A Novel Dental Sealant Containing Dimethylaminohexadecyl Methacrylate Suppresses the Cariogenic Pathogenicity of Streptococcus mutans Biofilms.

Cariogenic oral biofilms are strongly linked to dental caries around dental sealants. Quaternary ammonium monomers copolymerized with dental resin systems have been increasingly explored for modulation of biofilm growth. Here, we investigated the effect of dimethylaminohexadecyl methacrylate (DMAHDM) on the cariogenic pathogenicity of Streptococcus mutans (S. mutans) biofilms. DMAHDM at 5 mass% was incorporated into a parental formulation containing 20 mass% nanoparticles of amorphous calcium phosphate (NACP). S. mutans biofilms were grown on the formulations, and biofilm inhibition and virulence properties were assessed. The tolerances to acid stress and hydrogen peroxide stress were also evaluated. Our findings suggest that incorporating 5% DMAHDM into 20% NACP-containing sealants (1) imparts a detrimental biological effect on S. mutans by reducing colony-forming unit counts, metabolic activity and exopolysaccharide synthesis; and (2) reduces overall acid production and tolerance to oxygen stress, two major virulence factors of this microorganism. These results provide a perspective on the value of integrating bioactive restorative materials with traditional caries management approaches in clinical practice. Contact-killing strategies via dental materials aiming to prevent or at least reduce high numbers of cariogenic bacteria may be a promising approach to decrease caries in patients at high risk.

Experimental and statistical methods to evaluate antibacterial activity of a quaternary pyridinium salt on planktonic, biofilm-forming, and biofilm states.

Robust evaluation and comparison of antimicrobial technologies are critical to improving biofilm prevention and treatment. Herein, a multi-pronged experimental framework and statistical models were applied to determine the effects of quaternary pyridinium salt, 4-acetyl-1-hexadecylpyridin-1-ium iodide (QPS-1), on Streptococcus mutans in the planktonic, biofilm-forming and biofilm cell states. Minimum inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined via common methods with novel application of statistical approaches combining random effects models and interval censored data to estimate uncertainties. The MICs and MBCs for planktonic and biofilm-forming states ranged from 3.12 to 12.5 µg ml-1, with biofilm values only ≈ 8 times higher. Potent anti-biofilm activity and reactive structural features make QPS-1 a promising antibacterial additive for dental and potentially other biomedical devices. Together, the experimental framework and statistical models provide estimates and uncertainties for effective antimicrobial concentrations in multiple cell states, enabling statistical comparisons and improved characterization of antibacterial agents.

Effects of sample preparation on bacterial colonization of polymers.

Characterization of materials developed for medical usage frequently includes studies in which the materials are inoculated with bacteria in order to assess bacterial colonization and biofilm formation. Observed differences in bacterial growth are typically considered to be due to the material or the incubation conditions. To our knowledge, the method used to prepare the materials has generally not been considered with regard to its influence on bacterial colonization. The objective of this study was to determine the effects that various preparation methods exert on bacterial colonization of polymer disks. Polymer disks of the same dimethacrylate composition were photopolymerized: (1) between untreated glass slides, (2) between polyester release film, (3) between glass slides treated with an alkyl silane, (4) between glass slides treated with a perfluorinated silane, or (5) with one free surface in an argon-purged chamber. Surface chemistry was quantified using X-ray photoelectron spectroscopy, hydrophobicity was assessed by water contact angle, and topography was characterized using atomic force microscopy. The disks were inoculated with Streptococcus mutans for 4 h, fixed, and visualized using confocal laser scanning microscopy. Differences among all groups were found with regard to surface chemistry, hydrophobicity, topography, and bacteria morphology, density, and coverage, indicating that the method of sample preparation strongly affects both the surface properties and the initial bacterial colonization. Polymerization on untreated slides was selected as the preferred method of preparation due to minimal material transfer to the polymer and consistent, reproducible bacterial colonization.

Exploring cellular contact guidance using gradient nanogratings.

