Anti-biofilm Dentin Primer with Quaternary Ammonium and Silver Nanoparticles

Abstract

Antibacterial bonding agents could combat recurrent caries at the tooth-composite margins. The objectives of this study were to develop novel antibacterial dentin primers containing quaternary ammonium dimethacrylate (QADM) and nanoparticles of silver (NAg), and to investigate the effects on dentin bond strength and dental plaque microcosm biofilms for the first time. Scotchbond Multi-Purpose (“SBMP”) bonding agent was used. QADM and NAg were incorporated into SBMP primer, yielding 4 primers: SBMP primer (control), control + 10% QADM (mass), control + 0.05% NAg, and control + 10% QADM + 0.05% NAg. Human saliva was collected to grow microcosm biofilms. The NAg particle size (mean ± SD; n = 100) was 2.7 ± 0.6 nm. Dentin shear bond strengths (n = 10) with human third molars were approximately 30 MPa for all groups (p > 0.1). QADM-NAg-containing primer increased the bacteria inhibition zone by 9-fold, compared with control primer (p < 0.05). QADM-NAg-containing primer reduced lactic acid production and colony-forming units of total micro-organisms, total streptococci, and mutans streptococci by an order of magnitude. In conclusion, novel QADM-NAg-containing primers were strongly antibacterial without compromising dentin bond strength, and hence are promising to inhibit biofilms and secondary caries. The processing method of incorporating QADM and NAg together into the same primer produced the strongest antibacterial effect, which could have a wide applicability to other bonding systems.

Introduction

Composites are increasingly used due to their esthetics, direct-filling capability, and improved performance (Bayne et al., 1998; Lim et al., 2002; Xu et al., 2006; Drummond, 2008; Ferracane, 2011). Nonetheless, half of restorations fail within 10 years, replacing them consumes 50 to 70% of dentists’ time (Deligeorgi et al., 2001), and the main cause of failure is secondary caries (Sakaguchi, 2005). Composites accumulate more biofilms/plaques than other restoratives (Beyth et al., 2007), and biofilm acids can cause caries (Featherstone, 2004; ten Cate, 2006).

To address this problem, investigators developed novel antibacterial composites (Imazato, 2003; Beyth et al., 2006; Namba et al., 2009; Fan et al., 2011). Quaternary ammonium salt (QAS) monomers, such as 12-methacryloyloxydodecylpyridinium bromide (MDPB), were effective in providing antibacterial properties (Imazato, 2003). While previous QAS monomers were monomethacrylates, a quaternary ammonium dimethacrylate (QADM) was recently synthesized (Antonucci et al., 2011; Cheng et al., 2012). An advantage of quaternary ammonium methacrylates is that they are copolymerized with other monomers after curing and hence provide long-lasting antibacterial effects (Imazato, 2003). In addition, Ag is another important antibacterial agent (Slenters et al., 2008), which has shown a long-term antimicrobial effect (Damm et al., 2007) and can result in less bacterial resistance than antibiotics (Percival et al., 2005). Recent studies developed nanoparticles of silver (NAg) (Cheng YJ et al., 2011; Cheng L et al., 2012). However, QADM and NAg have not been incorporated into bonding agents.

Bonding agents enable composite to adhere to tooth structure (Spencer and Wang, 2002; Spencer et al., 2010; Pashley et al., 2011). It is desirable for bonding agents to be antibacterial, to inhibit recurrent caries at the tooth-composite margins (Imazato, 2003; Imazato et al., 2007; Li et al., 2009). Dentin primer has direct contact with tooth structure; hence it would be beneficial to use antibacterial primers. A literature search revealed only a few reports on antibacterial primers. MDPB (Imazato et al., 1997, 1998, 2007) and chlorhexidine (Hiraishi et al., 2010) were used to develop antibacterial primers. There has been no report on primers containing QADM or NAg.

The objectives of this study were to develop antibacterial primers incorporating QADM and NAg and to investigate biofilm viability and acid production using a dental plaque microcosm model. It was hypothesized that: (1) incorporating QADM or NAg into primer would not compromise dentin shear bond strength; (2) QADM-NAg-containing primer, in uncured or cured state, would be strongly antibacterial; and (3) combining QADM and NAg in primer would yield antibacterial properties stronger than those that would be achieved by the addition of each agent alone.

