Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions

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Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema

Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions Xianju Xie a,b , Lin Wang b,c , Dan Xing b,d , Ke Zhang a,b , Michael D. Weir b , Huaibing Liu e , Yuxing Bai a,∗∗ , Hockin H.K. Xu b,f,g,∗ a

Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China Department of Endodontics, Periodontics and Prosthodontics, University of Maryland Dental School, Baltimore, MD 21201, USA c VIP Integrated Department, Stomatological Hospital of Jilin University, Changchun, China d Department of Dentistry, China Rehabilitation Research Center, Beijing, China e L.D. Caulk Division, Dentsply Sirona Restorative, Milford, DE 19963, USA f Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA g Department of Mechanical Engineering, University of Maryland, Baltimore County, MD 21250, USA b

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective. A new adhesive containing nanoparticles of amorphous calcium phosphate (NACP)

Received 10 December 2016

with calcium (Ca) and phosphate (P) ion rechargeability was recently developed; however,

Accepted 9 March 2017

it was not antibacterial. The objectives of this study were to: (1) develop a novel adhesive

Available online xxx

with triple benefits of Ca and P ion recharge, protein-repellent and antibacterial functions

Keywords:

phorylcholine (MPC); and (2) investigate dentin bond strength, protein adsorption, Ca and P

via dimethylaminohexadecyl methacrylate (DMAHDM) and 2-methacryloyloxyethyl phosDental adhesive

ion concentration, microcosm biofilm response and pH properties.

Calcium phosphate nanoparticles

Methods. MPC, DMAHDM and NACP were mixed into a resin consisting of ethoxylated

Antibacterial

bisphenol A dimethacrylate (EBPADMA), pyromellitic glycerol dimethacrylate (PMGDM),

Protein repellent

2-hydroxyethyl methacrylate (HEMA) and bisphenol A glycidyl dimethacrylate (BisGMA).

Human saliva microcosm biofilm

Protein adsorption was measured using a micro bicinchoninic acid method. A human saliva

Caries inhibition

microcosm biofilm model was tested on resins. Colony-forming units (CFU), live/dead assay, metabolic activity, Ca and P ion concentration and biofilm culture medium pH were determined. Results. The adhesive with 5% MPC + 5% DMAHDM + 30% NACP inhibited biofilm growth, reducing biofilm CFU by 4 log, compared to control (p < 0.05). Dentin shear bond strengths were similar (p > 0.1). Biofilm medium became a Ca and P ion reservoir having ion concentration increasing with NACP filler level. The adhesive with 5% MPC + 5% DMAHDM + 30% NACP maintained a safe pH > 6, while commercial adhesive had a cariogenic pH of 4.

∗ Corresponding author at: Biomaterials and Tissue Engineering Division, Department of Endodontics, Periodontics and Prosthodontics, University of Maryland Dental School, Baltimore, MD 21201, USA. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Bai), [email protected] (H.H.K. Xu). http://dx.doi.org/10.1016/j.dental.2017.03.002 0109-5641/© 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Xie X, et al. Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.002

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Significance. The new adhesive with triple benefits of Ca and P ion recharge, protein-repellent and antibacterial functions substantially reduced biofilm growth, reducing biofilm CFU by 4 orders of magnitude, and yielding a much higher pH than commercial adhesive. This novel adhesive is promising to protect tooth structures from biofilm acids. The method of using NACP, MPC and DMAHDM is promising for application to other dental materials to combat caries. © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Because of their esthetics and direct-filling capability, composites and adhesives are widely used to restore tooth cavities [1–4]. However, dental resins were shown to accumulate more biofilms and plaques than amalgams and glass ionomer restorations [5]. The acid production by biofilms can decrease the local pH to a cariogenic range of 5–4, which could lead to tooth structure demineralization and secondary caries formation [6]. Recurrent caries is the main reason for restoration failures, and replacement of the failed restorations accounts for 50–70% of all restorations performed [7]. The toothrestoration bonded interface has been identified as the weak link [7,8]. A strong and durable adhesion to dental hard tissues is a key factor in the success of the restoration [9–12]. The mechanism of dentin bonding involves the infiltration of adhesive monomers into a demineralized dentin collagen matrix and the formation of the hybrid layer (HL) [9–12]. The adhesive is not only a connection between the tooth structure and the restorative composite, it also serves as a barrier to protect the demineralized collagen scaffold from the acidic and enzymatic attacks of the oral bacteria, enzymes and fluids [10,13]. Clinically, residual bacteria could exist in the prepared tooth cavity. In addition, microleakage could allow bacteria to invade the tooth-restoration interfaces. Therefore, it is desirable for the adhesive to be antibacterial to inhibit recurrent caries at the margins [14–17]. For this purpose, quaternary ammonium methacrylates (QAMs) were incorporated into dental resins to achieve antibacterial activities to combat biofilm growth and acid production [14,18]. Resins containing 12-methacryloyloxydodecylpyridinium bromide (MDPB) had a potent antibacterial function [14]. Recently, a new dimethylaminohexadecyl methacrylate (DMAHDM) was synthesized and incorporated into composites and bonding agents, achieving strong inhibition against oral biofilms [19]. In addition, oral bacteria attach to dental resins through a layer of adsorbed salivary proteins on the resin surface, which is a prerequisite for bacterial adhesion and biofilm growth [20]. Therefore, rendering the resin protein-repellent would help to repel bacteria attachment. Indeed, studies showed that a protein-repellent agent 2-methacryloyloxyethyl phosphorylcholine (MPC) could be incorporated into resins to repel proteins and bacteria [21]. Another approach for caries-inhibition is to incorporate calcium phosphate (CaP) particles into resins to promote remineralization and suppress demineralization [22–24]. Adhesives containing CaP particles could remineralize the remnants of tooth lesions in the cavity as well as the acid-