Nanoscale surface features that mimic extracellular matrix are critical environmental cues for cell contact guidance and are vital in advanced medical devices in order to manipulate cell behaviors. Among them, nanogratings (line-and-space gratings) are common platforms to study geometric effects on cell contact guidance, especially cell alignment, but generally are one pattern height per platform. In this study, we developed a strategy to fabricate controlled substrates with a wide range of pattern shapes and surface chemistries and to separate surface chemistry and topography effects. As a demonstration of this strategy, six nanograting platforms on three materials were fabricated and applied to examine and differentiate the effects of surface topography and surface chemistry on cell contact guidance of murine preosteoblasts. All of the six platforms contained the same gradient in pattern height (0 to ≈350 nm). They were prepared using nanoimprint lithography and annealing for thermoplastic materials (low molecular weight polystyrene (PS) and polymethylmethacrylate (PMMA)) and photoimprint for a thermoset material (a cross-linked dimethacrylate (DMA)). Each material contains two platforms that are only different in line-and-space pitch (420 or 800 nm). The DMA nanogratings had a reverse line-and-space profile to those of the PS and PMMA nanogratings. Using these platforms, a full range of cell alignment, from randomly orientated to completely parallel to the grating direction was achieved. Results from focal adhesion assays and scanning electronic microscopy indicated a change in cell-substrate contact from a noncomposite state (full contact) to a composite state (partial contact between cell and substrate) as pattern height increased. These gradient platforms allowed for the separation of surface chemistry and surface topography to provide insight into the mechanisms responsible for cell contact guidance on nanopatterned surfaces.

How we are assessing the developing antibacterial resin-based dental materials? A scoping review.

OBJECTIVES: To identify antibacterial additives and screening/assessment approaches used to evaluate the antibacterial activity of resin-based restorative dental materials containing these additives. DATA: In vitro studies that compared the antibacterial effects of resin-based restorative dental materials with and without antibacterial additives were screened. Risk bias was assessed, and the following data were extracted: antibacterial additive, parental dental material, curing mode, bacterial growth outcome assessment, samples used as a substrate for bacterial growth, inoculum complexity, and culture time as an indicator of biofilm maturity. SOURCE: Arksey and O'Malley's five stages framework using Medline (OVID), EMBASE, and Scopus (Elsevier) databases guided this review. STUDY SELECTION: From 6503 studies initially identified, 348 studies were considered eligible for full-text screening, and 153 were included for data extraction. Almost all studies have a high sampling bias related to both sample size and blindness. Quaternary ammonium monomers were the most investigated additive (45 %), and the most prevailing parental material was resin composite (49 %). There was extensive methodological heterogeneity among the studies for outcome assessment with the majority using resin composite disks (78 %), mono-species Streptococcus mutans as the inoculum (54 %), and a relatively short period of biofilm growth (≤24 h). CONCLUSION: The findings herein present the urgent need for improved biological efficacy studies in this important and exciting field. There is a need for efforts to improve study designs to mimic the oral environment in vivo and to develop standardized methods to help understand and optimize these materials. CLINICAL SIGNIFICANCE: Most studies that incorporate antibacterial additives into resin-based materials claim promising results by bacterial reduction. However, these results should be interpreted with caution due to significant variation in the methods applied for quantifying bacterial growth, the frequent lack of complexity in the biofilms, and the often-short duration of biofilm growth.

Effect of dental monomers and initiators on Streptococcus mutans oral biofilms.

OBJECTIVE: Resin-based composites are known to elute leachables that include unincorporated starting materials. The objective of this work was to determine the effect of common dental monomers and initiators on Streptococcus mutans biofilm metabolic activity and biomass. METHODS: S. mutans biofilms were inoculated in the presence of bisphenol A glycerolate dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), camphorquinone (CQ), and ethyl 4-(dimethylamino)benzoate (4E) at 0.01µg/mL up to 500µg/mL, depending on the aqueous solubility of each chemical. Biofilms were evaluated at 4h and 24h for pH (n=3-8), biomass via crystal violet (n=12), metabolic activity via tetrazolium salt (n=12), and membrane permeability for selected concentrations via confocal microscopy (n=6). Parametric and non-parametric statistics were applied. RESULTS: 500µg/mL TEGDMA reduced 24h metabolic activity but not biomass, similar to prior results with leachables from undercured BisGMA-TEGDMA polymers. 50µg/mL BisGMA reduced biofilm biomass and activity, slightly delayed the pH drop, and decreased the number of cells with intact membranes. 100µg/mL CQ delayed the pH drop and metabolic activity at 4h but then significantly increased the 24h metabolic activity. 4E had no effect up to 10µg/mL. SIGNIFICANCE: Monomers and initiators that leach from resin composites affect oral bacterial biofilm growth in opposite ways. Leachables, which can be released for extended periods of time, have the potential to alter oral biofilm biomass and activity and should be considered in developing and evaluating new dental materials.

