Effect of a novel quaternary ammonium silane cavity disinfectant on durability of resin–dentine bond

Abstract

Objective

The present study examined the effect of a quaternary ammonium silane (QAS) cavity disinfectant on the viability of human dental pulp cells, dentine bond durability and nanoleakage of simplified etch-and-rinse adhesives.

Methods

Etched dentine surface of third molars were randomly divided into two adhesive groups, Adper™ Single Bond 2 and Prime & Bond ® NT™. For each adhesive, the teeth were randomly assigned to five cavity disinfectant groups ( N = 6): Group 1: deionised water (control); Group 2: 2% chlorhexidine (CHX); Group 3: 2% QAS; Group 4: 5% QAS and Group 5: 10% QAS. The cavity disinfectants were applied on etched dentine surfaces for 20 s, followed by adhesive application. The bonded teeth were sectioned for bond strength testing at 24 h, 6 months and 12 months. Viability of human dental pulpal cells was examined using MTT assay. Bond strength data were analysed using 3-way ANOVA and Tukey test. Interfacial nanoleakage was evaluated after 24 h and 12 months and analysed using Kruskal–Wallis test.

Results

Significant differences in bond strength were observed for the factors disinfectants ( p < 0.001) and time ( p < 0.001); while the factor, adhesive, was not significantly different ( p = 0.203). The 2% QAS cavity disinfectant preserved bond strength of both adhesives and reduced interfacial nanoleakage after 12 months. Cell viability was the lowest for 2% CHX, followed by 2% QAS and the control.

Conclusions

The 2% QAS cavity disinfectant demonstrated greater cell viability compared to 2% CHX, with no adverse effect on immediate bond strength and preserved bond stability over time.

Clinical significance

Incorporation of 2% quaternary ammonium silane cavity disinfectant in the resin–dentine bonding protocol enhances the success rate of bonded restorations.

Introduction

The integrity of the adhesive bond between the tooth substrate and resin is pivotal to the durability of resin-based restorations. Despite the popularity of tooth-coloured restorations, secondary caries at tooth-restoration margins is still the primary reason for replacement of resin composite restorations . Incomplete removal of caries-infected dentine during cavity preparation may result in entrapment of bacteria within the cavity. Residual bacterial colonies within the smear layer and dentinal tubules produce degenerative products, such as lipoteicholic acids and endotoxins that diffuse into the dental pulp, causing irritation and inflammation . Proliferation of remnant bacteria may result in leakage at restorative margins, causing secondary caries and restoration failure over time . Hence, disinfection of a cavity preparation with an antimicrobial agent has been recommended prior to the restorative procedures .

Chlorhexidine (CHX) is commonly used as a cavity disinfectant to eliminate residual bacteria in caries-affected dentine following mechanical caries removal . Chlorhexidine is a potent and broad spectrum antimicrobial agent against oral bacteria, notably Streptococcus mutans . Being an inhibitor of dentine matrix-bound matrix metalloproteinases and cysteine cathepsins , CHX plays an important role in preserving the integrity of resin–dentine bond over time. The substantivity of CHX is due to its ability to bind to tissues surfaces . However, CHX only binds electrostatically to dentine collagen and may be displaced by competing cations from dentinal fluid and saliva , thereby compromising its antimicrobial and protease inhibitory effects . Furthermore, CHX has been shown to exert dose-dependent, mild transdentinal toxic effects on odontoblast-like cells . Hence, there is a need to look for alternative antibacterial cavity cleansers to inhibit oral biofilms and caries.

The quaternary ammonium compound, 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride (SiQAC; C 26 H 58 ClNO 3 Si; CAS Registry Number 27668-52-6), is commonly used as antimicrobial coatings for medical devices and garment fabrics because of its low in vivo toxicity profile . Being a trialkoxysilane, SiQAC possesses hydrolysable and condensable methoxy groups, which enable it to covalently attach to other alkoxysilanes or silanol-containing substrate surfaces via the formation of siloxane bridges . The antimicrobial property of SiQAC may be attributed to its long, lipophilic C18 alkyl chain. The latter penetrates bacterial cell membrane and causes cell death via a contact-killing mechanism . Recently, SiQAC has been coupled to other trialkoxysilanes with methacryloxy or epoxy functionalities via sol–gel platform chemistry that utilises tetraethoxysilane (TEOS) or dimethyldiethoxysilane as the anchoring unit . Such sol–gel reactions between one tetraalkoxy silane and two trialkoxysilanes generated a host of antimicrobial quaternary ammonium silane (QAS) molecules with methyacryloxy or epoxy functionalities that can copolymerise with methacrylate or epoxy resins.

