Abstract
Objective
Antibacterial bonding agents are promising to hinder the residual and invading bacteria at the tooth–restoration interfaces. The objectives of this study were to develop an antibacterial bonding agent by incorporation of quaternary ammonium dimethacrylate (QADM) and nanoparticles of silver (NAg), and to investigate the effect of QADM-NAg adhesive and primer on dentin bond strength and plaque microcosm biofilm response for the first time.
Methods
Scotchbond Multi-Purpose adhesive and primer were used as control. Experimental adhesive and primer were made by adding QADM and NAg into control adhesive and primer. Human dentin shear bond strengths were measured ( n = 10). A dental plaque microcosm biofilm model with human saliva as inoculum was used to investigate biofilm metabolic activity, colony-forming unit (CFU) counts, lactic acid production, and live/dead staining assay ( n = 6).
Results
Adding QADM and NAg into adhesive and primer did not compromise the dentin shear bond strength which ranged from 30 to 35 MPa ( p > 0.1). Scanning electron microscopy (SEM) examinations revealed numerous resin tags, which were similar for the control and the QADM and NAg groups. Adding QADM or NAg markedly reduced the biofilm viability, compared to adhesive control. QADM and NAg together in the adhesive had a much stronger antibacterial effect than using each agent alone ( p < 0.05). Adding QADM and NAg in both adhesive and primer had the strongest antibacterial activity, reducing metabolic activity, CFU, and lactic acid by an order of magnitude, compared to control.
Significance
Without compromising dentin bond strength and resin tag formation, the QADM and NAg containing adhesive and primer achieved strong antibacterial effects against microcosm biofilms for the first time. QADM-NAg adhesive and primer are promising to combat residual bacteria in tooth cavity and invading bacteria at the margins, thereby to inhibit secondary caries. QADM and NAg incorporation may have a wide applicability to other dental bonding systems.
1
Introduction
Of the total 166 million restorations placed in 2005 in the United States, 52.5 million (31.6%) were amalgams, 77.3 million (46.6%) were composites, and 36.2 million (21.8%) were crowns . Resin composites are increasingly used because of their esthetics and improved performance . Advances in filler compositions and polymer matrix have enhanced the composite properties . One main drawback is that composites tend to accumulate more biofilms/plaques in vivo than other restorative materials . Replacement of failed restorations accounts for 50–70% of all restorative dentistry , and replacement dentistry costs $5 billion per year in the U.S. . The main reason for restoration failure is secondary caries at the restoration margins, caused by acid production by biofilms . Therefore, antibacterial composites were developed to inhibit biofilms and caries . Quaternary ammonium salt (QAS) monomers such as 12-methacryloyloxydodecylpyridinium bromide (MDPB) were copolymerized in resins to yield antibacterial activities . Recently, a quaternary ammonium dimethacrylate (QADM) was synthesized to possess strong antibacterial properties without compromising the mechanical properties of the resin . In other studies, antibacterial nanocomposites containing nanoparticles of silver (NAg) were developed . However, there has been no report on incorporating QADM and NAg into dental adhesives.
Composite restorations are bonded to tooth structure via adhesives . Extensive studies have been performed to improve, characterize and understand enamel and dentin bonding . It is desirable for the adhesive to be antibacterial to inhibit recurrent caries at the tooth–composite margins . Residual bacteria could exist in the prepared tooth cavity, and microleakage could allow bacteria to invade the tooth–restoration interface. Adhesives that are antibacterial in the cured state could help inhibit the growth of residual and invading bacteria . Indeed, MDPB-containing adhesives markedly inhibited the Streptococcus mutans ( S. mutans ) growth . Another study developed an antibacterial adhesive containing methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB) . Cetylpyridinium chloride (CPC) was also incorporated into a resin with bacteriastatic activity . Besides the adhesive resin, it is also beneficial for the dentin primer to be antibacterial because it directly contacts the tooth structure . A primer containing MDPB achieved potent antibacterial effects . Another primer contained chlorhexidine with antimicrobial activities . There have been only a few reports on the development of antibacterial adhesives and primers. There has been no report on antibacterial adhesive and primer that incorporate QADM and NAg.
