Novel bioactive root canal sealer to inhibit endodontic multispecies biofilms with remineralizing calcium phosphate ions

Abstract

Objective

The objectives of this study were to: (1) develop a bioactive endodontic sealer via dimethylaminohexadecyl methacrylate (DMAHDM), 2-methacryloyloxyethyl phosphorylcholine (MPC) and nanoparticles of amorphous calcium phosphate (NACP) for the first time; and (2) evaluate inhibition of early-stage and mature multispecies endodontic biofilm, bond strength to root canal dentine, and calcium (Ca) and phosphate (P) ion release.

Methods

A series of bioactive endodontic sealers were formulated with DMAHDM, MPC, and NACP. Root dentine bond strength was measured via a push-out test. Three endodontic strains, Enterococcus faecalis , Actinomyces naeslundii , and Fusobacterium nucleatum , were grown on endodontic sealer disks to form multispecies biofilms. Biofilms were grown for 3 days (early) and 14 days (mature). Colony-forming units (CFU), live/dead assay, metabolic activity and polysaccharide were determined. Ca and P ion release from endodontic sealer was measured.

Results

Incorporating DMAHDM, MPC and NACP did not decrease the push-out bond strength ( p > 0.1). Adding DMAHDM and MPC reduced endodontic biofilm CFU by 3 log. DMAHDM or MPC each greatly decreased the biofilm CFU ( p < 0.05). Endodontic sealer with DMAHDM + MPC had much greater killing efficacy than DMAHDM or MPC alone ( p < 0.05). Endodontic sealer with DMAHDM + MPC had slightly lower, but not significantly lower, Ca and P ion release compared to that without DMAHDM + MPC ( p > 0.1).

Conclusions

A novel bioactive endodontic sealer was developed with potent inhibition of multispecies endodontic biofilms, reducing biofilm CFU by 3 log, while containing NACP for remineralization and possessing good bond strength to root canal dentine walls.

Clinical significance

The new bioactive endodontic sealer is promising for endodontic applications to eradicate endodontic biofilms and strengthen root structures. The combination of DMAHDM, MPC and NACP may be applicable to other preventive and restoration resins.

Introduction

The ultimate goal of endodontic treatment is to eliminate the bacterial infection in the root canal system, preventing microorganisms from impairing periapical healing or even contributing to the development of apical lesions . However, inability to sufficiently disinfect the root canal system leads to failure or persistent apical pathosis . The anatomic complexity of the root canal makes complete debridement of bacteria almost impossible, even if conventional methods of chemomechanical debridement are performed to the highest technical standards . Unfortunately, the persistence of microorganisms in the root canal system following root canal treatment can lead to post-treatment diseases . Therefore, it would be highly desirable to develop a strongly-antibacterial root canal sealer that can kill endodontic pathogens. An endodontic sealer with potent antimicrobial functions may help eliminate residual microorganisms in the canal and kill new invading pathogens.

Root canal infections are characterized by microbial biofilms that adhere to the root canal dentine and extend to the apical foramina and in some cases beyond . Endodontic treatment failures are frequently associated with gram-positive aerobic and facultative microorganisms. The presence of Enterococcus faecalis ( E. faecalis ) in failed endodontic treatment is extensively covered in the literature and is rarely detected in primary infected and untreated cases. This Gram-positive coccus E. faecalis has the ability to invade dentinal tubules and withstand prolonged nutritional deprivation, and thus are difficult to eradicate. In addition, one cannot discount the presence or significance of other microorganisms belonging to the genera Actinomyces , Fusobacterium and Prevotella , which have been frequently detected in endodontic treatment failures .

Root filling materials with antimicrobial properties may contribute to eliminate residual microorganisms unaffected by both chemomechanical preparation and intracanal medication . Furthermore, they may limit the ingress of microorganisms from saliva, impeding or at least retarding the complete recontamination of the root canal after saliva challenge . Nowadays, dental resins are increasingly used in tooth restorations . Among them, resin-based bondable endodontic sealers have been used in clinical treatments to seal the interfaces in order to minimize re-infection in the root canals . However, a major challenge with bonding inside root canals is the shrinkage stress created on the canal walls of these long, narrow “cavities” during the polymerization of the resin sealer . This could lead to microleakage, which could allow the invasion of endodontic pathogens to cause secondary infections. Therefore, it would be highly desirable to develop an antibacterial root canal sealer. In previous studies, quaternary ammonium monomers (QAMs) such as 12-methacryloyloxydodecylpyridinium bromide (MDPB) were copolymerized in resins to yield antibacterial properties based on the “contact-killing” mechanism . Recently, a bonding agent containing a new dimethylaminohexadecyl methacrylate (DMAHDM) showed the greatest antibiofilm activity among several tested antibacterial agents .

