2.1

Synthesis of new quaternary ammonium methacrylates (QAMs)

A modified Menschutkin reaction approach was used to synthesize the new QAMs. This method uses a tertiary amine group to react with an organo-halide, as described in previous studies . A benefit of this reaction is that the reaction products are generated at virtually quantitative amounts and require minimal purification . In the present study, 2-bromoethyl methacrylate (BEMA) was the organo-halide. N,N-dimethylaminohexane (DMAH) and 1-(dimethylamino)docecane (DMAD) were the two tertiary amines.

The scheme of synthesis of dimethylaminohexane methacrylate (DMAHM) is shown in Fig. 1 A . Ten mmoles of DMAH (Tokyo Chemical Industry, Tokyo, Japan), 10 mmol of BEMA (Monomer-Polymer and Dajac Labs, Trevose, PA), and 3 g of ethanol were added to a 20 mL scintillation vial with a magnetic stir bar. The vial was capped and stirred at 70 °C for 24 h. After the reaction was complete, the ethanol solvent was removed via evaporation at room temperature over several days. This yielded DMAHM as a clear liquid.

A modified Menschutkin reaction was used to synthesize new antibacterial monomers: (A) DMAHM and (B) DMADDM. DMAH = N,N-dimethylaminohexane. BEMA = 2-bromoethyl methacrylate. DMAHM = dimethylaminohexane methacrylate. DMAD = 1-(dimethylamino)docecane. DMADDM = dimethylaminododecyl methacrylate. EtOH = anhydrous ethanol. The number of the alkyl chain length units was 6 for DMAHM and 12 for DMADDM.

The scheme of synthesis of dimethylaminododecyl methacrylate (DMADDM) is shown in Fig. 1 B. In a 20 mL scintillation vials were added 10 mmol of DMAD (Tokyo Chemical Industry), 10 mmol of BEMA, and 3 g of ethanol. A magnetic stir bar was added, and the vial was capped and stirred at 70 °C for 24 h. After the reaction was complete, the solvent was removed via evaporation. The number of the alkyl chain length units was 6 for DMAHM and 12 for DMADDM ( Fig. 1 ).

To characterize the reaction products, Fourier transform infrared spectroscopy (FTIR, Nicolet 6700, Thermo Scientific, Waltham, MA) was used. FTIR spectra of the starting materials and the viscous products were collected between two KBr windows in the 4000–400 cm ⁻¹ region with 128 scans at 4 cm ⁻¹ resolution . Water and CO ₂ bands were removed from all spectra by subtraction. ¹ H NMR spectra (GSX 270, JEOL, Peabody, MA) of the starting materials and products were taken in deuterated chloroform at a concentration of approximately 3%. All spectra were run at room temperature, 15 Hz sample spinning, 45° tip angle for the observation pulse, and a 10 s recycle delay, for 64 scans .

2.2

Minimum inhibitory concentration (MIC) and bactericidal concentration (MBC)

MIC is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism . When the antibacterial agent is added into a bacterial solution at the MIC, not all the bacteria are killed, but the concentration of bacteria is so low that the solution appears clear without turbidity . When the MBC of an antibacterial agent is used in a bacterial solution, all the bacteria will be killed; if this solution is placed on agar plates, there will be no bacteria colonies after incubation. MIC and MBC were measured using S. mutans (ATCC 700610, UA159, American Type Culture, Manassas, VA). The use of S. mutans was approved by the University of Maryland. S. mutans is a cariogenic, aerotolerant anaerobic bacterium and the primary causative agent of dental caries . MIC and MBC were determined via serial microdilution assays . Unpolymerized DMAHM or DMADDM monomer was dissolved in brain heart infusion (BHI) broth (BD, Franklin Lakes, NJ) to give a final concentration of 200 mg/mL. From these starting solutions, serial two fold dilutions were made into 1 mL volumes of BHI broth. Fifteen microliters of stock S. mutans was added to 15 mL of BHI broth with 0.2% sucrose and incubated at 37 °C with 5% CO ₂ . Overnight cultures of S. mutans were adjusted to 2 × 10 ⁶ CFU/mL with BHI broth, and 50 μL of inocula was added to each well of a 96-well plate containing 50 μL of a series of antibacterial monomer dilution broths. BHI broth with 1 × 10 ⁶ CFU/mL bacteria suspension without antibacterial agent served as negative control. Chlorhexidine diacetate (CHX) (Sigma, St. Louis, MO) served as positive control. The previously synthesized QADM served as an antibacterial monomer control. After incubation at 37 °C in 5% CO ₂ for 48 h, the wells were read for turbidity, referenced by the negative and positive control wells. MIC was determined as the endpoint (the well with the lowest antibacterial agent concentration) where no turbidity could be detected with respect to the controls . To determine MBC, an aliquot of 50 μL from each well without turbidity was inoculated on BHI agar plates and incubated at 37 °C in 5% CO ₂ for 48 h. MBC was determined as the lowest concentration of antibacterial agent that produced no colonies on the plate. The tests were performed in triplicate .

