Designing Multiagent Dental Materials for Enhanced Resistance to

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Designing multi#agent dental materials for enhanced resistance to biofilm damage at the bonded interface Mary Anne Sampaio de Melo, Santiago Orrego, Michael D. Weir, Huakun H.K. Xu, and Dwayne D. Arola ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01923 • Publication Date (Web): 15 Apr 2016 Downloaded from http://pubs.acs.org on April 19, 2016

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ACS Applied Materials & Interfaces 1

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Designing multi‐‐agent dental materials for enhanced resistance to biofilm damage at the bonded interface Mary Anne Melo1,#, Santiago Orrego2#, Michael D. Weir3, Huakun H. K. Xu3, Dwayne D. Arola4 1

Division of Operative Dentistry, Department of General Dentistry, School of Dentistry,

University of Maryland Baltimore, Baltimore, MD, USA 2

Department of Mechanical Engineering, Johns Hopkins University, Whiting School of

Engineering, Baltimore, MD, USA 3

Biomaterials and Tissue Engineering Division, Department of Endodontics,

Prosthodontics and Periodontics, School of Dentistry, University of Maryland Baltimore, Baltimore, MD, USA 4

Department of Materials Science & Engineering, College of Engineering, University of

Washington, Seattle, WA, USA #

Shared equally in the development of this manuscript and are co-first authors.

Correspondence: Mary Anne S. Melo (Email: [email protected]), Division of Operative Dentistry, Department of General Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA MeSH Keywords: Anti-Bacterial Agents, Ammonium Compounds, Biofilms, Dentin, Nanoparticles, Dental Caries, Amorphous calcium phosphate.

1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces

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ABSTRACT The oral environment is considered to be an asperous environment for restored tooth structure. Recurrent dental caries is a common cause of failure of tooth-colored restorations. Bacterial acids, microleakage, and cyclic stresses can lead to deterioration of the polymeric resin–tooth bonded interface. Research on the incorporation of cuttingedge anticaries agents for the design of new, long-lasting, bioactive resin-based dental materials is demanding and provoking work. Released antibacterial agents as silver nanoparticles

(NAg),

non-released

antibacterial

macromolecules

(DMAHDM,

dimethylaminohexadecyl methacrylate) and released acid neutralizer amorphous calcium phosphate nanoparticles (NACP) haveshown potential as individual and dual anticaries approaches. In this study, these agents were synthesized, and a prospective combination was incorporated into all the dental materials required to perform a composite restoration: dental primer, adhesive, and composite. We focused on combining different dental materials loaded with multi-agents to improve the durability of the complex dental bonding interface. A combined effect of bacterial acid attack and fatigue on the bonding interface simulated the harsh oral environment. Human salivaderived oral biofilm was grown on each sample prior to the cyclic loading. The oral biofilm viability during the fatigue performance was monitored by the live-dead assay. Damage of the samples that developed during the test was quantified from the fatigue life distributions. Results indicate that the resultant multi-agent dental composite materials were able to reduce the acidic impact of the oral biofilm, thereby improving the strength and resistance to fatigue failure of the dentin-resin bonded interface. In summary, this study shows that dental restorative materials containing multiple therapeutic agents of different chemical characteristics can be beneficial towards improving resistance to mechanical and acidic challenges in oral environments.


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1. Introduction Over the past decades, polymer resin-based dental materials have been used widely in restorative dentistry for composite restorations due to their versatile and aesthetic properties.1 Due to their polymeric matrix, they are potential sources of carbon and energy for microorganisms including oral bacteria and fungi.2 In several ways, biodegradation by acid and mechanical fatigue by mastication are the most critical factors in the long-term clinical performance of composite restorations.3-5 The major reasons for failure are recurrent caries and restoration fracture. These threats support the development of dental restorative materials that mimic the mechanical behavior of the natural tooth, while also resisting chemical attack by lactic acid from oral bacteria and external sources.4 Although in materials science the term composite is given to any material consisting of two different constituents, in restorative dentistry this term has been applied to a particular group of materials universally used for tooth-colored restorations: dental primers, adhesives, and resin composites. They are composite materials consisting of mostly inorganic fillers and additives that are bound together with a polymer matrix and are responsible for adhesion to the tooth structure and reconstruction of missing tissue.1 Biofilm accumulation and cariogenic challenge are conditions that play a critical role in the progression of dental caries.5 Acid-producing oral bacteria known as cariogenic bacteria can infiltrate the margins promoting demineralization of the tooth6 and degradation of the resin-based dental composites, the bonding adhesives and the interface.7 Since oral bacteria may attack the bonding materials, recent strategies have been developed to fight bacteria and resist the formation of biofilms on the dental materials.5 Adding nanoparticles of silver and the use of antimicrobial monomers have been highlighted as potent antibacterial approaches. 8 Silver nanoparticles (NAg) exhibit a unique set of properties with advantage of greater surface area-to-volume ratio. This feature results in a higher proportion of material exposed for the potential reaction, which requires a lower concentration of nanosilver to reach the same antibacterial effect of silver on the micro scale. For dental materials, it can achieve a strong antibacterial effect while using a relatively low filler



