Abstract
Objectives
This work aimed (1) to develop polyacid formulations by the one-step photoreduction of silver nanoparticles (AgNP) in a polyacrylate solution of conventional glass ionomer cement (GIC), imparting antibacterial activity; and (2) to evaluate handling and mechanical properties of experimental ionomers in comparison to a commercially available conventional GIC.
Methods
Formulations with increasing sub-stoichiometric amounts of AgNO 3 were monitored during continuous UV light exposure by UV–vis spectroscopy and analyzed by transmission electron microscopy. The resulted synthesis of formulations containing small and disperse spherical nanoparticles (∼6 nm) were used to design the experimental nano-silver glass ionomer cements (NanoAg-GIC). The cements were characterized as to net setting time and compressive strength according to ISO 9917-1:2007 specifications. The antibacterial activity of these cements was assessed by Ag + diffusion tests on nutritive agar plates ( E. coli ) and by MTT assay ( S. mutans).
Results
The higher concentration of silver (0.50% by mass) in the matrix of NanoAg-GIC allowed viable net setting time and increased in 32% compressive strength of the experimental cement. All groups containing AgNP induced statistically significant E. coli growth inhibition zones ( p -value <.05), indicating diffusion of Ag + ions on the material surroundings. Metabolic activity of S. mutans grown on NanoAg-GIG with higher concentration of silver was significantly affected compared to control ( p -value <.01).
Conclusions
Silver nanoparticles one-step preparation in polyacrylate solution allowed the production of highly bioactive water-based cements within suitable parameters for clinical use and with large potential of dental and biomedical application.
1
Introduction
Conventional glass ionomer cement (GIC) is a composite material formed bacid-basic reaction among aqueous solutions of poly (acrylic acid) (PAA) and ion-leachable glass powder [ ]. This material is defined as bioactive because of exchanging ions with its environment [ ], ]. The bioactivity of these cements compared to resin-based materials is increased by the hydrophilic character of PAA, used in applications involving materials that must be permeable to water and ions [ , ]. By combining the water solubility of both PAA and ion-leachable glasses, GIC forms a highly bioactive water-based restorative material with potential for therapeutic ion supply [ ], , ]. Because of these properties, GIC has a specific application on early intervention of cavitated caries, playing an important role on management of high-risk patients [ ,[ ]. Serving the same purpose, cariostatic solutions with silver and fluoride ions – e.g. Silver Diammine Fluoride (SDF) or Nano-silver Fluoride ® (NSF) – induce reduction on clinical counts of Streptococcus mutans (SM) [ ]. Therefore, silver nanoparticles being imparted to GIC composition would promote a quick boost of silver and fluoride ions to be exchanged with carious dentin, being a strategic material for the management of cavitated caries decay in high-risk patients.
Polymer/silver nanocomposites have been widely investigated as antibacterial materials for dental [ , ] and biomedical applications [ , ], such as adhesives and resin-based restoratives materials, bone cements, wound dressings and coatings for antibacterial applications. Silver nanoparticles (AgNP) exhibit high activity against a broad range of microorganisms within a wide therapeutic window [ ]. Particle size, concentration and time dependent toxicity of AgNP have been discussed in literature [ , , ]. However, insertion of nanoparticles in polymer matrix reduces toxicity, preventing direct uptake of particles by mammalian cells [ ]. Nanocomposites release silver ions due to the high surface area of nanosized silver (Ag 0 ) exposed in the material, AgNPs being slowly oxidized into Ag 2 O in aerobic conditions. In addition, releasing rate can be augmented by acidic environment caused by adhered bacteria [ ], turning these nanocomposites into smart-surface materials with responsive rate of silver ions release under bacteria’s pathogenic action. Moreover, silver concentrations in nanocomposites as anti-caries agents (3.5 × 10 −3 M in NSF ® ) proved to be effective in hardening and arresting dentine caries as compared to routinely used SDF [ ], ], without staining the dental tissue black and had no metallic taste, minimizing the concentration dependent toxicity of ionic silver [ ].