Materials & Methods

Scotchbond Multi-Purpose (referred to as “SBMP”) (3M, St. Paul, MN, USA) was used as the parent bonding agent to test the effect of QDMA-NAg incorporation. The purpose was to develop a model system, and the method of QADM-NAg incorporation could be applied to other bonding agents. SBMP etchant contained 37% phosphoric-acid. SBMP primer contained 35 to 45% 2-hydroxyethylmethacrylate (HEMA), 10 to 20% copolymer of acrylic/itaconic acids, and 40 to 50% water. SBMP adhesive contained 60 to 70% BisGMA and 30 to 40% HEMA. QADM and NAg were incorporated into SBMP primer. SBMP etchant and adhesive were not modified.

Bis(2-methacryloyloxy-ethyl) dimethylammonium bromide (QADM) was recently synthesized (Antonucci et al., 2011). Ten mmol of 2-(N,N-dimethylamino)ethyl methacrylate (Sigma-Aldrich, St. Louis, MO, USA) and 10 mmol of 2-bromoethyl methacrylate (Monomer-Polymer Labs, Trevose, PA, USA) were combined in ethanol and stirred at 60°C for 24 hrs. The solvent was then evaporated, yielding the QADM (Cheng et al., 2012). QADM was mixed with the SBMP primer at QADM/(primer + QADM) = 10wt%. This was selected because preliminary study showed that 10% of QADM in the primer provided antibacterial activity without compromising the dentin bond strength, while 20% QADM in primer decreased the bond strength.

Silver 2-ethylhexanoate (Strem, Newburyport, MA, USA) of 0.08 g was dissolved into 1 g of 2-(tert-butylamino)ethyl methacrylate (TBAEMA, Sigma). TBAEMA could facilitate Ag-salt dissolution in resin (Cheng YJ et al., 2011). This Ag solution was mixed into SBMP primer at 0.05wt% of silver 2-ethylhexanoate, because preliminary study indicated that this concentration had no adverse effect on the dentin bond strength and color of the primer.

Hence, 4 primers were tested: (1) SBMP primer (control); (2) control primer + 10% QADM (termed “10QADM”); (3) control primer + 0.05% NAg (termed “0.05NAg”); and (4) control primer + 10% QADM + 0.05% NAg (termed “10QADM + 0.05NAg”).

We used a transmission electron microscope (TEM, Tecnai-T12, FEI, Hillsboro, OR, USA) to examine NAg in resin. Following a previous study (Cheng YJ et al., 2011), a thin sheet of mica was partially split, and the Ag-resin monomer was placed in the gap. The resin in the mica was pressed to form a film and photo-cured. The resin film was used for TEM examination.

Extracted caries-free human third molars were ground on 320-grit SiC paper until occlusal enamel was removed. The dentin was etched with 37% phosphoric acid for 15 sec (Antonucci et al., 2009). A primer was applied, and the solvent was removed with an air stream. The adhesive was applied and light-cured for 10 sec (Optilux-VCL401, Demetron, Danbury, CT, USA).

A stainless-steel iris having a central opening (diameter = 4 mm, thickness = 1.5 mm) was held against the adhesive-treated dentin (Antonucci et al., 2009). The opening was filled with a composite (TPH, Caulk/Dentsply, Milford, DE, USA) and light-cured for 60 sec. Specimens were stored in water at 37°C for 24 hrs. Dentin shear bond strength, S_D, was measured (Antonucci et al., 2009). A chisel was aligned parallel to the composite-dentin interface and loaded via a Universal Testing Machine (MTS, Eden Prairie, MN, USA) at 0.5 mm/min until the composite-dentin bond failed. S_D = 4P/(πd²), where P is load-at-failure, and d is composite diameter.

The bonded tooth was cut through the center longitudinally, and the sections were polished with SiC papers up to 4,000 grit. Following a previous study (Imazato et al., 2007), the polished surface was treated with 50% phosphoric acid and 10% NaOCl. Specimens were gold-coated and examined via scanning electron microscopy (SEM, Quanta-200, FEI).

Saliva was collected from a healthy donor having natural dentition without active caries and not having used antibiotics within the preceding 3 mos. The donor did not brush teeth for 24 hrs and abstained from food/drink intake for 2 hrs prior to donating saliva (Cheng L et al., 2011).