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level of NAg in the resin. A low filler level of NAg in the resin is desirable to maintain good mechanical properties and esthetics, as a high NAg concentration in the resin could compromise its color. Broad optical properties depending on the nanoparticle diameter, the refractive index near the nanoparticle surface and aggregation are also beneficial features of this material. In addition, ease of synthesis and chemical reactiveness with simplified functionalization has facilitated the integration of nanosilver into the dental materials field.9 Dental nanocomposites, primers, and adhesive containing Ag nanoparticles that yield a potent antibacterial activity by this releasekilling approach have been recently reported.10 Very recently, a series of novel quaternary ammonium monomers (QAM), including the new dimethylaminohexadecyl methacrylate (DMAHDM) presenting an alkyl chain length of 16-carbon, were synthesized and incorporated into resin-based dental material formulations including primer, adhesive and composite as another antibacterial strategy.11

Similar

to

commercially

available

dental

adhesive,

12-

methacryloyloxydodecylpyridinium bromide (MDPB), these monomers can form covalent bonds with the polymer matrix and be immobilized in the resin-based materials, characterizing the non-released, contact-killing approach of this agent.12 Biologically, NAg and DMAHDM show highly effective antimicrobial properties against cariogenic oral bacteria via their capacity for killing bacteria by controlled chemical-release and by contact, respectively. Used together, they may offer a potent strategy to address the persistent challenge of obtaining effective and long-lasting antibacterial resin-based materials without compromising the quality and longevity of the bonded interface.13 There have also been active investigations in the design of dental filling materials with the addition of amorphous calcium phosphate nanofillers (NACP) for increased release of calcium and phosphate ions for remineralization.14 NACP possesse acid neutralization capacity and appear to reduce moderately bacteria growth. The calcium and phosphate ion releases are not expected to have antibacterial properties, but it demonstrates that the NACP rapidly increase the pH of a lactic acid. 15 A combined multi-modality treatment with agents of different therapeutic effects has been of particular interest in the dental caries management/dental materials field


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because prevalent diseases such as dental caries cannot be treated with only one type of anticaries therapy due to its pathological complexities. A combination of adequately selected agents can bring synergistic or additive effects, even at low dose, especially with the increased release capabilities of nanomaterials. Thus, this innovative strategy to materials design may revolutionize the traditional infrastructure of dental materials. Restorative materials that possess antibacterial properties and inhibit bacterial growth on the restoration could be advantageous to prevent secondary caries as an additional strategy against dental caries.16 The influence of these materials on the interface durability and in response to subcritical cyclic loading is important to predicting their clinical behavior. In this study, we investigate the durability of resin–dentin interfaces involving multi-agent dental composite materials when exposed to Oral multispecies biofilm challenge under cycling conditions that produce fluctuating stresses. The cyclic nature of loading may culminate in fatigue failure, the most clinically relevant simulated in the vitro scenario. It was hypothesized that: (1) incorporating multiagents into a set of dental restorative

materials would not compromise the initial

ﬂexural strength, and ﬂexural fatigue strength of the bonded dentin-composite interface; and (2) multi-agents containing dental materials would be able to inhibit biofilm formation and protect the interface from acidic degradation promoted by bacterial acids. This study is, to the best of our knowledge, the first example of functional evaluation of multi-agent dental composite materials considering the bonding interface. Figure 1 illustrates the general anticaries (anti-dental caries) strategy using multi-agents dental composite materials.



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Figure 1. General anticaries strategy using multi-agents containing dental composite materials. Nanosized particles facilitate the release of both nanosilver (NAg) and amorphous calcium phosphate (NACP) that allowing their diffusion in a surrounding environment. NAg acts as a released antibacterial agent and NACP acts as an acid neutralizer. The new dimethylaminohexadecyl methacrylate (DMAHDM) can inactivate caries-related bacteria by contact without leaching from dental materials. Note that DMAHDM is non-volatile, chemically stable and sustain long-term antibacterial activity.