etched dentin, and hence are promising to improve the longevity of the restorations [25,26]. Recently, bonding agents containing nanoparticles of amorphous calcium phosphate (NACP) were developed [27,28]. These bonding agents could release high levels of Ca and P ions to induce remineralization and combat caries [27,28]. The addition of NACP did not negatively affect the dentin bond strength [27,28]. Due to their small particle sizes, the NACP readily flowed with bonding agent into dentinal tubules to form resin tags [27]. The NACP adhesive was “smart” because it could substantially increase the Ca and P ion release at a low cariogenic pH when these ions would be most needed to combat caries [28]. For both total-etch and self-etch bonding systems, the bonding stability is limited by the degradation of the HL [13,29,30]. The Ca and P ion release from adhesive may be highly beneficial and can serve as seed crystals to facilitate remineralization in HL and at the tooth-restoration margins [13,25]. Thus, the CaP adhesive may protect the exposed collagen within the bonded interface and improve the bonding stability and durability [13,31]. Therefore, the NACP-containing adhesive with Ca and P ion release could be meritorious in protecting the weak link of the tooth restoration. However, the Ca and P ion release from CaP resins lasted for only a couple of months and then diminished over time [22,32,33]. Recently, a rechargeable CaP resin was developed with long-term Ca and P ion release for the first time [34]. Its Ca and P ion recharge and re-release were sustained, showing no decrease in ion release with increasing the number of recharge/re-release cycles. However, while NACP resins have remineralization and acid neutralizing capabilities, they are not antibacterial [27]. To date, there has been no report on the incorporation of DMAHDM and MPC into the rechargeable NACP adhesive to achieve both protein-repellent, antibacterial and long-term CaP ion recharge and re-release capabilities. Therefore, the objectives of this study were to develop a novel bioactive adhesive for caries-inhibition by incorporating DMAHDM and MPC into a rechargeable NACP adhesive, and to investigate the dentin bond strength, protein adsorption, biofilm response and pH properties. It was hypothesized that: (1) Incorporating MPC, DMAHDM and NACP into the adhesive would yield dentin bond strength similar to a commercial control adhesive; (2) Incorporating MPC, DMAHDM into the rechargeable NACP adhesive would greatly decrease protein-adsorption, biofilm growth and viability; (3) Increasing NACP filler level in the resin would increase the pH and Ca and P ion concentrations in the biofilm culture medium.

Please cite this article in press as: Xie X, et al. Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.002

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2.

Materials and methods

2.1.