2.3

Processing of DMADDM–NACP nanocomposite

A spray-drying technique as described previously was used to make NACP (Ca ₃ [PO ₄ ] ₂ ). Calcium carbonate (CaCO ₃ , Fisher, Fair Lawn, NJ) and dicalcium phosphate anhydrous (CaHPO ₄ , Baker Chemical, Phillipsburg, NJ) were dissolved into an acetic acid solution to obtain final Ca and P ionic concentrations of 8 mmol/L and 5.333 mmol/L, respectively. This resulted in a Ca/P molar ratio of 1.5, the same as that for ACP. This solution was sprayed into a heated chamber, and an electrostatic precipitator (AirQuality, Minneapolis, MN) was used to collect the dried particles. This method produced NACP with a mean particle size of 116 nm, as measured in a previous study .

Because Section 2.2 showed that DMADDM had a much greater antibacterial potency than DMAHM and QADM, DMADDM was used for incorporation into the NACP nanocomposite to obtain antibacterial properties. BisGMA (bisphenol glycidyl dimethacrylate) and TEGDMA (triethylene glycol dimethacrylate) (Esstech, Essington, PA) were mixed at a mass ratio = 1:1, and rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate (mass fractions). DMADDM was mixed with the photo-activated BisGMA-TEGDMA resin at the following DMADDM/(BisGMA-TEGDMA + DMADDM) mass fractions: 0%, 2.5%, 5%, 7.5% and 10%, yielding five groups of resin, respectively. The 10% mass fraction followed previous studies . The other mass fractions enabled the investigation of the relationship between DMADDM mass fraction and antibacterial efficacy. A dental barium boroaluminosilicate glass of a median particle size of 1.4 μm (Caulk/Dentsply, Milford, DE) was silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n-propylamine . The NACP and glass particles were mixed into each resin, at the same filler level of 70% by mass, with 20% of NACP and 50% of glass . Because the resin mass fraction was 30% in the composite, the five DMADDM mass fractions in the composite were 0%, 0.75%, 1.5%, 2.25% and 3%, respectively.

Six composites were tested: five NACP nanocomposites at the five DMADDM mass fractions described above, and a commercial control composite. Renamel (Cosmedent, Chicago, IL) served as a control composite. It consisted of nanofillers of 20–40 nm in size, at 60% filler level in a multifunctional methacrylate ester resin. For mechanical testing, each paste was placed into rectangular molds of 2 mm × 2 mm × 25 mm. For biofilm experiments, each paste was placed into disk molds of 9 mm in diameter and 2 mm in thickness. The specimens were photo-cured (Triad 2000, Dentsply, York, PA) for 1 min on each side. The specimens were then incubated in distilled water at 37 °C for 24 h prior to mechanical or biofilm testing.

2.4

Mechanical testing

A computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) was used to fracture the specimens in three-point flexure using a span of 10 mm and a crosshead speed of 1 mm/min. Flexural strength S was measured as: S = 3 P _max L /(2 bh ² ), where P _max is the load-at-failure, L is span, b is specimen width and h is specimen thickness. Elastic modulus E was measured as: E = ( P / d )( L ³ /[4 bh ³ ]), where load P divided by displacement d is the slope in the linear elastic region of the load–displacement curve. The specimens were taken out of the water and fractured within several minutes while still being wet .