Salivary protein coating could decrease the efficacy of “contact-inhibition” . Therefore, efforts were made to develop protein-repellent functions . 2-methacryloyloxyethyl phosphorylcholine (MPC) is a methacrylate with phospholipid polar groups and is a common biopolymer . Polymers containing MPC are known to reduce protein adsorption and bacterial adhesion . Indeed, MPC-containing adhesives showed strong protein-repellent properties and inhibited cariogenic bacteria . In addition, it would be beneficial for the new endodontic sealer to be able to release calcium (Ca) and phosphate (P) ions in order to remineralize root dentine and strengthen the root structures. Recently, a novel Ca and P ion releasing bonding agent was developed containing nanoparticles of amorphous calcium phosphate (NACP) for remineralization .

However, to date, there has been no report on endodontic sealer containing MPC and DMAHDM, and the effects on multispecies endodontic biofilms. Furthermore, there has been no report on antibacterial endodontic sealer that also possesses Ca and P ion release capabilities. Therefore, the objectives of this study were to: (1) develop a novel bioactive endodontic sealer incorporating DMAHDM, MPC and NACP; and (2) investigate its inhibition against multispecies endodontic biofilms and its release of Ca and P ions for the first time. The following hypotheses were tested: (1) incorporation of DMAHDM, MPC and NACP would not compromise the pull-out bond strength to root dentine of the new endodontic sealer; (2) DMAHDM + MPC would possess much greater killing efficacy against endodontic multispecies biofilms than MPC or DMAHDM alone; (3) the sealer with DMAHDM + MPC would possess the same killing efficacy against early-stage and mature endodontic biofilms; (4) the new endodontic sealer would release high levels of Ca and P ions.

Materials and methods

Fabrication of endodontic sealer containing DMAHDM, MPC and NACP

The experimental primer contained pyromellitic dianhydride glycerol dimethacrylate (PMGDM, Esstech, Essington, PA) and 2-hydroxyethyl methacrylate (HEMA, Esstech) at a mass ratio 10:3, with 50% acetone solvent (all by mass), following a previous study . The experimental adhesive contained PMGDM, ethoxylated bisphenol A dimethacrylate (EBPADMA, Sigma–Aldrich, St. Louis, MO), HEMA and bisphenol A glycerolate dimethacrylate (BisGMA, Esstech) at 45/40/10/5 ratio (referred to as “PEHB”), following a previous study . As the photo-initiator, 1% phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide (BAPO, Sigma–Aldrich) was added to the adhesive. This adhesive was used because a previous study showed that the incorporation of NACP into this adhesive yielded substantial release of calcium (Ca) and phosphate (P) ions . PEHB was used as the resin matrix for endodontic sealer into which antibacterial and remineralizing agents were added.

NACP were incorporated into the adhesive, because NACP could release Ca and P ions to remineralize the demineralized tooth and protect the tooth structures . NACP were synthesized using a spray-drying technique . Briefly, calcium carbonate and dicalcium phosphate were dissolved in acetic acid to produce Ca and P concentrations of 8 mmol/L and 5.333 mmol/L, respectively, thus yielding a Ca/P molar ratio of 1.5, the same as that for ACP [Ca 3 (PO 4 ) 2 ]. This solution was sprayed into a heated chamber of the spray-drying machine. An electrostatic precipitator collected the dried particles. This method produced NACP with a mean particle size of 116 nm . NACP were mixed into endodontic sealer at a NACP filler level of 30% by mass. The 30% NACP was selected because a previous study achieved a high level of Ca and P ion release using this formulation .

DMAHDM was synthesized using a modified Menschutkin reaction in which a tertiary amine group was reacted with an organohalide . 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 ethanol in 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, colourless, and viscous liquid . DMAHDM was mixed into the primer at a DMAHDM/(primer + DMAHDM) mass fraction of 5%. Similarly, DMAHDM was mixed into the PEHB sealer at DMAHDM/(sealer + DMAHDM) = 5%. The 5% DMAHDM was selected following a previous study .

MPC was obtained commercially (Sigma–Aldrich) which was synthesized via a method reported previously . MPC powder was incorporated into primer at MPC/(primer + MPC) mass fractions of 0%, 1.5%, 3%, and 4.5%. Similarly, MPC was mixed into PEHB sealer at MPC/(sealer + MPC) mass fractions of 0%, 1.5%, 3%, and 4.5%. MPC mass fractions > 4.5% were not used due to dentine bond strength loss in preliminary studies.

