Material and methods

A commercial RMGIC (Fuji ORTHO LC; GC Corporation, Tokyo, Japan), called “Fuji,” was used as the parent system. Silver 2-ethylhexanoate (Strem Chemicals, Newburyport, Mass) of 0.1 g was dissolved into 0.9 g of 2-(tert-butylamino)ethyl methacrylate (TBAEMA; Sigma-Aldrich, St Louis, Mo). TBAEMA improved the solubility by forming silver-nitrogen bonds with silver ions to facilitate silver salt to dissolve in the resin solution. Previous studies showed that NAg sizes ranging from 2 to 5 nm could be used. In our study, measurement of 100 particles yielded 2.7 ± 0.6 nm (mean and standard deviation). TBAEMA contains reactive methacrylate groups that can be chemically bonded in the resin upon photopolymerization. The silver solution was incorporated into Fuji at silver 2-ethylhexanoate/(Fuji + silver 2-ethylhexanoate) mass fractions of 0.05% and 0.1%. Preliminary study indicated that adding 0.15% or more of NAg turned the Fuji to a yellow-brown color.

The Fuji cement was used without added NAg as a control. A commercially available composite that does not release fluoride (Transbond XT; 3M Unitek, Monrovia, Calif) was used as a second control. Transbond XT was selected because it has been widely tested in several studies and has been shown to have suitable enamel bond strength, but it has no antibacterial effects. The following 4 orthodontic adhesives were investigated: (1) Transbond XT, control (TB control); (2) Fuji + 0% NAg (Fuji control); (3) Fuji + 0.05% NAg; and (4) Fuji + 0.1% NAg.

Eighty extracted maxillary first premolars were randomly divided into 4 groups of 20 each. Each tooth was embedded vertically in a self-curing acrylic resin (Lang Dental Manufacturing, Wheeling, Ill), taking into account the buccal axes of the clinical crowns, so that their labial surfaces would be parallel to the force during the shear bond testing. Metal premolar orthodontic brackets were used. The mean base surface area was 9.63 mm ² .

For group 1, the enamel was etched for 30 seconds with 37% phosphoric acid (Scotchbond; 3M ESPE, St Paul, Minn) and then rinsed for 10 seconds. Each tooth was dried with a stream of air until a chalky white appearance was observed. Transbond XT primer (3M Unitek) was applied to the etched surfaces in a thin, uniform coat. Then, Transbond XT light-cured adhesive paste (3M Unitek) was applied to the bracket base and pushed against the enamel surface. A bracket placement pliers was used to hold and keep the bracket in position on the center of the enamel surface. A 300-g force was applied vertically (buccolingually) on the bracket for 5 seconds using a force gauge (Correx, Bern, Switzerland) to ensure uniform bonding pressure and adhesive thickness. Excess adhesive around the bracket base was removed with a clinical probe, and then the specimens were photo-cured (Demetron VCL 401; Kerr, Orange, Calif) for a total of 40 seconds. The curing light was held against the bracket and the tooth on the mesial aspect for 20 seconds followed by 20 seconds against the distal aspect at a constant distance of 3 mm and at a 45° angle to the enamel surface.

For groups 2 through 4, we used RMGICs that, according to the manufacturer and the literature, can be used for bonding brackets without acid etching. Hence, the bonding procedure consisted of pumicing the enamel surface for 10 seconds with flour pumice, followed by a rinse of 10 seconds with water. Each tooth was then wiped with a moist cotton roll to ensure that the bonding surface was not desiccated, and excess water was removed. Then RMGIC adhesive paste was applied to the bracket base, and the bracket was positioned and bonded to the enamel, with the same force to maintain uniform bonding pressure and adhesive thickness; then the bracket was light cured for a total of 40 seconds as described above.

After the bonding procedures, each group was randomly divided into 2 groups of 10 each. The samples were then stored in distilled water at 37°C for 1 day or 30 days before the shear bond testing. Shear bond strength was measured as previously described. Briefly, a chisel was connected with a computer-controlled universal testing machine (MTS Systems, Eden Prairie, Minn), and the chisel tip was positioned on the upper part of the bracket base. An occlusal-gingival load (1 kN; speed, 0.5 mm/min) was applied to the bracket, producing a shear load at the bracket-tooth interface until the bond failed. A 0.019 × 0.025-in stainless steel wire was ligated into each bracket slot to reduce deformation of the bracket during debonding. The shear bond strength equals the debonding force divided by the bracket surface area.