Development of bioactive bonding agents

The parent primer contained pyromellitic glycerol dimethacrylate (PMGDM) (Esstech, Essington, PA) and 2hydroxyethyl methacrylate (HEMA) (Esstech) at a mass ratio 3.3/1, with 50% acetone solvent (all mass fractions) [35,36]. This primer is referred to as “PM primer”. The adhesive consisted of 44.5% of PMGDM, 39.5% of ethoxylated bisphenol A dimethacrylate (EBPADMA) (Sigma-Aldrich, St, Louis, MO), 10% of HEMA and 5% of bisphenol A glycidyl dimethacrylate (BisGMA) (Esstech) [34]. 1% of phenylbis (2,4,6trimethylbenzoyl) phosphine oxide (Sigma–Aldrich) was added to enable light cure [34]. PMGDM and EBPADMA were used because they had cytotoxicity similar to other dental dimethacrylates, but significantly less than the cytotoxicity of BisGMA [36]. In addition, PMGDM is an acidic adhesive monomer [37], and can chelate with calcium ions from the recharging solution to render the resin rechargeable. HEMA was added to improve the flowablity and hydrophilicity, following a previous study [23]. BisGMA was added because it could improve the cross-linkage of monomers and the bonding properties of the adhesive [38]. This parent adhesive is referred to as “PEHB”. DMAHDM was synthesized using a modified Menschutkin reaction where a tertiary amine group was reacted with an organo-halide [35]. A benefit of this reaction is that there action products are generated at virtually quantitative amounts and require minimal purification. Briefly, 10 mmol of 2-(dimethylamino) ethyl methacrylate (DMAEMA, Sigma-Aldrich) and 10 mmol of 1-bromohexadecane (BHD, TCI America, Portland, OR) were combined with 3 g of ethanolin a 20 mL scintillation vial. The vial was stirred at 70 ◦ C for 24 h. The solvent was then removed via evaporation, yielding DMAHDM as a clear, colorless, and viscous liquid [35]. To make the primer antibacterial, DMAHDM was mixed at DMAHDM/(PM primer + DMAHDM) = 5% by mass. The 5% was selected following a previous study [19]. Similarly, to make the adhesive antibacterial, DMAHDM was mixed into adhesive at DMAHDM/(PEHB + DMAHDM) = 5%. MPC was obtained commercially (Sigma-Aldrich) which was synthesized as previously described [39]. The PM primer was first mixed with DMAHDM as described above. Then 5% by mass of MPC was mixed with the PM-DMAHDM primer. Higher MPC mass fractions were not used due to a decrease in dentin bond strength when combined with DMAHDM in preliminary study. Similarly, 5% MPC was incorporated into the PEHB-DMAHDM adhesive. NACP [Ca3 (PO4 )2 ] were synthesized via a spry-drying technique as previously described [33,40]. Briefly, calcium carbonate and dicalcium phosphate anhydrous were dissolved into an acetic acid solution. The concentrations of Ca and P ions were 8 mmol/L and 5.333 mmol/L, respectively. The solution was sprayed into a heated chamber to evaporate the water and volatile acid. The dried NACP powders were collected using an electrostatic precipitator. This yielded NACP with a mean particle size of 116 nm [33]. NACP were incorporated into the adhesive at 0%, 20%, 30% and 40% filler mass fractions, fol-

3

lowing previous studies [27,41]. NACP levels greater than 40% were not used due to a decrease in dentin bond strength in preliminary study. A commercial bonding agent Prime & Bond NT (Dentsply, Milford, DE) served as a comparative control (denoted “commercial control”). According to the manufacturer, NT was a total-etching one-bottle bonding system and contained 30% typical methacrylates, <10% methyl methacrylate, and 60% acetone. NT was combined with a self-cure activator (SCA) at 1:1 ratio to enable dual-cure. Six groups were tested: (1) Commercial Prime & Bond NT (denoted “commercial control”). (2) PM primer and PEHB adhesive, no MPC, no DMAHDM, no NACP (denoted “Experimental control”). (3) Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, no NACP (denoted “MPC+DMAHDM”). (4) Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 20% NACP (denoted “MPC+DMAHDM+20NACP”). (5) Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 30% NACP (denoted “MPC+DMAHDM+30NACP”). (6) Primer contained 5% MPC and 5% DMAHDM. Adhesive contained 5% MPC and 5% DMAHDM, filled with 40% NACP (denoted “MPC+DMAHDM+40NACP”).

2.2.

Dentin shear bond strength testing

Extracted human third molars were stored in 0.01% thymol solution at 4 ◦ C. Each tooth was cut perpendicularly to the long axis of the tooth to expose the mid-coronal dentin using a low speed diamond saw (Isomet, Buehler, Lake Bluff, IL). The dentin surface was polished with 600-grit SiC paper. Then the dentin surface was etched with 37% phosphoric acid gel for 15 s and rinsed with water. A primer was applied with a brushtipped applicator for 15 s, and the dentin was gently blown with air for 5 s to remove the solvent. An adhesive was then applied and light-cured for 10 s with an Optilux curing unit (VCL 401, Demeron Kerr, Danbury, CT). A stainless-steel cylindrical mold (inner diameter = 4 mm, thickness = 1.5 mm) was placed on the adhesive-treated dentin surface. A composite (TPH, Caulk/Dentsply, Milford, DE) was filled into the mold and light-cured for 60 s. The bonded specimens were stored in distilled water at 37 ◦ C for 24 h. A chisel on a Universal Testing Machine (MTS, Eden Prairie, MN) was aligned to be parallel to the composite-dentin interface [27,28]. Load was applied at a cross-head speed of 0.5 mm/min until the bond failed. Dentin shear bond strength = 4P/(␲d2 ), where P is the load at failure, and d is the diameter of the composite [27,28]. Ten teeth were tested for each group.

