2.1

Antibacterial adhesive system containing DMADDM

Scotchbond Multi-Purpose bonding system (3 M, St. Paul, MN) was used as the parent bonding system and referred as “SBMP”. According to the manufacturer, SBMP etchant contained 37% phosphoric acid. SBMP primer single bottle contained 35–45% 2-hydroxyethylmethacrylate (HEMA), 10–20% copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contained 60–70% BisGMA and 30–40% HEMA.

DMADDM was a quaternary ammonium methacrylate, and was recently synthesized and incorporated into composites . The synthesis used a modified Menschutkin reaction, where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that the products are generated at virtually quantitative amounts and require minimal purification. Ten mmol of 1-(dimethylamino)docecane (Sigma, St. Louis, MO) and 10 mM of 2-bromoethyl methacrylate (BEMA, Monomer-Polymer and Dajec Labs, Trevose, PA) were combined with 3 g of ethanol in a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h. The solvent was then removed via evaporation, yielding DMADDM as a clear, colorless, and viscous liquid. DMADDM was mixed with SBMP primer at a DMADDM/(SBMP primer + DMADDM) mass fraction of 5%, following previous studies . The same 5% mass fraction of DMADDM was incorporated into SBMP adhesive.

2.2

Antibacterial adhesive system containing NAg

Silver 2-ethylhexanoate powder (Strem, New Buryport, MA) was dissolved in 2-(tert-butylamino)ethyl methacrylate (TBAEMA, Sigma) at 0.08 g of silver salt per 1 g of TBAEMA . TBAEMA was used because it improves the solubility by forming Ag N coordination bonds with Ag ions, thereby facilitating the Ag salt to dissolve in the resin solution. TBAEMA contains reactive methacrylate groups and therefore can be chemically incorporated into a dental resin upon photopolymerization . This method produced NAg with a mean silver particle size of 2.7 nm, which were well dispersed in the cured resin matrix . Ag was incorporated into SBMP primer at silver 2-ethylhexanoate/(primer + silver 2-ethylhexanoate) mass fraction of 0.1%, following a previous study . The same 0.1% mass fraction of silver was incorporated in SBMP adhesive.

Therefore, three groups were tested: (1) SBMP primer “P”, SBMP adhesive “A” (termed P&A control); (2) SBMP primer + 5% DMADDM, SBMP adhesive + 5% DMADDM (termed P&A + DMADDM); (3) SBMP primer + 0.1% NAg, SBMP adhesive + 0.1% NAg (termed P&A + NAg).

2.3

Resin specimen preparation

The cover of a sterile 96-well plate was used for specimen preparation . Ten μL of a primer was placed in the bottom of the dent of the cover. After drying with a stream of air, 10 μL of adhesive was applied and photo-polymerized for 20 s. Then, a composite (TPH, Caulk/Dentsply, Milford, DE) was placed and photo-cured for 1 min. This yielded a tri-layered primer/adhesive/composite disk of approximately 8 mm in diameter and 1 mm in thickness. The cured disks were immersed in water and agitated for 1 h to remove any uncured monomers, following previous studies . This treatment ensured that the subsequent antibacterial property measurement was due to the antibacterial resin, and not due to a short-term burst release of uncured monomers. The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC).

2.4

Saliva collection for the dental plaque microcosm model and salivary pellicle

The dental plaque microcosm model was approved by the University of Maryland. Human saliva was shown to be ideal for growing plaque microcosm biofilms in vitro, with the advantage of maintaining much of the complexity and heterogeneity of the dental plaque in vivo . Saliva was collected from ten healthy adult donors having natural dentition without active caries or periopathology, and without the use of antibiotics within the past 3 months . Participants did not brush teeth for 24 h and abstained from food/drink intake for at least 2 h prior to donating saliva. An equal amount of saliva from each of the ten donors was mixed together. Half of the saliva was diluted in sterile glycerol to a concentration of 30%, and stored at −80 °C to be used as biofilm inoculums . The other half of the saliva was used for creating salivary pellicles on resin surfaces. The saliva was centrifuged for 15 min at 12 kg to remove debris and then annealed for 30 min at 60 °C, to kill the bacteria but not denature the proteins. After filtration through a 0.45 μm cellulose acetate filter (Corning, New York, NY), the saliva with proteins but without bacteria was frozen at −80 °C for usage in salivary pellicle formation on resin surfaces .

2.5

Saliva pellicle treatment and types of growth medium

The tri-layered primer/adhesive/composite disks were divided into two groups. Group 1 received salivary pellicle coatings; group 2 had no salivary pellicles. Saliva treatment was performed by immersing the cured specimens in 1 mL of filter-sterilized saliva for 2 h at 37 °C; a previous study showed that this immersion resulted in salivary pellicles covering the specimen surface . The specimens were then used for bacteria inoculation.