Glass ionomers are hand-mixed to a paste consistency, with the relative proportion of powder to liquid varying in accordance to the particular clinical application [ ], ]. PAA aqueous solution, a component of conventional GIC, usually contains tartaric acid (TA) as a chelating agent, which allows setting reaction time control [ ]. In aqueous solution, PAA produces polyacrylate anions (PA − ) with uncoordinated carboxylate groups (COO − ), which can bind metallic cations such as silver salts (Ag + ) with high stability [ , ]. Indeed, carboxylic groups have been described as good assisting agents for Ag + reduction [ ].
In order to overcome agglomeration suffered by nanoparticles despite of homogenization step, when synthesis is achieved prior to the incorporation into a polymer matrix, an “ in situ” synthesis might be induced directly in the polymer solution. In the present study, an one-step synthesis of nanosilver in long-chain PAA aqueous solution was described as a convenient route for developing a potentially antibacterial water-based cement. Our strategy is based on the incorporation of Ag + ions through interaction with the carboxylic pendant groups of PAA, and further the photoreduction of Ag + ions to AgNP. Because of the bioactivity expected from these cements, it was hypothesized that it should be possible to impart significant silver antibacterial properties to the cement only by adding sub-stoichiometric amounts of silver salts to PAA aqueous solution originally used to formulate the product, without damage to the GIC properties. The synthesis, characterization of AgNP in the poly(acrylic acid) solution, and physico-chemical properties of the final glass-ionomer cements which allows its applicability in dental practice are detailed in the present paper. The antibacterial properties of silver nanoparticles based glass ionomers cements (NanoAg-GIC) are also validated against gram-negative Escherichia coli (general antibacterial validation) and gram-positive Streptococcus mutans (the primary causative agent in the formation of dental cavities).
2
Experimental section
2.1
Materials
Silver nitrate (AgNO 3 ) (99%), l -(+)-tartaric acid (TA) (99.5%) and poly(acrylic acid) (PAA) (M w ∼ 100,000, 35% by mass in H 2 O), all supplied by Sigma-Aldrich, were used as received. Concentration and average molecular weight of PAA solution were selected according to the effect of these parameters on the viscosity and compressive strength values of GIC [ , ]. The fluoro-alumino-silicate ionomer glass powder was supplied by Dental Physics Laboratory of the Queen Mary University of London (England).
2.2
AgNP synthesis and characterization
Formulations containing an increasing amount of AgNO 3 (0, 0.05, 0.10 or 0.50% by mass) and TA (5 or 10% by mass) were prepared by adding these components to a 35% (by mass) PAA aqueous solution under stirring at room temperature. Those concentrations of TA were chosen because 5 to 10% (by mass) is known to be the suitable range to control viability of GIC net setting reaction for dental applications [ ]. Then, formulations were submitted to UV irradiation for 30 to 90 min (254 nm, 2 × 30 W – Vilbert Lourmat). The optimal exposure time was determined by monitoring the optical properties of irradiated solutions by UV–vis Spectroscopy (LAMBDA 750 UV–vis/NIR Spectrophotometer, Perkin Elmer). After irradiation, formulations containing synthesized silver nanoparticles were named as PAA-TA/Ag. Morphological analysis of AgNP was performed by transmission electron microscopy (TEM) (Philips CM200 instrument) with LaB6 cathode at an accelerating voltage of 200 KV. The PAA-TA/Ag solutions were directly dropped on 400 mesh Holey Carbon grids (Ted Pella Inc.). Size distribution of silver particle diameter was obtained using Image J image processing software [ ].
2.3
Preparation of samples and net setting time of NanoAg-GIC
PAA-TA/Ag solutions were hand-mixed on a glass stab with the ionomer glass powder in the proportion of 3:7 (m/m). Depending on the amount of silver salt added in the PAA-TA/Ag formulations, NanoAg-GIC samples were sorted in four groups: A – Without Ag (negative control); B – Low Ag (0.05%); C – Medium Ag (0.10%); and D – High Ag (0.50%).
