2.1

Synthesis of DMAHDM and antibacterial bonding agent

An antimicrobial monomer, dimethylaminododecyl methacrylate (DMAHDM) with an alkyl chain length of 16, was recently synthesized . DMAHDM was made using a modified Menschutkin reaction, where a tertiary amine was reacted with an organo-halide . 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 were described recently .

Scotchbond Multi-Purpose (3 M, St. Paul, MN) was used as the parent system (referred as “SBMP”) to test the effect of DMAHDM incorporation. According to the manufacturer, SBMP primer contained 35–45% of HEMA, 10–20% of a copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contained 60–70% of bisphenol A diglycidyl methacrylate (BisGMA), 30–40% of 2-hydroxyethyl methacrylate (HEMA), tertiary amines and photo-initiator. DMAHDM was mixed into primer at DMAHDM/(SBMP primer + DMAHDM) mass fractions of 2.5%, 5%, 7.5%, and 10%. Similarly, DMAHDM was mixed into adhesive at DMAHDM/(SBMP adhesive + DMAHDM) mass fractions of 2.5%, 5%, 7.5%, and 10%. The 10% mass fraction followed previous studies . These four mass fractions and the control (0%) allowed the examination of the effect of surface charge density. Five groups were tested:

• (1)

  SBMP primer and adhesive (0% DMAHDM, referred to as “SBMP control”);

• (2)

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

• (3)

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

• (4)

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

• (5)

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

2.2

Resin specimen fabrication

Resin disks were made using the cover of a sterile 96-well plate as molds . Following previous studies, 10 μL of primer was brushed onto the bottom of a dent of approximately 8 mm in diameter . The primer was dried with a stream of air, and then 20 μL of adhesive was applied. A Mylar strip was used to cover the adhesive which was then light-cured for 20 s (Optilux VCL 401, Demetron Kerr, Danbury, CT). This yielded a cured resin disk of approximately 8 mm in diameter and 0.5 mm in thickness . The disks were removed from the cover of the 96-well plate, immersed in 200 mL of distilled water, and stirred via a magnetic stirrer at a speed of 100 rpm (Bellco Glass, Vineland, NJ) for 1 h to remove any uncured monomers, following a previous study . The purpose of this was to avoid complications from the release of uncured monomers which could have moderate antibacterial effects, so that the measured antibacterial properties were due to the polymerized QAM in the resin and not due to uncured monomer release during biofilm cultures. The disks were dried, sterilized in an ethylene oxide sterilizer (Anprolene AN 74i, Andersen, Haw River, NC), de-gassed for 7 d and then used in biofilm experiments.

2.3

Quaternary amine charge density of bonding agent containing DMAHDM

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 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 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, charge density was calculated as the total molecules of charge per exposed surface area. The surface area was equal to the summation of top, bottom and side areas, measured for each disk due to slight variations in disk sizes .

2.4

Confocal laser scanning microscopy (CLSM) analysis of 3D biofilms

The use of Streptococcus mutans ( S. mutans ) bacteria (ATCC700610, American Type, Manassas, VA) was approved by the University of Maryland Baltimore Institutional Review Board. A 15 μL of S. mutans stock bacteria was added to 15 mL of brain heart infusion broth (BHI, Becton, Sparks, MD) and incubated at 37 °C with 5% CO ₂ for 16 h. 150 μL of this S. mutans suspension was then diluted by 10-fold in a growth medium which consisted of BHI supplemented with 0.2% sucrose to form S. mutans inoculation medium of 1.5 mL .

Each resin disk was placed in a well of a 24-well plate and inoculated with 1.5 mL of the S. mutans inoculation medium. The samples were incubated at 5% CO ₂ and 37 °C. The medium consisted of BHI with 0.2% sucrose. After 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 and incubated for 24 h. This totaled two days of culture which was shown previously to form biofilms on dental resins . The biofilms on resin disks were washed with phosphate buffered saline (PBS) to remove the loose bacteria. Disks with adherent biofilms were stained using a BacLight live/dead 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 .