Tooth root push-out strength to test bonding to root canal dentine walls

The push-out bond strength test was conducted using 54 extracted human canines. The teeth collection was approved by the University of Maryland Baltimore Institutional Review Board. The crowns were removed at the cementoenamel junction with a water-cooled diamond saw (Isomet, Buehler, Lake Bluff, IL), and the roots were trimmed coronally to a uniform length of 17 mm. The working length was determined visually by subtracting 1 mm from the length of a size 10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) at the apical foramen. The canals were endodontically treated according to the crown-down technique using stainless steel K-files up to size 60 (Dentsply Maillefer) attached to an oscillating handpiece (NSK Nakanishi, Kanuma, Tochigi, Japan). A step-back preparation with size 70 and size 80 K-files and sizes 3, 4 and 5 Gates-Glidden drills (Dentsply Maillefer) was undertaken to complete the canal instrumentation. The canals were irrigated with 2 mL of 1% sodium hypochlorite at each change of file, rinsed with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) for 5 min and received a final flush with 5 mL of distilled water, followed by drying with absorbent paper points . The roots were then randomly assigned to nine groups ( n = 6) using the following root canal filling materials:

  • (1)

    Commercial sealer control group. Primer: Epiphany primer. Endodontic sealer: Epiphany sealer (Pentron Clinical Technologies, Wallingford, CT, USA).

  • (2)

    PEHB + NACP sealer control group. Primer: The experimental primer. Sealer: 70% PEHB + 30% NACP.

  • (3)

    DMAHDM group. Primer: 95% experimental primer + 5% DMAHDM. Sealer: 65% PEHB + 5% DMAHDM + 30% NACP (denoted “PEHB + NACP + 5DMAHDM”).

  • (4)

    1.5% MPC group. Primer: 98.5% experimental primer + 1.5% MPC. Sealer: 68.5% PEHB + 1.5% MPC + 30% NACP (denoted “PEHB + NACP + 1.5MPC”).

  • (5)

    3% MPC group. Primer: 97% experimental primer + 3% MPC. Sealer: 67% PEHB + 3% MPC + 30% NACP (denoted “PEHB + NACP + 3MPC”).

  • (6)

    4.5% MPC group. Primer: 95.5% experimental primer + 4.5% MPC. Sealer: 65.5% PEHB + 4.5% MPC + 30% NACP (denoted “PEHB + NACP + 4.5MPC”).

  • (7)

    DMAHDM + 1.5% MPC group. Primer: 93.5% experimental primer + 5% DMAHDM + 1.5% MPC. Sealer: 63.5% PEHB + 5% DMAHDM + 1.5% MPC + 30% NACP (denoted “PEHB + NACP + 5DMAHDM + 1.5MPC”).

  • (8)

    DMAHDM + 3% MPC group. Primer: 92% experimental primer + 5% DMAHDM + 3% MPC. Adhesive: 62% PEHB + 5% DMAHDM + 3% MPC + 30% NACP (denoted “PEHB + NACP + 5DMAHDM + 3MPC”).

  • (9)

    DMAHDM + 4.5% MPC group. Primer: 90.5% experimental primer + 5% DMAHDM + 4.5% MPC. Sealer: 60.5% PEHB + 5% DMAHDM + 4.5% MPC + 30% NACP (denoted “PEHB + NACP + 5DMAHDM + 4.5MPC”).

The commercial control was included for comparative purpose for pull-out strength. According to the manufacturer, the dual-cure methacrylate resin-based sealer Epiphany consists of a mixture of urethane dimethacrylate (UDMA), polyethylene dimethacrylate, bisphenol-A-glycidyldimethacrylate (BisGMA), ethoxylated BisGMA, barium sulphate, silica, calcium hydroxide, bismuth oxide, photoinitiators stabilizers and pigments.

The sealer was applied to the canal with a lentulo spiral (Dentsply Maillefer) attached to a low-speed handpiece (NSK Nakanishi) to avoid bubble formation. Specimens were light-cured for 40 s to create a coronal seal following manufacturer’s recommendations. All roots were coded and placed in 100% humidity for 48 h prior to testing.

For preparation of the specimens, the roots were fixed on acrylic plates with wax and were sectioned in a water-cooled precision saw (Isomet, Buehler, Lake Bluff, IL). Nine slices (1 mm thick) were obtained from each root (three per root third). The first slice of each third was used for the push-out test. Each root section was then subjected to a compressive loading via a Universal Testing Machine (5500R, MTS, Cary, NC) at a crosshead speed of 1 mm/min using a 0.8-mm diameter stainless steel cylindrical plunger. The plunger tip was positioned so that it only contacted the filling material. Push-out bond strength = debond force/area, where the area (of the bonded interface) is the root canal perimeter times the tested root length.

Specimen fabrication for protein adsorption and biofilm experiments

The push-out strength results showed that PEHB + 5DMAHDM + 4.5MPC had a lower strength than other groups. Therefore, it was not included in the subsequent protein-repellent test. In addition, preliminary study showed that 1.5% MPC was not as effective as 3% MPC to repel proteins. Hence, the following five groups were tested in protein and biofilm experiments: (commercial sealer control; PEHB + NACP sealer control; PEHB + NACP + 5DMAHDM; PEHB + NACP + 3MPC; PEHB + NACP + 5DMAHDM + 3MPC).