After debonding, each enamel surface was observed with a stereomicroscope (Zoom 2000; Leica Microsystems, Wetzlar, Germany) with 10 times magnification, and the adhesive remnant index (ARI) score was assessed, as defined by Årtun and Bergland. These scores quantified the remaining resin on the enamel surface to assess the area where the fracture had occurred during the shear bond testing. The following scores were used: 0, no adhesive remaining on the enamel; 1, less than half of the adhesive remaining on the enamel; 2, more than half of the adhesive remaining on the enamel; and 3, all adhesive remaining on the enamel, with a distinct impression of the bracket base.

For the biofilm experiments, each adhesive paste was placed into a disk mold of 9 mm in diameter and 2 mm in thickness. They were light-cured for 40 seconds on each side. The disks were then sterilized with ethylene oxide and degassed for 3 days.

Saliva was collected from 10 healthy adult donors with natural dentition and no active caries or periopathology, and who had not used antibiotics within the last 3 months. The donors did not brush their teeth for 24 hours and abstained from food and drink for 2 hours before donating the saliva. The saliva was added to a growth medium as the inoculum. The growth medium contained mucin (type II, porcine, gastric) at a concentration of 2.5 g per liter; bacteriologic peptone, 2.0 g per liter; tryptone, 2.0 g per liter; yeast extract, 1.0 g per liter; sodium chloride, 0.35 g per liter; potassium chloride, 0.2 g per liter; calcium chloride, 0.2 g per liter; cysteine hydrochloride, 0.1 g per liter; hemin, 0.001 g per liter; and vitamin K1, 0.0002 g per liter at a pH of 7. First, 1.5 mL of the inoculum was added to each well of the 24-well plates containing a resin disk and incubated at 37°C in 5% carbon dioxide for 8 hours. Second, the disks were transferred to new 24-well plates filled with fresh medium and incubated for 16 hours. Third, after 16 hours, the disks were transferred to new 24-well plates with fresh medium and incubated for 24 hours. This totaled 2 days of culture, and microcosm biofilms were formed as shown previously.

The 2-day biofilms on the disks and the 8-hour planktonic bacteria in the growth medium were used in the biofilm activity experiments. The planktonic bacteria in the growth medium were not collected at 24 or 48 hours because the disks were transferred to new 24-well plates filled with fresh medium, and the different amount of bacteria adhering on the disks would result in different numbers of planktonic bacteria in the growth medium.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Disks with 2-day biofilms were transferred to new 24-well plate, with 1 mL of MTT dye in each well. Separately, the collected medium with planktonic bacteria from each well was transferred to a tube containing 100 μL of MTT dye. All specimens were incubated at 37°C in 5% carbon dioxide for 1 hour. Then the biofilm specimens were transferred to a new 24-well plate. The planktonic bacteria were collected by centrifugation at 5 kg for 4 minutes. An aliquot of 1 mL of dimethyl sulfoxide was added to solubilize the formazan crystals. After incubation for 20 minutes in the dark, 200 μL of the dimethyl sulfoxide solution was transferred to a 96-well plate, and the absorbance at 540 nm was measured via a microplate reader (SpectraMax M5; Molecular Devices, Sunnyvale, Calif). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk.

Disks with 2-day biofilms were rinsed with cysteine peptone water to remove loose bacteria and placed in a new 24-well plate. Separately, the planktonic bacteria from each well were transferred to a tube and collected by centrifugation at 5 kg for 4 minutes. An aliquot of 1.5 mL of buffered peptone water supplemented with 0.2% sucrose was added to each well or tube. Samples were incubated at 37°C in 5% carbon dioxide for 3 hours to allow the bacteria to produce acid. The buffered peptone water solutions were then stored for lactate analysis. Lactate concentrations in the buffered peptone water solutions were determined using an enzymatic (lactate dehydrogenase) method. The microplate reader was used to measure the absorbance at 340 nm (optical density) for the collected buffered peptone water solutions. Standard curves were prepared using a lactic acid standard (Supelco, Bellefonte, Pa).

Disks with 2-day biofilms were transferred into tubes with 2 mL of cysteine peptone water, and the biofilms were harvested by sonication and vortexing (Fisher, Pittsburgh, Pa). Separately, the colony-forming unit counts of the planktonic bacteria from the medium were also measured. Three types of agar plates were used to measure the colony-forming units. First, tryptic soy blood agar culture plates were used to determine total microorganisms. Second, mitis salivarius agar culture plates containing 15% sucrose were used to determine the total streptococci. Third, mitis salivarius agar culture plates plus 0.2 units of bacitracin per milliliter were used to determine mutans streptococci. The bacterial suspensions were serially diluted and spread onto agar plates for the colony-forming unit analysis.