Two types of growth medium were used to culture the bacteria. The first was Brain Heart Infusion broth which contained no salivary proteins (BHI, Becton Dickinson, Sparks, MD), following previous studies . The second was an artificial saliva named the McBain medium, with the purpose to mimic saliva and enable the maintenance of complex, stable salivary microcosms . McBain medium contained mucin (type II, porcine, gastric) at a concentration of 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl ₂ , 0.2 g/L; cysteine hydrochloride, 0.1 g/L; haemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7 . Mucin is similar to that in natural saliva and can cover the resin specimen surfaces similar to natural saliva. Mucin accounts for about 26% of salivary proteins in natural saliva. Bacteriological peptone, tryptone and yeast extract provided nutrition for bacterial growth.

2.6

Dental plaque microcosm biofilm culture

Each resin disk, with or without salivary pellicle, was placed into a well of 24-well plates, with the primer surface on the top. The saliva-glycerol stock was added, with 1:50 final dilution, to either the BHI or the McBain medium as inoculum. An inoculum of 1.5 mL was added to each well, and incubated in 5% CO ₂ at 37 °C for 8 h. The disks were then transferred to new 24-well plates with fresh medium. After 16 h, the disks were transferred to new 24-well plates with fresh medium and incubated for 24 h. This constituted 2 days of incubation, which was shown to form plaque microcosm biofilms on resin disks .

2.7

Live/dead bacterial viability assay

Specimens with 2-day biofilms were washed with phosphate buffered saline (PBS). Then, the biofilms were stained using a live/dead BacLight bacterial kit (Molecular Probes, Eugene, OR). Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. Six specimens were examined for each group with confocal laser scanning microscopy (CLSM 510, Carl Zeiss, Thornwood, NY) . A magnification of 200 was used in taking the images. The green channel was with 488 nm excitation and 514 nm emission. The red channel was with 543 nm excitation and 570 nm emission.

2.8

MTT metabolic assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan . Resin disk with 2-day biofilm was transferred into new 24-well plate with 1 mL of MTT dye (0.5 mg/mL MTT in PBS) in each well and incubated at 37 °C in 5% CO ₂ for 1 h. During this process, metabolically active bacteria reduced the MTT to purple formazan. After 1 h, the biofilm specimens were transferred to a new 24-well plate. An aliquot of 1 mL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. After incubation in the dark for 20 min, 200 μL of the DMSO solution was transferred to a 96-well plate, and the absorbance at 540 nm (optical density OD540) was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnvale, CA) .

2.9

Lactic acid production and colony-forming unit (CFU) counts

Disks with 2-day biofilms were rinsed in cysteine peptone water (CPW) to remove loose bacteria and then placed in a new 24-well plate. An aliquot of 1.5 mL of buffered peptone water (BPW) supplemented with 0.2% sucrose was added into each well. The relatively high buffer capacity of BPW prevented the pH from becoming significantly acidic, as a low pH would hinder bacterial acid production. Biofilm were incubated at 5% CO ₂ and 37 °C for 3 h to allow bacteria to produce acid. After 3 h, the BPW solutions were stored for lactate analysis. Lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method . The microplate reader was used to measure the absorbance at 340 nm for the BPW solutions. Standard curves were prepared using a lactic acid standard (Supelco Analytical, Bellefonte, PA) .

Disks with biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing (Fisher, Pittsburgh, PA) . Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine total microorganisms . Second, mitis salivarius agar (MSA) culture plates, containing 15% sucrose, were used to determine total streptococci . This is because MSA contains selective agents crystal violet, potassium tellurite and trypan blue, which inhibit most gram-negative bacilli and most gram-positive bacteria except streptococci, thus enabling streptococci to grow . Third, cariogenic mutans streptococci are known to be resistant to bacitracin, and this property is often used to isolate mutans streptococci from the highly heterogeneous oral microflora. Hence, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine mutans streptococci . The bacterial suspensions were serially diluted and spread onto agar plates for CFU analysis .

2.10

Statistical analysis

All data collected from this research were first checked for normal distribution with the Kolmogorov–Smirnov test and tested for homogeneity with the Levene’s test. For MTT metabolic assay and acid production study, inter-group differences were estimated by a statistical analysis of variance (ANOVA) for factorial models; individual groups were compared with Fisher’s protected least-significant difference test. For CFU, the values were first transformed by Log10 to normalize the data distribution and then subjected to ANOVA and Fisher’s protected least-significant difference test. Statistical analyses were performed by SPSS 13.0 software (SPSS Inc., Chicago, IL) at a significance level of p < 0.05.