2.3. Measurement of protein adsorption onto resin surface The cover of a sterile 96-well plate (Costar, Corning Inc., Corning, NY) was used as molds to fabricate resin disks following a previous study [21]. Briefly, 10 ␮L of a primer was placed in the bottom of each dent of the 96-well plate. After dry-

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ing with a stream of air, 20 ␮L of adhesive was applied to the dent and photo-polymerized for 30 s (Optilux), using a Mylar strip covering to obtain a disk of approximately 8 mm in diameter and 0.5 mm in thickness. The cured resin disks were immersed in 200 mL of distilled water and magnetically stirred with a bar at a speed of 100 rpm for 1 h to remove any uncured monomers [42]. The disks were then sterilized with ethylene oxide (Anprolene AN 74i, Andersen, Haw River, NC) and de-gassed for 3 days [21]. Protein adsorption on resin disks was determined using a micro bicinchoninic acid (BCA) method [43,44]. Each disk was immersed in phosphate buffered saline (PBS) for 2 h, and then immersed in 4.5 g/L bovine serum albumin (BSA, SigmaAldrich) solution at 37 ◦ C for 2 h [43,44]. The disks were rinsed with fresh PBS by stirring at a speed of 300 rpm for 5 min, and then immersed in a solution of 1% sodium dodecylsulfate (SDS) in PBS and sonicated for 20 min to detach the BSA adsorbed on the disk [43,44]. A protein analysis kit (micro BCA protein assay kit, Fisher Scientific, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. Briefly, 25 ␮L of the SDS solution and 200 ␮L of the BCA reagent were mixed into the wells of a 96-well plate and incubated at 60 ◦ C for 30 min [43,44]. The absorbance at 562 nm was measured via a microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA). Standard curves were prepared using the BSA standard [43,44].

2.4. Saliva collection and dental plaque microcosm biofilm formation The dental plaque microcosm model was approved by University of Maryland Institutional Review Board. Saliva is ideal for growing microcosm biofilms in vitro, with the advantage of maintaining much of the complexity and heterogeneity of dental plaque in vivo [45]. Saliva was collected from ten healthy donors having natural dentition without active caries, and not having used antibiotics within the past 3 months. The donors did not brush teeth for 24 h and abstained from food and drink intake for 2 h prior to donating saliva [46]. An equal volume of saliva from each of the ten donors was combined to form the saliva sample. The saliva was diluted in sterile glycerol to a concentration of 70%, and stored at −80 ◦ C for subsequent use [45]. The saliva–glycerol stock was added, with 1:50 final dilution, into a McBain artificial saliva growth medium as inoculum [46]. This 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; CaCl2 , 0.2 g/L; cysteine hydrochloride, 0.1 g/L; hemin, 0.001 g/L; vitamin K1 , 0.0002 g/L, at pH 7 [47]. 2% sucrose was added to this medium. 1.5 mL of inoculum was added to each well of 24-well plates with a resin disk, and incubated at 37 ◦ C in 5% CO2 for 8 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for 16 h. Then, the disks were transferred to new 24-well plates with fresh medium and incubated for another 24 h. This totaled 48 h of culture, which was previously shown to form relatively mature dental plaque microcosm biofilms on resins [46].

2.5.

Live/dead staining of biofilms

Resin disks with 2-day biofilms were washed with PBS and stained using the BacLight live/dead kit (Molecular Probes, Eugene, OR) [46]. Live bacteria were stained with Syto 9 to produce a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to produce a red fluorescence. The stained disks were examined using an inverted epifluorescence microscope (Eclipse TE2000-S, Nikon, Melville, NY).

2.6.

MTT assay of metabolic activity of biofilms

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) assay was used to evaluate the metabolic activity of biofilms on the disks [46]. MTT is a colorimetric assay that measures the enzymatic reduction of MTT, a yellow tetrazole, to formazan. Disks with 2-day biofilms were transferred to a new 24-well plate, and 1 mL of MTT dye was added to each well and incubated at 37 ◦ C in 5% CO2 for 1 h. Then the disks were transferred to a new 24-well plate, and 1 mL of dimethyl sulfoxide (DMSO) was added to resolve the formazan crystals. The plate was incubated for 20 min with gentle mixing in the dark. Then, 200 mL of the DMSO solution from each well was collected, and its absorbance at 540 nm was measured via a microplate reader (SpectraMax M5). A higher absorbance is related to a higher formazan concentration, which indicates a higher metabolic activity in the biofilm on the disk [46].

2.7.

Colony-forming unit (CFU) counts

Disks with 2-day biofilms were transferred into tubes with 2 mL of CPW, and the biofilms were harvested by sonication and vortexing [46]. Three types of agar plates were prepared. First, tryptic soy blood agar culture plates were used to determine the total microorganisms [47]. Second, mitis salivarius agar (MSA) culture plates containing 15% sucrose were used to determine the total streptococci [48]. This is because MSA contains selective agents including crystal violet, potassium tellurite and trypan blue, which inhibit most Gram-negative bacilli and most Gram-positive bacteria except streptococci, thus enabling the streptococci to grow [48]. Third, MSA agar culture plates plus 0.2 units of bacitracin per mL was used to determine the mutans streptococci [47]. The bacterial suspensions were serially diluted, spread onto agar plates and incubated at 37 ◦ C in 5% CO2 for 24 h. The number of colonies that grew was counted and used, along with the dilution factor, to calculate the CFU on each resin disk [46].

