2.1

Development of new QAM and antibacterial bonding agent

Dimethylaminododecyl methacrylate (DMAHDM) with an alkyl chain length of 16 was synthesized using 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 virtually quantitative amounts and require minimal purification . Briefly, 10 mmol of 1-(dimethylamino)docecane (Sigma, St. Louis, MO) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR) 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 DMAHDM as a clear, colorless, and viscous liquid. Details of this method have been described recently .

Scotchbond Multi-Purpose adhesive and primer (referred as “SBMP”) (3M, St. Paul, MN) were used as the parent bonding system to test the effect of incorporation of antibacterial agent. According to the manufacturer, SBMP adhesive contained 60–70% of bisphenol A diglycidyl methacrylate (BisGMA) and 30–40% of 2-hydroxyethyl methacrylate (HEMA), tertiary amines and photo-initiator. SBMP primer contained 35–45% of HEMA, 10–20% of a copolymer of acrylic and itaconic acids, and 40–50% water. DMAHDM was mixed with SBMP primer at DMAHDM/(SBMP primer + DMAHDM) mass fraction of 2.5%, 5%, 7.5%, and 10%. Similarly, DMAHDM was mixed with SBMP adhesive at DMAHDM/(SBMP adhesive + DMAHDM) mass fraction of 2.5%, 5%, 7.5%, and 10%. The 10% mass fraction followed previous studies . Therefore, five groups were tested:

• (1)

  Unmodified SBMP primer and adhesive (referred to as “SBMP control”);

• (2)

  SBMP primer + 2.5% DMAHDM, SBMP adhesive + 2.5% DMAHDM (“SBMP + 2.5% DMAHDM”);

• (3)

  SBMP primer + 5% DMAHDM, SBMP adhesive + 5% DMAHDM (“SBMP + 5% DMAHDM”);

• (4)

  SBMP primer + 7.5% DMAHDM, SBMP adhesive + 7.5% DMAHDM (“SBMP + 7.5% DMAHDM”);

• (5)

  SBMP primer + 10% DMAHDM, SBMP adhesive + 10% DMAHDM (“SBMP + 10% DMAHDM”).

2.2

Microtensile dentin bond strength

Human third molars were collected under a protocol approved by the University of Maryland Baltimore. 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 the mid-coronal 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 corresponding 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 0.9 mm × 0.9 mm composite-dentin beams . Eight teeth were used for each bonding agent group ( n = 8). Five beams were randomly selected from the beams of each tooth, yielding 40 beams for each group. Each beam was stressed to failure in uniaxial tension using 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 .

2.3

Quaternary amine charge density of bonding agent containing DMAHDM

The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used for fabricating resin disk specimens . Ten μL of a primer was placed in the bottom of each dent. After drying with a stream of air, 20 μL of adhesive was applied to the dent and photo-polymerized for 10 s (Optilux VCL 401) using a Mylar strip covering to obtain a specimen disk of 8 mm in diameter and 0.5 mm in thickness. The cured disks were immersed in water and agitated for 1 h to remove uncured monomers, following a previous study . The density of quaternary ammonium groups present on the polymer surfaces was quantified using a fluorescein dye method . Resin disks of each bonding agent group were placed in a 48-well plate. Fluorescein sodium salt (200 μL of 10 mg/mL in deionized (DI) water) was added into each well, and specimens were left for 10 min at room temperature in the dark. After removing the fluorescein solution and rinsing extensively with DI water, each sample was placed in a new well, and 200 μL of 0.1% (by mass) of cetyltrimethylammonium chloride (CTMAC) in DI water was added. Samples were shaken for 20 min at room temperature in the dark to desorb the bound dye. The CTMAC solution was supplemented with 10% (by volume) of 100 mM phosphate buffer at pH 8. This was prepared with 0.94 mg/mL monosodium phosphate-monohydrate and 13.2 mg/mL disodium phosphate-anhydrous in DI water. Sample absorbance was read at 501 nm using a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA) . The fluorescein concentration was calculated using Beers Law and an extinction coefficient of 77 mM ⁻¹ cm ⁻¹ . Using a ratio of 1:1 for fluorescein molecules to the accessible quaternary ammonium groups, the surface charge density was calculated as the total molecules of charge per exposed surface area (the summation of top, bottom and side areas, measured independently for each polymer disk due to slight variations in disk sizes). Six replicates were tested for each group.

