The purpose of this study was to incorporate EgMA, an antibacterial monomer into two commercial dental adhesive systems for their application in endodontic restoration with the aim to disinfect the root canal space before curing and to inhibit bacterial growth on their surfaces after being cured.
EgMA monomer was added at 20% wt. into the formulation of the single-component self-etch, Clearfil Universal Bond™ (CUB) and into the catalyst and the adhesive components of the total-etch Adper Scotchbond-multipurpose™ (SBMP) adhesive systems. The degree of conversion (DC) was calculated from FTIR spectra, glass transition temperature (Tg) determined by DSC, water sorption and solubility were measured gravimetrically, and surface free energy (SFE) via contact angle measurements. The bonding performance to coronal and middle root canal dentin was assessed through push-out bond strength after filling the canals with a composite core material and the surface integrity was observed using SEM and confocal laser scanning microscopy (CLSM). The standard agar diffusion test (ADT) was used to identify the sensitivity of three endodontically pathogenic bacteria, Enterococcus faecalis , Streptococcus mutans and Propionibacterium acnes to uncured EgMA modified adhesives. Multispecies biofilm model from these strains was grown on the disc surface of cured adhesives and investigated using quantitative microbial culture and CLSM with live/dead staining. MTT assay was also used to determine the cytotoxicity of these adhesives.
The incorporation of EgMA lowered polymerization exotherm and enhanced the hydrophobic character of these adhesives, without changing the DC and Tg in comparison to the controls (without EgMA). The total push-out bond strengths of the EgMA-containing adhesives were not significantly different from those of the controls (p > 0.05). The modification of self-etch adhesive system enhanced the bond strength in the middle region of the roots canal. SEM of debonded specimens and CLSM examination showed the integrity of the resin-dentin interfaces. For all three bacteria tested, the sizes of the inhibition zones produced by uncured EgMA modified adhesives were significantly greater (p < 0.05) than those of the controls. The results of biofilm inhibition tests showed less CFU for total bacteria on bonding agents with EgMA compared to the control materials (p < 0.05). The modification at 20% monomer concentration had no adverse effects on cytocompatibility of both adhesives tested.
The inclusion of EgMA endows dental adhesives with effective antibacterial effects without influencing their curing properties, bonding ability to root canal dentin, and cytotoxicity against human gingival fibroblasts, indicating the usefulness of their application in endodontic restorations.
Bonding of posts to root canal dentin is still a challenge due to the reduced number of dentinal tubules in the apical third of the root , limited access and visibility. Furthermore, the large configuration factor (C-factor) of the endodontic cavity results in a high contraction stress, that can exceed the bond strength, increasing the risk of voids and microgaps within the cement interface with subsequent bonding failure and microleakage . Microleakage can cause new bacterial invasion of the root canal space also, complete removal of bacteria from the root canal system following the cleaning and shaping of the root canal is at present impossible to achieve . Residual bacteria often remain in the tubules, which may repopulate the root canal and jeopardise clinical performance and longevity of the endodontic restoration. Therefore, imparting an antibacterial function to dental restorative materials, and in particular to the dental adhesives as they directly contact tooth structure and infiltrates into dentinal tubules is expected to disinfect the cavity, lowering the risk of reinfection and secondary caries.
Several attempts to produce dental adhesives with antibacterial activity have been made either by the addition of soluble antimicrobial agents, such as chlorhexidine, or immobilisation of antibacterial components in the resin matrix . However, the release of antibacterial agent could cause an adverse effect on mechanical properties, toxicity and short-term antibacterial effectiveness whilst, the immobilisation of antimicrobial agents prevents or reduces colonisation of contacted bacteria without leaching out from the material, resulting in long-lasting antibacterial activity without adverse effects on mechanical properties and bonding characteristics . A number of ionic mono and di-methacrylate monomers containing quaternary ammonium groups have been incorporated into existing dental dimethacrylate-based monomers demonstrating bactericidal activity. For instance, Clearfil protect bond, which contains methacryloyloxydodecylpyridinium bromide (MDPB), and dental adhesives with methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB) have been found to exhibit an inhibitory effect on the growth of Streptococcus mutans . However, some of the quaternary ammonium based monomers exhibit miscibility problems with hydrophobic dimethacrylates . In addition, incorporation of these monomers at high concentrations to obtain reliable antibacterial effects results in adverse effects on mechanical properties and unwanted release of the monomers into the surrounding tissues .