2.8. Ca and P ion concentration measurement of the biofilm culture medium The experimental control and commercial control groups were not included in Ca and P ion concentration measurements because they did not release Ca and P ions. The biofilm culture media after 72 h of incubation for the other four groups were collected and centrifuged at 12000 rpm for 5 min (Eppendorf Centrifuge 5415, Brinkmann, Westbury, NY). Then, 1 mL supernatant was used and analyzed for Ca and P concen-

Please cite this article in press as: Xie X, et al. Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.002

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0

MPC+DMAHDM+40NACP

5

MPC+DMAHDM+30NACP

10

MPC+DMAHDM+20NACP

15

MPC+DMAHDM

20

Experimental control

b

Fig. 1 – Dentin shear bond strength using extracted human teeth, tested after storage in water for 24 h (mean ± sd; n = 10) Bars with dissimilar letters indicate values that are significantly different from each other (p < 0.05).

trations via a spectrophotometric method (DMS-80 UV–vis, Varian, Palo Alto, CA) using known standards and calibration curves [22,49].

2.9.

pH of biofilm culture medium

Each resin disk was placed in a well and 1.5 mL of inoculum was added to each well of 24-well plates. They were incubated at 37 ◦ C in 5% CO2 for 24 h as described above. Then, the disks with adherent biofilms were transferred to new 24well plates with fresh medium, and the pH measurement was started. The pH of the culture medium was measured from 24 h to 72 h using a pH meter (Accumet Excel XL25, Fisher, Pittsburgh, PA) [50]. The pH measurements were not collected for the initial 0–24 h of culture, because the planktonic bacteria in the medium would interfere with the pH. By placing disks with adherent biofilms in new wells with fresh medium and measuring the pH from 24 h to 72 h, it enabled the measured pH to be related to the biofilm on the resin [50]. The pH data were recorded once every hour from 24 h to 32 h of the incubation. Then no pH measurement was made at night for 16 h. The pH measurement was re-started the next morning every two hours from 48 h to 60 h of the incubation. The last pH measurement was made in the next morning at 72 h of the incubation.

2.10.

Statistical analysis

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

3.

Results

Dentin shear bond strength results are plotted in Fig. 1 (mean ± sd; n = 10). Different NACP filler levels had a significant effect on dentin bond strength (p < 0.05). The adhesives

a

a

5 4 3 2 1 0

MPC+DMAHDM+40NACP

25

6

MPC+DMAHDM+30NACP

a,b

MPC+DMAHDM+20NACP

a

MPC+DMAHDM

30

7

Experimental control

a

Commercial control

a a

Commercial control

Dentin Shear Bond Strength (MPa)

35

Protein Adsorption on adhesive disks (µg/cm2)

d e n t a l m a t e r i a l s x x x ( 2 0 1 7 ) xxx–xxx

b

b

b

b

Fig. 2 – Protein adsorption onto adhesive resin disk surfaces (mean ± sd; n = 6) Bars with dissimilar letters indicate values that are significantly different from each other (p < 0.05).

with NACP filler level of 20–30% had dentin bond strengths matching the controls (p > 0.1). The dentin bond strength of the adhesive with 40% NACP filler was significantly lower (p < 0.05). The results of protein adsorption onto the resin disk surfaces are plotted in Fig. 2 (mean ± sd; n = 6). Experimental control and commercial control had similar protein adsorption (p > 0.1). All the adhesives containing MPC had much less protein adsorption (p < 0.05). The NACP filler level had no significant effect on protein adsorption (p > 0.1). Representative live/dead staining images of 2-day biofilms on resins are shown in Fig. 3. In (A) and (B), experimental control and commercial control adhesives were nearly fully covered by live bacteria. In contrast, images (C–F) showed much less bacterial adhesion, and the biofilms consisted of primarily dead bacteria with red and yellow staining. The MTT metabolic activity of 2-day biofilms on resins is plotted in Fig. 4 (mean ± sd; n = 6). Experimental control and commercial control were similar (p > 0.1). Adding MPC and DMAHDM to the adhesive progressively reduced the metabolic activity of biofilms (p < 0.05). The NACP filler level had no significant effect on the metabolic activity of biofilms (p > 0.1). Fig. 5 plots the CFU counts of 2-day biofilms on resins: (A) Total microorganisms, (B) total streptococci, and (C) mutans streptococci (mean ± sd; n = 6). Experimental control and commercial control had similar CFU (p > 0.1). The total microorganisms, total streptococci and mutans streptococci on adhesives containing MPC and DMAHDM were almost 4 orders of magnitude less than those of controls. NACP filler level had no effect on CFU (p > 0.1). The Ca and P ion concentrations in the biofilm culture medium are plotted in Fig. 6 (mean ± sd; n = 6). The ion concentrations significantly increased when the NACP filler level was increased from 20% to 40% (p < 0.05). These results demonstrate that the vicinity of a NACP-containing resin would have a reservoir of Ca and P ions to promote remineralization. The pH values of the culture medium with biofilms on resins are plotted in Fig. 7 (mean ± sd; n = 6). The pH decreased

