2.1

NACP and glass filler particles

NACP [Ca ₃ (PO ₄ ) ₂ ] was synthesized via a spray-drying technique as recently described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution to obtain Ca and PO ₄ ionic concentrations of 8 mmol/L and 5.333 mmol/L, respectively, yielding a Ca/P molar ratio of 1.5. This solution was sprayed into a heated chamber, and an electrostatic precipitator was used to collect the dried particles. This yielded NACP with a mean particle size of 116 nm . As a co-filler to reinforce the composite, a barium boroaluminosilicate glass of a mean particle size of 1.4 μm (Caulk/Dentsply, Milford, DE) was silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine. These fillers were used in the composites as described below.

2.2

QADM resin composites

BisGMA (bisphenol glycidyl dimethacrylate) and TEGDMA (triethylene glycol dimethacrylate) (Esstech, Essington, PA) were mixed at a mass ratio of 1:1, and rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4- N , N -dimethylaminobenzoate. The QADM was bis(2-methacryloyloxy-ethyl) dimethyl-ammonium bromide, which was synthesized as described recently . Its synthesis employed a modified Menschutkin reaction in which a tertiary amine group was reacted with an organo-halide. 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 Labs, Trevose, PA) were combined with 3 g of ethanol in a 20-mL scintillation vial. After stirring at 60 °C for 24 h, the solvent was removed and the QADM was obtained in the form of a clear, colorless and viscous liquid. This method is desirable because the reaction products were generated at quantitative amounts and required no further purification. The QADM was mixed with the photo-activated BisGMA–TEGDMA (referred to as BT). Previous studies used a single QADM mass fraction of 20% . The present study tested the following QADM/(BT + QADM) mass fractions: 0%, 20%, 40%, and 50%. Each resin was mixed with 30% NACP and 35% glass fillers, with a total filler level of 65%, to yield a cohesive paste. Because there was 35% resin in the composite, the QADM mass fractions in the composite were: 0%, 7%, 14%, and 17.5%.

For mechanical testing, the paste was placed into rectangular molds of 2 × 2 × 25 mm. For biofilm testing, disk molds of 9 mm in diameter and 2 mm in thickness were used. A commercial composite, Renamel (Cosmedent, Chicago, IL), was also tested. It consisted of nanofillers of 20–40 nm with 60% fillers in a multifunctional methacrylate ester resin. Renamel is designated as “composite control”. All the specimens were photo-polymerized (Triad 2000, Dentsply, York, PA) for 1 min on each side. This yielded five composites: composite control, NACP + 0% QADM, NACP + 7% QADM, NACP + 14% QADM, NACP + 17.5% QADM. Six specimens per composite were used for each test ( n = 6).

2.3

Mechanical properties

A computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) was used. Composite specimens were immersed in distilled water at 37 °C for 1 d, and then fractured in three-point flexure with a 10 mm span at a crosshead-speed of 1 mm/min. The specimens were wet and not dried, and were fractured within a few minutes after being taken out of the water. Flexural strength ( S ) was calculated as: S = 3 P _max L /(2 bh ² ), where P _max is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus ( E ) was calculated as: E = ( P / d )( L ³ /[4 bh ³ ]), where load P divided by displacement d is the slope in the linear elastic region.

2.4

Saliva collection for biofilm inoculum

The microcosm model of this study was approved by the University of Maryland. Saliva is ideal for growing dental 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 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/drink intake for 2 h prior to donating saliva. Stimulated saliva was collected during parafilm chewing and was kept on ice. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C .

2.5

Dental plaque microcosm biofilm formation

The saliva–glycerol stock was added, with 1:50 final dilution, into the growth medium as inoculum. The growth 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; hemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7 . Composite disks were sterilized in ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC). 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. Then, the disks were 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 totals 48 h of incubation, which was adequate to form plaque microcosm biofilms .

2.6

Live/dead assay and bacterial viability

After 48 h growth, the plaque microcosm biofilms 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. Disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).