The NanoAg-GIC samples obtained were placed in plexiglass circular mould (10 mm × 2 mm) and measured according to ISO 9917-1:2007 specifications [ ]. The net setting time was recorded as the time elapsed between the end of mixing and the time when the needle fails to make a complete circular indentation in the cement.
2.4
Analysis of the silver content by inductively coupled plasma – optical emission spectrometry (ICP-OES)
Samples of each group (A, B, C, and D) depending on having or not been previously immersed in deionized water were named “After-release” (AR) and “Pre-release” (PR), respectively. AR samples were immersed in deionized water 1 h after preparation, and incubated for 24 h at 37 °C in triplicate. PR and AR samples were ground into powder and prepared according to annex G of ISO 9917-1:2007 [ ] for determination of trace metals by the analytical technique Inductively Coupled Plasma – Optical Emission Spectrometry ICP-OES. Analyses were performed on Optima 2100DV (Perkin Elmer). The operating conditions were as following: RF Power of 1300 W, nebulizer gas flow rate of 0.80 L min −1 , plasma gas flow rate of 15 L min −1 and sample gas flow rate of 1.50 L min −1 .
2.5
Compressive strength test
NanoAg-GIC groups (A, B, C, and D) were compared to Vitro Molar™ (Nova DFL, Rio de Janeiro, Brazil), as a reference of commercially available cement. Within 60 s after the end of mixing, specimens were packed to a slight excess in the PMMA mould with internal dimensions of (6.0 ± 0.1) mm high and (4.0 ± 0.1) mm diameter. One hour after the end of mixing, samples were removed from the moulds and immersed in milli-Q water at (37 ± 1) °C. The cylindrical specimens (n = 8) of each group were slightly dried 24 h after incubation with a sheet of damp filter paper (Whatman No. 1), and a compressive load was applied along the long axis of the specimen, at a cross-head speed of (0.75 ± 0.30) mm min −1 , using a ProLine (Zwick Roell) tester machine with load cell of 1000 N. The compressive strength, σ, in MPa of each specimen was determined as indicated in ISO 9917:2007 [ ].
2.6
Bacterial diffusion test
Bacterial cultures of Escherichia coli ( E.coli ) SCC1 strain [ ] were spread from the stock solution (stored at −80 °C) onto a lysogeny broth (LB) agar. After 24 h of incubation at 30 °C, 1 to 3 colonies were inoculated in 10 mL of LB broth, resulting in a preculture that was further grown overnight (∼14 h). A second preculture, by inoculating 10% (v/v) of the last preculture in fresh broth, was incubated for 4 h at 30 °C, and the bacterial suspension was adjusted to 10 8 cell m L −1 . 100 μL was spread on LB agar-supplemented growth medium (15 g L −1 ) to form a thin bacterial film.
The disks (10 mm × 2 mm) of NanoAg-GIC (groups B, C and D) and negative control (Without Ag − group A) were used for bacterial tests one hour after mixing, due to ion-releasing properties expected from the first 24 h after manipulation of these cements. Sterilized disks and the positive control (filter paper disks with 10 μL of 2% vol. clorhexidine digluconate – CHD) were immediately placed in contact with inoculated agar plates and incubated for 24 h at 30 °C. Inhibition zones and samples diameters were measured to allow further determination of inhibition areas, calculated by subtracting specimens’ area values from the total area of bacterial inhibition growth. Results are given as the average of the three replicates.
2.7
MTT metabolic activity of Streptococcus mutans ( S. mutans )
Bacterial cultures of S. mutans strain (ATCC 25175) were spread from the stock solution (stored at −20 °C) onto Columbia Blood agar (enriched and selective medium). After 48 h of incubation at 37 °C, 3 to 5 colonies were inoculated in 5 mL of saline Müeller Hinton (saline solution [ ], as displayed in Table 1 ) resulting in a preculture further grown overnight (∼16 h). A second preculture in fresh broth added of 2% (v/v) of saccharose was incubated for 3 h at 37 °C. Reaching the log phase, inoculum was adjusted to 10 7 cell mL −1 .
