Background
The high incidence of white spot lesions among orthodontic patients highlights the need for strategies to prevent mineral loss in high-risk areas. Silver phosphate (Ag 3 PO 4 ) nanoparticles offer multifunctional potential by inhibiting biofilm formation and increasing phosphate availability. This study aimed to develop an orthodontic resin with the addition of Ag 3 PO 4 and evaluate its physicochemical and biological properties.
Methods
Ag 3 PO 4 nanoparticles were synthesized through coprecipitation and characterized. The particles were incorporated into orthodontic resins at concentrations of 0.5, 1, and 2 wt%. Physicochemical and biological properties were assessed with polymerization kinetics, microhardness, cytotoxicity, shear bond strength, antibacterial activity against Streptococcus mutans, and mineral deposition over time.
Results
Ag 3 PO 4 reduced bacterial biofilm and enhanced mineral deposition. The G 1% showed significant antibacterial activity immediately after application, whereas the G 2% maintained effectiveness after 6 months. Microhardness values remained stable, although a higher concentration slightly decreased bond strength. No cytotoxicity was observed at any concentration, and phosphate deposition was detected with higher intensity on the surface of specimens that were loaded with higher concentrations of Ag 3 PO 4.
Conclusions
Ag 3 PO 4 particles were successfully produced and incorporated into orthodontic resins, with the 1 wt% concentration maintaining the resin’s physical properties and bonding ability without cytotoxic effect. The addition of Ag 3 PO 4 promoted the antimicrobial effect and phosphate deposition, supporting a multifunctional strategy to control biofilm formation and promote phosphate deposition around orthodontic appliances.
Highlights
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Ag 3 PO 4 nanoparticles were added to orthodontic resins.
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G 1% had initial antibacterial effects; G 2% maintained it for 6 months.
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No cytotoxicity and adequate bond strength were observed.
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Ag 3 PO 4 promoted remineralizing capacity in orthodontic resins.
White spot lesions (WSLs) are early signs of dental caries and a common complication of orthodontic treatment, resulting from biofilm accumulation around brackets, which leads to enamel demineralization. , Maintaining oral hygiene around bonding devices is challenging, even for cooperative patients, and Streptococcus mutans accumulation disrupts the balance between demineralization and remineralization. The bonding interface of orthodontic appliances is a critical site for WSLs, and the incorporation of antibacterial and remineralizing agents, such as ion-releasing particles, contact-killing monomers, and material coatings, has been proposed. Despite the development of new appliances, bonding techniques, and materials, the incidence of WSLs in orthodontic patients remains high. ,
With a high surface area to volume ratio, nanoparticles can enhance their interaction with bacterial cells and the potential release of minerals. Silver-based nanoparticles are among the most widely used antibacterial agents. ,,, Silver nanoparticles, silver core–shells, , and silver-loaded mesoporous materials are used for a lasting antibacterial effect with limited cytotoxicity, effectively reducing the viability of S mutans in orthodontic resins. Combined with this effect, the incorporation of mineral sources into the particles could enhance remineralization and prevent tissue loss. As supersaturated levels of calcium and phosphate ions around the enamel are essential for mineral nucleation in these sites, phosphate-releasing materials could be used as a source for remineralization in orthodontic applications.
Silver phosphate (Ag 3 PO 4 ) particles combine antibacterial and remineralizing effects in a multifunctional particle. , Although silver damages the DNA and membrane proteins of bacteria, phosphate may act as a protective barrier for mineral loss. Integrating antibacterial and remineralizing agents into a single multifunctional particle simplifies the system, reduces interfaces, and ensures uniform dispersion within orthodontic resins, overcoming challenges associated with multiagent bonding approaches. The effect of Ag 3 PO 4 in orthodontic resins, their ability to control bacteria viability, and promote mineral deposition have not been addressed before. Therefore, this study aimed to develop an orthodontic resin with the addition of Ag 3 PO 4 and evaluate its physicochemical and biological properties. The null hypothesis was that the addition of Ag 3 PO 4 would not influence the physicochemical and biological properties of orthodontic resins.
Material and methods
Ag 3 PO 4 particles were synthesized by chemical precipitation (CP). Aqueous solutions of 1 × 10 –3 mol diammonium hydrogen phosphate and 3 × 10 –3 mol silver nitrate (Neon Comercial Ltda, São Paulo, Brazil) were mixed to create a solid yellow precipitate that was washed, centrifuged, and dried at 60°C for 24 hours. The Ag 3 PO 4 chemical structure was assessed with Raman spectroscopy (Senterra, Bruker Optik GmbH, Ettlingen, Germany). Scanning electron microscopy (SEM) (Supra 35- VP Carl Zeiss FE-SEM; Jena, Germany) was performed at 5.00 kV, with energy-dispersive x-ray spectroscopy for elemental mapping. Laser diffraction (Mastersizer2000; Malvern Instruments, Worcestershire, United Kingdom) was used to determine the particle diameter and specific surface area. The crystalline phase and purity of the particles were assessed with x-ray diffraction (XRD) (D/Max-2500PC; Rigaku, Japan).