Resin disks were made using the cover of a 96-well plate (Costar, Corning, Corning, NY) as moulds following a previous study . Ten μL of primer was placed at the bottom of each dent in the 96-well plate. After drying with a stream of air, 20 μL of adhesive was placed into the dent and photo-polymerized for 30 s (Demetron VCL401), using a mylar strip covering to obtain a disk of approximately 8 mm in diameter and 0.5 mm in thickness. The cured disks were immersed in water and stirred with a magnetic bar at 100 rpm for 1 h to remove any uncured monomers, following a previous study . The disks were sterilized with ethylene oxide (AnproleneAN 74i, Andersen, Haw River, NC) and de-gassed for 7 days.

Protein adsorption onto endodontic sealer resin surfaces

Protein adsorption on resin disks was determined using a micro bicinchoninic acid (BCA) method . Thirty resin disks were fabricated, with six disks for each group. Each disk was immersed in phosphate buffered saline (PBS) for 2 h, and then immersed in 4.5 g/L bovine serum albumin (BSA, Sigma–Aldrich) solution at 37 °C for 2 h . A protein analysis kit (micro BCA assay, Fisher, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. 25 μL of the SDS solution and 200 μL of the BCA reagent were mixed into the wells of a 96-well plate and incubated at 60 °C for 30 min. 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 .

Endodontic bacteria strains and culture media

The use of all bacterial species was approved by University of Maryland Baltimore Institutional Review Board. All species were obtained from the American Type Culture Collection (ATCC, Manassas, VA): Actinomyces naeslundii ( A. naeslundii , ATCC12104); Fusobacterium nucleatum ( F. nucleatum , ATCC25586); Enterococcus faecalis ( E. faecalis , ATCC4083). E. faecalis , A. naeslundii , and F. nucleatum were grown in tryptic soy broth (TSB, Sigma–Aldrich) supplemented with yeast extract (5 g/L), l -cysteine hydrochloride (0.5 g/L), hemin (5 mg/L) and menadione (1 mg/L) at 37 °C anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ), following previous studies . For each species, the inoculum was adjusted to 10 8 colony-forming unit counts CFU/mL for future biofilm formation, based on the standard curve of OD 600nm versus the CFU/mL for each species, as in a previous study . A. naeslundii , F. nucleatum and E. faecalis were selected to develop a robust endodontic three-species biofilm model, which was validated in a previous study . These three species represented the early, middle and late colonizers of endodontic biofilms, respectively.

Multispecies endodontic biofilm formation on endodontic sealer disks

Bacteria grow aggregated on canal surfaces to form a biofilm, and this biofilm enables the bacteria to survive antimicrobial insults . The establishment of the biofilm architecture follows a sequence of events, going from initial attachment of a single cell to formation of a three-dimensional mature biofilm . The early and mature phases of a biofilm were respectively characterized with a thin biofilm formation and a mushroom-shaped biofilm architecture . To determine the relationship between bactericidal efficacy and biofilm formation, early-stage (3-day) and mature (14-day) multispecies endodontic biofilms were tested . 100 μL of A. naeslundii , F. nucleatum and E. faecalis suspensions were mixed and pipetted into 30 mL TSB supplemented medium to form a mixed species suspension. Sterile saliva pellicle-coated resin disks were transferred to a new 24-well plate. Each well was inoculated with 1.5 mL of the mixed bacterial suspension and incubated in an anaerobic condition at 37 °C for 3 days and 14 days, respectively, representing the early-stage and mature endodontic biofilms . The fresh TSB-supplemented medium was changed every 24 h.

Live/dead bacteria imaging

Resin disks with 3-day or 14-day biofilms were washed with cysteine peptone water (CPW) to remove the non-adherent bacteria. Live/dead bacterial kit (Molecular Probes, Eugene, OR) was used following the manufacturer’s instructions. Live bacteria were stained with SYTO 9 to emit a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to emit a red fluorescence. Biofilms were examined with an inverted epifluorescence microscope (TE2000-S, Nikon, Melville, NY).

Colony-forming unit (CFU) counts

For CFU counts, twelve resin disks were made for each endodontic sealer, with six disks for each biofilm stage. Biofilms were formed by culturing for 3 days and 14 days as described above. Disks were transferred into vials with 2 mL CPW, and the biofilms were harvested by scraping and sonication/vortexing (Fisher, Pittsburg, PA). Tryptic soy blood agar plates (supplemented with 5 g/L yeast extract, 0.5 g/L l -cysteine hydrochloride, 5 mg/L hemin, 1 mg/L menadione, 5% sheep blood) were used, following ATCC instructions. Biofilm suspensions were serially diluted, spread onto agar plate and incubated at 37 °C anaerobically for 72 h . Then, the number of colonies was counted by a colony counter (Reichert, NY), which was used with the dilution factor to calculate the CFU counts .