| Saline solution for preparation of MH broth, reagents per liter (L −1 ): | |
|---|---|
| KCl | 1.1 g |
| CaCl 2 | 110 mg |
| MgCl 2 ·6H 2 O | 40 mg |
| NaCl | 0.6 g |
| NH 4 Cl | 0.1 g |
| KH 2 PO 4 | 0.5 g |
| K 2 HPO 4 ·3H 2 O | 0.7 g |
One hour after the end of mixing, sterilized disks (10 mm × 2 mm) of NanoAg-GIC (groups B, C, and D) and negative control (Without Ag – group A) were placed into a 6-well plate and further incubated in 3 mL of inoculum for 24 h at 37 °C in 5–10% CO 2 atmosphere (candle jar). Disks containing 24 h S. mutans- biofilm on the surfaces were transferred to a 24-well plate, with 1 mL of MTT dye (0.5 mg mL −1 in PBS) in each well and incubated at 37 °C for 1 h. S. mutans -biofilm specimens were further transferred to a new 24-well plate and aliquots of 1 mL of dimethyl sulfoxide (DMSO) were added to solubilize the formazan crystals for 20 min in the dark. DMSO solution was transferred to a quartz cell and the absorbance at 540 nm was measured [ ].
2.8
Statistical analyses
One-way ANOVA followed by post hoc Tukey’s test were used for statistical analysis and confidence intervals of 95% ( p -value <.05) were considered to be significant.
2
Experimental section
2.1
Materials
Silver nitrate (AgNO 3 ) (99%), l -(+)-tartaric acid (TA) (99.5%) and poly(acrylic acid) (PAA) (M w ∼ 100,000, 35% by mass in H 2 O), all supplied by Sigma-Aldrich, were used as received. Concentration and average molecular weight of PAA solution were selected according to the effect of these parameters on the viscosity and compressive strength values of GIC [ , ]. The fluoro-alumino-silicate ionomer glass powder was supplied by Dental Physics Laboratory of the Queen Mary University of London (England).
2.2
AgNP synthesis and characterization
Formulations containing an increasing amount of AgNO 3 (0, 0.05, 0.10 or 0.50% by mass) and TA (5 or 10% by mass) were prepared by adding these components to a 35% (by mass) PAA aqueous solution under stirring at room temperature. Those concentrations of TA were chosen because 5 to 10% (by mass) is known to be the suitable range to control viability of GIC net setting reaction for dental applications [ ]. Then, formulations were submitted to UV irradiation for 30 to 90 min (254 nm, 2 × 30 W – Vilbert Lourmat). The optimal exposure time was determined by monitoring the optical properties of irradiated solutions by UV–vis Spectroscopy (LAMBDA 750 UV–vis/NIR Spectrophotometer, Perkin Elmer). After irradiation, formulations containing synthesized silver nanoparticles were named as PAA-TA/Ag. Morphological analysis of AgNP was performed by transmission electron microscopy (TEM) (Philips CM200 instrument) with LaB6 cathode at an accelerating voltage of 200 KV. The PAA-TA/Ag solutions were directly dropped on 400 mesh Holey Carbon grids (Ted Pella Inc.). Size distribution of silver particle diameter was obtained using Image J image processing software [ ].
2.3
Preparation of samples and net setting time of NanoAg-GIC
PAA-TA/Ag solutions were hand-mixed on a glass stab with the ionomer glass powder in the proportion of 3:7 (m/m). Depending on the amount of silver salt added in the PAA-TA/Ag formulations, NanoAg-GIC samples were sorted in four groups: A – Without Ag (negative control); B – Low Ag (0.05%); C – Medium Ag (0.10%); and D – High Ag (0.50%).
The NanoAg-GIC samples obtained were placed in plexiglass circular mould (10 mm × 2 mm) and measured according to ISO 9917-1:2007 specifications [ ]. The net setting time was recorded as the time elapsed between the end of mixing and the time when the needle fails to make a complete circular indentation in the cement.