An experimental methacrylate-based resin composite was formulated, as shown in Table I , with 75 wt% bisphenol-A-glycidyl-methacrylate (BisGMA), 25 wt% triethylene glycol dimethacrylate (TEGDMA), and 1 mol% camphorquinone (CQ), and 1 mol% ethyl-4-dimethylamino benzoate (EDAB) was used as a photoinitiator. The inorganic phase constituted 85 wt% of the final composite. Quartz (80 wt%) and colloidal silica (5 wt%) were used after silanization with 3-(trimethoxysilyl)propyl methacrylate (5 wt%; Merck KGaA; Darmstadt, Germany). Ag 3 PO 4 was incorporated at 0.5, 1, or 2 wt%, relative to the total composite mass. The control formulation contained no Ag 3 PO 4.
Table I
Orthodontic resin formulation and concentrations of components
| Component | Relative amount | |
|---|---|---|
| Organic matrix | BisGMA | 11.25 wt% |
| TEGDMA | 3.75 wt% | |
| Initiator system | CQ | 1 mol% |
| EDAB | 1 mol% | |
| BHT | 0.01 wt% | |
| Inorganic fillers | Quartz particles | 80 wt% |
| Colloidal silica | 5 wt% | |
| Ag 3 PO 4 | 0.5, 1.0, or 2.0 wt% |
Fourier transform infrared spectroscopy (FTIR- Vertex 70; Bruker Optics, Ettingen, Germany) was used to evaluate resins during their polymerization at 1000 mW/cm 2 for 20 seconds (Valo Cordless; Ultradent, Utah). The resins (n = 3; 0.35 × 5 mm) were placed on a reflectance device, and the analysis was performed for 40 seconds at 4 cm -1 resolution. The degree of conversion (DC) and the rate of polymerization [Rp max(s-1) ] were calculated based on the peak height in the 1640 cm -1 (C=C) and 1610 cm -1 (C-C) bands. Polymerization curves were submitted to a sigmoidal curve fitting (Sigma Plot version 12.0; Systat Software Inc, San Jose, Calif).
Specimens (n = 3; 0.35 × 5.0 mm) were polished using number 1200 silicon carbide papers before being submitted to 5 Knoop indentations each in a microhardness tester (HMV 2; Shimadzu, Tokyo, Japan) using a 10 g load for 10 seconds (Knoop hardness number 1 [KNH1]). The specimens were immersed in 70% ethanol for 2 hours, and the measurement was repeated to record the Knoop hardness number 2 (KNH2). The percentage differences between KNH1 and KNH2 were used to calculate the differential Knoop hardness number percentage (ΔKNH%) for softening solvent analysis.
Primary gingival fibroblasts were obtained from a healthy patient after ethical approval (No. 83291824.0.0000.5347). Specimens (n = 5; 0.35 × 5 mm) were immersed in culture media for 24 hours, and this extract was used to treat cells for 72 hours. For the MTT assay, after 68 hours of culture, cells were incubated with 50 μL of 0.5 mg/mL MTT solution for 4 hours. Formazan crystals were dissolved, and the absorbance was measured at 570 nm in a spectrophotometer (Multiskan GO; Thermo Fisher Scientific, Waltham, Mass). For the Sulforhodamine B (SRB) assay, the cells were fixed and stained with a 0.4% SRB solution after 72 hours of culture. The cells were quantified at 560 nm. Cell viability was expressed as a percentage based on absorbance relative to cells cultured in control media.
Forty-eight bovine incisors were selected for shear bond strength (SBS) testing. Their crowns were sectioned, and the labial face was polished with number 600 silicon carbide, etched with 37% phosphoric acid (Scotchbond Universal Etchant; Solventum, Maplewood, Minn) for 30 seconds, rinsed with water, and air-dried. Maxillary central incisor metal brackets (Base area: 11.65 mm 2; Roth Max; Morelli, São Paulo, Brazil) were bonded to the teeth under a 300 g force load and then light-cured for 10 seconds on each side of the bracket. The specimens were stored in distilled water for 24 hours, and the SBS test was performed using a knife-edged chisel with a crosshead speed of 1 mm/min. The adhesive remnant index (ARI) was evaluated with a stereomicroscope (10x).