2.5

Dental plaque microcosm biofilm and live/dead assay

The use of the dental plaque microcosm biofilm model with human saliva as inoculum was approved by the University of Maryland. Saliva was collected from a healthy adult donor following a previous study . The donor had natural dentition without active caries or periopathology, and without the use of antibiotics within the last 3 months. The donor did not brush teeth for 24 h and abstained from food or drink intake for at least 2 h prior to donating saliva . Stimulated saliva was collected during parafilm chewing and kept on ice. The saliva was diluted in sterile glycerol to a concentration of 70% saliva and 30% glycerol . The saliva–glycerol stock was added, with 1:50 final dilution, into the growth medium as inoculum. The growth medium was the McBain artificial saliva medium, which 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 K ₁ , 0.0002 g/L, at pH 7 . A 1.5 mL of inoculum was measured to contain total microorganisms of (2.4 ± 0.2) × 10 ⁶ CFU, which included total streptococci of (1.0 ± 0.1) × 10 ⁵ CFU, and mutans streptococci of (3.3 ± 0.8) × 10 ⁴ CFU. The composite disks were sterilized in ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC). The 1.5 mL of inoculum was added to each well of 24-well plates with a composite disk, and incubated in 5% CO ₂ at 37 °C for 8 h.

The disks were then transferred to new 24-well plates filled with fresh medium and incubated. After 16 h, the disks were transferred to new 24-well plates with fresh medium and incubated for 24 h. This totaled 48 h of incubation, which was shown to be adequate to form dental plaque microcosm biofilms on resins .

After 48 h of growth, the microcosm biofilms adherent on the disks were gently washed three times with phosphate buffered saline (PBS), and then stained using the BacLight live/dead bacterial viability kit (Molecular Probes, Eugene, OR) . Live bacteria were stained with Syto 9 to produce a green fluorescence, and bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. The stained disks were examined using a confocal laser scanning microscopy (CLSM 510, Carl Zeiss, Thornwood, NY).

2.6

MTT assays

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed according to previous studies . It is a colorimetric method that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Briefly, disks with 48-h biofilms were rinsed with PBS and transferred to 24 well plates. Then, 1 mL of MTT dye (0.5 mg/mL MTT in PBS) was added to each well and incubated for 1 h. The disks were transferred to new 24-well plates, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min in the dark. Then, 200 μL of the DMSO solution from each well was transferred to a 96-well plate, and the absorbance at 540 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnvale, CA) .

2.7

Lactic acid production and CFU counts

Composite disks with 48-h biofilms were rinsed in cysteine peptone water (CPW) to remove the loose bacteria. Each disk was placed in a new 24-well plate and 1.5 mL of buffered peptone water (BPW) supplemented with 0.2% sucrose . The samples were incubated in 5% CO ₂ at 37 °C for 3 h to allow the biofilms to produce acid. The BPW solutions were then stored for lactate analysis. Lactate concentrations were determined using an enzymatic (lactate dehydrogenase) method according to previous studies . 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) .

Composite disks with 2-day biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication (3510R-MTH, Branson, Danbury, CT) for 5 min, followed by vortexing at 2400 rpm for 30 s using a vortex mixer (Fisher Scientific, Pittsburgh, PA). Three types of agar plates were used to assess the microorganism viability after serial dilution in CPW: Mitis salivarius agar (MSA) culture plates, containing 15% sucrose, to determine total streptococci ; MSA agar culture plates plus 0.2 units of bacitracin per mL, to determine mutans streptococci ; and Tryptic Soy Blood Agar culture plates to determine total microorganisms .

One-way analysis of variance (ANOVA) was performed to detect the significant effects of the variables. Tukey’s multiple comparison test was used at a p value of 0.05.