2.4
Analysis of the silver content by inductively coupled plasma – optical emission spectrometry (ICP-OES)
Samples of each group (A, B, C, and D) depending on having or not been previously immersed in deionized water were named “After-release” (AR) and “Pre-release” (PR), respectively. AR samples were immersed in deionized water 1 h after preparation, and incubated for 24 h at 37 °C in triplicate. PR and AR samples were ground into powder and prepared according to annex G of ISO 9917-1:2007 [ ] for determination of trace metals by the analytical technique Inductively Coupled Plasma – Optical Emission Spectrometry ICP-OES. Analyses were performed on Optima 2100DV (Perkin Elmer). The operating conditions were as following: RF Power of 1300 W, nebulizer gas flow rate of 0.80 L min −1 , plasma gas flow rate of 15 L min −1 and sample gas flow rate of 1.50 L min −1 .
2.5
Compressive strength test
NanoAg-GIC groups (A, B, C, and D) were compared to Vitro Molar™ (Nova DFL, Rio de Janeiro, Brazil), as a reference of commercially available cement. Within 60 s after the end of mixing, specimens were packed to a slight excess in the PMMA mould with internal dimensions of (6.0 ± 0.1) mm high and (4.0 ± 0.1) mm diameter. One hour after the end of mixing, samples were removed from the moulds and immersed in milli-Q water at (37 ± 1) °C. The cylindrical specimens (n = 8) of each group were slightly dried 24 h after incubation with a sheet of damp filter paper (Whatman No. 1), and a compressive load was applied along the long axis of the specimen, at a cross-head speed of (0.75 ± 0.30) mm min −1 , using a ProLine (Zwick Roell) tester machine with load cell of 1000 N. The compressive strength, σ, in MPa of each specimen was determined as indicated in ISO 9917:2007 [ ].
2.6
Bacterial diffusion test
Bacterial cultures of Escherichia coli ( E.coli ) SCC1 strain [ ] were spread from the stock solution (stored at −80 °C) onto a lysogeny broth (LB) agar. After 24 h of incubation at 30 °C, 1 to 3 colonies were inoculated in 10 mL of LB broth, resulting in a preculture that was further grown overnight (∼14 h). A second preculture, by inoculating 10% (v/v) of the last preculture in fresh broth, was incubated for 4 h at 30 °C, and the bacterial suspension was adjusted to 10 8 cell m L −1 . 100 μL was spread on LB agar-supplemented growth medium (15 g L −1 ) to form a thin bacterial film.
The disks (10 mm × 2 mm) of NanoAg-GIC (groups B, C and D) and negative control (Without Ag − group A) were used for bacterial tests one hour after mixing, due to ion-releasing properties expected from the first 24 h after manipulation of these cements. Sterilized disks and the positive control (filter paper disks with 10 μL of 2% vol. clorhexidine digluconate – CHD) were immediately placed in contact with inoculated agar plates and incubated for 24 h at 30 °C. Inhibition zones and samples diameters were measured to allow further determination of inhibition areas, calculated by subtracting specimens’ area values from the total area of bacterial inhibition growth. Results are given as the average of the three replicates.
2.7
MTT metabolic activity of Streptococcus mutans ( S. mutans )
Bacterial cultures of S. mutans strain (ATCC 25175) were spread from the stock solution (stored at −20 °C) onto Columbia Blood agar (enriched and selective medium). After 48 h of incubation at 37 °C, 3 to 5 colonies were inoculated in 5 mL of saline Müeller Hinton (saline solution [ ], as displayed in Table 1 ) resulting in a preculture further grown overnight (∼16 h). A second preculture in fresh broth added of 2% (v/v) of saccharose was incubated for 3 h at 37 °C. Reaching the log phase, inoculum was adjusted to 10 7 cell mL −1 .
| Saline solution for preparation of MH broth, reagents per liter (L −1 ): | |
|---|---|
| KCl | 1.1 g |
| CaCl 2 | 110 mg |
| MgCl 2 ·6H 2 O | 40 mg |
| NaCl | 0.6 g |
| NH 4 Cl | 0.1 g |
| KH 2 PO 4 | 0.5 g |
| K 2 HPO 4 ·3H 2 O | 0.7 g |
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