Using similar platform chemistry, an ethanol- or acetone-soluble, fully-hydrolysed, partially-condensed version of QAS has been synthesised by the authors. The synthesis involves reaction of TEOS with 3-(triethoxysilyl)-propyldimethyloctadecyl ammonium chloride (i.e. the ethoxy version of SiQAC, abbreviated as Et-SiQAC). Because the hydrolysis by-product of this sol–gel reaction is ethanol instead of methanol, the QAS (codenamed K21; CAS Registry Number 1566577-36-3; IUPAC name: 1-octadecanaminium, N,N′-[[3,3-bis [[[3-(dimethyloctadecylammonio) propyl] dihydroxysilyl] oxy]-1,1,5,5,-tetrahydroxy-1,5-trisiloxanediyl] di-3,1-propanediyl] bis [N,N-dimethyl] chloride (1:4); Fig. 1 A) may be used, without further purification, as an intra-oral cavity disinfectant. Unlike the SiQAC or Et-SiQAC molecule, inclusion of TEOS as the network-forming agent enables a three-dimensional organically modified silicate to be produced by condensation of additional tetra- and triethoxysilane molecules with remnant silanol groups within the molecule.

Fig. 1
Proposed chemical formula of the quaternary ammonium silane molecule K21. (For interpretation of the references to color in the text, the reader is referred to the web version of this article.)
Reproduced with permission from KHG fiteBac Technology, Marietta, GA, USA

When the QAS cavity disinfectant is applied to smear layer-depleted dentine, progressive condensation of the 3-D organically-modified silicate network may result in occlusion of hybrid layers and dentinal tubules that are not completely infiltrated by dentine bonding agents, with the potential of reducing nanoleakage and dentine hypersensitivity. Based on the contact-killing antimicrobial activities exhibited by SiQAC-derived macromolecules , it is anticipated that the QAS cavity disinfectant may be used on caries-infected dentine , caries-affected dentine, as well as sound dentine, where ingress of bacteria may occur via leaking cavosurface margins. Being a molecule without methacryloxy functional groups, the QAS cavity disinfectant may be used for disinfecting cavities to be restored by amalgam or glass ionomer cements.

However, the use of cavity disinfectants that do not copolymerise with dentine adhesives may influence the bond strength of those dentine adhesives . It is not known if the QAS cavity disinfectant has any adverse effect on dental pulp cells and dentine bonding after it is applied to acid-etched dentine. Accordingly, prior to clinical evaluation, the effect of QAS cavity disinfectant on the viability of human dental pulp cells, resin–dentine bond durability and nanoleakage should be characterised. Thus, the objectives of the present study were to evaluate the cytotoxicity of QAS on human dental pulp cells. The effect of dentine pre-treatment with different concentrations of the QAS cavity disinfectant on immediate, long-term bond strength and nanoleakage of simplified etch-and-rinse adhesives were also examined. The null hypotheses tested were that (i) there is no difference in cytotoxicity between QAS and CHX, (ii) pre-treatment of dentine with QAS does not affect the immediate dentine bond strength and (iii) pre-treatment of dentine with QAS has no effect in preventing degradation of the resin–dentine interface over time.

Materials and methods

Synthesis

The experimental versions of QAS cavity disinfectant examined in the present study were synthesised by sol–gel reaction between 1 mole of TEOS (Mw 208) and 4 moles of Et-SiQAC (Mw 538). In a typical synthesis, 2.08 g of TEOS was blended with 29.89 g of Et-SiQAC (72 wt% of Et-SiQAC dissolved in ethanol) and 5 mL of ethanol (to render the blend more homogeneous). Hydrolysis was initiated by the addition of 10.08 g of 0.02 M HCl-acidified water (pH 1.66, representing 3.5 times the stoichiometric molar concentration of water required, to ensure complete hydrolysis). Hydrolysis and condensation of the two ethoxysilanes was monitored by Fourier transform infrared spectroscopy .

Following completion of the hydrolysis reaction (approximately 3 h), a yellow solution mixture was obtained, the IR spectrum of which was characterised by the presence of silanol groups, ethanol and water . The yellow solution mixture was then maintained at 80 °C for 6 h to remove as much as possible the reaction by-products (ethanol and water), until a pale yellow rubbery solid material was produced. This yellow solid ( Fig. 1 B) represented a partially-condensed form of the K21 molecule shown in Fig. 1 A. Full condensation could be achieved by adding an alkali solution to raise the pH of the solution to 7.2, , wherein an ethanol-insoluble precipitate was progressively produced. This procedure was omitted during the synthesis phase and complete hydrolysis was subsequently achieved after the material was applied on dentine (to be reported in Section 2.3 ). The as-synthesised QAS solid was dissolved immediately in absolute ethanol to produce a 50% QAS solution. The 50% solution was further diluted with absolute ethanol to produce 2%, 5%, and 10% (w/v) QAS solutions. The three QAS solutions were stored in airtight vials and stored at 4 °C until use.

