2.1

Antibacterial bonding agents containing QADM or NAg

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

Bis(2-methacryloyloxyethyl) dimethylammonium bromide, a quaternary ammonium dimethacrylate (QADM), was recently synthesized . Its synthesis employed a modified Menschutkin reaction, where a tertiary amine group was reacted with an organo-halide. A benefit of this reaction is that the reaction products are generated at quantitative amounts and require minimal purification . Briefly, 10 mmol of 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA, Sigma, St. Louis, MO) and 10 mmol of 2-bromoethyl methacrylate (BEMA, Monomer-Polymer and Dajec, Trevose, PA) were combined with 3 g of ethanol in a 20 mL scintillation vial. The vial was stirred at 60 °C for 24 h. The solvent was then removed, yielding QADM as a clear, colorless, and viscous liquid. QADM was mixed with SBMP adhesive at QADM/(SBMP adhesive + QADM) mass fraction of 10%, following a previous study . The same 10% mass fraction was used in SBMP primer .

Silver 2-ethylhexanoate salt (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 improved the solubility by forming Ag N bonds with Ag ions to facilitate Ag salt to dissolve in resin solution. TBAEMA contains reactive methacrylate groups which can be chemically bonded in the resin upon photopolymerization. Ag was incorporated into SBMP primer at a silver 2-ethylhexanoate/(primer + silver 2-ethylhexanoate) mass fraction of 0.05%, following a previous study . The same 0.05% mass fraction was used in SBMP adhesive.

Transmission electron microscopy (TEM) was used to examine the NAg in resin, following a recent method . Briefly, a thin sheet of mica was partially split and the Ag-containing resin was placed in the gap. The resin-mica sandwich was pressed with an applied load of 2.7 × 10 ⁷ N to form a thin sheet of resin in between the two mica layers . The resin was photo-cured for 1 min. The mica sheet was then split apart after 1 day to expose the polymerized film. An ultrathin layer of carbon was vacuum-evaporated onto the resin (Electron Microscopy Sciences, Hatfield, PA). The carbon-coated sample was then partially submerged in distilled water in order to float the thin film onto the water’s surface. A copper grid was then used to retrieve the film. A high-resolution TEM (Tecnai T12, FEI, Hillsboro, OR) was used at an accelerating voltage of 120 kV. Images were collected and the NAg size was measured using AMT V600 image analysis software (Advanced Microscopy Techniques, Woburn, MA) .

2.2

Microtensile dentin bond strength

Three primer/adhesive groups were tested: (1) SBMP primer, SBMP adhesive (termed SBMP control); (2) primer + 10% QADM, adhesive + 10% QADM (termed SBMP + QADM); (3) primer + 0.05% NAg, adhesive + 0.05% NAg (termed SBMP + NAg).

Human third molars were collected with donor consent under a protocol approved by the University of Maryland. The roots of teeth were removed via a water-cooled cutting saw (Isomet, Buehler, Lake Bluff, IL) . The occlusal one-third of the tooth crown was removed to expose midcoronal dentin. The dentin surface was polished with 600-grit SiC paper, etched with 37% phosphoric acid gel for 15 s, and rinsed with distilled water. A primer was applied and the solvent was removed with a stream of air for 5 s. Then the adhesive was applied and light-cured for 10 s (Optilux VCL 401, Demetron Kerr, Danbury, CT). A composite (TPH, Caulk/Dentsply, Milford, DE) was applied and light-cured for 60 s . After storage in de-ionized water at 37 °C for 24 h, each bonded tooth was vertically sectioned into slabs with a 0.9 mm thickness . The central slab was used for microscopy. The other slabs were sectioned into 0.9 mm × 0.9 mm composite–dentin beams . Eight teeth were used for each bonding agent group. Five beams were obtained from each tooth, yielding 40 beams for each group ( n = 40). Each beam was stressed to failure under uniaxial tension in a computer-controlled Universal Testing Machine (MTS, Eden Prairie, MN) at a cross-head speed of 1 mm/min . The load-at-failure divided by the cross-sectional area at the site of failure yielded the microtensile dentin bond strength .