XTT metabolic assay

Twelve disks of each sealer were made for metabolic assay, with six disks for each biofilm stage. The 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay were conducted to measure the metabolic activity of biofilms as described previously . Disks with 3-day and 14-day biofilms were washed using CPW, followed by transferring into new 24-well plates with 1 mL XTT working regent (0.5 mg/mL XTT and 1 μM menadione), then cultured for 2 h at 37 °C. The absorbance at OD 492 nm was measured via the microplate reader (SpectraMax M5) . The conversion of the XTT substrate to a soluble coloured formazan product correlates with cell viability. A higher absorbance is related to a higher metabolic activity in the biofilm adherent on the resin disks .

Measurement of polysaccharide production by biofilms on endodontic sealers

For polysaccharide, twelve disks of each endodontic sealer were made with six disks for each biofilm stage. The water-insoluble polysaccharide in the extracellular polymeric substance (EPS) of biofilms was determined using a phenol-sulfuric acid method . Each disc with 3-day and 14-day biofilm was immersed in a vial with 2 mL CPW, and the biofilm was collected by sonication/votexing. Centrifugation yielded a precipitate, which was rinsed with PBS and resuspended in 1 mL of de-ionized water. Then, 1 mL of 6% phenol solution was added to the vial, followed by 5 mL of 95–97% sulfuric acid . The vial was incubated for 30 min. Then, 100 μL of the solution was transferred into a 96-well plate. The amount of polysaccharide in biofilms was determined by measuring the absorbance at OD 490 nm with the microplate reader. Five glucose concentrations of 0, 5, 10, 20, 50 and 100 mg/mL were used as standard in the conversion of the OD readings to the polysaccharide concentrations .

Calcium (Ca) and phosphate (P) ion release measurement

The ion releases from PEHB + NACP + 5DMAHDM + 3MPC and PEHB + NACP control were measured to investigate the effect of adding DMAHDM and MPC on ion release. A sodium chloride (NaCl) solution (133 mmol/L) was buffered to three different pH values: pH 4 with 50 mmol/L lactic acid, pH 5.5 with 50 mmol/L acetic acid, and pH 7 with 50 mmol/L HEPES . As in previous studies , three specimens of 2 × 2 × 12 mm were immersed in 50 mL of solution yielding a specimen volume/solution of 2.9 mm 3 /mL. This was similar to a specimen volume per solution of about 3.0 mm 3 /mL in a previous study . For each solution, the concentrations of Ca and P ions released from the specimens were measured at 1, 3, 7, 14, 21, and 28 days. At each time, aliquots of 0.5 mL were removed and replaced by fresh solution. The aliquots were analyzed for Ca and P ions via a spectrophotometric method (DMS-80 UV-visible, Varian Palo Alto, CA) using known standards and calibration curves, as described previously .

Statistical analysis

All data were checked for normal distribution with the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) was performed to evaluate differences in push-out bonding strength. Two-way ANOVA was used to assess differences in biofilms and resins. Post hoc multiple comparisons were performed using Tukey’s honestly significant difference test. Statistical analyses were performed by SPSS 19.0 (SPSS, Chicago, IL) at alpha of 0.05.

Materials and methods

Fabrication of endodontic sealer containing DMAHDM, MPC and NACP

The experimental primer contained pyromellitic dianhydride glycerol dimethacrylate (PMGDM, Esstech, Essington, PA) and 2-hydroxyethyl methacrylate (HEMA, Esstech) at a mass ratio 10:3, with 50% acetone solvent (all by mass), following a previous study . The experimental adhesive contained PMGDM, ethoxylated bisphenol A dimethacrylate (EBPADMA, Sigma–Aldrich, St. Louis, MO), HEMA and bisphenol A glycerolate dimethacrylate (BisGMA, Esstech) at 45/40/10/5 ratio (referred to as “PEHB”), following a previous study . As the photo-initiator, 1% phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide (BAPO, Sigma–Aldrich) was added to the adhesive. This adhesive was used because a previous study showed that the incorporation of NACP into this adhesive yielded substantial release of calcium (Ca) and phosphate (P) ions . PEHB was used as the resin matrix for endodontic sealer into which antibacterial and remineralizing agents were added.

NACP were incorporated into the adhesive, because NACP could release Ca and P ions to remineralize the demineralized tooth and protect the tooth structures . NACP were synthesized using a spray-drying technique . Briefly, calcium carbonate and dicalcium phosphate were dissolved in acetic acid to produce Ca and P concentrations of 8 mmol/L and 5.333 mmol/L, respectively, thus yielding a Ca/P molar ratio of 1.5, the same as that for ACP [Ca 3 (PO 4 ) 2 ]. This solution was sprayed into a heated chamber of the spray-drying machine. An electrostatic precipitator collected the dried particles. This method produced NACP with a mean particle size of 116 nm . NACP were mixed into endodontic sealer at a NACP filler level of 30% by mass. The 30% NACP was selected because a previous study achieved a high level of Ca and P ion release using this formulation .