Macrophage response to methacrylate conversion using a gradient approach.

Incomplete conversion, an ongoing challenge facing photopolymerized methacrylate-based polymers, affects leachables as well as the resulting polymer network. As novel polymers and composites are developed, methods to efficiently screen cell response to these materials and their properties, including conversion, are needed. In this study, an in vitro screening methodology was developed to assess cells cultured directly on cross-linked polymer networks. A gradient in methacrylate double bond conversion was used to increase the experimental throughput. A substrate of 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl] propane (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) was prepared with a conversion ranging from 43.0% to 61.2%. Substrates aged for 7 days had no significant differences in surface roughness or hydrophilicity as a function of conversion. Leachables were detectable for at least 7 days using UV absorption, but their global cytotoxicity was insignificant after 5 days of aging. Thus, RAW 264.7 macrophage-like cells were cultured on aged substrates to evaluate the cell response to conversion, with possible contributions from the polymer network and local leachables. Conversions of 45% and 50% decreased viability (via calcein/ethidium staining) and increased apoptosis (via annexin-V staining). No significant changes (p>0.05) in tumor necrosis factor-alpha and interleukin-1beta gene expression, as measured by quantitative, real-time reverse transcription-polymerase chain reaction, were seen as conversion increased. Thus, conversions greater than 50% are recommended for equimolar BisGMA/TEGDMA. The ability to distinguish cell response as a function of conversion is useful as an initial biological screening platform to optimize dental polymers.

Two-dimensional gradient platforms for rapid assessment of dental polymers: a chemical, mechanical and biological evaluation.

OBJECTIVES: The increased usage of composite dental restorations underscores the need for continued improvements in material properties. Well-controlled sample fabrication and reproducible methods to quantify and compare material properties will accelerate material design and optimization. Our objectives were to fabricate samples encompassing a range of processing parameters and develop techniques that systematically quantify multiple properties of these samples, thus reducing sample-to-sample variation while concurrently testing numerous processing conditions. METHODS: Gradient samples were prepared to evaluate the effects of composition and irradiation time. Comonomer ratio of 2,2-bis[p-2'-hydroxy-3'-methacryloxypropoxy]-phenyl]propane (BisGMA) and triethylene glycol dimethacrylate (TEGDMA) was varied discretely, and irradiation time was varied continuously across each composition. Degree of conversion was measured using infrared spectroscopy, mechanical properties were evaluated using nanoindentation, and cell viability and density were quantified using fluorescence microscopy. RESULTS: Higher BisGMA contents increased elastic moduli while higher TEGDMA contents increased conversions. Cell response depended only on irradiation time and not composition, with conversions of at least 52% and 60% required for unaffected viability and cell density, respectively. A single composition-irradiation combination to achieve all of the 'best' properties (highest conversion, highest elastic modulus, lack of cytotoxicity) was not identified, illustrating the necessity of testing all combinations for multiple relevant properties. SIGNIFICANCE: Simultaneously screening composition and conversion increased the experimental throughput and allowed for the quantification of chemical, mechanical, and biological properties in a controlled, reproducible fashion. This 2D gradient approach is useful for optimizing compositions and processing parameters to achieve the desired combination of properties.

Biofilm over teeth and restorations: What do we need to know?