2.1

Synthesis of new quaternary ammonium methacrylates (QAMs)

A modified Menschutkin reaction approach was used to synthesize the new QAMs. This method uses a tertiary amine group to react with an organo-halide, as described in previous studies . A benefit of this reaction is that the reaction products are generated at virtually quantitative amounts and require minimal purification . In the present study, 2-bromoethyl methacrylate (BEMA) was the organo-halide. N,N-dimethylaminohexane (DMAH) and 1-(dimethylamino)docecane (DMAD) were the two tertiary amines.

The scheme of synthesis of dimethylaminohexane methacrylate (DMAHM) is shown in Fig. 1 A . Ten mmoles of DMAH (Tokyo Chemical Industry, Tokyo, Japan), 10 mmol of BEMA (Monomer-Polymer and Dajac Labs, Trevose, PA), and 3 g of ethanol were added to a 20 mL scintillation vial with a magnetic stir bar. The vial was capped and stirred at 70 °C for 24 h. After the reaction was complete, the ethanol solvent was removed via evaporation at room temperature over several days. This yielded DMAHM as a clear liquid.

A modified Menschutkin reaction was used to synthesize new antibacterial monomers: (A) DMAHM and (B) DMADDM. DMAH = N,N-dimethylaminohexane. BEMA = 2-bromoethyl methacrylate. DMAHM = dimethylaminohexane methacrylate. DMAD = 1-(dimethylamino)docecane. DMADDM = dimethylaminododecyl methacrylate. EtOH = anhydrous ethanol. The number of the alkyl chain length units was 6 for DMAHM and 12 for DMADDM.

The scheme of synthesis of dimethylaminododecyl methacrylate (DMADDM) is shown in Fig. 1 B. In a 20 mL scintillation vials were added 10 mmol of DMAD (Tokyo Chemical Industry), 10 mmol of BEMA, and 3 g of ethanol. A magnetic stir bar was added, and the vial was capped and stirred at 70 °C for 24 h. After the reaction was complete, the solvent was removed via evaporation. The number of the alkyl chain length units was 6 for DMAHM and 12 for DMADDM ( Fig. 1 ).

To characterize the reaction products, Fourier transform infrared spectroscopy (FTIR, Nicolet 6700, Thermo Scientific, Waltham, MA) was used. FTIR spectra of the starting materials and the viscous products were collected between two KBr windows in the 4000–400 cm ⁻¹ region with 128 scans at 4 cm ⁻¹ resolution . Water and CO ₂ bands were removed from all spectra by subtraction. ¹ H NMR spectra (GSX 270, JEOL, Peabody, MA) of the starting materials and products were taken in deuterated chloroform at a concentration of approximately 3%. All spectra were run at room temperature, 15 Hz sample spinning, 45° tip angle for the observation pulse, and a 10 s recycle delay, for 64 scans .

2.2

Minimum inhibitory concentration (MIC) and bactericidal concentration (MBC)

MIC is the lowest concentration of an antimicrobial agent that will inhibit the visible growth of a microorganism . When the antibacterial agent is added into a bacterial solution at the MIC, not all the bacteria are killed, but the concentration of bacteria is so low that the solution appears clear without turbidity . When the MBC of an antibacterial agent is used in a bacterial solution, all the bacteria will be killed; if this solution is placed on agar plates, there will be no bacteria colonies after incubation. MIC and MBC were measured using S. mutans (ATCC 700610, UA159, American Type Culture, Manassas, VA). The use of S. mutans was approved by the University of Maryland. S. mutans is a cariogenic, aerotolerant anaerobic bacterium and the primary causative agent of dental caries . MIC and MBC were determined via serial microdilution assays . Unpolymerized DMAHM or DMADDM monomer was dissolved in brain heart infusion (BHI) broth (BD, Franklin Lakes, NJ) to give a final concentration of 200 mg/mL. From these starting solutions, serial two fold dilutions were made into 1 mL volumes of BHI broth. Fifteen microliters of stock S. mutans was added to 15 mL of BHI broth with 0.2% sucrose and incubated at 37 °C with 5% CO ₂ . Overnight cultures of S. mutans were adjusted to 2 × 10 ⁶ CFU/mL with BHI broth, and 50 μL of inocula was added to each well of a 96-well plate containing 50 μL of a series of antibacterial monomer dilution broths. BHI broth with 1 × 10 ⁶ CFU/mL bacteria suspension without antibacterial agent served as negative control. Chlorhexidine diacetate (CHX) (Sigma, St. Louis, MO) served as positive control. The previously synthesized QADM served as an antibacterial monomer control. After incubation at 37 °C in 5% CO ₂ for 48 h, the wells were read for turbidity, referenced by the negative and positive control wells. MIC was determined as the endpoint (the well with the lowest antibacterial agent concentration) where no turbidity could be detected with respect to the controls . To determine MBC, an aliquot of 50 μL from each well without turbidity was inoculated on BHI agar plates and incubated at 37 °C in 5% CO ₂ for 48 h. MBC was determined as the lowest concentration of antibacterial agent that produced no colonies on the plate. The tests were performed in triplicate .