For scanning electron microscopy (SEM), the composite–dentin slabs were polished, and treated with 37% phosphoric acid gel for 15 s, and then 5.25% NaOCl for 10 min . Specimens were dehydrated using increasing ethanol concentrations of 50%, 70%, 85% and 100% . Specimens were sputter-coated with gold and examined via SEM (Quanta 200, FEI, Hillsboro, OR). For TEM, thin composite–dentin sections of an approximate thickness of 120 μm were cut and fixed with 2% paraformaldehyde and 2.5% glutaraldehyde, following a previous study . Samples were embedded in epoxy (Spurr’s, Electron Microscopy Sciences, PA). Ultra-thin sections (approximate thickness = 100 nm) were cut using a diamond knife (Diatome, Bienne, Switzerland) with an ultra-microtome (EM-UC7, Leica, Germany). The non-demineralized sections were examined in TEM (Tecnai-T12, FEI).

2.3

Cytotoxicity of adhesive eluent with human gingival fibroblasts

The cover of a sterile 96-well plate was used for specimen preparation . Ten microliter of a primer was placed in the bottom of the dent. After drying with a stream of air, 20 μL of adhesive was applied. A micro applicator with a small brush tip (Benda, Centrix, Shelton, CT) was used to mix the primer and adhesive together, which was then photo-polymerized for 20 s using a Mylar strip covering. This yielded a cured primer/adhesive disk of approximately 8 mm in diameter and 0.5 mm in thickness. The primer/adhesive disk was well cured, and after the cured samples were removed from the 96-well plate, there was no primer left on the well cover as confirmed with optical microscopy. After sterilization with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC), the disk was immersed in 10 mL fibroblast medium (FM, ScienCell, San Diego, CA) and agitated for 24 h at 37 °C to obtain eluent from the disks . Cytotoxicity was then tested using the original extract solution along with a series of dilutions . The original extract was diluted with fresh FM at dilutions of 2-fold (1 part of original extract + 1 part of fresh FM), 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, and 128-fold, which were then used for the fibroblast cytotoxicity test.

Human gingival fibroblasts (HGF, ScienCell, San Diego, USA) were cultured in FM supplemented with 2% fetal bovine serum, 100 IU/mL penicillin and 100 IU/mL streptomycin. The protocol of using HGF was approved by the University of Maryland. A seeding density of 4000 cells/well was used in 96-well plates . After 24 h of incubation at 37 °C with 5% CO ₂ in air, the culture medium was removed and replaced with 100 μL of the adhesive eluent at one of the aforementioned dilution folds. The cells were cultured for another 48 h, and then 20 μL of sterile filtered MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) solution at a concentration of 5 mg/mL was added to each well. After incubating in a darkroom for 4 h, the unreacted dye was removed and 150 μL/well of dimethylsulfoxide (DMSO, Sigma) was added . The solution absorbance was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnvale, CA) at 492 nm . FM without any resin eluent was used to culture fibroblasts as control, and its absorbance was set as 100%. The fibroblast viability for cells cultured with eluents = absorbance with eluents/absorbance of control .

2.4

Live/dead assay of S. mutans on primer/adhesive/composite tri-layer disks

The cover of a 96-well plate was used for specimen preparation . As in Section 2.3 , primer and adhesive were applied and photo-polymerized. Then a composite (TPH) was placed and photo-cured for 1 min to obtain a disk of 8 mm in diameter and 1 mm in thickness. Disks were immersed in water and agitated for 1 h to remove any uncured monomer, following a previous study . The disks were sterilized with ethylene oxide (Anprolene AN 74i).