DMAHDM was synthesized using a modified Menschutkin reaction in which a tertiary amine group was reacted with an organohalide . 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 ethanol in 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, colourless, and viscous liquid . DMAHDM was mixed into the primer at a DMAHDM/(primer + DMAHDM) mass fraction of 5%. Similarly, DMAHDM was mixed into the PEHB sealer at DMAHDM/(sealer + DMAHDM) = 5%. The 5% DMAHDM was selected following a previous study .

MPC was obtained commercially (Sigma–Aldrich) which was synthesized via a method reported previously . MPC powder was incorporated into primer at MPC/(primer + MPC) mass fractions of 0%, 1.5%, 3%, and 4.5%. Similarly, MPC was mixed into PEHB sealer at MPC/(sealer + MPC) mass fractions of 0%, 1.5%, 3%, and 4.5%. MPC mass fractions > 4.5% were not used due to dentine bond strength loss in preliminary studies.

Tooth root push-out strength to test bonding to root canal dentine walls

The push-out bond strength test was conducted using 54 extracted human canines. The teeth collection was approved by the University of Maryland Baltimore Institutional Review Board. The crowns were removed at the cementoenamel junction with a water-cooled diamond saw (Isomet, Buehler, Lake Bluff, IL), and the roots were trimmed coronally to a uniform length of 17 mm. The working length was determined visually by subtracting 1 mm from the length of a size 10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) at the apical foramen. The canals were endodontically treated according to the crown-down technique using stainless steel K-files up to size 60 (Dentsply Maillefer) attached to an oscillating handpiece (NSK Nakanishi, Kanuma, Tochigi, Japan). A step-back preparation with size 70 and size 80 K-files and sizes 3, 4 and 5 Gates-Glidden drills (Dentsply Maillefer) was undertaken to complete the canal instrumentation. The canals were irrigated with 2 mL of 1% sodium hypochlorite at each change of file, rinsed with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) for 5 min and received a final flush with 5 mL of distilled water, followed by drying with absorbent paper points . The roots were then randomly assigned to nine groups ( n = 6) using the following root canal filling materials:

  • (1)

    Commercial sealer control group. Primer: Epiphany primer. Endodontic sealer: Epiphany sealer (Pentron Clinical Technologies, Wallingford, CT, USA).

  • (2)

    PEHB + NACP sealer control group. Primer: The experimental primer. Sealer: 70% PEHB + 30% NACP.

  • (3)

    DMAHDM group. Primer: 95% experimental primer + 5% DMAHDM. Sealer: 65% PEHB + 5% DMAHDM + 30% NACP (denoted “PEHB + NACP + 5DMAHDM”).

  • (4)

    1.5% MPC group. Primer: 98.5% experimental primer + 1.5% MPC. Sealer: 68.5% PEHB + 1.5% MPC + 30% NACP (denoted “PEHB + NACP + 1.5MPC”).

  • (5)

    3% MPC group. Primer: 97% experimental primer + 3% MPC. Sealer: 67% PEHB + 3% MPC + 30% NACP (denoted “PEHB + NACP + 3MPC”).

  • (6)

    4.5% MPC group. Primer: 95.5% experimental primer + 4.5% MPC. Sealer: 65.5% PEHB + 4.5% MPC + 30% NACP (denoted “PEHB + NACP + 4.5MPC”).

  • (7)

    DMAHDM + 1.5% MPC group. Primer: 93.5% experimental primer + 5% DMAHDM + 1.5% MPC. Sealer: 63.5% PEHB + 5% DMAHDM + 1.5% MPC + 30% NACP (denoted “PEHB + NACP + 5DMAHDM + 1.5MPC”).

  • (8)

    DMAHDM + 3% MPC group. Primer: 92% experimental primer + 5% DMAHDM + 3% MPC. Adhesive: 62% PEHB + 5% DMAHDM + 3% MPC + 30% NACP (denoted “PEHB + NACP + 5DMAHDM + 3MPC”).

  • (9)

    DMAHDM + 4.5% MPC group. Primer: 90.5% experimental primer + 5% DMAHDM + 4.5% MPC. Sealer: 60.5% PEHB + 5% DMAHDM + 4.5% MPC + 30% NACP (denoted “PEHB + NACP + 5DMAHDM + 4.5MPC”).

The commercial control was included for comparative purpose for pull-out strength. According to the manufacturer, the dual-cure methacrylate resin-based sealer Epiphany consists of a mixture of urethane dimethacrylate (UDMA), polyethylene dimethacrylate, bisphenol-A-glycidyldimethacrylate (BisGMA), ethoxylated BisGMA, barium sulphate, silica, calcium hydroxide, bismuth oxide, photoinitiators stabilizers and pigments.