Eugenol is a well-known antimicrobial essential oil, which is used in combination with zinc oxide in different dental applications such as temporary filling materials and root canal sealers and is very effective against a range of oral bacteria . The main disadvantage of eugenol-containing materials is the fact that they inhibit the polymerization reaction of methacrylate resins due to remaining free eugenol. Eugenyl methacrylate (EgMA) is an eugenol derivative that is able to copolymerize with other methacrylate monomers after curing and immobilises the antibacterial eugenol moieties in the polymer backbone without the inhibitory effect characteristic of the phenol group . The authors have previously reported that the incorporation of EgMA was effective in providing resin composite materials with intrinsically antibacterial activity against a range of oral bacteria commonly associated with the failure of coronal and endodontic restorations . This effect based on the strong antibacterial activity of EgMA monomer . In addition, the immobilisation of eugenol is advantageous as it avoids the migration of this molecule into the surrounding tissues and improves its hydrolytic stability.
Thus, the aim of the study was to investigate the efficacy of the modified dental adhesives via the inclusion of the eugenol methacrylate derivative. The influence of this monomer on curing properties, Tg, wettability, water sorption, bonding ability and cytotoxicity of these modified bonding agents are reported.
Materials and methods
EgMA incorporation into bonding agents
Two commercial adhesives, Clearfil Universal Bond™ (CUB) and Adper Scotchbond™ multi-purpose plus (SBMP) adhesives were used in this study as parent bonding systems to test the effects of incorporation of the antibacterial monomer. Their manufacturers and chemical composition are presented in Table 1 . EgMA monomer was synthesized via a method reported previously by Rojo et al. . A stock solution of EgMA monomer and CQ/EDAB (0.5 wt./0.5 wt.) both from Sigma–Aldrich (Company Ltd., Dorset, UK) was added at 20% by weight into the formulation of single-component CUB and into the adhesive/catalyst components of SBMP to prepare two modified experimental adhesives, designated respectively, as Mod.CUB and Mod.SBMP ( Table 1 ). The selection of this percentage was based on pilot study that showed that the addition of 20 wt.% EgMA into Bis-GMA/HEMA (70/30 wt.%) blend, a commonly used dental adhesive resin formulation, had no adverse effects on degree of monomer conversion and Tg of the polymers.
|Boding agent||Manufacturers (patch number)||Code||Composition|
|Clearfil Universal Bond™ (self-etch)||Kuraray, Tokyo, Japan (1562R041R)||CUB||MDP, Bis-GMA, HEMA, hydrophilic aliphatic dimethacrylate, colloidal silica, silane coupling agent, CQ, ethanol, water|
|Modified Clearfil Universal Bond™||Mod.CUB||CBU + 20 wt.% stock solution of EgMA a|
|Adper Scotchbond™ multi-purpose plus (total-etch)||3 M ESPE, St. Paul, MN, USA (N662538)||SBMP||Etchant: 35% phosphoric acid gel|
|Activator: ethyl alcohol, sodium benzenesulfinate|
|Primer: water, HEMA, copolymer of acrylic, itaconic acids|
|Adhesive: Bis-GMA, HEMA, tertiary amines, photi-initiator|
|Catalyst: Bis-GMA, HEMA, benzoyl peroxide|
|Modified Adper Scotchbond™ multi-purpose plus||Mod.SBMP||SBMP Etchant|
|SBMP Adhesive + 20 wt.% stock solution of EgMA a|
|SBMP Catalyst + 20 wt.% stock solution of EgMA a|
For the solvated one-bottle, CUB control and modified CUB adhesives, the solvent was evaporated under reduced pressure in a dark container until the resin reached a constant mass as solvent evaporation was assumed to be complete and then carefully placed into different moulds. For total-etch SBMP control and modified SBMP adhesives, the activator and primer were first smeared on moulds, dried with a gentle stream of air, then equal masses from adhesive and catalyst components were mixed and applied. For water sorption, solubility, surface contact angle, cytotoxicity and biofilm inhibition tests, resin discs of each material were produced in Teflon mould (10 mm diameter, 1 mm thick). After filling the mould, the discs were covered with glass slides, to exclude atmospheric oxygen, and then cured by visible light for 40 s, using a dental curing unit (Optilux, Demetron Res Crop, Danbury, USA) with an irradiance of 600 mW cm −2 . After removing the specimen from the mould, light-curing was repeated on the opposite surface for another 40 s.