The use of S. mutans (ATCC700610, American Type, Manassas, VA) was approved by the University of Maryland. S. mutans is a cariogenic bacterium and is the primary causative agent of caries. S. mutans was cultured overnight at 37 °C in Brain Heart Infusion (BHI, Becton, Sparks, MD) in an anaerobic atmosphere. Bacterial suspension obtained was adjusted to an optical density (OD) of 0.5 at 600 nm for further usage . The resin disks were placed, with the primer surface facing up, in a 24-well plate with 2 mL of BHI supplemented with 0.2% sucrose. Bacterial suspension was diluted by 1:100, and then 10 μL of the suspension was inoculated in each well. After 24-h, the biofilm on the disk was used in the following experiments, and the planktonic bacteria in the medium were also collected separately .

Disks with 24-h biofilms were washed with phosphate buffered saline (PBS). The bacteria were stained using a live/dead 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. Separately, the planktonic bacteria in the medium were collected, centrifuged at 5 kg for 4 min, and similarly live/dead stained. Each test was done at n = 6. The stained specimens were examined with an epifluorescence microscope (TE2000-S, Nikon, Melville, NY) .

2.5

MTT metabolic activity of S. mutans

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. Briefly, disks with 1-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% 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. The planktonic bacteria were collected by centrifugation at 5 kg for 4 min. An aliquot of 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals . After incubation for 20 min in the dark, 200 μL of the DMSO solution was transferred to a 96-well plate, and the absorbance at 540 nm was measured via a microplate reader (SpectraMax M5) . A higher absorbance indicates a higher formazan concentration, which in turn indicates more metabolic activity of the bacteria. Six replicates were tested for each group ( n = 6).

2.6

Lactic acid production and colony-forming units (CFUs)

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

Bacteria in biofilms on disks were harvested by sonicating (3510R, Branson, Danbury, CT) and vortexing (Fisher, Pittsburgh, PA). Separately, the CFU counts of the planktonic bacteria from the medium were also measured. The bacterial suspensions were serially diluted, and spread onto BHI agar plates for CFU analysis ( n = 6) following previous studies .

2.7

gtf gene expression of S. mutans

S. mutans can synthesize extracellular glucans by glucosyltransferases (GTFs), which is important for bacterial cell adhesion and biofilm formation . The GTF expression was shown to involve three types of genes: gtfB , gtfC , and gtfD . These gene expressions were measured here because it is desirable for the antibacterial primer/adhesive to hinder these gene expressions, thereby suppressing glucans synthesis, biofilm formation, and secondary caries . To collect sufficient amount of bacteria for RNA extraction and gene expression analysis, larger primer/adhesive disks were fabricated. Disks were fabricated in the covers of 12-well plates using 50 μL of primer, 50 μL of adhesive, and a composite (TPH). This yielded a primer/adhesive/composite tri-layer disk of 20 mm in diameter and 1 mm in thickness, while otherwise following the same method described in Section 2.3 . Each disk was placed in a 6-well plate with 5 mL of BHI supplemented with 0.2% sucrose. Bacterial suspension was diluted by 1:100, then 50 μL of the suspension was inoculated in each well and incubated for 24 h.

Biofilm on each disk and the corresponding bacteria in medium were used separately for gene expression measurement. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR, 7900HT, Applied Biosystems, Foster City, CA) was used. Total RNA for S. mutans was isolated with a TRIzol Max bacterial RNA isolation kit (Invitrogen, CA) according to the manufacturer’s instructions . cDNA was synthesized by mixing total RNA (2 μg) and high-capacity cDNA reverse transcription kit (Applied Biosystems) in a 20 μL reaction volume . RT-PCR was carried out with a Taqman fast universal PCR master mix (Applied Biosystems). Oligonucleotide primers and probes for gtfB , gtfC , gtfD and 16S rRNA (a housekeeping gene as an internal control) were used. The expression levels of all genes were normalized with the amplification of the 16S rRNA gene of S. mutans as an internal standard . Three separate experiments each with duplicates were performed (total n = 6).

2.8

Statistical analysis

All data were first checked for normal distribution with the Kolmogorov–Smirnov test and for homogeneity with Levene’s test. 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. Statistical analyses were performed by SPSS 13.0 software (SPSS, Chicago, IL) at a significance level of p < 0.05.