Disks with 48-h biofilms were rinsed with PBS and then immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C. The specimens were rinsed with PBS, subjected to graded ethanol dehydrations, and rinsed twice with 100% hexamethyldisilazane. The specimens were then sputter-coated with gold and examined via scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR).

2.7

MTT assay of metabolic activity

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to examine the metabolic activity of biofilms . MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Each disk with 48-h biofilm was transferred to a new 24-well plate, then 1 mL of MTT dye (0.5 mg/mL MTT in PBS) was added to 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 disks were transferred to a new 24-well plate, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min with gentle mixing at room temperature in the dark. After mixing via pipetting, 200 μL of the DMSO solution from each well 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). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk.

2.8

Colony forming unit (CFU) counts and lactic acid production

Each disk with 48-h biofilm was rinsed with cysteine peptone water (CPW) to remove loose bacteria. The disks were transferred to 24-well plates containing buffered peptone water (BPW) plus 0.2% sucrose, and incubated in 5% CO ₂ at 37 °C for 3 h to allow the biofilms to produce acid. Subsequently, the BPW solutions were stored for lactate analysis.

The disks with biofilms were then transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing at the maximum speed for 20 s using a vortex mixer (Fisher, Pittsburgh, PA). Three types of agar plates were used to measure the CFU counts to assess the microorganism viability. 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 is 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 purpose of measuring these three CFU counts was to provide antibacterial information on not only the total microorganisms, but also mutans streptococci, in the plaque microcosm biofilm. The mutans streptococci group consists of S. mutans and S. sobrinus , both species playing a key role in tooth decay.

Lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method, following previous studies . The microplate reader was used to measure the absorbance at 340 nm (optical density OD ₃₄₀ ) for the collected BPW solutions. Standard curves were prepared using a lactic acid standard (Supelco, Bellefonte, PA).

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

2.1

NACP and glass filler particles

NACP [Ca ₃ (PO ₄ ) ₂ ] was synthesized via a spray-drying technique as recently described . Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution to obtain Ca and PO ₄ ionic concentrations of 8 mmol/L and 5.333 mmol/L, respectively, yielding a Ca/P molar ratio of 1.5. This solution was sprayed into a heated chamber, and an electrostatic precipitator was used to collect the dried particles. This yielded NACP with a mean particle size of 116 nm . As a co-filler to reinforce the composite, a barium boroaluminosilicate glass of a mean particle size of 1.4 μm (Caulk/Dentsply, Milford, DE) was silanized with 4% 3-methacryloxypropyltrimethoxysilane and 2% n -propylamine. These fillers were used in the composites as described below.

2.2

QADM resin composites

BisGMA (bisphenol glycidyl dimethacrylate) and TEGDMA (triethylene glycol dimethacrylate) (Esstech, Essington, PA) were mixed at a mass ratio of 1:1, and rendered light-curable with 0.2% camphorquinone and 0.8% ethyl 4- N , N -dimethylaminobenzoate. The QADM was bis(2-methacryloyloxy-ethyl) dimethyl-ammonium bromide, which was synthesized as described recently . Its synthesis employed a modified Menschutkin reaction in which a tertiary amine group was reacted with an organo-halide. 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 Labs, Trevose, PA) were combined with 3 g of ethanol in a 20-mL scintillation vial. After stirring at 60 °C for 24 h, the solvent was removed and the QADM was obtained in the form of a clear, colorless and viscous liquid. This method is desirable because the reaction products were generated at quantitative amounts and required no further purification. The QADM was mixed with the photo-activated BisGMA–TEGDMA (referred to as BT). Previous studies used a single QADM mass fraction of 20% . The present study tested the following QADM/(BT + QADM) mass fractions: 0%, 20%, 40%, and 50%. Each resin was mixed with 30% NACP and 35% glass fillers, with a total filler level of 65%, to yield a cohesive paste. Because there was 35% resin in the composite, the QADM mass fractions in the composite were: 0%, 7%, 14%, and 17.5%.