OBJECTIVE: The goal of this manuscript is to provide an overview of biofilm attributes and measurement approaches in the context of studying biofilms on tooth and dental material surfaces to improve oral health. METHODS: A historical perspective and terminology are presented, followed by a general description of the complexity of oral biofilms. Then, an approach to grouping measurable biofilm properties is presented and considered in relation to biofilm-material interactions and material design strategies to alter biofilms. Finally, the need for measurement assurance in biofilm and biofilm-materials research is discussed. RESULTS: Biofilms are highly heterogeneous communities that are challenging to quantify. Their characteristics can be broadly categorized into constituents (identity), quantity, structure, and function. These attributes can be measured over time and in response to substrates and external stimuli. Selecting the biofilm attribute(s) of interest and appropriate measurement methods will depend on the application and, in the case of antimicrobial therapies, the strategic approach and expected mechanism of action. To provide measurement assurance, community accepted protocols and guidelines for minimum data and metadata should be established and broadly applied. Consensus standards may help to streamline testing and demonstration of product claims. SIGNIFICANCE: Understanding oral biofilms and their interactions with tooth and dental material surfaces holds great promise for enabling improvements in oral and overall human health. Both substrate and biofilm properties should be considered to develop a more thorough understanding of the system.

Automation of antimicrobial activity screening.

Manual and automated methods were compared for routine screening of compounds for antimicrobial activity. Automation generally accelerated assays and required less user intervention while producing comparable results. Automated protocols were validated for planktonic, biofilm, and agar cultures of the oral microbe Streptococcus mutans that is commonly associated with tooth decay. Toxicity assays for the known antimicrobial compound cetylpyridinium chloride (CPC) were validated against planktonic, biofilm forming, and 24 h biofilm culture conditions, and several commonly reported toxicity/antimicrobial activity measures were evaluated: the 50 % inhibitory concentration (IC50), the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC). Using automated methods, three halide salts of cetylpyridinium (CPC, CPB, CPI) were rapidly screened with no detectable effect of the counter ion on antimicrobial activity.

Novel Dental Cement to Combat Biofilms and Reduce Acids for Orthodontic Applications to Avoid Enamel Demineralization.

Orthodontic treatments often lead to biofilm buildup and white spot lesions due to enamel demineralization. The objectives of this study were to develop a novel bioactive orthodontic cement to prevent white spot lesions, and to determine the effects of cement compositions on biofilm growth and acid production. 2-methacryloyloxyethyl phosphorylcholine (MPC), nanoparticles of silver (NAg), and dimethylaminohexadecyl methacrylate (DMAHDM) were incorporated into a resin-modified glass ionomer cement (RMGI). Enamel shear bond strength (SBS) was determined. Protein adsorption was determined using a micro bicinchoninic acid method. A dental plaque microcosm biofilm model with human saliva as inoculum was used to investigate metabolic activity, colony-forming units (CFU) and lactic acid production. Incorporating 3% of MPC, 1.5% of DMAHDM, and 0.1% of NAg into RMGI, and immersing in distilled water at 37 °C for 30 days, did not decrease the SBS, compared to control (p > 0.1). RMGI with 3% MPC + 1.5% DMAHDM + 0.1% NAg had protein amount that was 1/10 that of control. RMGI with triple agents (MPC + DMAHDM + NAg) had much stronger antibacterial property than using a single agent or double agents (p < 0.05). Biofilm CFU on RMGI with triple agents was reduced by more than 3 orders of magnitude, compared to commercial control. Biofilm metabolic activity and acid production were also greatly reduced. In conclusion, adding MPC + DMAHDM + NAg in RMGI substantially inhibited biofilm viability and acid production, without compromising the orthodontic bracket bond strength to enamel. The novel bioactive cement is promising for orthodontic applications to hinder biofilms and plaque buildup and enamel demineralization.

Primer containing dimethylaminododecyl methacrylate kills bacteria impregnated in human dentin blocks.