2. MATERIALS & METHODS 2.1 Chemicals and Reagents. Commercially available materials 2-(tert-butylamino) ethyl methacrylate (TBAEMA); 2-(dimethylamino) ethyl methacrylate (DMAEMA), Camphorquinone

(CQ);

ethyl

4-(diamethylamino)

benzoate

(4E);

3

methacryloxypropyltrimethoxysilane and 2% n-propylamine were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and used without further purification. Bisphenol A glycidyl dimethacrylate (Bis-GMA) and triethylene glycol dimethacrylate (TEGDMA) were obtained from Esstech (Essington, PA, USA) and used as received. Silver 2ethylhexanoate powder was acquired from Strem (New Buryport, MA, USA). 1-


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bromohexadecane was supplied by BHD (TCI America, Portland, OR). Barium boroaluminosilicate glass particles were obtained from Caulk/ Dentsply (Milford, DE, USA). Calcium carbonate (CaCO3) and dicalcium phosphate anhydrous (CaHPO4) were provided, respectively, by Fisher (Fair Lawn, NJ) and Baker Chemical Co. (Phillipsburg, NJ). 2.2 Synthesis of nanosilver. In situ preparation of silver nanoparticles using a dimetacrylate polymer matrix as the stabilizing agent was used in this study. Briefly, Silver 2-ethylhexanoate powder was dissolved in TBAEMA at 1: 10 ratio for silver salt / TBAEMA following previous studies.17,18 The reaction was allowed to proceed for 30 min under continuous stirring at a speed of 200 rpm until the color of the mixture was completely clear. TBAEMA was used as dispersant because it improves the solubility by forming Ag-N coordinate bonds with Ag ions, thereby facilitating the Ag salt to dissolve in the resin solution.19 In addition, it contains reactive methacrylate groups and can be chemically incorporated into a resin upon photopolymerization. This method allowed a suitable dispersion of NAg particles with a mean size of 2.7 nm into the resin.17 The size and morphology of the silver nanoparticles dispersed in TBAEMA were investigated by high-resolution transmission electron microscopy (HR-TEM) (FEI Company, Hillsboro, OR) at an operating voltage of 120 kV. 2.3 Synthesis of DMAHDM. Dimethylaminohexadecyl methacrylate (DMAHDM) showed an alkyl chain length of 16 carbon and was synthesized through a modified Menschutkin reaction method, which used a tertiary amine group to react with an organohalide,

following

previous

studies.20

In

detail,

2-(dimethylamino)

ethyl

methacrylate (DMAEMA) and 10 mmol of 1- bromohexadecane were combined in a 20 mL tared scintillation vial equipped with a magnetic stir bar. The reaction was carried out for 24h at 70°C. After the reaction was complete, the ethanol solvent was removed via evaporation, yielding > 95% DMAHDM as a limpid, crystal clear, and fluidic final product. 2.4 Synthesis of NACP. A spray-drying technique was used to fabricate NACP as previously described.21 In summary, calcium carbonate (CaCO3) and dicalcium phosphate anhydrous (CaHPO4,) were dissolved in an acetic acid solution to obtain final Ca and PO4 ionic concentrations of 8 mmol/L and 5.333 mmol/L, respectively. The



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resulting 1.5 ratio is similar to micro sized Ca/P molar ratio. The process was initiated by atomizing/spraying of a Ca/P suspension of micro-sized droplets followed by a drying process in a heated chamber, resulting in the production of solid particles. An electrostatic precipitator (Air Quality, Minneapolis, MN) was used to collect the dried particles. This process produced NACP with a mean particle size of 116 nm, as measured in a previous study.22 TEM was performed to examine the NACP in the resin. 2.5 Multi-agent loading-dental materials. The antibacterial agents DMAHDM and NAg wereincorporated into a commercially available dental bonding system (Scotchbond Multi-Purpose, 3M, St. Paul, MN, USA).14 The antibacterial agents DMAHDM and NAg carried on the Ag-TBAEMA concentrated solution were incorporated into both components at mass fractions of 5% and 0.1%, respectively.23 A dental composite was formulated to include the antibacterial agents as well as the NACP. The resin matrix content was 50:50 wt % Bis-GMA: TEGDMA with 0.2 wt % CQ and 0.8 wt % 4E as the photoinitiator system. The DMAHDM and NAg were incorporated at mass fractions of 5% and 0.1% into BisGMA-TEGMA resin. The filler content

represents

boroaluminosilicate

65% glass

mass

fraction

particles

of

the

(Ø1.4µm)

dental

composite.