Microtensile bond strength and nanoleakage evaluation

Tooth preparation

One hundred and seventeen freshly extracted, sound human third molars (age range 21–34 years) that had been stored in chloramine T solution at 4 °C for no more than three months after extraction were used in the present study. The teeth were collected after the patient’s informed consent was obtained under a protocol approved by the Institutional Review Board of The University of Hong Kong (UW 14-406). Sixty teeth were used for microtensile bond strength testing and twenty teeth for nanoleakage evaluation. Eighteen teeth were used to evaluate the effect of QAS condensation on etched dentine and the resin–dentine interface using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Nineteen teeth were used for cell viability assay.

The occlusal enamel was cut perpendicular to the longitudinal axis of each tooth with a slow-speed diamond-impregnated disc (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. The sectioning procedure exposed a flat mid-coronal dentine surface (1 mm below the dentinoenamel junction). The exposed dentine was polished wet with 180-grit silicon carbide paper to create a standardised smear layer. The specimens were ultrasonically rinsed in deionised water for 5 min.

Bonding procedures

The exposed dentine surface of each tooth was acid-etched with 32% phosphoric acid (Uni-Etch, Bisco Inc., Schaumburg, IL, USA) for 15 s, rinsed with deionised water for 15 s and kept visibly moist. The teeth were randomly divided into two groups according to the adhesive employed: Adper™ Single Bond 2 (SB, 3M ESPE, St. Paul, MN, USA) and Prime & Bond ® NT™ (PB, Dentsply DeTrey, 78467 Konstanz, Germany). The compositions of the two simplified etch-and-rinse adhesives are shown in Table 1 . The specimens from each adhesive group were blot-dried and further randomly assigned to one of the following five subgroups for dentine pre-treatment with 2% CHX or QAS cavity disinfectants ( N = 6):

  • Group 1: deionised water (control)

  • Group 2: 2% chlorhexidine (CHX, Millipore Sigma, St. Louis, MO, USA)

  • Group 3: 2% QAS

  • Group 4: 5% QAS

  • Group 5: 10% QAS

Table 1
Composition of dental adhesives tested in the present study.
Adhesive Composition
Adper™ Single Bond 2 Bis-GMA
HEMA
Copolymer of acrylic and itaconic acids
Glycerol 1,3-dimethacrylate
Diurethane dimethacrylate
Silane-treated silica
Ethanol
Water
Prime & Bond ® NT™ Di- and trimethacrylate resins
PENTA
Nanofillers – amorphous silicon dioxide
Photoinitiators
Stabilizers
Cetylamine hydrofluoride
Acetone
Abbreviations : Bis-GMA, bisphenol A glycidyl dimethacrylate; HEMA, 2-hydroxyethyl methacrylate; PENTA, dipentaerythritol penta acrylate monophosphate.

The etched dentine surfaces were pre-treated with the cavity disinfectants or deionised water using a sterilised micro brush. The solutions were left undisturbed for 20 s and then gently air-dried. The adhesives were applied to the dentine surface according to the manufacturers’ instructions. Briefly, after applying two consecutive coats of adhesive to the demineralised dentine surface for 10 s with a fully saturated applicator; the coats were gently air-dried for 5 s to evaporate the solvent and light-cured at room temperature using a light-emitting diode curing unit (Elipar S10, 3M ESPE) for 15 s with an output intensity of 600 mW/cm 2 . Resin composite build-ups were performed with a light-cured resin composite (Filtek Z250, 3M ESPE) in four 1-mm thick increments. Each increment was individually light-cured for 20 s. The bonded specimens were stored in distilled water at 37 °C for 24 h.

Microtensile bond strength

The bonded teeth were sectioned occluso-gingivally into 0.9 mm × 0.9 mm composite-dentine beams, according to the non-trimming version of the microtensile test. The beams were randomly assigned for microtensile bond strength (μTBS) evaluation after 24 h, 6 months or 12 months of storage in artificial saliva at 37 °C, following the protocol of Pashley et al. . The artificial saliva contained (mmoles/L): CaCl 2 (0.7), MgCl 2 ·6H 2 O (0.2), KH 2 PO 4 (4.0), KCl (30), NaN 3 (0.3), and HEPES buffer. The storage medium was changed once every week.

At each time point, each beam was attached to a Bencor Multi-T device (Danville Engineering, San Ramon, CA, USA) with cyanoacrylate adhesive (Zapit, Dental Ventures of North America, Corona, CA, USA) and stressed to failure under tension with a universal testing machine (Model 4440, Instron, Inc., Canton, MA, USA) at a crosshead speed of 1 mm/min. After the test, the specimens were removed from the jig using a scalpel blade. The cross-sectional area of each specimen at the site of fracture was measured to the nearest 0.01 mm with a pair of digital callipers (Model CD-6BS; Mitutoyo, Tokyo, Japan). The μTBS values were expressed in MPa by dividing the force at debonding with the cross-sectional surface area.

