2.1

AgNP synthesis and characterisation

Synthesis of α-lipoic acid-capped AgNPs was as previously described [ , ]. Briefly, two solutions were formed, each containing docusate sodium salt (0.33 M, AOT ≥ 96%, Cat.No.86140; Sigma Aldrich, Missouri, USA) in n -heptane (40 mL, Cat No.H350-1; Fischer Scientific, New Hampshire, USA). An aqueous solution of silver nitrate (0.13 M, 1.6 mL; Cat.No.10224350; Fisher Scientific) was added to one AOT/heptane solution, forming a microemulsion (EM), and an aqueous solution of sodium borohydride (1.84 M, 1.6 mL; NaBH ₄ crystalline 98–99%, Cat.No. ICN10289425; Fischer Scientific) was added to the other AOT/heptane solution, forming a second EM. The two EMs were combined by dropwise addition and stirred in the dark for 1 h. Subsequently, the capping agent, α-lipoic acid (0.08 mM in 0.25 mL ethanol), was added to the combined EMs and stirred for 2 min. A phase separation was induced in the AgNP-containing microemulsion using an acetone: methanol mixture (1:1). The AgNPs were removed from the interface and washed using ethanol to remove any excess capping agent. The ethanol-AgNP mixture was centrifuged at 8000 × g for 5 min. The ethanol supernatant was removed from the AgNP solid and fresh ethanol (1 mL) was added. The AgNPs were temporarily resuspended by manual agitation and recentrifuged (8000 × g for 5 min). The ethanol resuspension and centrifugation were repeated three times. The ethanol was then removed and the AgNP solid dried at room temperature in the dark. The AgNPs were dispersed in deionised H ₂ O (pH adjusted to 10.5 with ammonium hydroxide) and centrifuged (16,000 × g for 45 min). The supernatant containing the smallest AgNPs remaining in suspension was transferred to a fresh tube, centrifuged again and the supernatant recovered.

The α-lipoic acid-functionalised AgNPs were analysed in suspension at RT by dynamic light scattering (DLS) using a Zetasizer Nano (ZS model, Malvern Instruments; Malvern, UK), with detection angle of 173°, and a 3 mW He-Ne laser operating at a wavelength of 633 nm. Measurements were repeated four times, with a minimum equilibration time of 120 s. The mean hydrodynamic diameter (Z-average; Z-Avg) and polydispersity index (PDI) values were obtained from correlation functions using the “multiple narrow modes” algorithm based on a non-negative least square fit (Zetasizer software v. 6.20; Malvern Instruments; United Kingdom).

AgNPs were prepared for transmission electron microscopy (TEM) by depositing colloidal samples (10 μL) onto plasma glowed carbon-coated (400 – mesh) copper grids. After 60 s, the excess volume was removed with filter paper and the sample air dried. Images were obtained using a CM100 BioTWIN transmission electron microscope (Philips/FEI Corporation; Eindhoven, Holland) equipped with a LaB6 emitter and a MegaView III digital camera (Olympus Corporation; Tokyo, Japan). Nanoparticle size was determined by a particle analysis programme (Fiji is Just Image J, Soft Imaging System GmbH).

Inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine the silver concentration of the α-lipoic acid-stabilised AgNP suspension and silver, sodium, calcium, and strontium concentrations from GIC leachates. Samples were prepared by addition of quartz distilled HNO ₃ (0.1 mL) to 0.5 mL of sample in a Teflon digestion vessel (Cat. No. 010-500-264; SPS Science, Quebec, Canada). The samples were then digested at 100 °C for 1 h reducing the volumes to ≈ 0.2 mL. Sample volumes were increased to 35 mL with deionised H ₂ O. Samples were then analysed with an ICP-MS instrument (Model 7900, Agilent, Technologies; California, USA) using a low matrix tune to determine the metal concentration.

2.2

AgNP-modified GIC preparation

The commercial GICs used were Fuji IX GP (Shade A3, Lot: 1711091; GC, Tokyo, Japan), Ketac™ Universal Aplicap™ Molar (Shade A3, Lot: 3257151; 3M-ESPE, St Paul, USA) and Riva Selfcure (Shade A3, Lot: C1711091F; SDI, Bayswater, VIC, AU). Sintered silver-containing GICs were GC Miracle Mix (Lot: 1806191), Ketac™ Silver Aplicap™ (Lot: 3297033), and Riva Silver (Lot: G1707061EG). α-Lipoic acid-coated AgNPs (6, 10 or 24 μg silver) were added to the liquid phase of each GIC capsule after removal of the capsule plunger or side hatch. The plunger/side hatch was then replaced, pushed flush into the capsule, and the contents of the capsule were mixed at high speed on a Caulk Vari-Mix III amalgamator (Dentsply; Pennsylvania, USA) for the time specified by the respective GIC manufacturer. GICs were then extruded into the moulds appropriate for each assay. Specimens were allowed to set for 1 h before analysis.

2.3

Antibiofilm effects of GICs

Streptococcus mutans (UA159) was obtained from the Department of Oral Sciences, University of Otago, New Zealand, and was regularly sub-cultured under anaerobic conditions (GasPak EZ anaerobe container system, BD Biosciences, New Jersey, USA) on Columbia blood agar (CBA; Cat No: 1100; Fort Richard, Mt. Wellington, New Zealand) at 37 °C. Brain heart infusion (BHI) (Cat No: 237500; Difco Laboratories, Detroit, MI, USA) was inoculated from a CBA culture and incubated in 5% CO ₂ , at 37 °C for 16 h.