2.3

Processing of DMADDM–NACP nanocomposite

A spray-drying technique as described previously was used to make NACP (Ca ₃ [PO ₄ ] ₂ ). Calcium carbonate (CaCO ₃ , Fisher, Fair Lawn, NJ) and dicalcium phosphate anhydrous (CaHPO ₄ , Baker Chemical, Phillipsburg, NJ) were dissolved into an acetic acid solution to obtain final Ca and P ionic concentrations of 8 mmol/L and 5.333 mmol/L, respectively. This resulted in a Ca/P molar ratio of 1.5, the same as that for ACP. This solution was sprayed into a heated chamber, and an electrostatic precipitator (AirQuality, Minneapolis, MN) was used to collect the dried particles. This method produced NACP with a mean particle size of 116 nm, as measured in a previous study .

Because Section 2.2 showed that DMADDM had a much greater antibacterial potency than DMAHM and QADM, DMADDM was used for incorporation into the NACP nanocomposite to obtain antibacterial properties. BisGMA (bisphenol glycidyl dimethacrylate) and TEGDMA (triethylene glycol dimethacrylate) (Esstech, Essington, PA) were mixed at a mass ratio = 1:1, and rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4-N,N-dimethylaminobenzoate (mass fractions). DMADDM was mixed with the photo-activated BisGMA-TEGDMA resin at the following DMADDM/(BisGMA-TEGDMA + DMADDM) mass fractions: 0%, 2.5%, 5%, 7.5% and 10%, yielding five groups of resin, respectively. The 10% mass fraction followed previous studies . The other mass fractions enabled the investigation of the relationship between DMADDM mass fraction and antibacterial efficacy. A dental barium boroaluminosilicate glass of a median particle size of 1.4 μm (Caulk/Dentsply, Milford, DE) was silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n-propylamine . The NACP and glass particles were mixed into each resin, at the same filler level of 70% by mass, with 20% of NACP and 50% of glass . Because the resin mass fraction was 30% in the composite, the five DMADDM mass fractions in the composite were 0%, 0.75%, 1.5%, 2.25% and 3%, respectively.

Six composites were tested: five NACP nanocomposites at the five DMADDM mass fractions described above, and a commercial control composite. Renamel (Cosmedent, Chicago, IL) served as a control composite. It consisted of nanofillers of 20–40 nm in size, at 60% filler level in a multifunctional methacrylate ester resin. For mechanical testing, each paste was placed into rectangular molds of 2 mm × 2 mm × 25 mm. For biofilm experiments, each paste was placed into disk molds of 9 mm in diameter and 2 mm in thickness. The specimens were photo-cured (Triad 2000, Dentsply, York, PA) for 1 min on each side. The specimens were then incubated in distilled water at 37 °C for 24 h prior to mechanical or biofilm testing.