The objectives of this study were to develop an antibacterial bonding agent by incorporation of QADM and NAg, and to investigate the effect of QADM-NAg adhesive and primer on dentin bond strength and plaque microcosm biofilm response for the first time. A human saliva microcosm biofilm model was used to evaluate the antibacterial properties. It was hypothesized that: (1) QADM and NAg incorporation would not decrease the bond strength to human dentin, compared to control without QADM and NAg; (2) The adhesive with QADM and NAg would greatly decrease the biofilm viability, metabolic activity and lactic acid production; (3) Combining QADM and NAg together in the adhesive would achieve a stronger antibacterial capability than using each agent alone, and adding QADM and NAg into both adhesive and primer would further increase the antibacterial potency.
2
Materials and methods
2.1
QADM incorporation
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. The purpose was to investigate a model system, and then the method of QADM and NAg incorporation could be applied to other adhesive systems. According to the manufacturer, SBMP etchant contains 37% phosphoric acid. SBMP primer single bottle contains 35–45% 2-Hydroxyethylmethacrylate (HEMA), 10–20% copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contains 60–70% BisGMA and 30–40% HEMA.
Bis(2-methacryloyloxyethyl) dimethylammonium bromide was a quaternary ammonium dimethacrylate (QADM), and was recently synthesized and incorporated into dental composites . The synthesis of QADM was performed 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 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. The vial was stirred at 60 °C for 24 h. The solvent was then removed, yielding QADM as a clear, colorless, and viscous liquid. The QADM was mixed with the SBMP adhesive or primer at a QADM mass fraction of 10%. QADM mass fractions of 20% or higher were not used due to a decrease in dentin bond strength in preliminary study.
2.2
NAg incorporation
Silver 2-ethylhexanoate powder (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, following previous studies . TBAEMA was used because it improves the solubility by forming Ag-N coordination bonds with Ag ions, thereby facilitating the Ag salt to dissolve in the resin solution. TBAEMA was selected since it contains reactive methacrylate groups and therefore can be chemically incorporated into a dental resin upon photopolymerization . This method produced NAg with a mean particle size of 2.7 nm that were well dispersed in the resin matrix . The Ag solution was mixed with SBMP adhesive at silver 2-ethylhexanoate mass fractions of 0.05% and 0.1%. Ag mass fractions of 0.15% or higher were not used due to a decrease in dentin bond strength.
2.3
Dentin Shear bond testing and SEM examination
As listed in Table 1 , six groups were used for dentin shear bond strength testing. The purpose of groups 1–3 was to investigate the effects of QADM or NAg individually. The purpose of 3 and 4 was to examine the effect of NAg mass fraction. The purpose of comparing 2, 3 and 5 was to examine the effect of combining QADM and NAg together in the same adhesive. The purpose of comparing 5 with 6 was to investigate the effects of adding QADM and NAg into both the adhesive and the primer on dentin bond strength and biofilm response.
| Group | Adhesive resin | Dentin primer | Group name |
|---|---|---|---|
| 1 | Control | Control | Control |
| 2 | Control + 10% QADM | Control | A + 10QADM |
| 3 | Control + 0.05% NAg | Control | A + 0.05NAg |
| 4 | Control + 0.1% NAg | Control | A + 0.1NAg |
| 5 | Control + 10% QADM + 0.05% NAg | Control | A + 10QADM + 0.05NAg |
| 6 | Control + 10% QADM + 0.05% NAg | Control + 10% QADM + 0.05% NAg | A&P + 10QADM + 0.05NAg |
a The control adhesive was the SBMP adhesive without modification. The control primer was the SBMP primer without modification. QADM = quaternary ammonium dimethacrylate. NAg = nanoparticles of silver. In the “Group name” column, A = adhesive resin. P = primer.