Uncured primers were tested by an agar diffusion test (ADT). Saliva was added to growth medium containing mucin (concentration = 2.5 g/L), bacteriological peptone (concentration = 2.0 g/L), tryptone (concentration = 2.0 g/L), yeast extract (concentration = 1.0 g/L), NaCl (concentration = 0.35 g/L), KCl (concentration = 0.2 g/L), CaCl₂ (concentration = 0.2 g/L), and cysteine hydrochloride (concentration = 0.1 g/L) (pH = 7.0) (McBain, 2009). The inoculum was incubated (37°C, 5% CO₂) for 24 hrs. Three types of media were used. First, tryptic soy blood agar plates were used to determine total micro-organisms (Cheng L et al., 2011). Second, mitis salivarius agar (MSA) plates, containing 15% sucrose, were used to determine total streptococci. Third, MSA plus 0.2 units of bacitracin/mL was used to determine mutans streptococci.

Bacteria suspension of 0.4 mL was poured onto each agar plate (diameter = 90 mm, thickness = 4 mm). Then, a 30-µL quantity of primer was impregnated into a sterile paper disk (diameter = 9 mm, thickness = 1.5 mm) (Imazato et al., 2006). The primer-impregnated paper disk (referred to as “disk”) was placed on a plate with bacteria and incubated for 48 hrs. Bacteria inhibition zone size = (Outer diameter of inhibition zone – disk diameter)/2.

Cured specimens were fabricated as schematically shown in Fig. 3A, following a previous study (Imazato et al., 1998). A polyethylene mold (inner diameter = 9 mm, thickness = 2 mm) was situated on a glass slide. Primer was brushed on the glass slide. After the specimen was dried with a stream of air, SBMP adhesive was applied and cured for 20 sec with Optilux. Then, composite TPH was placed to fill the mold and light-cured for 1 min. Specimens were agitated in water to remove uncured monomers (Imazato et al., 1998).

Figure 3.

Cured primers with biofilms for live/dead staining. (A) Schematic of biofilm on the primer surface of a layered specimen. (B-E) Representative live/dead staining images. Live bacteria were stained green and dead bacteria were stained red. Live and dead bacteria close to (or on the top of) each other stained yellow/orange. Control primer was covered with primarily live bacteria. 10QADM, 0.05NAg, and 10QADM+0.05NAg primers had much more staining of compromised bacteria. (F) Live bacteria area fraction = area of green staining/total area of the image. Six specimens were used for each primer (n = 6). Three randomly chosen fields of view for each specimen yielded 18 images per primer. Values with dissimilar letters are significantly different (p < 0.05).

Each specimen was placed into a well of 24-well plates, and 1.5 mL inoculum was added. A 2-day incubation formed mature biofilms (Cheng L et al., 2011). Specimens were then live-/ dead-stained (Molecular Probes, Eugene, OR, USA) and examined by epifluorescence microscopy (TE2000-S, Nikon, Melville, NY, USA). The area of green staining (live bacteria) was computed with the use of NIS Elements imaging software (Nikon). The area fraction of live bacteria = green staining area/total area of the image.

For acid production, specimens with 2-day biofilms were transferred to 24-well plates containing buffered-peptone water (BPW) plus 0.2% sucrose, and incubated for 3 hrs to allow biofilms to produce acid. Lactate concentrations were determined by an enzymatic method (Cheng L et al., 2011). A microplate reader (SpectraMax, Molecular Devices, Sunnyvale, CA, USA) was used to measure the 340-nm absorbance. Standard curves were prepared by a lactic acid standard (Supelco, Bellefonte, PA, USA).

For colony-forming units (CFU), specimens with 2-day biofilms were transferred into tubes with 2-mL cysteine-peptone water, and biofilms were harvested by sonication/vortexing (Fisher, Pittsburgh, PA, USA). Three types of agar plates were used: Tryptic soy blood plates to determine total micro-organisms; MSA plus 15% sucrose to determine total streptococci; and MSA plus 0.2 units of bacitracin/mL to determine mutans streptococci.

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

Results

TEM examination showed NAg sizes ranging from 2 to 5 nm (Fig. 1A). Measurement of 100 particles (mean ± SD) yielded 2.7 ± 0.6 nm. Dentin shear bond strength (Fig. 1B) ranged from 30 to 35 MPa, which was not compromised by the addition of QADM-NAg into primer (p > 0.1) (Figs. 1C, 1D). SEM examination of dentin-adhesive interfaces revealed numerous resin tags “T”. There was no noticeable difference among the 4 groups.

Figure 1.