2.4

Bacteria early-attachment on bonding agent disks

Resin disk of 8 mm in diameter and 0.5 mm in thickness were sterilized with ethylene oxide (AnproleneAN 74i, Andersen, Haw River, NC) and de-gassed for 7 days. The use of Streptococcus mutans ( 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. The bacterial suspension was adjusted to an optical density (OD) of 0.06 at 600 nm for further usage. The overnight cultured bacterial suspension was diluted in fresh BHI until the OD value equaled to 0.06. Based on preliminary study on the relationship between OD value and CFU, the density of OD 0.06 was equivalent to a density of 10 ⁷ S. mutans . Resin disks were placed in a 24-well plate with 1.5 mL of bacteria suspension, and incubated at 37 °C and 5% CO ₂ for 4 h, which was suitable for examining bacterial early attachment . Disks were washed three times to remove nonadherent bacteria, fixed with 37 mg/mL formaldehyde, and stained for 1 h with 1 μmol/L SYTOX green (Invitrogen, Carlsbad, CA) . The stained disks were examined with an epifluorescence microscope (TE2000-S, Nikon, Melville, NY). Three disks were evaluated for each group. Five images were collected at random locations on each disk, yielding 15 images per group. Image J software (NIH) was used to measure the area coverage by bacteria in each image. The area coverage was normalized as a percentage of the SBMP control.

2.5

Anti-biofilm effect of bonding agent containing DMAHDM

Resin disks were placed in a 24-well plate with 1.5 mL of BHI supplemented with 0.2% sucrose as the culture medium. S. mutans bacterial suspension was diluted by 1:100, and then 10 μL of the suspension was inoculated into each well . After incubating in 5% CO ₂ at 37 °C for 8 h, the disks were 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 another 24 h. This constituted a total of 2 days of culture, which was shown previously to form mature biofilms on resins .

Disks with the 2-day biofilms were transferred into tubes with 2 mL of cysteine peptone water (CPW), and the biofilms were harvested by sonication (3510R-MTH, Branson, Danbury, CT) for 3 min, and then vortexing at maximum speed for 20 s using a vortex mixer (Fisher, Pittsburgh, PA). The bacterial suspensions thus obtained were serially diluted, spread onto agar plates for CFUs analysis . Six replicates were tested for each group.

2.6

Statistical analysis

All data were checked for normal distribution with the Kolmogorov–Smirnov test and were checked for homogeneity with the 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 pre-set alpha of 0.05.

2.1

Development of new QAM and antibacterial bonding agent

Dimethylaminododecyl methacrylate (DMAHDM) with an alkyl chain length of 16 was synthesized using 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 virtually quantitative amounts and require minimal purification . Briefly, 10 mmol of 1-(dimethylamino)docecane (Sigma, St. Louis, MO) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR) 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 DMAHDM as a clear, colorless, and viscous liquid. Details of this method have been described recently .

Scotchbond Multi-Purpose adhesive and primer (referred as “SBMP”) (3M, St. Paul, MN) were used as the parent bonding system to test the effect of incorporation of antibacterial agent. According to the manufacturer, SBMP adhesive contained 60–70% of bisphenol A diglycidyl methacrylate (BisGMA) and 30–40% of 2-hydroxyethyl methacrylate (HEMA), tertiary amines and photo-initiator. SBMP primer contained 35–45% of HEMA, 10–20% of a copolymer of acrylic and itaconic acids, and 40–50% water. DMAHDM was mixed with SBMP primer at DMAHDM/(SBMP primer + DMAHDM) mass fraction of 2.5%, 5%, 7.5%, and 10%. Similarly, DMAHDM was mixed with SBMP adhesive at DMAHDM/(SBMP adhesive + DMAHDM) mass fraction of 2.5%, 5%, 7.5%, and 10%. The 10% mass fraction followed previous studies . Therefore, five groups were tested:

• (1)

  Unmodified SBMP primer and adhesive (referred to as “SBMP control”);

• (2)

  SBMP primer + 2.5% DMAHDM, SBMP adhesive + 2.5% DMAHDM (“SBMP + 2.5% DMAHDM”);

• (3)

  SBMP primer + 5% DMAHDM, SBMP adhesive + 5% DMAHDM (“SBMP + 5% DMAHDM”);

• (4)

  SBMP primer + 7.5% DMAHDM, SBMP adhesive + 7.5% DMAHDM (“SBMP + 7.5% DMAHDM”);

• (5)

  SBMP primer + 10% DMAHDM, SBMP adhesive + 10% DMAHDM (“SBMP + 10% DMAHDM”).