Antibacterial dimethylaminododecyl methacrylate (DMADDM) was recently synthesized. The objectives of this study were to: (1) investigate antibacterial activity of DMADDM-containing primer on Streptococcus mutans impregnated into dentin blocks for the first time, and (2) compare the antibacterial efficacy of DMADDM with a previous quaternary ammonium dimethacrylate (QADM). Scotchbond Multi-Purpose (SBMP) bonding agent was used. DMADDM and QADM were mixed into SBMP primer. Six primers were tested: SBMP control primer P, P+2.5% DMADDM, P+5% DMADDM, P+7.5% DMADDM, P+10% DMADDM, and P+10% QADM. S. mutans were impregnated into human dentin blocks, and each primer was applied to dentin to test its ability to kill bacteria in dentinal tubules. Bacteria in dentin were collected via a sonication method, and the colony-forming units (CFU) and inhibition zones were measured. The bacterial inhibition zone of P+10% DMADDM was 10 times that of control primer (P<0.05). CFU in dentin with P+10% DMADDM was reduced by three orders of magnitude, compared with control. DMADDM had a much stronger antibacterial effect than QADM, and antibacterial efficacy increased with increasing DMADDM concentration. Dentin shear bond strengths were similar among all groups (P>0.1). In conclusion, antibacterial DMADDM-containing primer was validated to kill bacteria inside dentin blocks, possessing a much stronger antibacterial potency than the previous QADM. DMADDM-containing bonding agent was effective in eradicating bacteria in dentin, and its efficacy was directly proportional to DMADDM mass fraction. Therefore, DMADDM may be promising for use in bonding agents as well as in other restorative and preventive materials to inhibit bacteria.

Microstructure and mechanical properties of in situ Streptococcus mutans biofilms.

Insight into live microbial biofilm microstructure and mechanical properties and their interactions with the underlying substrate can lead to the development of new remedial strategies and/or materials. Here we report mechanical properties of dental pathogenic Streptococcus mutans biofilms, grown on a polystyrene-coated plate of a shear rheometer in physiologically relevant conditions, precisely controlled in a custom built bioreactor. In situ measurements demonstrated the importance of microstructure and composition of extracellular polymeric substances on the biofilm modulus. The biofilms behave like a weak gel with storage moduli higher than loss moduli. The simple but robust experimental technique presented here can easily be extended to other biofilm-material systems.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films.

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-ß and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation.

Antibacterial activity and ion release of bonding agent containing amorphous calcium phosphate nanoparticles.

OBJECTIVE: Recurrent caries at the margins is a primary reason for restoration failure. The objectives of this study were to develop bonding agent with the double benefits of antibacterial and remineralizing capabilities, to investigate the effects of NACP filler level and solution pH on Ca and P ion release from adhesive, and to examine the antibacterial and dentin bond properties. METHODS: Nanoparticles of amorphous calcium phosphate (NACP) and a quaternary ammonium monomer (dimethylaminododecyl methacrylate, DMADDM) were synthesized. Scotchbond Multi-Purpose (SBMP) primer and adhesive served as control. DMADDM was incorporated into primer and adhesive at 5% by mass. NACP was incorporated into adhesive at filler mass fractions of 10%, 20%, 30% and 40%. A dental plaque microcosm biofilm model was used to test the antibacterial bonding agents. Calcium (Ca) and phosphate (P) ion releases from the cured adhesive samples were measured vs. filler level and solution pH of 7, 5.5 and 4. RESULTS: Adding 5% DMADDM and 10-40% NACP into bonding agent, and water-aging for 28 days, did not affect dentin bond strength, compared to SBMP control at 1 day (p>0.1). Adding DMADDM into bonding agent substantially decreased the biofilm metabolic activity and lactic acid production. Total microorganisms, total streptococci, and mutans streptococci were greatly reduced for bonding agents containing DMADDM. Increasing NACP filler level from 10% to 40% in adhesive increased the Ca and P ion release by an order of magnitude. Decreasing solution pH from 7 to 4 increased the ion release from adhesive by 6-10 folds. SIGNIFICANCE: Bonding agents containing antibacterial DMADDM and remineralizer NACP were formulated to have Ca and P ion release, which increased with NACP filler level from 10% to 40% in adhesive. NACP adhesive was "smart" and dramatically increased the ion release at cariogenic pH 4, when these ions would be most-needed to inhibit caries. Therefore, bonding agent containing DMADDM and NACP may be promising to inhibit biofilms and remineralize tooth lesions thereby increasing the restoration longevity.

Effects of dual antibacterial agents MDPB and nano-silver in primer on microcosm biofilm, cytotoxicity and dentine bond properties.