GIC specimens were prepared as 10 mm diameter × 2 mm discs (N = 3 × 3 GIC groups × 3 AgNP concentrations, plus controls). Specimens were sequentially finished on a TegraPol-21 polisher (Struers; Copenhagen, Denmark) with silicon carbide (1200, 2000, 4000 grit) for 30 s each side and sonicated in an ultrasonic cleaner for 5 min between polishing with each grit size. Samples were autoclaved at 121 °C for 15 min. Sterile GIC specimens were placed into the wells of a 24 well plate (CELLSTAR, Greiner Bio-One International; Kremsmünster, Austria) each containing BHI (1 mL) supplemented with 1% sucrose. Each well was inoculated with the bacterial culture (10 μL) deriving an initial absorbance (A600) of 0.01. The plate was placed in a rotary shaker incubator (37 °C, 60 rpm) for 24 h, after which the GIC specimens were aseptically removed and placed into wells containing BHI-sucrose (1 mL), and incubated at 37 °C for 24 h. The transfer of GIC specimens and re-incubation was repeated to produce a 72 h cultured biofilm. GIC specimens were dip-washed three times in phosphate-buffered saline (PBS) to remove non-adherent bacteria.

The biofilm/GIC specimens were treated with SYTO 9 and propidium iodide (Live/Dead™ Bac Light™ bacterial viability kit, Cat. No: L7012, Invitrogen, Thermo Fischer Scientific; Waltham, Massachusetts, USA) and remained at room temperature in the dark for 15 min, as per manufacturer’s instructions. The stained biofilms were gently washed with PBS, excess liquid wicked away, and samples were kept moist during imaging. Stained biofilms were examined with a LSM 710 confocal laser-scanning microscope (Carl Zeiss; Jena, Germany) and digital images captured with Zeiss Zen software, 2009 (3 images per specimen, 3 specimens per group). Three-dimensional stack images of the biofilms were analysed with Comstat2 (Molecular Microbial Ecology Group, Denmark) to quantify live, dead and total biovolumes. The biovolume (μm ³ /μm ² ) is the number of biomass pixels of each stacked image multiplied by the voxel size, and divided by the substratum area of the image [ ]. Percentage biofilm reduction was calculated as a percentage of the non-modified GIC total biofilm biovolume.

2.4

Compressive strength

The compressive strength of GICs was measured according to the standard ISO 9917:2007 with size modification of the GIC specimens. Cylindrical GIC specimens (2 mm diameter × 3 mm) were prepared in Teflon moulds with a set time of 1 h, after which they were removed and immersed in deionised H ₂ O at 37 °C for 24 h. Excess deionised H ₂ O was wicked from the cylindrical specimens immediately before placement on the mechanical testing platform. A compressive load was aligned to the long axis of the specimen at a cross-head speed of 1 mm min ⁻¹ , using a Universal Testing Machine (Instron 3369; Instron, Norwood, MA, USA) with a 500 N load cell. Deionised H ₂ O (pH 10.5, 5 μL)-modified GICs were used as the background control.

2.5

Flexural strength

Rectangular GIC bar specimens (20 mm × 2.0 mm × 2.0 mm) were prepared in Teflon moulds with a set time of 1 h, after which they were removed and immersed in deionised H ₂ O at 37 °C for 24 h. Deionised H ₂ O (pH 10.5, 5 μL)-modified GICs were used as the background control. Three-point bend tests were carried out on the GIC bar specimens using a universal testing machine with a 100 N load cell equipped with Bluehill 3 modular Software (Instron 3369; Instron, Norwood, MA,USA) and using a self-aligning three-point bending jig (flexural strength of ceramics test fixture ASTM C 1161, configuration A, fixture number WTF-CF-43; Wyoming Test Fixtures, USA) at a crosshead speed of 1 mm min ⁻¹ until the specimen fractured (ISO 9917:2007); the maximum force, flexural stress and strain were recorded. Prior to testing, each specimen was measured using a digital calliper and the dimensions entered into the Bluehill 3 software controlling the universal testing machine. Flexural strength (σ) was calculated using Eq. (1) , where F = the maximum load (N), L is the distance between the supports on which the sample is placed (20 mm), b is the width of the specimen (2 mm), and h is the height of the specimen (2 mm).

2.6

Colour measurement of GICs

Colour changes were characterised on GIC specimens prepared as discs (10 mm diameter × 2 mm) using the VITA Easyshade® V (VITA Zahnfabrik; Bad Säckingen, Germany), which utilises the Commission International d‘Eclairage L*a*b* colour space (CIE L*a*b*) compared to a white calibration control. All commercial GIC products were A3 classical tooth shade. GIC specimens were prepared in Teflon moulds and their colour immediately assessed (t = 0). Specimens were then placed in deionised H ₂ O at 37 °C for a further 14 days and measurements were taken at day one and 14. Colour differences arising from the incorporation of AgNPs were expressed as ΔE* (Eq. (2) ) where <SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='ΔL*’>Δ?*ΔL*
Δ L *
is the difference of whiteness or brightness, <SPAN role=presentation tabIndex=0 id=MathJax-Element-3-Frame class=MathJax style="POSITION: relative" data-mathml='Δa*’>Δ?*Δa*
Δ a *
was the difference in redness (positive a*) or greenness (negative a*), and <SPAN role=presentation tabIndex=0 id=MathJax-Element-4-Frame class=MathJax style="POSITION: relative" data-mathml='Δb*’>Δ?*Δb*
Δ b *
is the difference in yellowness (positive b*) or blueness (negative b*).

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