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Fig. 3 – Representative live/dead staining images of 2-day biofilms on adhesive resin disks: (A) Commercial control adhesive, (B) experimental control, (C) PEHB+MPC+DMAHDM, (D) PEHB+MPC+DMAHDM+20NACP, (E) PEHB+MPC+DMAHDM+30NACP, (F) PEHB+MPC+DMAHDM+40NACP. Live bacteria were stained green, and dead bacteria were stained red. When live and dead bacteria were in close proximity or on the top of each other, the staining had yellow or orange colors. (For interpretation of the references to color in this figure legend and in the text, the reader is referred to the web version of this article.) Please cite this article in press as: Xie X, et al. Novel dental adhesive with triple benefits of calcium phosphate recharge, protein-repellent and antibacterial functions. Dent Mater (2017), http://dx.doi.org/10.1016/j.dental.2017.03.002

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MPC+DMAHDM+20NACP

MPC+DMAHDM+30NACP

MPC+DMAHDM+40NACP

MPC+DMAHDM

109 108 107 106 105

d

d

MPC+DMAHDM+40NACP

(B) MPC+DMAHDM+30NACP

c

MPC+DMAHDM+20NACP

c

d

d

104 109

(C)

e

e

f

f

108 107 106 105 104

MPC+DMAHDM+40NACP

The present study developed a novel bioactive adhesive with triple benefits of Ca and P ion rechargeability, protein-repellent and antibacterial functions to inhibit secondary caries at the tooth-restoration bonded interface. Previous studies showed that PEHB resin containing NACP could be repeated recharged to have durable Ca and P ion release; however, the rechargeable resin was not antibacterial or protein-repellent. In the present study, the hypotheses were proven that the rechargeable PEHB-NACP adhesive with 5% MPC and 5% DMAHDM yielded dentin bond strength similar to controls; biofilm growth was greatly reduced on the PEHB-NACP-MPC-DMAHDM resin; and the biofilm culture medium contained a high concentration of Ca and P ion reservoir and the stead-state pH was greatly increased from a cariogenic pH of 4 for the controls to a safe pH of above 6. The novel adhesive greatly reduced protein adsorption and biofilm growth, decreased biofilm CFU by almost 4 log. These results, along with a previous study showing that the NACP-containing resin could be repeatedly recharged to have long-term Ca and P ion release [34], indicate the advantage of combining protein-repellent, antibacterial, acid neutralization, high pH, and CaP remineralization capabilities. Therefore, the novel PEHB adhesive with 5% MPC, 5%

b

MPC+DMAHDM+30NACP

Discussion

b

MPC+DMAHDM+20NACP

4.

b

1010

MPC+DMAHDM

with increasing time due to acid production by the biofilms, and reached a steady-state pH of 4.0 for commercial control, 4.1 for experimental control, 5.6 for MPC+DMAHDM, and 5.8 for MPC+DMAHDM+20NACP. The steady-state pH values of MPC+DMAHDM, MPC+DMAHDM+20NACP and two controls are significantly different from each other (p < 0.05). The pH for MPC+DMAHDM+30NACP decreased with time and reached 6, then recovered to 6.5 at 72 h. The pH for MPC+DMAHDM+40NACP decreased with time and reached 6.1, then recovered to 6.7. The pH values at 72 h for MPC+DMAHDM+30NACP and MPC+DMAHDM+40NACP are not significantly different from each other (p > 0.1).

b 105

MPC+DMAHDM

Fig. 4 – MTT metabolic activity of 2-day biofilms on adhesive resin disks (mean ± sd; n = 6). Values with dissimilar letters are significantly different from each other (p < 0.05).

Experimental control

b

106

Experimental control

b

107

Experimental control

b

108

Commercial control

b

109

Commercial control

0

(A)

a

a 1010

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0.3

MPC+DMAHDM+20NACP

0.4

MPC+DMAHDM

0.5

Experimental control

0.6

Total Microorganisms (CFU/disk)

0.7

Total Streptococci (CFU/disk)

a

Mutans Streptococci (CFU/disk)

0.8

1011 a

Commercial control

Biofilm Metabolic Activity (A540/cm2)

0.9

f

f

103

Fig. 5 – CFU of 2-day biofilms on adhesive resin disks: (A) Total microorganisms, (B) total streptococci, and (C) mutans streptococci (mean ± sd; n = 6). Note the log scale in y axis. In each plot, values with dissimilar letters are significantly different (p < 0.05).

