPs identified so far, upregulation of MMP-8 and MMP-9 has been associated with periodontitis and peri-implantitis (Araújo et al., 2011;Franco et al., 2017;Al-Majid et al., 2018;Checchi et al., 2020), and other studies have reported that these two MMPs are linked to disease progression and bone loss (Arakawa et al., 2012;Al-Majid et al., 2018). Elevated levels of MMP-8 and MMP-9 are found in periodontal tissues where the disease is established, potentially indicating the severity and progression of the pathology (Franco et al., 2017;Al-Majid et al., 2018;Checchi et al., 2020). Moreover, MMP-8 has been implicated in bone loss in patients with severe peri-implantitis (Arakawa et al., 2012;Al-Majid et al., 2018). Hashimoto et al. (2016) evaluated cytotoxicity, genotoxicity, and MMP production of gold and platinum nanoparticles on human cells were evaluated, along with their effect on dental resin properties (Hashimoto et al., 2016). The authors demonstrated that gold nanoparticles inhibited MMP production without causing cell damage, which is an interesting characteristic considering that MMP production can contribute to the failure of dental restorations (Hashimoto et al., 2016). Therefore, therapies that can reduce the production of MMP-8 and MMP-9 may be effective in preventing peri-implant disease.
The favorable biological responses of the α-Ag 2 WO 4 , β-Ag 2 MoO 4 , and α-AgVO 3 microcrystals in the present investigation, along with studies highlighting their antimicrobial properties, suggest that these microcrystals are promising candidates as coating materials for dental and medical devices.

Conclusion
In conclusion, α-Ag 2 WO 4 (7.81 μg/mL), β-Ag 2 MoO 4 (15.62 μg/ mL), and α-AgVO 3 (3.9 μg/mL and 15.62 μg/mL) demonstrated low cytotoxicity to THP-1 and macrophage-like cells over a sufficiently long period to measure potential damage. Additionally, these microcrystals increased the production of O 2 − and modulated cytokines and MMP production in a cell phenotype-dependent manner. The data presented here indicated that α-Ag 2 WO 4 (7.81 μg/mL), β-Ag 2 MoO 4 (15.62 μg/mL), and α-AgVO 3 (3.9 μg/ mL and 15.62 μg/mL) are capable of modulating immune response by either increasing or decreasing the production of key proinflammatory cytokines. Thus, the potential future applications of these microcrystals in the dental and medical fields appear promising and warrant further evaluation.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.