The sealer was applied to the canal with a lentulo spiral (Dentsply Maillefer) attached to a low-speed handpiece (NSK Nakanishi) to avoid bubble formation. Specimens were light-cured for 40 s to create a coronal seal following manufacturer’s recommendations. All roots were coded and placed in 100% humidity for 48 h prior to testing.

For preparation of the specimens, the roots were fixed on acrylic plates with wax and were sectioned in a water-cooled precision saw (Isomet, Buehler, Lake Bluff, IL). Nine slices (1 mm thick) were obtained from each root (three per root third). The first slice of each third was used for the push-out test. Each root section was then subjected to a compressive loading via a Universal Testing Machine (5500R, MTS, Cary, NC) at a crosshead speed of 1 mm/min using a 0.8-mm diameter stainless steel cylindrical plunger. The plunger tip was positioned so that it only contacted the filling material. Push-out bond strength = debond force/area, where the area (of the bonded interface) is the root canal perimeter times the tested root length.

Specimen fabrication for protein adsorption and biofilm experiments

The push-out strength results showed that PEHB + 5DMAHDM + 4.5MPC had a lower strength than other groups. Therefore, it was not included in the subsequent protein-repellent test. In addition, preliminary study showed that 1.5% MPC was not as effective as 3% MPC to repel proteins. Hence, the following five groups were tested in protein and biofilm experiments: (commercial sealer control; PEHB + NACP sealer control; PEHB + NACP + 5DMAHDM; PEHB + NACP + 3MPC; PEHB + NACP + 5DMAHDM + 3MPC).

Resin disks were made using the cover of a 96-well plate (Costar, Corning, Corning, NY) as moulds following a previous study . Ten μL of primer was placed at the bottom of each dent in the 96-well plate. After drying with a stream of air, 20 μL of adhesive was placed into the dent and photo-polymerized for 30 s (Demetron VCL401), using a mylar strip covering to obtain a disk of approximately 8 mm in diameter and 0.5 mm in thickness. The cured disks were immersed in water and stirred with a magnetic bar at 100 rpm for 1 h to remove any uncured monomers, following a previous study . The disks were sterilized with ethylene oxide (AnproleneAN 74i, Andersen, Haw River, NC) and de-gassed for 7 days.

Protein adsorption onto endodontic sealer resin surfaces

Protein adsorption on resin disks was determined using a micro bicinchoninic acid (BCA) method . Thirty resin disks were fabricated, with six disks for each group. Each disk was immersed in phosphate buffered saline (PBS) for 2 h, and then immersed in 4.5 g/L bovine serum albumin (BSA, Sigma–Aldrich) solution at 37 °C for 2 h . A protein analysis kit (micro BCA assay, Fisher, Pittsburgh, PA) was used to determine the BSA concentration in the SDS solution. 25 μL of the SDS solution and 200 μL of the BCA reagent were mixed into the wells of a 96-well plate and incubated at 60 °C for 30 min. 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 .

Endodontic bacteria strains and culture media

The use of all bacterial species was approved by University of Maryland Baltimore Institutional Review Board. All species were obtained from the American Type Culture Collection (ATCC, Manassas, VA): Actinomyces naeslundii ( A. naeslundii , ATCC12104); Fusobacterium nucleatum ( F. nucleatum , ATCC25586); Enterococcus faecalis ( E. faecalis , ATCC4083). E. faecalis , A. naeslundii , and F. nucleatum were grown in tryptic soy broth (TSB, Sigma–Aldrich) supplemented with yeast extract (5 g/L), l -cysteine hydrochloride (0.5 g/L), hemin (5 mg/L) and menadione (1 mg/L) at 37 °C anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ), following previous studies . For each species, the inoculum was adjusted to 10 8 colony-forming unit counts CFU/mL for future biofilm formation, based on the standard curve of OD 600nm versus the CFU/mL for each species, as in a previous study . A. naeslundii , F. nucleatum and E. faecalis were selected to develop a robust endodontic three-species biofilm model, which was validated in a previous study . These three species represented the early, middle and late colonizers of endodontic biofilms, respectively.

Multispecies endodontic biofilm formation on endodontic sealer disks

Bacteria grow aggregated on canal surfaces to form a biofilm, and this biofilm enables the bacteria to survive antimicrobial insults . The establishment of the biofilm architecture follows a sequence of events, going from initial attachment of a single cell to formation of a three-dimensional mature biofilm . The early and mature phases of a biofilm were respectively characterized with a thin biofilm formation and a mushroom-shaped biofilm architecture . To determine the relationship between bactericidal efficacy and biofilm formation, early-stage (3-day) and mature (14-day) multispecies endodontic biofilms were tested . 100 μL of A. naeslundii , F. nucleatum and E. faecalis suspensions were mixed and pipetted into 30 mL TSB supplemented medium to form a mixed species suspension. Sterile saliva pellicle-coated resin disks were transferred to a new 24-well plate. Each well was inoculated with 1.5 mL of the mixed bacterial suspension and incubated in an anaerobic condition at 37 °C for 3 days and 14 days, respectively, representing the early-stage and mature endodontic biofilms . The fresh TSB-supplemented medium was changed every 24 h.