Degree of conversion
The degree of conversion of each adhesive was analysed before and after cure using FTIR spectroscopy (ATR accessory, Spectrum one, Perkin Elmer, Waltham, MA, USA). The spectra of the polymer were obtained by light-curing one drop of each adhesive between two translucent Mylar strips, pressed to produce a very thin film. Five cured specimens of each group were tested 10 min after curing and after 24 h storage at 37 °C. The degree of cure was determined using Eq. (1) :
Degree of conversion ( % ) = [ 1 − ( A 1637 / A 1608 ) p o l y m e r ) ( A 1637 / A 1608 ) m o n o m e r ) ] × 100
where A1637 and A1608 correspond to the absorbance of the aliphatic υ C = C peak registered at 1637 cm −1 and to the aromatic υ C = C peak registered at 1608 cm −1 respectively before and after polymerization.
A thermocouple (1.3 mm diameter) fitted out to a high-sensitivity temperature recorder (KM1242, Herts, UK) was used to measure the polymerization temperature. The wire was placed centrally in a cylindrical Teflon mould (4 mm diameter, 12 mm depth) filled with each adhesive material and its stripped ends were levelled with the material’s surface to be irradiated. The materials were polymerized for 40 s from one side and the maximum temperature was reported during the polymerization cycle. Five measurements were done for each tested material at 23 °C.
Differential scanning calorimetry (DSC) was carried out using a Perkin Elmer machine (Waltham, MA, USA) to determine the glass transition temperature of the cured adhesives. Samples of about 10 mg were heated from 0 °C to 230 °C at the rate of 20 °C/min in an inert N 2 atmosphere. Three samples from each formulation were tested.
Measurement of contact angle and surface free energy (SFE)
The contact angle θ and SFE ( Y s ) were evaluated on bonding surface discs using the sessile drop method as described in our previous study .
Water sorption and solubility
Water sorption and solubility were determined according to the ISO specification 4049. Five resin discs (10 mm diameter, 1 mm thick) of each adhesive material were immersed in 10 ml distilled water and weighted at noted interval during the 28 days immersion period.
The mass change percentage was calculated using Eq. (2) :
where Mi is the initial mass of the specimen and Ms is the mass of saturated specimen at the end of the immersion period.
The specimens were dry-stored again at 37 °C and reweighed using approximately the same time intervals until a constant dry mass (Md) was obtained.
Sorption (SR) and solubility (SL) in μg/mm 3 were calculated based on the percentage of mass gain or loss during the sorption and desorption cycles using the following equations:
Push-out bond strength test
Thirty-six human single-rooted premolars extracted after obtaining an informed consent of the patients and following a protocol approved by an institutional review board were used in this study (Research Ethics Committee Reference Number14/LO/0123). All teeth were stored at 4 °C in distilled water and used within one month. Teeth were randomly and equally assigned to 4 groups based on the adhesive materials used in this study (n = 9), three specimens from each group were reserved for confocal microscopy analysis. The crown was sectioned at the cemento-enamel junction using a low speed; water-cooled diamond saw microtome (Isomet 1000, Buehler, Lake Bluff, IL, USA). The teeth were endodontically treated with nickel–titanium rotary instruments (Protaper; Dentsply Maillefer, Ballaigues, Switzerland) and 1% sodium hypochlorite irrigation. The canal was filled with gutta-percha and calcium hydroxide endodontic sealer (Sealapex, Kerr, SpA, Salerno, Italy) using the lateral condensation technique. The prepared roots were mounted vertically in acrylic resin block using an aligning device. After 24 h storage at 37 °C in relative humidity, the first 8 mm of the canal was shaped with a cylindrical flat end diamond bur (Komet 837/016, Lemgo, Germany) so that a standardised cavity of 2 mm in diameter was prepared in the coronal and middle portion of the root canal.