2.1

Antibacterial adhesive system containing DMADDM

Scotchbond Multi-Purpose bonding system (3 M, St. Paul, MN) was used as the parent bonding system and referred as “SBMP”. According to the manufacturer, SBMP etchant contained 37% phosphoric acid. SBMP primer single bottle contained 35–45% 2-hydroxyethylmethacrylate (HEMA), 10–20% copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contained 60–70% BisGMA and 30–40% HEMA.

DMADDM was a quaternary ammonium methacrylate, and was recently synthesized and incorporated into composites . The synthesis used a modified Menschutkin reaction, where a tertiary amine group was reacted with an organo-halide . A benefit of this reaction is that the products are generated at virtually quantitative amounts and require minimal purification. Ten mmol of 1-(dimethylamino)docecane (Sigma, St. Louis, MO) and 10 mM of 2-bromoethyl methacrylate (BEMA, Monomer-Polymer and Dajec Labs, Trevose, PA) were combined with 3 g of ethanol in a 20 mL scintillation vial. The vial was stirred at 70 °C for 24 h. The solvent was then removed via evaporation, yielding DMADDM as a clear, colorless, and viscous liquid. DMADDM was mixed with SBMP primer at a DMADDM/(SBMP primer + DMADDM) mass fraction of 5%, following previous studies . The same 5% mass fraction of DMADDM was incorporated into SBMP adhesive.

2.2

Antibacterial adhesive system containing NAg

Silver 2-ethylhexanoate powder (Strem, New Buryport, MA) was dissolved in 2-(tert-butylamino)ethyl methacrylate (TBAEMA, Sigma) at 0.08 g of silver salt per 1 g of TBAEMA . TBAEMA was used because it improves the solubility by forming Ag N coordination bonds with Ag ions, thereby facilitating the Ag salt to dissolve in the resin solution. TBAEMA contains reactive methacrylate groups and therefore can be chemically incorporated into a dental resin upon photopolymerization . This method produced NAg with a mean silver particle size of 2.7 nm, which were well dispersed in the cured resin matrix . Ag was incorporated into SBMP primer at silver 2-ethylhexanoate/(primer + silver 2-ethylhexanoate) mass fraction of 0.1%, following a previous study . The same 0.1% mass fraction of silver was incorporated in SBMP adhesive.

Therefore, three groups were tested: (1) SBMP primer “P”, SBMP adhesive “A” (termed P&A control); (2) SBMP primer + 5% DMADDM, SBMP adhesive + 5% DMADDM (termed P&A + DMADDM); (3) SBMP primer + 0.1% NAg, SBMP adhesive + 0.1% NAg (termed P&A + NAg).

2.3

Resin specimen preparation

The cover of a sterile 96-well plate was used for specimen preparation . Ten μL of a primer was placed in the bottom of the dent of the cover. After drying with a stream of air, 10 μL of adhesive was applied and photo-polymerized for 20 s. Then, a composite (TPH, Caulk/Dentsply, Milford, DE) was placed and photo-cured for 1 min. This yielded a tri-layered primer/adhesive/composite disk of approximately 8 mm in diameter and 1 mm in thickness. The cured disks were immersed in water and agitated for 1 h to remove any uncured monomers, following previous studies . This treatment ensured that the subsequent antibacterial property measurement was due to the antibacterial resin, and not due to a short-term burst release of uncured monomers. The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC).

2.4

Saliva collection for the dental plaque microcosm model and salivary pellicle

The dental plaque microcosm model was approved by the University of Maryland. Human saliva was shown to be ideal for growing plaque microcosm biofilms in vitro, with the advantage of maintaining much of the complexity and heterogeneity of the dental plaque in vivo . Saliva was collected from ten healthy adult donors having natural dentition without active caries or periopathology, and without the use of antibiotics within the past 3 months . Participants did not brush teeth for 24 h and abstained from food/drink intake for at least 2 h prior to donating saliva. An equal amount of saliva from each of the ten donors was mixed together. Half of the saliva was diluted in sterile glycerol to a concentration of 30%, and stored at −80 °C to be used as biofilm inoculums . The other half of the saliva was used for creating salivary pellicles on resin surfaces. The saliva was centrifuged for 15 min at 12 kg to remove debris and then annealed for 30 min at 60 °C, to kill the bacteria but not denature the proteins. After filtration through a 0.45 μm cellulose acetate filter (Corning, New York, NY), the saliva with proteins but without bacteria was frozen at −80 °C for usage in salivary pellicle formation on resin surfaces .

2.5

Saliva pellicle treatment and types of growth medium

The tri-layered primer/adhesive/composite disks were divided into two groups. Group 1 received salivary pellicle coatings; group 2 had no salivary pellicles. Saliva treatment was performed by immersing the cured specimens in 1 mL of filter-sterilized saliva for 2 h at 37 °C; a previous study showed that this immersion resulted in salivary pellicles covering the specimen surface . The specimens were then used for bacteria inoculation.