OBJECTIVES: The objective of this study was to investigate the effects of dentine primer containing dual antibacterial agents, namely, 12-methacryloyloxydodecylpyridinium bromide (MDPB) and nanoparticles of silver (NAg), on dentine bond strength, dental plaque microcosm biofilm response, and fibroblast cytotoxicity for the first time. METHODS: Scotchbond Multi-Purpose (SBMP) was used as the parent bonding agent. Four primers were tested: SBMP primer control (referred to as "P"), P+5% MDPB, P+0.05% NAg, and P+5% MDPB+0.05% NAg. Dentine shear bond strengths were measured using extracted human teeth. Biofilms from the mixed saliva of 10 donors were cultured to investigate metabolic activity, colony-forming units (CFU), and lactic acid production. Human fibroblast cytotoxicity of the four primers was tested in vitro. RESULTS: Incorporating MDPB and NAg into primer did not reduce dentine bond strength compared to control (p>0.1). SEM revealed well-bonded adhesive-dentine interfaces with numerous resin tags. MDPB or NAg each greatly reduced biofilm viability and acid production, compared to control. Dual agents MDPB+NAg had a much stronger effect than either agent alone (p<0.05), increasing inhibition zone size and reducing metabolic activity, CFU and lactic acid by an order of magnitude, compared to control. There was no difference in cytotoxicity between commercial control and antibacterial primers (p>0.1). CONCLUSIONS: The method of using dual agents MDPB+NAg in the primer yielded potent antibacterial properties. Hence, this method may be promising to combat residual bacteria in tooth cavity and invading bacteria at the margins. The dual agents MDPB+NAg may have wide applicability to other adhesives, composites, sealants and cements to inhibit biofilms and caries.

Effect of polymer degree of conversion on Streptococcus mutans biofilms.

Biofilm-material interactions are increasingly recognized as critical to success of some materials/devices and failure of others. We use a model system of dental monomers, salivary pellicles, and oral biofilms to demonstrate for the first time that degree of conversion of cross-linked dimethacrylate polymers alters biofilm metabolic activity. This response is due primarily to leachable release (not surface chemistry) and is complex, with no changes in some biofilm measurements (i.e., biomass), and time- and leachable-dependent responses in others (i.e., metabolic activity). These results highlight the need for considering biofilm-material interactions when designing/evaluating new materials.

Antibacterial and physical properties of calcium-phosphate and calcium-fluoride nanocomposites with chlorhexidine.

OBJECTIVES: Previous studies have developed calcium phosphate and fluoride releasing composites. Other studies have incorporated chlorhexidine (CHX) particles into dental composites. However, CHX has not been incorporated in calcium phosphate and fluoride composites. The objectives of this study were to develop nanocomposites containing amorphous calcium phosphate (ACP) or calcium fluoride (CaF(2)) nanoparticles and CHX particles, and investigate Streptococcus mutans biofilm formation and lactic acid production for the first time. METHODS: Chlorhexidine was frozen via liquid nitrogen and ground to obtain a particle size of 0.62 µm. Four nanocomposites were fabricated with fillers of: nano ACP; nano ACP+10% CHX; nano CaF(2); nano CaF(2)+10% CHX. Three commercial materials were tested as controls: a resin-modified glass ionomer, and two composites. S. mutans live/dead assay, colony-forming unit (CFU) counts, biofilm metabolic activity, and lactic acid were measured. RESULTS: Adding CHX fillers to ACP and CaF(2) nanocomposites greatly increased their antimicrobial capability. ACP and CaF(2) nanocomposites with CHX that were inoculated with S. mutans had a growth medium pH>6.5 after 3 d, while the control commercial composites had a cariogenic pH of 4.2. Nanocomposites with CHX reduced the biofilm metabolic activity by 10-20 folds and reduced the acid production, compared to the controls. CFU on nanocomposites with CHX were three orders of magnitude less than that on commercial composite. Mechanical properties of nanocomposites with CHX matched a commercial composite without fluoride. SIGNIFICANCE: The novel calcium phosphate and fluoride nanocomposites could be rendered antibacterial with CHX to greatly reduce biofilm formation, acid production, CFU and metabolic activity. The antimicrobial and remineralizing nanocomposites with good mechanical properties may be promising for a wide range of tooth restorations with anti-caries capabilities.