Failure mode

After bond strength testing, the dentine side of the fractured beams were mounted on aluminium stubs, sputter-coated with gold/palladium and examined using a scanning electron microscope (SEM; Hitachi S-3400N, Hitachi High Technologies America, Inc., Schaumburg, IL, USA) operated at 15 kV to determine the mode of failure. The failure modes were classified as (i) adhesive failure, if the fracture site was within the adhesive, (ii) mixed failure, if the fracture site extended into either the resin composite or dentine, (iii) cohesive failure in resin composite and (iv) cohesive failure in dentine.

Nanoleakage

Twenty teeth ( N = 2) were used for interfacial nanoleakage evaluation for each time period tested. The teeth were pre-treated with the cavity disinfectants, bonded with the simplified etch-and-rinse adhesives and stored in artificial saliva at 37 °C for evaluation at 24 h or 12 months, as previously described for bond strength testing. At each time point, two coats of fast-drying nail varnish were applied 1 mm from the bonded interfaces. After drying of the nail varnish, the specimens were immersed in 50 wt% of ammoniacal silver nitrate solution (pH 9.5) according to the method by Tay et al. .

After 24 h, the specimens were rinsed and washed with deionised water for 5 min and placed in a photodeveloping solution (Kodak Professional Dektol Developer, Rochester, New York, USA) for 8 h under fluorescent light to facilitate reduction of diamine silver ions into metallic silver grains. The specimens were subsequently retrieved from the solution and polished with diamond pastes (6 μm, 3 μm, 1 μm; Buehler Ltd., Lake Bluff, IL, USA) using a polishing cloth. After the nail varnish was carefully removed, the specimens were ultrasonically cleaned for 10 min, air-dried and mounted on aluminium stubs. The specimens were then stored in a desiccator for 24 h. After carbon-coating, the specimens were examined using SEM at 15 kV in the back-scattered mode, focusing on the silver tracer expression along the resin–dentine interface. Forty images of the resin–dentine interfaces were obtained from each group at 500× magnification. The extent of silver deposition along the interface was evaluated by two examiners separately. The amount of silver uptake was scored on a scale of 0–4 using the classification method by Saboia et al. : 0, no nanoleakage; 1, <25% nanoleakage; 2, 25 ≤ 50% nanoleakage; 3, 50 ≤ 75% nanoleakage, 4 > 75% nanoleakage.

QAS condensation

Scanning electron microscopy

Because condensation of acid-catalysed, hydrolysed SiQAC occurs after its pH was adjusted to 7.0 , it was anticipated that neutralisation of the QAS solution by the buffering action of dentine would lead to condensation of a 3-D network of QAS within the dentinal tubules. Accordingly, the exposed dentine surfaces of 4 teeth ( N = 2) were acid-etched with 32% phosphoric acid (Uni-Etch, Bisco Inc.) for 15 s, rinsed with deionised water for 15 s and kept visibly moist. Two percent QAS or 10% QAS was applied to the acid-etched dentine for 20 s. Following evaporation of the ethanol solvent from the QAS solution, no dentine adhesive was applied to the QAS-treated dentine. Whilst this procedure deviated from the dentine bonding procedures described in subsequent sections, the objective was to prevent resin tags produced by a polymerised dentine adhesive from interfering with the identification of condensed QAS within the dentinal tubules. Two parallel grooves were made on external surfaces in mesiodistal direction of each tooth to facilitate split-fracture. Final separation was made using chisel and hammer. The specimens were mounted on aluminium stubs with double-sided conductive tape, sputter-coated with gold/palladium and examined using SEM.

The remaining four teeth ( N = 2) were similarly prepared and bonded with SB and PB following the application of 2% or 10% QAS. The bonded interfaces were similarly evaluated using SEM. The bonded specimens were stored in water for 24 h and split-fractured, in the mesiodistal direction. The splitted fragments were mounted on aluminium stubs, sputtered with gold/palladium and examined with SEM at 10 kV.

Confocal laser scanning microscopy

Non-destructive identification of QAS within the resin–dentine interface was performed using 0.1 wt % aqueous solution of sodium fluorescein (46960 Bioreagent, Millipore Sigma) . The fluoroscein was selected to trace the location of QAS within the hybrid layer and along the dentinal tubules. Two teeth were bonded with SB following the application of 2% QAS as previously described. The bonded specimens were sectioned into 0.5 mm thick mesio-distal slabs using a slow-speed water-cooled diamond saw. Two slabs from the centre of each tooth were selected. The slabs were slightly polished with wet 1200-grit silicon carbide paper for 30 s, ultrasonicated for 10 min and immersed in 0.1 wt% fluorescein for 24 h. The slabs were then rinsed with deionised water and examined using confocal laser scanning microscope (CLSM; Leica Fluoview FV 1000, Olympus, Tokyo, Japan) equipped with a 60×/1.4 NA oil immersion lens using 488 nm argon/helium and a 633 nm krypton ion laser illumination both in reflection and fluorescence modes. Reflected and fluorescence signals were detected with a photomultiplier tube to a depth of 20 μm and then converted to single-projection images for better visualization and qualitative analysis . Two images were obtained for each slab and the images representing the most common features regarding QAS location along the bonded interfaces.