2.1

Antibacterial bonding agents containing QADM or NAg

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

Bis(2-methacryloyloxyethyl) dimethylammonium bromide, a quaternary ammonium dimethacrylate (QADM), was recently synthesized . Its synthesis employed a modified Menschutkin reaction, where a tertiary amine group was reacted with an organo-halide. A benefit of this reaction is that the reaction products are generated at quantitative amounts and require minimal purification . Briefly, 10 mmol of 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA, Sigma, St. Louis, MO) and 10 mmol of 2-bromoethyl methacrylate (BEMA, Monomer-Polymer and Dajec, Trevose, PA) were combined with 3 g of ethanol in a 20 mL scintillation vial. The vial was stirred at 60 °C for 24 h. The solvent was then removed, yielding QADM as a clear, colorless, and viscous liquid. QADM was mixed with SBMP adhesive at QADM/(SBMP adhesive + QADM) mass fraction of 10%, following a previous study . The same 10% mass fraction was used in SBMP primer .

Silver 2-ethylhexanoate salt (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 improved the solubility by forming Ag N bonds with Ag ions to facilitate Ag salt to dissolve in resin solution. TBAEMA contains reactive methacrylate groups which can be chemically bonded in the resin upon photopolymerization. Ag was incorporated into SBMP primer at a silver 2-ethylhexanoate/(primer + silver 2-ethylhexanoate) mass fraction of 0.05%, following a previous study . The same 0.05% mass fraction was used in SBMP adhesive.

Transmission electron microscopy (TEM) was used to examine the NAg in resin, following a recent method . Briefly, a thin sheet of mica was partially split and the Ag-containing resin was placed in the gap. The resin-mica sandwich was pressed with an applied load of 2.7 × 10 ⁷ N to form a thin sheet of resin in between the two mica layers . The resin was photo-cured for 1 min. The mica sheet was then split apart after 1 day to expose the polymerized film. An ultrathin layer of carbon was vacuum-evaporated onto the resin (Electron Microscopy Sciences, Hatfield, PA). The carbon-coated sample was then partially submerged in distilled water in order to float the thin film onto the water’s surface. A copper grid was then used to retrieve the film. A high-resolution TEM (Tecnai T12, FEI, Hillsboro, OR) was used at an accelerating voltage of 120 kV. Images were collected and the NAg size was measured using AMT V600 image analysis software (Advanced Microscopy Techniques, Woburn, MA) .

2.2

Microtensile dentin bond strength

Three primer/adhesive groups were tested: (1) SBMP primer, SBMP adhesive (termed SBMP control); (2) primer + 10% QADM, adhesive + 10% QADM (termed SBMP + QADM); (3) primer + 0.05% NAg, adhesive + 0.05% NAg (termed SBMP + NAg).

Human third molars were collected with donor consent under a protocol approved by the University of Maryland. The roots of teeth were removed via a water-cooled cutting saw (Isomet, Buehler, Lake Bluff, IL) . The occlusal one-third of the tooth crown was removed to expose midcoronal dentin. The dentin surface was polished with 600-grit SiC paper, etched with 37% phosphoric acid gel for 15 s, and rinsed with distilled water. A primer was applied and the solvent was removed with a stream of air for 5 s. Then the adhesive was applied and light-cured for 10 s (Optilux VCL 401, Demetron Kerr, Danbury, CT). A composite (TPH, Caulk/Dentsply, Milford, DE) was applied and light-cured for 60 s . After storage in de-ionized water at 37 °C for 24 h, each bonded tooth was vertically sectioned into slabs with a 0.9 mm thickness . The central slab was used for microscopy. The other slabs were sectioned into 0.9 mm × 0.9 mm composite–dentin beams . Eight teeth were used for each bonding agent group. Five beams were obtained from each tooth, yielding 40 beams for each group ( n = 40). Each beam was stressed to failure under uniaxial tension in a computer-controlled Universal Testing Machine (MTS, Eden Prairie, MN) at a cross-head speed of 1 mm/min . The load-at-failure divided by the cross-sectional area at the site of failure yielded the microtensile dentin bond strength .