S mutans (NCTC 10449) was used to assess the antibacterial activity immediately (baseline) and after 6 months of storage in distilled water at 37°C (aged). Specimens (n = 6; 0.35 × 5 mm) were prepared for each analysis and immersed in a brain-heart infusion broth with 1 wt% sucrose and 10% S mutans inoculum for 24 hours at 37°C. For biofilm analysis, biofilms were removed from the specimens, diluted to 10 -6, plated on a brain-heart infusion broth, and incubated for 48 hours. Cells in a free-floating state were considered planktonic bacteria, and their evaluation was performed by collecting only the broth that was diluted, plated, and incubated for 48 hours. Colony-forming units (CFUs) were counted and transformed to log 10 CFU/mL.
The mineral deposition was analyzed by Raman spectroscopy (Senterra-Bruker Optics) in 100 equidistant points in a standardized area (1 × 1 mm) in the center of the specimens (0.35 × 5 mm). After an initial analysis, samples were immersed in artificial saliva, prepared with calcium chloride dihydrate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, and Tris, with pH adjusted to 7. The Raman measurements were repeated at 7 and 14 days after immersion. The phosphate ion (PO 4 3−) (960 cm -1) and C=C (1613 cm -1) intensities were integrated at each point. The PO 4 3− was normalized to the C=C values. A map was created using ratios in which higher PO 4 3− ratios were plotted as orange and red areas in the mapping using Sigma Plot (version 12.0; Systat Software Inc).
Statistical analysis
Data normality was analyzed using the Shapiro-Wilk test. The KNH1 and KNH2 data were analyzed with a paired Student t test. One-way analysis of variance was applied for the DC, cytotoxicity, KNH1, ΔKNH%, and SBS test results. Two-way analysis of variance was used for antimicrobial analysis. The Tukey honest significant difference test was used as a post-hoc test. Sigma Plot (version 12.0; Systat Software Inc) was used, adopting a significance level of 5%.
Results
Ag 3 PO 4 particles were successfully synthesized through the CP method. The Raman spectroscopy (Senterra-Bruker Optics) ( Fig 1 , A ) showed intense bands at 907 cm -1, and at 997 cm -1, it attributed to the symmetrical and asymmetrical stretching vibrations of the phosphate (PO 4 ) cluster. XRD showed a body-centered cubic structure with space group P4̅3n International Centre for Diffraction Data Database; No. 14000), related to diffraction signals in the crystallographic planes of silver oxide and PO 4 tetrahedral clusters, confirming the formation of a well-crystallized Ag 3 PO 4 phase without impurities ( Fig 1 , B ). Energy-dispersive x-ray spectroscopy mapping ( Fig 1 , C ) shows the homogeneous distribution of silver and phosphorus, whereas SEM images ( Fig 1 , D ) showed irregular and rounded shape particles that presented a median diameter of 109 nm ( Fig 1 , E ).
Ag 3 PO 4 characterization: A, Raman spectroscopy; B, XRD diffractogram; C, Energy-dispersive x-ray spectroscopy maps; D, Representative SEM images (50,000X); E, Laser diffraction analysis.
Ag 3 PO 4 addition decreased the DC and Rp max(s-1) ( Table II ). The G ctrl showed the highest DC (51.09% ± 2.21%), whereas resins with Ag 3 PO 4 presented lower values combined with a reduced slope in the DC curve ( Fig 2 , A ) and a reduced peak in the Rp max(s-1) ( Fig 2 , B ). The highest R pmax(s-1) was observed for the G ctrl (0.78 ± 0.07). For softening in the solvent test, all groups showed a reduction from KNH1 to KNH2 after ethanol immersion, with ΔKNH% ranging 44.33%-60.39% ( Table II ). No cytotoxicity was observed ( Fig 3 , A ). For the SRB assay, the viability of cells was close to 100% for all groups, whereas MTT values ranged from 111.45 ± 14.33 to 137.53 ± 14.82 (β <0.2) ( Fig 3 , B ).
Table II
Mean and standard deviation of microhardness value of the model resins before (KNH1) and after immersion in solvent (KNH2), the variation of microhardness values (ΔKNH [%]), and DC and polymerization rate of experimental orthodontic resins
| Groups | KNH1 | KNH2 | ΔKNH (%) | DC (%) | Rp max (s -1) |
|---|---|---|---|---|---|
| G ctrl | 15.89 ± 4.76 Aa | 5.82 ± 0.83 b | 59.97 ± 17.34 A | 51.09 ± 2.21 A | 0.78 ± 0.07 A |
| G 0.5% | 15.63 ± 5.01 Aa | 7.16 ± 0.62 b | 47.91 ± 19.6 A | 38.04 ± 3.85 B | 0.55 ± 0.14 B |
| G 1% | 16.88 ± 3.26 Aa | 7.25 ± 1.14 b | 60.39 ± 7.37 A | 29.75 ± 1.47 C | 0.36 ± 0.02 C |
| G 2% | 13.35 ± 2.20 Aa | 7.20 ± 0.98 b | 44.33 ± 21.26 A | 27.03 ± 4.90 C | 0.36 ± 0.04 C |
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