The biofilms were investigated using a 3D model as previously described . The fluorescence was examined using a confocal laser scanning microscope (CLSM, LSM510, Carl Zeiss, Thornwood, NY). Green fluorescence was provided with an argon laser (488-nm laser excitation), and red fluorescence was given with a helium-neon laser (543 nm laser excitation). Images were taken from the bottom of the biofilm that was in contact with the resin disk surface, and then section by section toward the top surface of the biofilm. The biofilm section parallel to the resin surface was referred to as the x-y plane, and the direction perpendicular to the resin surface was called the z axis . For each biofilm, 10 planes at equal distances along the z axis were imaged . These 2D sections were stacked and reconstructed to form a 3D image of the biofilm using the IMARIS software (Bitplane, Saint Paul, MN) . The biofilm images were analyzed using a software (bioImageL, Faculty of Odontology, Malmö University, Malmö, Sweden) . The bioImageL software is based on color segmentation algorithms written in MATLAB (MathWorks, Natick, MA) and can produce information of the structure and spatial differences in biofilm. The biofilm is characterized by parameters including biofilm thickness, green-stained live bacteria volume, red-stained dead bacteria volume, as well as the live and dead bacteria coverage on each two-dimensional x-y section along the biofilm thickness .

2.5

Statistical analysis

Statistical analyses were performed using SPSS 17.0 software (SPSS, Chicago, IL). Two-way analysis of variance (ANOVA) was used to examine the data in Fig. 6 on the effects of resin surface charge density and location in 3D biofilm. One way ANOVA was used to analyze all other data. Tukey’s multiple comparison tests were performed to detect significant effects of the variables using a p value of 0.05.

Charge density and biofilm thickness on resins: (A) Quaternary amine surface charge density of cured bonding agent resin vs. DMAHDM mass fraction, and (B) biofilm thickness grown for two days on resin (mean ± sd; n = 6). Surface charge density significantly increased with increasing DMAHDM mass fraction. Biofilm thickness decreased with increasing DMAHDM mass fraction. In each plot, values with dissimilar letters are significantly different from each other (p < 0.05).

Representative CLSM images of 3D biofilms cultured for 2 days on bonding agents: (A) SBMP control, (B) SBMP + 2.5DMAHDM, (C) SBMP + 5DMAHDM, (D) SBMP + 7.5DMAHDM, (E) SBMP + 10DMAHDM. The x and y axes are parallel to the resin surface. The z axis is perpendicular to the resin surface. Live bacteria were stained green, and bacteria with compromised membranes were stained red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Biofilm volume cultured for 2 days on bonding agents: (A) Live biofilm volume, and (B) dead biofilm volume (mean ± sd; n = 6). In each plot, values with dissimilar letters are significantly different from each other (p < 0.05).

Representative 2D live/dead images of cross-sectioned biofilm in the x-y plane. All five bonding agents were tested; shown here are three examples: (A) SBMP control, (B) SBMP + 5DMAHDM, (C) SBMP+10DMAHDM. The top labels indicate the materials. The left labels indicate the location of the section in the biofilm: The top biofilm surface, the middle section, and the bottom section (near the resin surface) of the biofilm. Live bacteria were stained green. Bacteria with compromised membranes were stained red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Bacterial viability distribution in 3D biofilm (mean ± sd; n = 6): (A) SBMP control, (B) SBMP + 2.5DMAHDM, (C) SBMP + 5DMAHDM, (D) SBMP + 7.5DMAHDM, (E) SBMP + 10DMAHDM. Percentage of live bacteria = live bacteria area/(live bacteria area + dead bacteria area). Percentage of live bacteria is plotted vs. the location of the 2D cross-section in the 3D biofilm at a distance measured from the resin surface.

Percentage of live bacteria in 3D biofilm vs. resin surface charge density (mean ± sd; n = 6). Two surfaces were measured: Top biofilm surface, and bottom biofilm surface. Percentage of live bacteria decreased with increasing charge density for both surfaces. Percentage of live bacteria is lower at biofilm bottom, and higher in biofilm top surface. Percentage of live bacteria approached 0 for the entire biofilm on SBMP + 10DMAHDM.

2.1

Synthesis of DMAHDM and antibacterial bonding agent

An antimicrobial monomer, dimethylaminododecyl methacrylate (DMAHDM) with an alkyl chain length of 16, was recently synthesized . DMAHDM was made using a modified Menschutkin reaction, where a tertiary amine was reacted with an organo-halide . 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 were described recently .

Scotchbond Multi-Purpose (3 M, St. Paul, MN) was used as the parent system (referred as “SBMP”) to test the effect of DMAHDM incorporation. According to the manufacturer, SBMP primer contained 35–45% of HEMA, 10–20% of a copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contained 60–70% of bisphenol A diglycidyl methacrylate (BisGMA), 30–40% of 2-hydroxyethyl methacrylate (HEMA), tertiary amines and photo-initiator. DMAHDM was mixed into primer at DMAHDM/(SBMP primer + DMAHDM) mass fractions of 2.5%, 5%, 7.5%, and 10%. Similarly, DMAHDM was mixed into adhesive at DMAHDM/(SBMP adhesive + DMAHDM) mass fractions of 2.5%, 5%, 7.5%, and 10%. The 10% mass fraction followed previous studies . These four mass fractions and the control (0%) allowed the examination of the effect of surface charge density. Five groups were tested:

• (1)

  SBMP primer and adhesive (0% DMAHDM, referred to as “SBMP control”);

• (2)

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

• (3)

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

• (4)

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

• (5)

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

2.2

Resin specimen fabrication

Resin disks were made using the cover of a sterile 96-well plate as molds . Following previous studies, 10 μL of primer was brushed onto the bottom of a dent of approximately 8 mm in diameter . The primer was dried with a stream of air, and then 20 μL of adhesive was applied. A Mylar strip was used to cover the adhesive which was then light-cured for 20 s (Optilux VCL 401, Demetron Kerr, Danbury, CT). This yielded a cured resin disk of approximately 8 mm in diameter and 0.5 mm in thickness . The disks were removed from the cover of the 96-well plate, immersed in 200 mL of distilled water, and stirred via a magnetic stirrer at a speed of 100 rpm (Bellco Glass, Vineland, NJ) for 1 h to remove any uncured monomers, following a previous study . The purpose of this was to avoid complications from the release of uncured monomers which could have moderate antibacterial effects, so that the measured antibacterial properties were due to the polymerized QAM in the resin and not due to uncured monomer release during biofilm cultures. The disks were dried, sterilized in an ethylene oxide sterilizer (Anprolene AN 74i, Andersen, Haw River, NC), de-gassed for 7 d and then used in biofilm experiments.

2.3

Quaternary amine charge density of bonding agent containing DMAHDM

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 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 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, charge density was calculated as the total molecules of charge per exposed surface area. The surface area was equal to the summation of top, bottom and side areas, measured for each disk due to slight variations in disk sizes .

2.4

Confocal laser scanning microscopy (CLSM) analysis of 3D biofilms

The use of Streptococcus mutans ( S. mutans ) bacteria (ATCC700610, American Type, Manassas, VA) was approved by the University of Maryland Baltimore Institutional Review Board. A 15 μL of S. mutans stock bacteria was added to 15 mL of brain heart infusion broth (BHI, Becton, Sparks, MD) and incubated at 37 °C with 5% CO ₂ for 16 h. 150 μL of this S. mutans suspension was then diluted by 10-fold in a growth medium which consisted of BHI supplemented with 0.2% sucrose to form S. mutans inoculation medium of 1.5 mL .

Each resin disk was placed in a well of a 24-well plate and inoculated with 1.5 mL of the S. mutans inoculation medium. The samples were incubated at 5% CO ₂ and 37 °C. The medium consisted of BHI with 0.2% sucrose. After 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 and incubated for 24 h. This totaled two days of culture which was shown previously to form biofilms on dental resins . The biofilms on resin disks were washed with phosphate buffered saline (PBS) to remove the loose bacteria. Disks with adherent biofilms were stained using a BacLight live/dead 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 .

The biofilms were investigated using a 3D model as previously described . The fluorescence was examined using a confocal laser scanning microscope (CLSM, LSM510, Carl Zeiss, Thornwood, NY). Green fluorescence was provided with an argon laser (488-nm laser excitation), and red fluorescence was given with a helium-neon laser (543 nm laser excitation). Images were taken from the bottom of the biofilm that was in contact with the resin disk surface, and then section by section toward the top surface of the biofilm. The biofilm section parallel to the resin surface was referred to as the x-y plane, and the direction perpendicular to the resin surface was called the z axis . For each biofilm, 10 planes at equal distances along the z axis were imaged . These 2D sections were stacked and reconstructed to form a 3D image of the biofilm using the IMARIS software (Bitplane, Saint Paul, MN) . The biofilm images were analyzed using a software (bioImageL, Faculty of Odontology, Malmö University, Malmö, Sweden) . The bioImageL software is based on color segmentation algorithms written in MATLAB (MathWorks, Natick, MA) and can produce information of the structure and spatial differences in biofilm. The biofilm is characterized by parameters including biofilm thickness, green-stained live bacteria volume, red-stained dead bacteria volume, as well as the live and dead bacteria coverage on each two-dimensional x-y section along the biofilm thickness .

2.5

Statistical analysis

Statistical analyses were performed using SPSS 17.0 software (SPSS, Chicago, IL). Two-way analysis of variance (ANOVA) was used to examine the data in Fig. 6 on the effects of resin surface charge density and location in 3D biofilm. One way ANOVA was used to analyze all other data. Tukey’s multiple comparison tests were performed to detect significant effects of the variables using a p value of 0.05.

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