Extracted caries-free human third molars were cleaned and stored in 0.01% thymol solution. Flat mid-coronal dentin surfaces were prepared by cutting off the tips of molar crowns with a diamond saw (Isomet, Buehler, Lake Bluff, IL). Each tooth was embedded in a poly-carbonate holder (Bosworth, Skokie, IL) and ground perpendicular to the longitudinal axis on 320-grit silicon carbide paper until the occlusal enamel was completely removed. As shown schematically in Fig. 1 A , the dentin surface was etched with 37% phosphoric acid gel for 15 s and rinsed with distilled water for 15 s, following a previous study . The primer was applied with a brush-tipped applicator and rubbed in for 15 s. 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 stainless-steel iris, having a central opening with a diameter of 4 mm and a thickness of 1.5 mm, was held against the adhesive-treated dentin surface. The central opening was filled with a composite (TPH, Caulk/Dentsply, Milford, DE), and light-cured for 60 s. The bonded specimens were stored in distilled water at 37 °C for 24 h.
The dentin shear bond strength, S D , was measured as shown schematically in Fig. 1 B . The chisel was connected with a computer-controlled Universal Testing Machine (MTS, Eden Prairie, MN) and held parallel to the composite–dentin interface. Load was applied at a rate of 0.5 mm/min until the bond failed. S D was calculated as: S D = 4 P /( πd 2 ), where P is the load at failure, and d is the diameter of the composite. Ten teeth were tested for each group ( n = 10).
The bonded tooth was cut through the center in the longitudinal direction via the diamond saw (Isomet) with copious water. Three specimens were prepared for each group. The sectioned surface was polished with increasingly finer SiC paper up to 4000 grit. Following a previous study , the polished surface was treated with 50% phosphoric acid for 30 s, then with 10% NaOCl for 2 min. After being thoroughly rinsed with water for 10 min, the specimens were air dried and then sputter-coated with gold. The dentin-adhesive bonded interfaces were then examined via scanning electron microscopy (SEM, Quanta 200, FEI, Hillsboro, OR).
2.4
Saliva collection for plaque microcosm model
The dental plaque microcosm model was approved by the University of Maryland. Human saliva was shown to be ideal for growing plaque microcosm biofilms in vitro , with the advantage of maintaining much of the complexity and heterogeneity of the dental plaque in vivo . The saliva for biofilm inoculums was collected from a healthy adult donor having natural dentition without active caries or periopathology, and without the use of antibiotics within the last 3 months, following a previous study . The donor did not brush teeth for 24 h and abstained from food/drink intake for at least 2 h prior to donating saliva. Stimulated saliva was collected during parafilm chewing and kept on ice. Saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 °C .
2.5
Specimen fabrication for biofilm experiments
Layered disk specimens for biofilm experiments were fabricated following previous studies . A polyethylene disk mold (inner diameter = 9 mm, thickness = 2 mm) was situated on a glass slide. For groups 1–5, each adhesive was applied into the mold to cover the glass slide. Then, a composite (TPH) was placed onto the adhesive to fill the disk mold and light-cured for 1 min. For group 6, the primer was first applied into the mold to cover the glass slide. After drying with a stream of air, the adhesive was applied and cured for 20 s with Optilux. Then, a composite (TPH) was placed on the adhesive to fill the disk mold and light-cured for 1 min. The disks were immersed in sterile water and agitated for 1 h to remove any uncured monomer, following a previous study . The disks were then dried and sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC).
Six groups were tested in biofilm experiments. Groups 1–5 had specimens with adhesives types 1–5 covering the top surface of the composite disk, without primer, in order to test the antibacterial properties of the adhesives, as shown schematically in Fig. 3 A. Group 6 had the QADM-NAg primer covering the adhesive on the composite disk in order to test the antibacterial properties of the primer/adhesive combination, as shown schematically in Fig. 3 B.
2.6
MTT assay of metabolic activity
The saliva-glycerol stock was added, with 1:50 final dilution, to a 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; haemin, 0.001 g/L; vitamin K1, 0.0002 g/L, at pH 7 . The inoculum was cultured at 37 °C in an incubator containing 5% CO 2 for 24 h. Each disk specimen was placed into a well of 24-well plates, with the antibacterial surface on the top. 1.5 mL of inoculum was added to each well, and incubated in 5% CO 2 at 37 °C for 8 h. The disks were then transferred to new 24-well plates 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 2-day (d) incubation formed plaque microcosm biofilms as shown previously .
The MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan . Each disk with the 2-d 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.
2.7
Live/dead staining of biofilms
Microcosm biofilms were grown on the disks for 2 d as described in section 2.6. The biofilms on the disks were gently washed three times with phosphate buffered saline (PBS), and then stained using a 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 an epifluorescence microscope (TE2000-S, Nikon, Melville, NY) .
2.8
Lactic acid production and colony-forming unit (CFU) counts
Each disk with the 2-d 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. 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.
Disks with biofilms were transferred into tubes with 2 mL CPW, and the biofilms were harvested by sonication and vortexing via a vortex mixer (Fisher, Pittsburgh, PA). Three types of agar plates were used. 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 are 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 .
Lactate concentrations in the BPW solutions were determined using an enzymatic (lactate dehydrogenase) method, following a previous study . 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 analysis of variance (ANOVA) was performed to detect the significant effects of the variables. Tukey’s multiple comparison was used to compare the data at a p value of 0.05.
2
Materials and methods
2.1
QADM incorporation
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. The purpose was to investigate a model system, and then the method of QADM and NAg incorporation could be applied to other adhesive systems. According to the manufacturer, SBMP etchant contains 37% phosphoric acid. SBMP primer single bottle contains 35–45% 2-Hydroxyethylmethacrylate (HEMA), 10–20% copolymer of acrylic and itaconic acids, and 40–50% water. SBMP adhesive contains 60–70% BisGMA and 30–40% HEMA.
Bis(2-methacryloyloxyethyl) dimethylammonium bromide was a quaternary ammonium dimethacrylate (QADM), and was recently synthesized and incorporated into dental composites . The synthesis of QADM was performed 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 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. The vial was stirred at 60 °C for 24 h. The solvent was then removed, yielding QADM as a clear, colorless, and viscous liquid. The QADM was mixed with the SBMP adhesive or primer at a QADM mass fraction of 10%. QADM mass fractions of 20% or higher were not used due to a decrease in dentin bond strength in preliminary study.
2.2
NAg incorporation
Silver 2-ethylhexanoate powder (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, following previous studies . TBAEMA was used because it improves the solubility by forming Ag-N coordination bonds with Ag ions, thereby facilitating the Ag salt to dissolve in the resin solution. TBAEMA was selected since it contains reactive methacrylate groups and therefore can be chemically incorporated into a dental resin upon photopolymerization . This method produced NAg with a mean particle size of 2.7 nm that were well dispersed in the resin matrix . The Ag solution was mixed with SBMP adhesive at silver 2-ethylhexanoate mass fractions of 0.05% and 0.1%. Ag mass fractions of 0.15% or higher were not used due to a decrease in dentin bond strength.
2.3
Dentin Shear bond testing and SEM examination
As listed in Table 1 , six groups were used for dentin shear bond strength testing. The purpose of groups 1–3 was to investigate the effects of QADM or NAg individually. The purpose of 3 and 4 was to examine the effect of NAg mass fraction. The purpose of comparing 2, 3 and 5 was to examine the effect of combining QADM and NAg together in the same adhesive. The purpose of comparing 5 with 6 was to investigate the effects of adding QADM and NAg into both the adhesive and the primer on dentin bond strength and biofilm response.
| Group | Adhesive resin | Dentin primer | Group name |
|---|---|---|---|
| 1 | Control | Control | Control |
| 2 | Control + 10% QADM | Control | A + 10QADM |
| 3 | Control + 0.05% NAg | Control | A + 0.05NAg |
| 4 | Control + 0.1% NAg | Control | A + 0.1NAg |
| 5 | Control + 10% QADM + 0.05% NAg | Control | A + 10QADM + 0.05NAg |
| 6 | Control + 10% QADM + 0.05% NAg | Control + 10% QADM + 0.05% NAg | A&P + 10QADM + 0.05NAg |
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