Effect of NAg and QADM incorporation into primer on dentin bonding. (A) TEM image of nanoparticles of silver (NAg). NAg particle sizes ranged from 2 to 5 nm, with mean ± SD (n = 100) of 2.7 ± 0.6 nm. NAg particles were well-dispersed in the resin, without agglomeration. In (B), dentin shear bond strength was between 30 and 35 MPa. Ten teeth were tested for each group (n = 10). Bars with the same letter indicate similar values (p > 0.1). (C, D) SEM of adhesive-dentin interface. T, resin tags formed by adhesive filling into dentinal tubules. HL, hybrid layer between the adhesive and the underlying mineralized dentin. A mixture of long and short resin tags was observed. Some tags were shorter because, during sample preparation, the sectioned surface was not exactly parallel to the long axis of dentinal tubules; thus some tubules were intersected by the cutting and were shortened. Numerous and similar resin tags were found for all 4 groups.

Uncured QADM-NAg primers had a strong antibacterial activity (Fig. 2). Control primer had minimal inhibition zones (Fig. 2A). Primers 10QADM, 0.05NAg, and 10QADM+0.05NAg had much larger inhibition zones (Figs. 2B-2D). Inhibition zone sizes are plotted in Figs. 2E-2G for total micro-organisms, total streptococci, and mutans streptococci. Inhibition zone sizes for 10QADM+0.05NAg were 9-fold those of control (p < 0.05).

Figure 2.

Antibacterial activity of uncured primers in the agar disk diffusion test. (A-D) Control primer, 10QADM, 0.05NAg, and 10QADM+0.05NAg, respectively. Note a small inhibition zone for control, and much wider inhibition zones for primers with QADM and NAg. This example is for mutans streptococci. Total micro-organisms and total streptococci had similar results. (E-G) Inhibition zone data for total micro-organisms, total streptococci, and mutans streptococci, respectively. Each value is mean ± SD (n = 6). Bars with dissimilar letters indicate values that are significantly different (p < 0.05).

Biofilms were grown on cured primers (Fig. 3A). Live bacteria were stained green, and dead bacteria were stained red (Figs. 3B-3E). Yellow/orange colors appeared when live and dead bacteria were in close proximity. Control primer had full coverage of primarily live bacteria. 10QADM, 0.05NAg, and 10QADM+0.05NAg had much more red/yellow/orange staining, indicating a strong antibacterial activity. Adding QADM or NAg substantially reduced the live bacteria area fraction (Fig. 3F). Incorporation of QADM and NAg together had the strongest antibacterial activity among all groups.

The 10QADM, 0.05NAg, and 10QADM+0.05NAg reduced the CFU counts (Fig. 4A-4C). CFU on 10QADM+0.05NAg were an order of magnitude less than those on control primer. Lactic acid from biofilms (Fig. 4D) was reduced on 10QADM, 0.05NAg, and 10QADM + 0.05NAg, compared with control (p < 0.05). Adding QADM-NAg to primer imparted potent antibacterial effects. The strongest effects were achieved by combining QADM and NAg in the primer.

Figure 4.

Biofilm CFU counts and lactic acid production on specimens with cured primers. (A) CFU of total micro-organisms. (B) CFU of total streptococci. (C) CFU of mutans streptococci. (D) Lactic acid production. Each value is mean ± SD (n = 6). In each plot, values with dissimilar letters are significantly different (p < 0.05). Adding QADM or NAg to the primer imparted a potent antibacterial effect. The strongest antibacterial effect was achieved with QADM and NAg together in the same primer.

Discussion

This study investigated, for the first time, the effects of QADM-NAg incorporation into primer on shear bond strength and microcosm biofilm response. While residual bacteria in tooth cavity can lead to caries and pulp damage, primer comes into direct contact with the dentin and therefore could be an important vehicle for the delivery of antimicrobial agents. The new QADM-NAg-containing primer in the uncured state increased the microcosm inhibition zone by 9-fold, compared with commercial control primer. Control primer contained HEMA and copolymer of acrylic/itaconic acids, which could copolymerize with QADM and the Ag solution of 2-(tert-butylamino)ethyl methacrylate. The cured QADM-NAg-containing primer reduced biofilm CFU and lactic acid by an order of magnitude, compared with control. These strong antibacterial properties were achieved without compromising dentin bond strength. Furthermore, the method of combining QADM and NAg in the primer yielded the strongest antibacterial properties.