Live/dead bacteria imaging

Resin disks with 3-day or 14-day biofilms were washed with cysteine peptone water (CPW) to remove the non-adherent bacteria. Live/dead bacterial kit (Molecular Probes, Eugene, OR) was used following the manufacturer’s instructions. Live bacteria were stained with SYTO 9 to emit a green fluorescence. Bacteria with compromised membranes were stained with propidium iodide to emit a red fluorescence. Biofilms were examined with an inverted epifluorescence microscope (TE2000-S, Nikon, Melville, NY).

Colony-forming unit (CFU) counts

For CFU counts, twelve resin disks were made for each endodontic sealer, with six disks for each biofilm stage. Biofilms were formed by culturing for 3 days and 14 days as described above. Disks were transferred into vials with 2 mL CPW, and the biofilms were harvested by scraping and sonication/vortexing (Fisher, Pittsburg, PA). Tryptic soy blood agar plates (supplemented with 5 g/L yeast extract, 0.5 g/L l -cysteine hydrochloride, 5 mg/L hemin, 1 mg/L menadione, 5% sheep blood) were used, following ATCC instructions. Biofilm suspensions were serially diluted, spread onto agar plate and incubated at 37 °C anaerobically for 72 h . Then, the number of colonies was counted by a colony counter (Reichert, NY), which was used with the dilution factor to calculate the CFU counts .

XTT metabolic assay

Twelve disks of each sealer were made for metabolic assay, with six disks for each biofilm stage. The 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay were conducted to measure the metabolic activity of biofilms as described previously . Disks with 3-day and 14-day biofilms were washed using CPW, followed by transferring into new 24-well plates with 1 mL XTT working regent (0.5 mg/mL XTT and 1 μM menadione), then cultured for 2 h at 37 °C. The absorbance at OD 492 nm was measured via the microplate reader (SpectraMax M5) . The conversion of the XTT substrate to a soluble coloured formazan product correlates with cell viability. A higher absorbance is related to a higher metabolic activity in the biofilm adherent on the resin disks .

Measurement of polysaccharide production by biofilms on endodontic sealers

For polysaccharide, twelve disks of each endodontic sealer were made with six disks for each biofilm stage. The water-insoluble polysaccharide in the extracellular polymeric substance (EPS) of biofilms was determined using a phenol-sulfuric acid method . Each disc with 3-day and 14-day biofilm was immersed in a vial with 2 mL CPW, and the biofilm was collected by sonication/votexing. Centrifugation yielded a precipitate, which was rinsed with PBS and resuspended in 1 mL of de-ionized water. Then, 1 mL of 6% phenol solution was added to the vial, followed by 5 mL of 95–97% sulfuric acid . The vial was incubated for 30 min. Then, 100 μL of the solution was transferred into a 96-well plate. The amount of polysaccharide in biofilms was determined by measuring the absorbance at OD 490 nm with the microplate reader. Five glucose concentrations of 0, 5, 10, 20, 50 and 100 mg/mL were used as standard in the conversion of the OD readings to the polysaccharide concentrations .

Calcium (Ca) and phosphate (P) ion release measurement

The ion releases from PEHB + NACP + 5DMAHDM + 3MPC and PEHB + NACP control were measured to investigate the effect of adding DMAHDM and MPC on ion release. A sodium chloride (NaCl) solution (133 mmol/L) was buffered to three different pH values: pH 4 with 50 mmol/L lactic acid, pH 5.5 with 50 mmol/L acetic acid, and pH 7 with 50 mmol/L HEPES . As in previous studies , three specimens of 2 × 2 × 12 mm were immersed in 50 mL of solution yielding a specimen volume/solution of 2.9 mm 3 /mL. This was similar to a specimen volume per solution of about 3.0 mm 3 /mL in a previous study . For each solution, the concentrations of Ca and P ions released from the specimens were measured at 1, 3, 7, 14, 21, and 28 days. At each time, aliquots of 0.5 mL were removed and replaced by fresh solution. The aliquots were analyzed for Ca and P ions via a spectrophotometric method (DMS-80 UV-visible, Varian Palo Alto, CA) using known standards and calibration curves, as described previously .

Statistical analysis

All data were checked for normal distribution with the Kolmogorov–Smirnov test. One-way analysis of variance (ANOVA) was performed to evaluate differences in push-out bonding strength. Two-way ANOVA was used to assess differences in biofilms and resins. Post hoc multiple comparisons were performed using Tukey’s honestly significant difference test. Statistical analyses were performed by SPSS 19.0 (SPSS, Chicago, IL) at alpha of 0.05.

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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Novel bioactive root canal sealer to inhibit endodontic multispecies biofilms with remineralizing calcium phosphate ions
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