For scanning electron microscopy (SEM), the composite–dentin slabs were polished, and treated with 37% phosphoric acid gel for 15 s, and then 5.25% NaOCl for 10 min . Specimens were dehydrated using increasing ethanol concentrations of 50%, 70%, 85% and 100% . Specimens were sputter-coated with gold and examined via SEM (Quanta 200, FEI, Hillsboro, OR). For TEM, thin composite–dentin sections of an approximate thickness of 120 μm were cut and fixed with 2% paraformaldehyde and 2.5% glutaraldehyde, following a previous study . Samples were embedded in epoxy (Spurr’s, Electron Microscopy Sciences, PA). Ultra-thin sections (approximate thickness = 100 nm) were cut using a diamond knife (Diatome, Bienne, Switzerland) with an ultra-microtome (EM-UC7, Leica, Germany). The non-demineralized sections were examined in TEM (Tecnai-T12, FEI).

2.3

Cytotoxicity of adhesive eluent with human gingival fibroblasts

The cover of a sterile 96-well plate was used for specimen preparation . Ten microliter of a primer was placed in the bottom of the dent. After drying with a stream of air, 20 μL of adhesive was applied. A micro applicator with a small brush tip (Benda, Centrix, Shelton, CT) was used to mix the primer and adhesive together, which was then photo-polymerized for 20 s using a Mylar strip covering. This yielded a cured primer/adhesive disk of approximately 8 mm in diameter and 0.5 mm in thickness. The primer/adhesive disk was well cured, and after the cured samples were removed from the 96-well plate, there was no primer left on the well cover as confirmed with optical microscopy. After sterilization with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC), the disk was immersed in 10 mL fibroblast medium (FM, ScienCell, San Diego, CA) and agitated for 24 h at 37 °C to obtain eluent from the disks . Cytotoxicity was then tested using the original extract solution along with a series of dilutions . The original extract was diluted with fresh FM at dilutions of 2-fold (1 part of original extract + 1 part of fresh FM), 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, and 128-fold, which were then used for the fibroblast cytotoxicity test.

Human gingival fibroblasts (HGF, ScienCell, San Diego, USA) were cultured in FM supplemented with 2% fetal bovine serum, 100 IU/mL penicillin and 100 IU/mL streptomycin. The protocol of using HGF was approved by the University of Maryland. A seeding density of 4000 cells/well was used in 96-well plates . After 24 h of incubation at 37 °C with 5% CO ₂ in air, the culture medium was removed and replaced with 100 μL of the adhesive eluent at one of the aforementioned dilution folds. The cells were cultured for another 48 h, and then 20 μL of sterile filtered MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma) solution at a concentration of 5 mg/mL was added to each well. After incubating in a darkroom for 4 h, the unreacted dye was removed and 150 μL/well of dimethylsulfoxide (DMSO, Sigma) was added . The solution absorbance was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnvale, CA) at 492 nm . FM without any resin eluent was used to culture fibroblasts as control, and its absorbance was set as 100%. The fibroblast viability for cells cultured with eluents = absorbance with eluents/absorbance of control .

2.4

Live/dead assay of S. mutans on primer/adhesive/composite tri-layer disks

The cover of a 96-well plate was used for specimen preparation . As in Section 2.3 , primer and adhesive were applied and photo-polymerized. Then a composite (TPH) was placed and photo-cured for 1 min to obtain a disk of 8 mm in diameter and 1 mm in thickness. Disks were immersed in water and agitated for 1 h to remove any uncured monomer, following a previous study . The disks were sterilized with ethylene oxide (Anprolene AN 74i).