Two types of growth medium were used to culture the bacteria. The first was Brain Heart Infusion broth which contained no salivary proteins (BHI, Becton Dickinson, Sparks, MD), following previous studies . The second was an artificial saliva named the McBain medium, with the purpose to mimic saliva and enable the maintenance of complex, stable salivary microcosms . McBain medium contained mucin (type II, porcine, gastric) at a concentration of 2.5 g/L; bacteriological peptone, 2.0 g/L; tryptone, 2.0 g/L; yeast extract, 1.0 g/L; NaCl, 0.35 g/L, KCl, 0.2 g/L; CaCl ₂ , 0.2 g/L; cysteine hydrochloride, 0.1 g/L; haemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7 . Mucin is similar to that in natural saliva and can cover the resin specimen surfaces similar to natural saliva. Mucin accounts for about 26% of salivary proteins in natural saliva. Bacteriological peptone, tryptone and yeast extract provided nutrition for bacterial growth.

2.6

Dental plaque microcosm biofilm culture

Each resin disk, with or without salivary pellicle, was placed into a well of 24-well plates, with the primer surface on the top. The saliva-glycerol stock was added, with 1:50 final dilution, to either the BHI or the McBain medium as inoculum. An inoculum of 1.5 mL was added to each well, and incubated in 5% CO ₂ at 37 °C for 8 h. The disks were then transferred to new 24-well plates with fresh medium. After 16 h, the disks were transferred to new 24-well plates with fresh medium and incubated for 24 h. This constituted 2 days of incubation, which was shown to form plaque microcosm biofilms on resin disks .

2.7

Live/dead bacterial viability assay

Specimens with 2-day biofilms were washed with phosphate buffered saline (PBS). Then, the biofilms were stained using a live/dead BacLight bacterial kit (Molecular Probes, Eugene, OR). Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. Six specimens were examined for each group with confocal laser scanning microscopy (CLSM 510, Carl Zeiss, Thornwood, NY) . A magnification of 200 was used in taking the images. The green channel was with 488 nm excitation and 514 nm emission. The red channel was with 543 nm excitation and 570 nm emission.

2.8

MTT metabolic assay

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan . Resin disk with 2-day biofilm was transferred into new 24-well plate with 1 mL of MTT dye (0.5 mg/mL MTT in PBS) in each well and incubated at 37 °C in 5% CO ₂ for 1 h. During this process, metabolically active bacteria reduced the MTT to purple formazan. After 1 h, the biofilm specimens were transferred to a new 24-well plate. An aliquot of 1 mL of dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. After incubation in the dark for 20 min, 200 μL of the DMSO solution was transferred to a 96-well plate, and the absorbance at 540 nm (optical density OD540) was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnvale, CA) .

2.9

Lactic acid production and colony-forming unit (CFU) counts

Disks with 2-day biofilms were rinsed in cysteine peptone water (CPW) to remove loose bacteria and then placed in a new 24-well plate. An aliquot of 1.5 mL of buffered peptone water (BPW) supplemented with 0.2% sucrose was added into each well. The relatively high buffer capacity of BPW prevented the pH from becoming significantly acidic, as a low pH would hinder bacterial acid production. Biofilm were incubated at 5% CO ₂ and 37 °C for 3 h to allow bacteria to produce acid. After 3 h, the BPW solutions were stored for lactate analysis. Lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method . The microplate reader was used to measure the absorbance at 340 nm for the BPW solutions. Standard curves were prepared using a lactic acid standard (Supelco Analytical, Bellefonte, PA) .

Disks with biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing (Fisher, Pittsburgh, PA) . Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine total microorganisms . Second, mitis salivarius agar (MSA) culture plates, containing 15% sucrose, were used to determine total streptococci . This is because MSA contains selective agents crystal violet, potassium tellurite and trypan blue, which inhibit most gram-negative bacilli and most gram-positive bacteria except streptococci, thus enabling streptococci to grow . Third, cariogenic mutans streptococci are known to be resistant to bacitracin, and this property is often used to isolate mutans streptococci from the highly heterogeneous oral microflora. Hence, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine mutans streptococci . The bacterial suspensions were serially diluted and spread onto agar plates for CFU analysis .

2.10

Statistical analysis

All data collected from this research were first checked for normal distribution with the Kolmogorov–Smirnov test and tested for homogeneity with the Levene’s test. For MTT metabolic assay and acid production study, inter-group differences were estimated by a statistical analysis of variance (ANOVA) for factorial models; individual groups were compared with Fisher’s protected least-significant difference test. For CFU, the values were first transformed by Log10 to normalize the data distribution and then subjected to ANOVA and Fisher’s protected least-significant difference test. Statistical analyses were performed by SPSS 13.0 software (SPSS Inc., Chicago, IL) at a significance level of p < 0.05.