DMAHDM and 30% NACP is promising for tooth cavity restorations to resist secondary caries. In the oral environment, the adsorption of salivary proteins onto the teeth and material surfaces forms the acquired pellicle coating [51,52]. This salivary pellicle film provides the essential receptors allowing the adherence of bacteria [51,52]. Therefore, the adsorption of salivary proteins is a prerequisite for bacterial attachment and biofilm formation. Hence, a resin that can repel proteins has the potential to repel bacteria attachment and biofilm formation. Indeed, the present study showed that adding MPC into the adhesive reduced protein

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pH of Biofilm Culture Medium

MPC+DMAHDM +20NACP

0

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b

MPC+DMAHDM+40NACP

60

MPC+DMAHDM+30NACP

80

20

7

c

100

40

7.5

d

(A)

MPC+DMAHDM

Calcium Ion Concentration (mmol/L)

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MPC+DMAHDM+40NACP

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6

MPC+DMAHDM+30NACP MPC+DMAHDM+20NACP

5.5 MPC+DMAHDM 5

4.5 Experimental control 4

70

24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72

(B) h

60

20 10 0

e

f MPC+DMAHDM +20NACP

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MPC+DMAHDM+40NACP

40

MPC+DMAHDM+30NACP

g

50

MPC+DMAHDM

Phosphate Ion Concentration (mmol/L)

Commercial control 3.5

Fig. 6 – Ca and P ion concentrations of the 72 h biofilm culture medium: (A) Ca ion concentration, (B) P ion concentration (mean ± sd, n = 6). Values with dissimilar letters are significantly different from each other (p < 0.05).

adsorption by an order of magnitude. Previous studies showed that proteins were adsorbed preferentially to hydrophobic surfaces, while highly hydrophilic surfaces with MPC decreased protein adsorption and bacterial adhesion. MPC is one of the most common biocompatible and hydrophilic biomedical polymers [39,53]. Regarding the protein-repellent mechanism, it was reported that, in the hydrated MPC polymer, there is an abundance of free water but no bound water [39,53]. The bound water would result in protein adsorption [54]. On the other hand, the large number of free water around the phosphorylcholine group could detach proteins, thereby repelling protein adsorption [54]. MPC is non-toxic, and MPC-containing medical devices have been used clinically to repel proteins, for example, artificial blood vessels, implantable artificial hearts, and artificial lungs [43]. Recently, MPC was incorporated into a dentin bonding agent, achieving a strong protein-repellent ability [21]. Antibacterial adhesive can kill the residual bacteria in the prepared tooth cavity, and inhibit new bacteria invasion at

Biofilm Culture Time (h) Fig. 7 – Effect of NACP, MPC and DMAHDM on the pH of the culture medium with the dental plaque microcosm biofilms (mean ± sd, n = 6). The pH of the biofilm medium with PEHB+MPC+DMAHDM+30NACP and PEHB+MPC+DMAHDM+40NACP adhesives was maintained above 6 during the 72 h of culture. The pH of biofilm medium with commercial adhesive control was cariogenic and around pH 4 during 72 h of culture.

the tooth-restoration interface due to marginal leakage in vivo [42,55]. For these reasons, efforts have been devoted to developing antibacterial bonding agents [15,42]. The combination of MPC with DMAHDM in the NACP rechargeable adhesive achieved an even more dramatic reduction in biofilms. The mode of antibacterial action for QAMs is known to be contactinhibition [16,56]. When the negatively-charged bacterial cell contacts the positively-charged sites of QAMs, the electric balance of the cell membrane could be disturbed, leading to bacteria death [16,56]. This contact-killing efficacy is reduced if a salivary protein pellicle separates the antibacterial resin surface from the bacteria [57]. Indeed, several studies demonstrated that a saliva-derived protein film on the cationic antibacterial surface reduced the original bactericidal effect [57,58]. In the present study using both MPC and DMAHDM in the rechargeable adhesive, MPC can provide synergy to DMAHDM by greatly reducing the protein adsorption; this would in turn expose the resin surface to the bacteria, thereby enhancing the contact-killing efficacy of DMAHDM. This synergistic factor likely contributed to the almost 4 log reduction in biofilm CFU and a substantial increase in biofilm medium pH. The purpose of NACP incorporation was for the adhesive to obtain Ca and P ion release and remineralization capabilities. Previous studies showed that CaP-containing resins remineralized enamel and dentin lesions [22,24]. Recent studies showed that NACP-containing composites released high levels of Ca and P ions in a simulating cariogenic solution [33]. Furthermore, NACP nanocomposite rapidly neutralized a car-