For mechanical testing, the paste was placed into rectangular molds of 2 × 2 × 25 mm. For biofilm testing, disk molds of 9 mm in diameter and 2 mm in thickness were used. A commercial composite, Renamel (Cosmedent, Chicago, IL), was also tested. It consisted of nanofillers of 20–40 nm with 60% fillers in a multifunctional methacrylate ester resin. Renamel is designated as “composite control”. All the specimens were photo-polymerized (Triad 2000, Dentsply, York, PA) for 1 min on each side. This yielded five composites: composite control, NACP + 0% QADM, NACP + 7% QADM, NACP + 14% QADM, NACP + 17.5% QADM. Six specimens per composite were used for each test ( n = 6).

2.3

Mechanical properties

A computer-controlled Universal Testing Machine (5500R, MTS, Cary, NC) was used. Composite specimens were immersed in distilled water at 37 °C for 1 d, and then fractured in three-point flexure with a 10 mm span at a crosshead-speed of 1 mm/min. The specimens were wet and not dried, and were fractured within a few minutes after being taken out of the water. Flexural strength ( S ) was calculated as: S = 3 P _max L /(2 bh ² ), where P _max is the fracture load, L is span, b is specimen width and h is thickness. Elastic modulus ( E ) was calculated as: E = ( P / d )( L ³ /[4 bh ³ ]), where load P divided by displacement d is the slope in the linear elastic region.

2.4

Saliva collection for biofilm inoculum

The microcosm model of this study was approved by the University of Maryland. Saliva is ideal for growing dental 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 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/drink intake for 2 h prior to donating saliva. Stimulated saliva was collected during parafilm chewing and was kept on ice. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C .

2.5

Dental plaque microcosm biofilm formation

The saliva–glycerol stock was added, with 1:50 final dilution, into the growth medium as inoculum. The growth 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; hemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7 . Composite disks were sterilized in ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC). 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. Then, the disks were 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 totals 48 h of incubation, which was adequate to form plaque microcosm biofilms .

2.6

Live/dead assay and bacterial viability

After 48 h growth, the plaque microcosm biofilms 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. Disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).

Disks with 48-h biofilms were rinsed with PBS and then immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C. The specimens were rinsed with PBS, subjected to graded ethanol dehydrations, and rinsed twice with 100% hexamethyldisilazane. The specimens were then sputter-coated with gold and examined via scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR).

2.7

MTT assay of metabolic activity

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to examine the metabolic activity of biofilms . MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Each disk with 48-h biofilm was transferred to a new 24-well plate, then 1 mL of MTT dye (0.5 mg/mL MTT in PBS) was added to 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 disks were transferred to a new 24-well plate, 1 mL of dimethyl sulfoxide (DMSO) was added to solubilize the formazan crystals, and the plate was incubated for 20 min with gentle mixing at room temperature in the dark. After mixing via pipetting, 200 μL of the DMSO solution from each well 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). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk.

2.8

Colony forming unit (CFU) counts and lactic acid production

Each disk with 48-h biofilm was rinsed with cysteine peptone water (CPW) to remove loose bacteria. The disks were transferred to 24-well plates containing buffered peptone water (BPW) plus 0.2% sucrose, and incubated in 5% CO ₂ at 37 °C for 3 h to allow the biofilms to produce acid. Subsequently, the BPW solutions were stored for lactate analysis.

The disks with biofilms were then transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing at the maximum speed for 20 s using a vortex mixer (Fisher, Pittsburgh, PA). Three types of agar plates were used to measure the CFU counts to assess the microorganism viability. 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 is 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 purpose of measuring these three CFU counts was to provide antibacterial information on not only the total microorganisms, but also mutans streptococci, in the plaque microcosm biofilm. The mutans streptococci group consists of S. mutans and S. sobrinus , both species playing a key role in tooth decay.

Lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method, following previous studies . The microplate reader was used to measure the absorbance at 340 nm (optical density OD ₃₄₀ ) for the collected BPW solutions. Standard curves were prepared using a lactic acid standard (Supelco, Bellefonte, PA).

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