Cell viability

Preparation of dentine discs

Nine teeth were used to compare the cytotoxic effects of 2% QAS-treated, 2% CHX-treated and untreated (control) dentine specimens. The roots were removed using the low-speed, water-cooled diamond saw, and the occlusal enamel of each crown segment was cut to expose dentine. A 0.5 mm thick dentine disc was prepared from the mid-coronal dentine of each tooth and the enamel was removed from the sides to produce square-shaped dentine discs. A final disc thickness of about 0.4 mm was achieved by grinding the occlusal side with wet 320-grit silicon carbide paper. The thickness was evaluated using a pair of digital callipers with a precision of 0.01 mm.

Dentine permeability

Dentine permeability was evaluated to enable a homogenous distribution of the dentine discs into three groups ( N = 3). Dentine permeability was determined by removing the smear layer from both sides of the discs by applying 0.5 M ethylenediamine tetra acetic acid (pH 7.4) for 60 s. After rinsing, the discs were placed in a diffusion chamber connected to 180 cm column of water for 5 min. The movement of a micro-bubble introduced through a metallic cannula was recorded for 1 min. Calculation of the hydraulic conductance (Lp) of dentine was based on a mathematical equation “Lp = Jv/ At ( P )”, where Lp = hydraulic conductance in μL cm −2 , min −1 , H 2 O −1 , Jv = fluid flow in μL min, A = surface area of the dentine in cm 2 , t = time, and P = hydrostatic pressure applied in cm H 2 O . The dentine discs were then allocated into groups so that the mean hydraulic conductance was not statistically different among the three groups (one-way analysis of variance, p > 0.05). An area of 0.28 cm 2 was standardised using a metallic ring on the dentine disc, and a fresh smear layer was created using 600-grit silicon carbide paper for 10 s.

Culture of human dental pulpal cell line

Viability of human dental pulpal cells (hDPCs) was measured by monitoring their metabolic activity using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Ten human third molars were collected with written informed consent from patients (20–34 years of age) who have been planned to have those teeth extracted for orthodontic or therapeutic reasons. The teeth were mechanically fractured with surgical chisels longitudinally. Dental pulp tissues were obtained using forceps. The pulp tissues were stored in alpha Minimal Essential Medium (GIBCO Invitrogen Corp., Paisley, Scotland, UK), which had previously been supplemented with foetal-calf serum (FCS), penicillin (100 U/mL), streptomycin (100 mg/mL) and glutamine (2 mmol/L). The pulpal tissues were minced and cultured in 6-well plates for two weeks at 37 °C in a humidified atmosphere containing 5% CO 2 and 95% atmospheric air. The medium was changed twice a week.

The cultured cells were expanded to 4× dilution after achieving 80% confluency. Cells sub-cultured to the sixth or seventh passages were utilised for the experiment. The cells (3 × 10 4 ) were seeded on the pulpal side of the dentine discs (0.28 cm 2 ) in 24-well plates in an incubator with 5% CO 2 and 95% air at 37 °C. The discs were transferred back to the same wells to receive the QAS and CHX treatments.

Application of QAS and CHX

The occlusal side of the dentine disc was etched using 35% phosphoric acid (Scotchbond etchant, 3M ESPE) for 15 s, rinsed with deionised water and blot-dried using lint-free tissues. Two percent QAS was applied onto the occlusal side of the dentine discs using a sterile microbrush for 20 s, followed by blot-drying. Likewise, 2% CHX was applied, while no pre-treatment was performed in the control group. After treatment with the cavity disinfectants, the dentine discs were returned to the CO 2 incubator for an additional 24 h.

MTT assay

The hDPCs cultured on dentine discs were retrieved by washing with phosphate buffered saline and incubating with 500 μg/mL MTT for 4 h at 37 °C. The insoluble formazan produced by the cells was dissolved in 400 μL acidified isopropanol (0.04 N HCl). Three 100 μL aliquots of the dissolved formazan supernatants were transferred to a 96-well plate and cell viability was evaluated by spectrophotometry at 570 nm using a microplate reader (Thermo Plate, Nanshan District, Shenzhen, Guangdong, China). Twelve replicates were performed for each experimental group and the results were expressed as percentages of the MTT activity of hDPSCs grown on culture plates, which was normalised to present 100% cell viability.