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iogenic acid challenge and raised the pH from 4 to above 6 in the simulating cariogenic solution [33]. The results of the present study showed that NACP could also release high levels of Ca and P ions in biofilm culture medium. Higher NACP filler level provided higher Ca and P ion concentrations in the medium of 72 h incubation. The dentin bond strength at 30% of NACP fillers matched that of commercial control; however, that at 40% NACP was lower. Therefore, 30% of NACP appeared to be optimal for the rechargeable PEHB adhesive. In the oral environment, a local plaque pH of above 6 is considered to be the safe zone, pH of 6.0–5.5 can be potentially cariogenic, and pH of 5.5–4 is the cariogenic or danger zone for caries formation [6]. Therefore, it is highly beneficial to maintain the local pH to be above 6 to protect tooth structures and avoid demineralization. In the present study, first, the MPC+DMAHDM adhesive with biofilms had a pH higher than that of the experimental control and commercial control adhesive, likely because MPC repelled bacteria and decreased biofilm growth, leaving less biofilms on the resin disks to produce acid. Furthermore, DMAHDM in the adhesive killed bacteria. Second, the MPC+DMAHDM+NACP adhesive yielded a higher pH than MPC+DMAHDM adhesive, because NACP-containing materials had an acid neutralization effect [33]. Third, the pH of MPC+DMAHDM+30NACP was above 6, in the safe zone for tooth structures. Furthermore, the pH had slightly recover after 8 h incubation and reached 6.5 for 30% NACP adhesive at 72 h. The reason of this phenomenon maybe based on this mechanism. The living bacteria produced acid to decrease the pH of the biofilm culture medium at first. During this process, NACP neutralized the acid and slowed down the speed of pH decrease. After 8 h, the sucrose supplement in the biofilm culture medium may be exhausted and the acid production of the bacteria was diminished [59]. Then the NACP neutralized the remaining acid in the medium and raise pH. Further study should investigate the protection efficacy of PEHB+5MPC+5DMAHDM+30NACP for tooth structures against biofilms in a clinically relevant in vivo environment. The bioactive properties of the novel adhesive of the present study are expected to have long-term durability. First, a previous study showed that the MPC-polymerized surface was resistant to mechanical stresses caused by brushing [60]. In the present study, MPC was mixed into the adhesive, and copolymerized and immobilized in the PEHB resin. MPC was dispersed in the adhesive and not limited to the surface only. Therefore, the protein-repellent effect is expected to be durable and will not be lost by wear. Second, DMAHDM is also copolymerized with the resin matrix and will not be lost over time. Third, PMGDM is an acidic adhesive monomer with a carboxylate group, hence the PEHB resin can chelate with calcium ions from the exterior environment such as a ion recharge solution [34,37]. The PEHB-NACP adhesive could repeatedly recharge Ca and P ions into the adhesive to have long-term ion re-release [34]. Therefore, all three bioactive mechanisms (MPC, DMAHDM, and NACP) of the adhesive are expected to have long-term durability. Further study is needed to investigate the anti-biofilm and remineralization efficacy vs. long periods of time such as several years. Efforts are also needed to apply the PEHB with MPC+DMAHDM+NACP method to other bonding agents, cements, sealants and composites to inhibit biofilms, remineralize lesions and maintain a safe pH.

5.

9

Conclusion

The present study developed a novel adhesive with triple benefits of calcium phosphate ion recharge, protein-repellent and antibacterial functions. MPC and DMAHDM were incorporated into a NACP rechargeable adhesive to achieve proteinrepellent and antibacterial capabilities to combat biofilms and caries. The new bioactive adhesive showed a strong protein-repellent capability and substantially reduced bacteria attachment and viability, reducing the biofilm CFU by almost 4 log compared to commercial control. The bioactive adhesive with dental plaque microcosm biofilm culture maintained a pH of above 6, while commercial control adhesive had a cariogenic pH of 4. Therefore, the new adhesive with triple benefits is promising to protect the tooth structures and inhibit biofilms and caries formation. The method of combining NACP, MPC and DMAHDM for triple benefits is promising for application to a wide range of dental restorative and preventive materials to reduce plaque buildup and inhibit caries.

Acknowledgments We thank Dr. Laurence C. Chow, Dr. Mary Anne S. Melo and Dr. Ning Zhang for discussions. This study was supported by NIH R01 DE17974 (HX), National Natural Science Foundation of China 81200820 (XX) and 81400487 (LW), Beijing Nova Program xx2014B060 (XX), University of Maryland seed grant (HX), and University of Maryland School of Dentistry bridge fund (HX).

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