The dental adhesives were applied in accordance with the manufacturer’s instructions. For the CUB control and modified CUB self-etch adhesives, the bonding agent was applied to the entire root canal with the applicator brush and rubbed for 10 s, then dried with a gentle air blow for 10 s and light-cured for 40 s using dental light-curing unit at 600 mW cm −2 (Optilux, Demetron Res Crop., Danbury, USA). For the SBMP control and modified SBMP total-etch adhesives, the root dentin was acid-etched with 35% phosphoric acid gel for 15 s, rinsed for 20 s with water and dried with absorbent paper points (Dentsply/Maillefer, Petrópolis, RJ, Brazil). A layer of activator was applied into the root canal using disposable brushes, followed by air-drying for 5 s. The primer was applied and air-dried for 5 s. The adhesive and catalyst were mixed and applied with a fresh disposable brush and light-cured for 40 s. A dual-cure composite core (Clearfil™DC Core plus, Kuraray, Japan) was injected into the post-space and light-cured for 60 s. The root segment was then placed in individually labelled containers in relative humidity at 37 °C. After 24 h, the bonded roots were transversely sectioned to create 1.5 mm thick root slices, the thickness was verified using a digital electronic calliper, the top root slices were discarded to avoid the influence of excess material, producing four root slices from each root (2 coronal and 2 middle) for subsequent push-out bond strength tests. Each slice was marked with a permanent marker on its coronal aspect and sufficiently supported by a stainless steel jig with clearance for the dislodged core material. The push-out force was applied in an apical-coronal direction using a cylindrical plunger with a diameter of 1.8 mm attached to a universal testing machine (Instron model 5569A-Series Dual Column, High Wycombe, UK) at a crosshead speed of 0.5 mm/min until failure. The maximum load at failure was recorded in Newton (N) and was converted to MPa by dividing the applied load by the bonded area, using the following equation:
The modes of failure were examined visually using a stereomicroscope (WILD M32; Heerbrugg, Switzerland) at ×30 and classified as adhesive failure between core material and dentin, cohesive failure with complete dentin or core material cohesive failure and mixed failure with partial interfacial adhesive failure with the presence of core/dentin cohesive failure.
Furthermore, four representative debonded specimens per group that failed in mixed or adhesive modes were selected to analyse the ultramorphology of the fractured surface with SEM. The specimens were dried overnight, mounted on aluminium stubs with carbon cement, sputter-coated with gold and observed with a scanning electron microscope (SEM, Hitachi High Technologies, S-3500N) at an accelerating voltage of 10 KeV and increasing magnifications.
Confocal laser scanning microscopy (CLSM)-interface evaluation
For this analysis, 0.1 wt% fluorescein dye (Sigma–Aldrich, UK) was added to the single bottle component of CUB adhesives (control and modified) and to the adhesive and catalyst bottles of SBMP (control and modified) while, 0.1 wt% Rhodamine B (Rh B: Sigma–Aldrich, UK) was added into the primer bottle of Adper SBMP adhesives.
A further three specimens from each group were bonded, as previously described, with these labelled adhesive systems and employed for the confocal microscopy analysis.
After 24 h storage in 100% relative humidity, the specimens were longitudinally sectioned into two halves and polished using wet SiC abrasive papers of ascending grit #600–#2500 (Versocit; Struers) with final ultra-sonication treatment in a distilled water bath for 5 min. The microscopy examination was performed using a confocal laser scanning microscope (Leica SP2 CLSM; Leica, Heidelberg, Germany) equipped with a 63×/1.4 NA oil-immersion lens and using 488-nm argon/helium (fluorescein excitation) or 568-nm krypton (rhodamine excitation) laser illumination. The entire resin–dentin interface was completely investigated and three representative images of the most common distinguishing characteristics detected in each specimen were captured. All images were further reconstructed with Image J software.
Agar diffusion test (ADT)
The antibacterial activity of uncured adhesive resins was evaluated by agar diffusion test against Enterococcus faecalis , S. mutans and Propionibacterium acnes . The bacteria were revived from −80 °C and plated on FAA plates (Fastidious Anaerobe Agar with 5% Horse Blood-Lab M, UK). Bacterial test suspensions with a concentration of 6 × 10 5 colony forming units (CFU)/mL were prepared from the pre-cultures. An aliquot of 150 μL of the bacterial suspension was spread evenly throughout the FAA agar plate using sterile swabs. Under aseptic conditions, a 20 μL portion of each bonding agent was absorbed onto sterile paper discs (6 mm diameter, 1.5 mm thick, Schleicher & Schuell, Germany) and placed on the inoculated agar surface (n = 3). Pure eugenol was used as positive control. After anaerobic incubation of the plates at 37 °C for 48 h, the inhibition zones produced around the paper discs were measured [(Outer diameter of inhibition zone − paper disk diameter)/2].