silanized

with

Barium 4%-3

methacryloxypropyltrimethoxysilane and 2% n-propylamine were used as filler.21 NACP and glass particles were mixed into the resin to reach the mass fractions of 30% NACP and 35% glass fillers, rendering a workable viscous dental composite. 2.6 Preparation of the complex bonding tooth-composite interface. The Ethical Research Committee of University of Maryland Baltimore County approved the use of 45 human third molars in this study (protocol # (Y04DA23151). Rectangular beams of mid-coronal dentin were sectioned with a cross section of 2x2 mm2 and length of 12 mm according to Figure 2A. Following a previous study 24, twin bonded resin-dentin interface specimens were prepared. The dentin slabs were fixed in a customized metal mold with internal measures (length x width x depth) of 12 x 6 x 2 mm and introduced with top occlusal section of slabs oriented upward. This orientation resulted in dentinal tubules oriented parallel to the interface, which is similar to the orientation when a dental cavity is prepared. The dental materials without multi-agents were used as a reference (control). The bonding procedures were performed in the etch-and-rinse mode. First, the


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proximal area (side of slabs perpendicular to long axis of the tooth) were etched with 35% phosphoric acid for 15 s, rinsed with water for 15 s and then lightly dried with a delicate paper wiper, leaving the dentin moist and suitable for bonding. Second, the primer component was applied and rubbed for 20s. The removal of excess solvent element was promoted by gently air-dry for 5s. Third, the adhesive component was sequentially applied over the primed dentin surface and light-cured using a quartztungsten-halogen unit with peak emission wavelength= 460 nm and output intensity of 600 mW/cm2 (Optilux 501, Kerr, Danbury, CT, USA) for 10s. A single 1mm-thick layer of composite (experimental or control) was placed on the bonded dentin and light-cured for 40s. The composites were added by 1mm-increments to fill the mold completely. Both sides of the samples were light-cured for 1 min. After curing, the molded blocks were sectioned into twin interface specimens (Fig. 2B) and then immersed in water and agitated for 1 h to remove any uncured monomer.13 The samples were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC).



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Figure 2. Schematic diagrams for specimen preparation: (A) The section of coronal dentin to obtain the dentin beam; (B) schematic of placement of dentin beam in the mold and bonding procedures: primer and adhesive are spread onto the dentin and light-cured. The resin composite is applied by 1-mm increments on both sides of the dentin and, then, light-cured; the molded section was cut following the dashed lines to obtain two interface samples; (C) The bonding performance and durability was evaluated by quasistatic and cyclic loading using the four-point flexural configuration.


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2.7 Oral bacterial-induced biodegradation model. The dynamic human salivabased biofilm model was established for cariogenic biofilm growth over the samples. This model has been shown adequate to simulate the complex actions and diversification of dental plaque in vivo.25 This study was approved by the University of Maryland, and an all-embracing description of this model has been published previously.26 In short, human saliva was collected from adult volunteers used as inoculum with 1:50 final dilution. The nutrient medium used for biofilm growth was enriched with 0.2 % sucrose according to previous descriptions.10,14 The inoculum was cultured in an incubator (5% CO2, 37 °C) overnight. The oral biofilms were grown for up to 7 days under internment cariogenic condition. Development of microcosm biofilms on the samples was observed by live/dead assay according to a previously reported protocol.23 Counter samples were immersed in Hank’s balanced salt solution (HBSS), a neutral pH solution, at 37°C for 7 d to evaluate the effect of acidic biofilm attack. Another set of samples were also immersed in HBSS solution for 1 d to be referred as baseline data. 2.8 Mechanical quasi-static Testing. The specimens were subjected to quasi-static loading under a 4-point flexure configuration (Figure 2C) using a universal testing system (Instron E-1000, Norwood, MA, USA) with loading rate= 0.06 mm/min. The flexural strength of the specimens was calculated 27, according to 3Pl/bh2, where l is the distance from the interior and exterior supports (3 mm), P is the measured load, b the width and h the thickness of the sample. 2.9 Fatigue performance Testing. Fatigue testing was initiated using a maximum cyclic stress of approximately 90% of the flexural strength identified from the quasistatic experiments. Cyclic loading was conducted using frequency =4 Hz and stress ratio = 0.1. For successive specimens, the maximum cyclic load was decreased in increments until reaching a flexural stress at which specimens did not fail within 1.2 million cycles. The stress-life (S–N) fatigue distribution was evaluated by plotting the cyclic stress amplitude against the number of cycles to failure. The fatigue life distribution in each group was fitted using non-linear regression according to a Basquintype model