Statistical analysis

The bond strength data was expressed as means ± standard deviations and analysed using a statistical package (SigmaStat Version 20, SPSS, Chicago, IL, USA). Because the bond strength values were normally distributed (Shapiro–Wilk test) and homoscedastic (modified Levene test), the data were analysed by three-way analysis of variance (ANOVA) to examine the effects of “disinfectants”, “adhesives” and “time”, and the interaction of those factors on microtensile bond strength. Post hoc multiple comparisons were performed using Tukey’s test. For nanoleakage evaluation, the inter-observer reproducibility and the intra-observer reproducibility were evaluated using weighted Kappa ( κ w ) statistic. The nanoleakage scores were treated as ordinal data. The Cochran-Mantel-Haenszel (CMH) method was used to test significant differences of the five treatment groups (control, 2% CHX, 2% QAS, 5% QAS, 10% QAS) at the two time periods (24 h vs. 12 months). A significant level of α = 0.05 was used. Data for cell viability assay were analysed using one-way ANOVA followed by Tukey’s multiple comparison procedures.

Materials and methods

Synthesis

The experimental versions of QAS cavity disinfectant examined in the present study were synthesised by sol–gel reaction between 1 mole of TEOS (Mw 208) and 4 moles of Et-SiQAC (Mw 538). In a typical synthesis, 2.08 g of TEOS was blended with 29.89 g of Et-SiQAC (72 wt% of Et-SiQAC dissolved in ethanol) and 5 mL of ethanol (to render the blend more homogeneous). Hydrolysis was initiated by the addition of 10.08 g of 0.02 M HCl-acidified water (pH 1.66, representing 3.5 times the stoichiometric molar concentration of water required, to ensure complete hydrolysis). Hydrolysis and condensation of the two ethoxysilanes was monitored by Fourier transform infrared spectroscopy .

Following completion of the hydrolysis reaction (approximately 3 h), a yellow solution mixture was obtained, the IR spectrum of which was characterised by the presence of silanol groups, ethanol and water . The yellow solution mixture was then maintained at 80 °C for 6 h to remove as much as possible the reaction by-products (ethanol and water), until a pale yellow rubbery solid material was produced. This yellow solid ( Fig. 1 B) represented a partially-condensed form of the K21 molecule shown in Fig. 1 A. Full condensation could be achieved by adding an alkali solution to raise the pH of the solution to 7.2, , wherein an ethanol-insoluble precipitate was progressively produced. This procedure was omitted during the synthesis phase and complete hydrolysis was subsequently achieved after the material was applied on dentine (to be reported in Section 2.3 ). The as-synthesised QAS solid was dissolved immediately in absolute ethanol to produce a 50% QAS solution. The 50% solution was further diluted with absolute ethanol to produce 2%, 5%, and 10% (w/v) QAS solutions. The three QAS solutions were stored in airtight vials and stored at 4 °C until use.

Microtensile bond strength and nanoleakage evaluation

Tooth preparation

One hundred and seventeen freshly extracted, sound human third molars (age range 21–34 years) that had been stored in chloramine T solution at 4 °C for no more than three months after extraction were used in the present study. The teeth were collected after the patient’s informed consent was obtained under a protocol approved by the Institutional Review Board of The University of Hong Kong (UW 14-406). Sixty teeth were used for microtensile bond strength testing and twenty teeth for nanoleakage evaluation. Eighteen teeth were used to evaluate the effect of QAS condensation on etched dentine and the resin–dentine interface using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Nineteen teeth were used for cell viability assay.

The occlusal enamel was cut perpendicular to the longitudinal axis of each tooth with a slow-speed diamond-impregnated disc (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under water cooling. The sectioning procedure exposed a flat mid-coronal dentine surface (1 mm below the dentinoenamel junction). The exposed dentine was polished wet with 180-grit silicon carbide paper to create a standardised smear layer. The specimens were ultrasonically rinsed in deionised water for 5 min.

Bonding procedures

The exposed dentine surface of each tooth was acid-etched with 32% phosphoric acid (Uni-Etch, Bisco Inc., Schaumburg, IL, USA) for 15 s, rinsed with deionised water for 15 s and kept visibly moist. The teeth were randomly divided into two groups according to the adhesive employed: Adper™ Single Bond 2 (SB, 3M ESPE, St. Paul, MN, USA) and Prime & Bond ® NT™ (PB, Dentsply DeTrey, 78467 Konstanz, Germany). The compositions of the two simplified etch-and-rinse adhesives are shown in Table 1 . The specimens from each adhesive group were blot-dried and further randomly assigned to one of the following five subgroups for dentine pre-treatment with 2% CHX or QAS cavity disinfectants ( N = 6):

  • Group 1: deionised water (control)

  • Group 2: 2% chlorhexidine (CHX, Millipore Sigma, St. Louis, MO, USA)

  • Group 3: 2% QAS

  • Group 4: 5% QAS

  • Group 5: 10% QAS

Table 1
Composition of dental adhesives tested in the present study.
Adhesive Composition
Adper™ Single Bond 2 Bis-GMA
HEMA
Copolymer of acrylic and itaconic acids
Glycerol 1,3-dimethacrylate
Diurethane dimethacrylate
Silane-treated silica
Ethanol
Water
Prime & Bond ® NT™ Di- and trimethacrylate resins
PENTA
Nanofillers – amorphous silicon dioxide
Photoinitiators
Stabilizers
Cetylamine hydrofluoride
Acetone
Abbreviations : Bis-GMA, bisphenol A glycidyl dimethacrylate; HEMA, 2-hydroxyethyl methacrylate; PENTA, dipentaerythritol penta acrylate monophosphate.