Synthesis and characterization of dimethacrylates containing quaternary ammonium functionalities for dental applications.

OBJECTIVES: The widespread incidence of recurrent caries highlights the need for improved dental restorative materials. The objective of this study was to synthesize low viscosity ionic dimethacrylate monomers (IDMAs) that contain quaternary ammoniums groups (antimicrobial functionalities) and are compatible with existing dental dimethacrylate-based monomers. Such monomers have the potential to copolymerize with other methacrylate monomers and produce antibacterial polymers. METHODS: Two monomers (IDMA-1 and IDMA-2) were synthesized using the Menschutkin reaction and incorporated at 0-30% (by mass) into a 1:1 (by mass) bisphenol A glycerolate dimethacrylate (BisGMA):triethylene glycol dimethacrylate (TEGDMA) resin. Resin viscosity was quantified using rheology, and polymer degree of conversion (DC) and surface charge density were measured using Fourier transform infrared spectroscopy (FTIR) and fluorescein binding, respectively. Effects of IDMA-1 on initial attachment of Streptococcus mutans and on viability and metabolic activity (via reductase enzymes) of RAW 264.7 macrophage-like cells were quantified. RESULTS: IDMA-1 and IDMA-2 were prepared and characterized. IDMA-1 was miscible with BisGMA:TEGDMA and slightly increased the resin viscosity and DC. As expected, polymeric surface charge density increased with increasing IDMA-1. Incorporation of 10% IDMA-1 into BisGMA:TEGDMA reduced bacterial colonization without affecting viability or metabolic activity of mammalian cells. Increasing IDMA-1 up to 30% had no additional effect on bacterial coverage, but ≥20% IDMA-1 significantly reduced macrophage density, viability, and metabolic activity. Leachables from polymers containing IDMA-1 were not cytotoxic. SIGNIFICANCE: The Menschutkin reaction provides a facile, convenient means to synthesize new monomers with quaternary ammonium groups for dental and medical applications.

Antibacterial amorphous calcium phosphate nanocomposites with a quaternary ammonium dimethacrylate and silver nanoparticles.

OBJECTIVES: Calcium and phosphate ion-releasing resin composites are promising for remineralization. However, there has been no report on incorporating antibacterial agents to these composites. The objective of this study was to develop antibacterial and mechanically strong nanocomposites incorporating a quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP). METHODS: The QADM, bis(2-methacryloyloxyethyl) dimethylammonium bromide (ionic dimethacrylate-1), was synthesized from 2-(N,N-dimethylamino)ethyl methacrylate and 2-bromoethyl methacrylate. NAg was synthesized by dissolving Ag 2-ethylhexanoate salt in 2-(tert-butylamino)ethyl methacrylate. Mechanical properties were measured in three-point flexure with bars of 2 mm×2 mm×25 mm (n=6). Composite disks (diameter=9 mm, thickness=2 mm) were inoculated with Streptococcus mutans. The metabolic activity and lactic acid production of biofilms were measured (n=6). Two commercial composites were used as controls. RESULTS: Flexural strength and elastic modulus of NACP+QADM, NACP+NAg, and NACP+QADM+NAg matched those of commercial composites with no antibacterial property (p>0.1). The NACP+QADM+NAg composite decreased the titer counts of adherent S. mutans biofilms by an order of magnitude, compared to the commercial composites (p<0.05). The metabolic activity and lactic acid production of biofilms on NACP+QADM+NAg composite were much less than those on commercial composites (p<0.05). Combining QADM and NAg rendered the nanocomposite more strongly antibacterial than either agent alone (p<0.05). SIGNIFICANCE: QADM and NAg were incorporated into calcium phosphate composite for the first time. NACP+QADM+NAg was strongly antibacterial and greatly reduced the titer counts, metabolic activity, and acid production of S. mutans biofilms, while possessing mechanical properties similar to commercial composites. These nanocomposites are promising to have the double benefits of remineralization and antibacterial capabilities to inhibit dental caries.