The use of S. mutans (ATCC700610, American Type, Manassas, VA) was approved by the University of Maryland. S. mutans is a cariogenic bacterium and is the primary causative agent of caries. S. mutans was cultured overnight at 37 °C in Brain Heart Infusion (BHI, Becton, Sparks, MD) in an anaerobic atmosphere. Bacterial suspension obtained was adjusted to an optical density (OD) of 0.5 at 600 nm for further usage . The resin disks were placed, with the primer surface facing up, in a 24-well plate with 2 mL of BHI supplemented with 0.2% sucrose. Bacterial suspension was diluted by 1:100, and then 10 μL of the suspension was inoculated in each well. After 24-h, the biofilm on the disk was used in the following experiments, and the planktonic bacteria in the medium were also collected separately .

Disks with 24-h biofilms were washed with phosphate buffered saline (PBS). The bacteria were stained using a live/dead 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. Separately, the planktonic bacteria in the medium were collected, centrifuged at 5 kg for 4 min, and similarly live/dead stained. Each test was done at n = 6. The stained specimens were examined with an epifluorescence microscope (TE2000-S, Nikon, Melville, NY) .

2.5

MTT metabolic activity of S. mutans

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. Briefly, disks with 1-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% 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. The planktonic bacteria were collected by centrifugation at 5 kg for 4 min. An aliquot of 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals . After incubation for 20 min in the dark, 200 μL of the DMSO solution was transferred to a 96-well plate, and the absorbance at 540 nm was measured via a microplate reader (SpectraMax M5) . A higher absorbance indicates a higher formazan concentration, which in turn indicates more metabolic activity of the bacteria. Six replicates were tested for each group ( n = 6).

2.6

Lactic acid production and colony-forming units (CFUs)

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

Bacteria in biofilms on disks were harvested by sonicating (3510R, Branson, Danbury, CT) and vortexing (Fisher, Pittsburgh, PA). Separately, the CFU counts of the planktonic bacteria from the medium were also measured. The bacterial suspensions were serially diluted, and spread onto BHI agar plates for CFU analysis ( n = 6) following previous studies .

2.7

gtf gene expression of S. mutans

S. mutans can synthesize extracellular glucans by glucosyltransferases (GTFs), which is important for bacterial cell adhesion and biofilm formation . The GTF expression was shown to involve three types of genes: gtfB , gtfC , and gtfD . These gene expressions were measured here because it is desirable for the antibacterial primer/adhesive to hinder these gene expressions, thereby suppressing glucans synthesis, biofilm formation, and secondary caries . To collect sufficient amount of bacteria for RNA extraction and gene expression analysis, larger primer/adhesive disks were fabricated. Disks were fabricated in the covers of 12-well plates using 50 μL of primer, 50 μL of adhesive, and a composite (TPH). This yielded a primer/adhesive/composite tri-layer disk of 20 mm in diameter and 1 mm in thickness, while otherwise following the same method described in Section 2.3 . Each disk was placed in a 6-well plate with 5 mL of BHI supplemented with 0.2% sucrose. Bacterial suspension was diluted by 1:100, then 50 μL of the suspension was inoculated in each well and incubated for 24 h.

Biofilm on each disk and the corresponding bacteria in medium were used separately for gene expression measurement. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR, 7900HT, Applied Biosystems, Foster City, CA) was used. Total RNA for S. mutans was isolated with a TRIzol Max bacterial RNA isolation kit (Invitrogen, CA) according to the manufacturer’s instructions . cDNA was synthesized by mixing total RNA (2 μg) and high-capacity cDNA reverse transcription kit (Applied Biosystems) in a 20 μL reaction volume . RT-PCR was carried out with a Taqman fast universal PCR master mix (Applied Biosystems). Oligonucleotide primers and probes for gtfB , gtfC , gtfD and 16S rRNA (a housekeeping gene as an internal control) were used. The expression levels of all genes were normalized with the amplification of the 16S rRNA gene of S. mutans as an internal standard . Three separate experiments each with duplicates were performed (total n = 6).

2.8

Statistical analysis

All data were first checked for normal distribution with the Kolmogorov–Smirnov test and for homogeneity with Levene’s test. 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. Statistical analyses were performed by SPSS 13.0 software (SPSS, Chicago, IL) at a significance level of p < 0.05.