2.2

Microtensile dentin bond strength

Human third molars were collected under a protocol approved by the University of Maryland Baltimore. 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 the mid-coronal 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 corresponding 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 0.9 mm × 0.9 mm composite-dentin beams . Eight teeth were used for each bonding agent group ( n = 8). Five beams were randomly selected from the beams of each tooth, yielding 40 beams for each group. Each beam was stressed to failure in uniaxial tension using 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 .

2.3

Quaternary amine charge density of bonding agent containing DMAHDM

The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used for fabricating resin disk specimens . Ten μL of a primer was placed in the bottom of each dent. After drying with a stream of air, 20 μL of adhesive was applied to the dent and photo-polymerized for 10 s (Optilux VCL 401) using a Mylar strip covering to obtain a specimen disk of 8 mm in diameter and 0.5 mm in thickness. The cured disks were immersed in water and agitated for 1 h to remove uncured monomers, following a previous study . The density of quaternary ammonium groups present on the polymer surfaces was quantified using a fluorescein dye method . Resin disks of each bonding agent group were placed in a 48-well plate. Fluorescein sodium salt (200 μL of 10 mg/mL in deionized (DI) water) was added into each well, and specimens were left for 10 min at room temperature in the dark. After removing the fluorescein solution and rinsing extensively with DI water, each sample was placed in a new well, and 200 μL of 0.1% (by mass) of cetyltrimethylammonium chloride (CTMAC) in DI water was added. Samples were shaken for 20 min at room temperature in the dark to desorb the bound dye. The CTMAC solution was supplemented with 10% (by volume) of 100 mM phosphate buffer at pH 8. This was prepared with 0.94 mg/mL monosodium phosphate-monohydrate and 13.2 mg/mL disodium phosphate-anhydrous in DI water. Sample absorbance was read at 501 nm using a plate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA) . The fluorescein concentration was calculated using Beers Law and an extinction coefficient of 77 mM ⁻¹ cm ⁻¹ . Using a ratio of 1:1 for fluorescein molecules to the accessible quaternary ammonium groups, the surface charge density was calculated as the total molecules of charge per exposed surface area (the summation of top, bottom and side areas, measured independently for each polymer disk due to slight variations in disk sizes). Six replicates were tested for each group.

2.4

Bacteria early-attachment on bonding agent disks

Resin disk of 8 mm in diameter and 0.5 mm in thickness were sterilized with ethylene oxide (AnproleneAN 74i, Andersen, Haw River, NC) and de-gassed for 7 days. The use of Streptococcus mutans ( 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. The bacterial suspension was adjusted to an optical density (OD) of 0.06 at 600 nm for further usage. The overnight cultured bacterial suspension was diluted in fresh BHI until the OD value equaled to 0.06. Based on preliminary study on the relationship between OD value and CFU, the density of OD 0.06 was equivalent to a density of 10 ⁷ S. mutans . Resin disks were placed in a 24-well plate with 1.5 mL of bacteria suspension, and incubated at 37 °C and 5% CO ₂ for 4 h, which was suitable for examining bacterial early attachment . Disks were washed three times to remove nonadherent bacteria, fixed with 37 mg/mL formaldehyde, and stained for 1 h with 1 μmol/L SYTOX green (Invitrogen, Carlsbad, CA) . The stained disks were examined with an epifluorescence microscope (TE2000-S, Nikon, Melville, NY). Three disks were evaluated for each group. Five images were collected at random locations on each disk, yielding 15 images per group. Image J software (NIH) was used to measure the area coverage by bacteria in each image. The area coverage was normalized as a percentage of the SBMP control.

2.5

Anti-biofilm effect of bonding agent containing DMAHDM

Resin disks were placed in a 24-well plate with 1.5 mL of BHI supplemented with 0.2% sucrose as the culture medium. S. mutans bacterial suspension was diluted by 1:100, and then 10 μL of the suspension was inoculated into each well . After incubating in 5% CO ₂ at 37 °C for 8 h, the disks were 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 another 24 h. This constituted a total of 2 days of culture, which was shown previously to form mature biofilms on resins .

Disks with the 2-day biofilms were transferred into tubes with 2 mL of cysteine peptone water (CPW), and the biofilms were harvested by sonication (3510R-MTH, Branson, Danbury, CT) for 3 min, and then vortexing at maximum speed for 20 s using a vortex mixer (Fisher, Pittsburgh, PA). The bacterial suspensions thus obtained were serially diluted, spread onto agar plates for CFUs analysis . Six replicates were tested for each group.

2.6

Statistical analysis

All data were checked for normal distribution with the Kolmogorov–Smirnov test and were checked for homogeneity with the 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 pre-set alpha of 0.05.