28

σ = A (N)B. Selected samples were evaluated using scanning electron

microscopy (FEI, Nova NanoSEM 450, Hillsboro, OR) in secondary electron imaging



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mode with different magnifications at an accelerating voltage of 20 kV Before evaluation, the specimens were dehydrated in a standard ascending ethanol series (70100%) and then sputtered with gold palladium. 2.10 Statistical Analysis. All data are expressed as the means ± standard deviations (SD; n=15). Statistical comparisons were performed using one-way analysis of variance (ANOVA). The fatigue life distributions were compared using the Wilcoxon Rank Sum Test with α = 0.05 as the level of significance for all tests. Considering the sample size n=15, the approximation formula was also used to perform the test of the hypothesis. The flexure strengths were compared using a one-way ANOVA and Tukey’s HSD post hoc analysis.

3. RESULTS AND DISCUSSION The anticaries agents were synthesized, incorporated in three different dental materials, reproduced in a dentin-restoration margin as used clinically and submitted concurrently to comparative testing versus the materials with regular formulation under the most relevant oral challenges: bacterial/ masticatory. The silver nanoparticles were formed in the resin by simultaneous reduction of the silver salt and photopolymerization of the dimethacrylates. The TEM images in Figure 3 illustrate the morphology of nanosized agents NAg and NACP respectively. The silver nanoparticles are well scattered in the resin matrix with minimal appearance of nanoparticle aggregates (Figure 3A). The amorphous calcium phosphate nanoparticles had an average size of 112 nm (Figure 3B) and some agglomerates up to 200nm in size. The antibacterial agent, dimethylaminohexadecyl methacrylate (DMAHDM) was also synthesized. Characterization of the reaction product was confirmed via FTIR and NMR as previously reported.20


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Figure 3. A1: Representative TEM micrograph of the size and dispersion of silver nanoparticles in the resin matrix, A2: Solution of nanosilver dispersed in 2-(tert-butylamino) ethyl methacrylate (TBAEMA) as medium; B1: Representative TEM micrograph of ACP nanoparticles (NACP) synthesized using the spraydrying technique and collected via the electrostatic precipitator and B2: Schematic representation of amorphous calcium phosphate.



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Dental caries is a prevalent disease that cannot be treated with only one type of anticaries therapy due to its pathological complexities. Prospective multi-agent systems can be used to solve problems that are difficult or impossible for an individual antibacterial agent to solve. The antibacterial properties of both agents used in this study have been explored using different concentrations and a variety of dental materials.29,30 Previous studies have incorporated quaternary ammonium monomers into dental primer and adhesive.14 The antimicrobial mechanism of these monomers is purportedly derived from the interaction of positively charged sites of quaternary ammonium monomers resin to negatively charged bacterial membranes sites. This electrostatic interaction promotes unbalanced osmosis, which leads to bacteria death.31 DMAHDM was a recently developed quaternary ammonium monomer with an alkyl chain length of 16 carbon, showing a strong antibacterial effect. DMAHDM is a monomethacrylate, with reactive groups on one end of the molecule. This structure allows its incorporation into the resin with less negative impact on the mechanical properties.32 Recent work showed the significant antibacterial effect of composites containing DMAHDM.33 The lactic acid production was dramatically reduced.34 Lactic acid represents 70% of the organic acids present in the human oral biofilm. The antibacterial properties of NAg are associated with liberation of Ag+ ions to the bacteria environment where the small particle size facilitates penetration through the cell membranes to affect intracellular processes from inside.35,36 Previous studies also showed that NAg-containing dental adhesive and composites can have long-term antibacterial effects.30 The remineralizing agent, NACP, expresses an indirect effect.18 Previous studies showed that NACP composite releases calcium and phosphate ions that promote remineralization and neutralize acid challenges by increasing the cariogenic pH (

Designing Multiagent Dental Materials for Enhanced Resistance to

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