The etched dentine surfaces were pre-treated with the cavity disinfectants or deionised water using a sterilised micro brush. The solutions were left undisturbed for 20 s and then gently air-dried. The adhesives were applied to the dentine surface according to the manufacturers’ instructions. Briefly, after applying two consecutive coats of adhesive to the demineralised dentine surface for 10 s with a fully saturated applicator; the coats were gently air-dried for 5 s to evaporate the solvent and light-cured at room temperature using a light-emitting diode curing unit (Elipar S10, 3M ESPE) for 15 s with an output intensity of 600 mW/cm 2 . Resin composite build-ups were performed with a light-cured resin composite (Filtek Z250, 3M ESPE) in four 1-mm thick increments. Each increment was individually light-cured for 20 s. The bonded specimens were stored in distilled water at 37 °C for 24 h.

Microtensile bond strength

The bonded teeth were sectioned occluso-gingivally into 0.9 mm × 0.9 mm composite-dentine beams, according to the non-trimming version of the microtensile test. The beams were randomly assigned for microtensile bond strength (μTBS) evaluation after 24 h, 6 months or 12 months of storage in artificial saliva at 37 °C, following the protocol of Pashley et al. . The artificial saliva contained (mmoles/L): CaCl 2 (0.7), MgCl 2 ·6H 2 O (0.2), KH 2 PO 4 (4.0), KCl (30), NaN 3 (0.3), and HEPES buffer. The storage medium was changed once every week.

At each time point, each beam was attached to a Bencor Multi-T device (Danville Engineering, San Ramon, CA, USA) with cyanoacrylate adhesive (Zapit, Dental Ventures of North America, Corona, CA, USA) and stressed to failure under tension with a universal testing machine (Model 4440, Instron, Inc., Canton, MA, USA) at a crosshead speed of 1 mm/min. After the test, the specimens were removed from the jig using a scalpel blade. The cross-sectional area of each specimen at the site of fracture was measured to the nearest 0.01 mm with a pair of digital callipers (Model CD-6BS; Mitutoyo, Tokyo, Japan). The μTBS values were expressed in MPa by dividing the force at debonding with the cross-sectional surface area.

Failure mode

After bond strength testing, the dentine side of the fractured beams were mounted on aluminium stubs, sputter-coated with gold/palladium and examined using a scanning electron microscope (SEM; Hitachi S-3400N, Hitachi High Technologies America, Inc., Schaumburg, IL, USA) operated at 15 kV to determine the mode of failure. The failure modes were classified as (i) adhesive failure, if the fracture site was within the adhesive, (ii) mixed failure, if the fracture site extended into either the resin composite or dentine, (iii) cohesive failure in resin composite and (iv) cohesive failure in dentine.

Nanoleakage

Twenty teeth ( N = 2) were used for interfacial nanoleakage evaluation for each time period tested. The teeth were pre-treated with the cavity disinfectants, bonded with the simplified etch-and-rinse adhesives and stored in artificial saliva at 37 °C for evaluation at 24 h or 12 months, as previously described for bond strength testing. At each time point, two coats of fast-drying nail varnish were applied 1 mm from the bonded interfaces. After drying of the nail varnish, the specimens were immersed in 50 wt% of ammoniacal silver nitrate solution (pH 9.5) according to the method by Tay et al. .

After 24 h, the specimens were rinsed and washed with deionised water for 5 min and placed in a photodeveloping solution (Kodak Professional Dektol Developer, Rochester, New York, USA) for 8 h under fluorescent light to facilitate reduction of diamine silver ions into metallic silver grains. The specimens were subsequently retrieved from the solution and polished with diamond pastes (6 μm, 3 μm, 1 μm; Buehler Ltd., Lake Bluff, IL, USA) using a polishing cloth. After the nail varnish was carefully removed, the specimens were ultrasonically cleaned for 10 min, air-dried and mounted on aluminium stubs. The specimens were then stored in a desiccator for 24 h. After carbon-coating, the specimens were examined using SEM at 15 kV in the back-scattered mode, focusing on the silver tracer expression along the resin–dentine interface. Forty images of the resin–dentine interfaces were obtained from each group at 500× magnification. The extent of silver deposition along the interface was evaluated by two examiners separately. The amount of silver uptake was scored on a scale of 0–4 using the classification method by Saboia et al. : 0, no nanoleakage; 1, <25% nanoleakage; 2, 25 ≤ 50% nanoleakage; 3, 50 ≤ 75% nanoleakage, 4 > 75% nanoleakage.