Biofilm inhibition test
Three-species biofilms composed of E. faecalis , S. mutans and P. acnes were grown on cured resin adhesives discs to investigate their capacity to reduce or inhibit colony formation by these bacterial species. To establish the biofilm, the bacterial strains were cultured anaerobically at 37 °C in MACS-MG-1000-anaerobic workstation (80% nitrogen, 10% hydrogen, 10% carbon dioxide) on Fastidious Anaerobe Agar supplemented with 5% defibrinated horse blood (FAA, Lab M, Heywood, UK). An individual starter culture of each bacterial strain was transferred into 3 mL of modified fluid universal medium (mFUM) and incubated anaerobically at 37 °C for 3 h. The absorbance was adjusted with fresh mFUM to 0.5 at 540 nm to obtain 10 7 cells mL −1 using Labsystems iEMS Reader (MF, Basingstoke, UK).
All discs (n = 6, for each adhesive) were soaked in distilled water at 37 °C for 24 h to remove unpolymerized monomers and then sterilised by wiping with 70% ethanol in water and were exposed to UV radiation for 30 min. Discs were placed in 1 mL of mFUM contained in 24 well tray and pre-reduced in the anaerobic workstation.
The discs were then seeded with 400 μL (4 × 10 6 cells) of each of the three starter cultures. The biofilms were grown anaerobically with regular medium change every 24 h for the first 7 days. In order to nutritionally starve the biofilms, they were further grown anaerobically for another 7 days without medium change following the protocol previously mention by Niazi et al. . To enumerate the numbers of bacteria in the biofilms, each disc was placed in 1 mL of BHI (Brain-Heart Infusion Broth, Lab M) and vortex for 1 min to disperse the biofilm from the surface of the disc. After serial dilution in BHI, aliquots (100 μL) were plated onto duplicate FAA plates and incubated anaerobically for 7 days and colonies were counted.
Two discs from each group were gently washed twice with PBS to remove non-adherent cells and stained with Live/Dead BacLight bacterial viability kit (Invitrogen, Paisley, UK) and washed again before visualisation under a Leica SP2 confocal laser scanning microscope (CSLM). The quadrants of the biofilm on resin disc were demarcated by 4 marks made at the corners of the glass bottom of the 35 mm diameter Petri dishes (SLS, UK) by using a permanent marker. Biofilm structure was examined by three different areas in each quadrant of the biofilm. The mean percentages of dead (red) and live (green) biovolumes were analysed using bio Image_L .
MTT (methyl tetrazolium) assay was used to evaluate the cytotoxicity of adhesives with human gingival fibroblast (P8, ScienCell™ Res. Lab., UK) at 24 h and 48 h according to the International Standard ISO 10993-5. Adhesives eluents were obtained by immersing sterile disc samples in 3 mL of fibroblast medium (500 mL basal medium, 10 mL fetal bovine serum, 5 mL of fibroblast growth supplement and 5 mL of penicillin/streptomycin solution, ScienCell™, UK) within bijou vials, which were then placed onto a roller at room temperature. The supernatants were collected at 24 and 72 h time points and refrigerated at −20 °C to be used for cytotoxicity measurements.
HGF cells were cultured at 37 °C humidified atmosphere with 5% CO 2 to reach about 80% confluent, trypsinised and then seeded on a 96 well plate (100 μL/well) at a density of 1 × 10 4 cells/well. The cells were incubated at 37 °C, 5% CO 2 for 24 h to allow for cell to attach and acclimatisation prior to addition of the test eluents. After 24 h, the fibroblast media were removed from both plates and replaced with 100 μL of the leached eluents from adhesives. Untreated cells served as a negative control while positive control cells were treated with 10% v/v ethanol solution. Each group consisted of five replicate wells. Then the plates were incubated for 24 h or 48 h (exposure times), after which the test eluents were removed and replaced with 100 μL of MTT (5 mg/mL PBS) for 4 h. MTT solution was then removed and 100 μL dimethyl sulfoxide (DMSO) was added to each well. The plate was shaken for 5 min and the absorbance of the purple coloured solution was measured using a UV–vis spectrophotometer plate reader at wavelength 570 nm (Opsys MR, Dynex Technologies, Chantilly, VA, USA). Relative cell viability is then expressed as a percentage of untreated negative control reading. Each experiment was done in duplicates.
After analysing the normality of data distribution (Kolmogorov–Smirnov test), a Mann–Whitney (nonparametric) test or Independent t-test (for normally distributed values) was used to determine the effects of EgMA monomer addition on properties of commercial parent adhesives tested. A one-way (ANOVA) and Tukey’s post hoc test were also employed for the statistical evaluation of ADT and cytotoxicity data. In all tests, the level of significance was set at p < 0.05.