2.4

Mechanical testing

A computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) was used to fracture the specimens in three-point flexure using a span of 10 mm and a crosshead speed of 1 mm/min. Flexural strength S was measured as: S = 3 P _max L /(2 bh ² ), where P _max is the load-at-failure, L is span, b is specimen width and h is specimen thickness. Elastic modulus E was measured as: E = ( P / d )( L ³ /[4 bh ³ ]), where load P divided by displacement d is the slope in the linear elastic region of the load–displacement curve. The specimens were taken out of the water and fractured within several minutes while still being wet .

2.5

Dental plaque microcosm biofilm and live/dead assay

The use of the dental plaque microcosm biofilm model with human saliva as inoculum was approved by the University of Maryland. Saliva was collected from a healthy adult donor following a previous study . The donor had natural dentition without active caries or periopathology, and without the use of antibiotics within the last 3 months. The donor did not brush teeth for 24 h and abstained from food or drink intake for at least 2 h prior to donating saliva . Stimulated saliva was collected during parafilm chewing and kept on ice. The saliva was diluted in sterile glycerol to a concentration of 70% saliva and 30% glycerol . The saliva–glycerol stock was added, with 1:50 final dilution, into the growth medium as inoculum. The growth medium was the McBain artificial saliva medium, which 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 K ₁ , 0.0002 g/L, at pH 7 . A 1.5 mL of inoculum was measured to contain total microorganisms of (2.4 ± 0.2) × 10 ⁶ CFU, which included total streptococci of (1.0 ± 0.1) × 10 ⁵ CFU, and mutans streptococci of (3.3 ± 0.8) × 10 ⁴ CFU. The composite disks were sterilized in ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC). The 1.5 mL of inoculum was added to each well of 24-well plates with a composite disk, and incubated in 5% CO ₂ at 37 °C for 8 h.

The disks were then transferred to new 24-well plates filled with fresh medium and incubated. After 16 h, the disks were transferred to new 24-well plates with fresh medium and incubated for 24 h. This totaled 48 h of incubation, which was shown to be adequate to form dental plaque microcosm biofilms on resins .

After 48 h of growth, the microcosm biofilms adherent on the disks were gently washed three times with phosphate buffered saline (PBS), and then stained using the BacLight live/dead bacterial viability kit (Molecular Probes, Eugene, OR) . Live bacteria were stained with Syto 9 to produce a green fluorescence, and bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. The stained disks were examined using a confocal laser scanning microscopy (CLSM 510, Carl Zeiss, Thornwood, NY).

2.6

MTT assays

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed according to previous studies . It is a colorimetric method that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Briefly, disks with 48-h biofilms were rinsed with PBS and transferred to 24 well plates. Then, 1 mL of MTT dye (0.5 mg/mL MTT in PBS) was added to each well and incubated for 1 h. The disks were transferred to new 24-well plates, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min in the dark. Then, 200 μL of the DMSO solution from each well was transferred to a 96-well plate, and the absorbance at 540 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnvale, CA) .

2.7

Lactic acid production and CFU counts

Composite disks with 48-h biofilms were rinsed in cysteine peptone water (CPW) to remove the loose bacteria. Each disk was placed in a new 24-well plate and 1.5 mL of buffered peptone water (BPW) supplemented with 0.2% sucrose . The samples were incubated in 5% CO ₂ at 37 °C for 3 h to allow the biofilms to produce acid. The BPW solutions were then stored for lactate analysis. Lactate concentrations were determined using an enzymatic (lactate dehydrogenase) method according to previous studies . 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) .

Composite disks with 2-day biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication (3510R-MTH, Branson, Danbury, CT) for 5 min, followed by vortexing at 2400 rpm for 30 s using a vortex mixer (Fisher Scientific, Pittsburgh, PA). Three types of agar plates were used to assess the microorganism viability after serial dilution in CPW: Mitis salivarius agar (MSA) culture plates, containing 15% sucrose, to determine total streptococci ; MSA agar culture plates plus 0.2 units of bacitracin per mL, to determine mutans streptococci ; and Tryptic Soy Blood Agar culture plates to determine total microorganisms .

One-way analysis of variance (ANOVA) was performed to detect the significant effects of the variables. Tukey’s multiple comparison test was used at a p value of 0.05.