In previous studies, investigators used QAS monomers in one group of antibacterial resins (Imazato, 2003). The antimicrobial mechanism is that when the negatively charged bacterial cell comes into contact with the positively charged sites of QAS resin, the electrical balance of the cell membrane could be disturbed, and the bacterium could explode under its own osmotic pressure (Beyth et al., 2006; Namba et al., 2009). Uncured MDPB primer was antibacterial (Imazato et al., 1997), and its antibacterial effect was maintained after being photo-cured (Imazato et al., 1998). Antibacterial methacryloxylethyl-cetyl-dimethyl ammonium chloride and cetylpyridinium chloride were also investigated (Li et al., 2009; Namba et al., 2009; Xie et al., 2011). The QADM of the present study has unique features. First, its synthesis was straightforward, because the reaction products were generated at quantitative amounts and required no further purification (Antonucci et al., 2011). Second, while previous studies used QAS monomethacrylates, QADM is a dimethacrylate with reactive groups on both ends of the molecule. Hence, QADM could be incorporated into resin with a less negative impact on mechanical properties. Third, QADM is miscible with other dimethacrylates, with minimal monomer leachability due to reactive groups on both ends of the molecule, compared with monomethacrylates.

Another group of antibacterial resins incorporated Ag (Yamamoto et al., 1996; Yoshida et al., 1999; Fan et al., 2011; Cheng YJ et al., 2011). The antimicrobial mechanism was suggested to be Ag ions inactivating vital enzymes of bacteria, causing DNA to lose its replication ability, leading to cell death (Rai et al., 2009). Because QADM and NAg act via different mechanisms, there may be a synergistic antibacterial effect that is more than simply being additive. Further study is needed to investigate how QADM-NAg synergy influences the development, metabolism, and bacterial composition of dental plaque biofilm. While QADM relies on contact inhibition, further study is needed to determine if NAg is capable of long-distance killing of bacteria due to Ag ion release. A previous study showed that Ag-containing resins had long-term antibacterial effects (Yoshida et al., 1999). In the present study, the Ag salt was reduced to NAg in situ, avoiding the need for pre-fabricated nanoparticles to be mixed with resin, which could cause agglomeration. The small size of NAg could allow them to flow with primer into dentinal tubules to kill residual bacteria. The specific surface area A = 6/(ρd), where density ρ = 10.5 g/cm³ for Ag, and particle size d = 2.7 nm. Hence, A = 212 m²/g for NAg. In comparison, Ag particles with 1 µm size would have A = 0.57 m²/g. The high surface area of NAg provided potent antibacterial effects at a low filler level to avoid negative influence on color and mechanical properties. Indeed, the QADM-NAg-containing primer had esthetics/color similar to those of control primer. Another issue is the cytotoxicity of the antibacterial agents. A previous study showed that MDPB had a smaller negative effect on the proliferation, differentiation, and mineralization of odontoblast-like cells than did BisGMA (Nishida et al., 2010). Silver is known to have low toxicity and good biocompatibility with human cells (Slenters et al., 2008). While a similarly good biocompatibility is expected for the QADM-NAg-containing primer, further study is needed to determine its cytotoxicity.

Single-species bacteria models have been used to test antibacterial resins (Hiraishi et al., 2010; Antonucci et al., 2011; Cheng YJ et al., 2011). The present study used a microcosm model to test QADM-NAg-containing primers. Dental biofilm models can be divided into 3 categories: single-species, defined consortium, and microcosm (McBain, 2009). Plaque is a complicated ecosystem, with about 1,000 bacterial species (ten Cate, 2006). Hence, microcosm models could maintain the complexity/heterogeneity in vivo (McBain, 2009). Oral streptococci contain many groups, including the mutans streptococci group, which contains S. mutans and S. sobrinus, the major pathogens of caries. The QADM-NAg primer reduced total micro-organisms, total streptococci, and mutans streptococci by 10-fold. In vivo studies are needed to further investigate the antibacterial efficacy of the novel QADM-NAg primer.

In summary, this study formulated antibacterial primers by incorporating QADM and NAg, and investigated their effects on plaque microcosm biofilms for the first time. The results showed that: (1) QADM-NAg-containing primer did not decrease dentin bond strength; (2) QADM-NAg-containing primer had much larger bacteria inhibition zones, and greatly reduced biofilm viability, CFU, and lactic acid, compared with control; and (3) adding QADM and NAg together to primer produced the strongest antibacterial effects. QADM and NAg could have wide applications in other bonding systems. The novel antibacterial QADM-NAg-containing primer may be promising to inhibit biofilms and secondary caries.