QAS condensation

Scanning electron microscopy

Because condensation of acid-catalysed, hydrolysed SiQAC occurs after its pH was adjusted to 7.0 , it was anticipated that neutralisation of the QAS solution by the buffering action of dentine would lead to condensation of a 3-D network of QAS within the dentinal tubules. Accordingly, the exposed dentine surfaces of 4 teeth ( N = 2) were acid-etched with 32% phosphoric acid (Uni-Etch, Bisco Inc.) for 15 s, rinsed with deionised water for 15 s and kept visibly moist. Two percent QAS or 10% QAS was applied to the acid-etched dentine for 20 s. Following evaporation of the ethanol solvent from the QAS solution, no dentine adhesive was applied to the QAS-treated dentine. Whilst this procedure deviated from the dentine bonding procedures described in subsequent sections, the objective was to prevent resin tags produced by a polymerised dentine adhesive from interfering with the identification of condensed QAS within the dentinal tubules. Two parallel grooves were made on external surfaces in mesiodistal direction of each tooth to facilitate split-fracture. Final separation was made using chisel and hammer. The specimens were mounted on aluminium stubs with double-sided conductive tape, sputter-coated with gold/palladium and examined using SEM.

The remaining four teeth ( N = 2) were similarly prepared and bonded with SB and PB following the application of 2% or 10% QAS. The bonded interfaces were similarly evaluated using SEM. The bonded specimens were stored in water for 24 h and split-fractured, in the mesiodistal direction. The splitted fragments were mounted on aluminium stubs, sputtered with gold/palladium and examined with SEM at 10 kV.

Confocal laser scanning microscopy

Non-destructive identification of QAS within the resin–dentine interface was performed using 0.1 wt % aqueous solution of sodium fluorescein (46960 Bioreagent, Millipore Sigma) . The fluoroscein was selected to trace the location of QAS within the hybrid layer and along the dentinal tubules. Two teeth were bonded with SB following the application of 2% QAS as previously described. The bonded specimens were sectioned into 0.5 mm thick mesio-distal slabs using a slow-speed water-cooled diamond saw. Two slabs from the centre of each tooth were selected. The slabs were slightly polished with wet 1200-grit silicon carbide paper for 30 s, ultrasonicated for 10 min and immersed in 0.1 wt% fluorescein for 24 h. The slabs were then rinsed with deionised water and examined using confocal laser scanning microscope (CLSM; Leica Fluoview FV 1000, Olympus, Tokyo, Japan) equipped with a 60×/1.4 NA oil immersion lens using 488 nm argon/helium and a 633 nm krypton ion laser illumination both in reflection and fluorescence modes. Reflected and fluorescence signals were detected with a photomultiplier tube to a depth of 20 μm and then converted to single-projection images for better visualization and qualitative analysis . Two images were obtained for each slab and the images representing the most common features regarding QAS location along the bonded interfaces.

Cell viability

Preparation of dentine discs

Nine teeth were used to compare the cytotoxic effects of 2% QAS-treated, 2% CHX-treated and untreated (control) dentine specimens. The roots were removed using the low-speed, water-cooled diamond saw, and the occlusal enamel of each crown segment was cut to expose dentine. A 0.5 mm thick dentine disc was prepared from the mid-coronal dentine of each tooth and the enamel was removed from the sides to produce square-shaped dentine discs. A final disc thickness of about 0.4 mm was achieved by grinding the occlusal side with wet 320-grit silicon carbide paper. The thickness was evaluated using a pair of digital callipers with a precision of 0.01 mm.

Dentine permeability

Dentine permeability was evaluated to enable a homogenous distribution of the dentine discs into three groups ( N = 3). Dentine permeability was determined by removing the smear layer from both sides of the discs by applying 0.5 M ethylenediamine tetra acetic acid (pH 7.4) for 60 s. After rinsing, the discs were placed in a diffusion chamber connected to 180 cm column of water for 5 min. The movement of a micro-bubble introduced through a metallic cannula was recorded for 1 min. Calculation of the hydraulic conductance (Lp) of dentine was based on a mathematical equation “Lp = Jv/ At ( P )”, where Lp = hydraulic conductance in μL cm −2 , min −1 , H 2 O −1 , Jv = fluid flow in μL min, A = surface area of the dentine in cm 2 , t = time, and P = hydrostatic pressure applied in cm H 2 O . The dentine discs were then allocated into groups so that the mean hydraulic conductance was not statistically different among the three groups (one-way analysis of variance, p > 0.05). An area of 0.28 cm 2 was standardised using a metallic ring on the dentine disc, and a fresh smear layer was created using 600-grit silicon carbide paper for 10 s.

Culture of human dental pulpal cell line

Viability of human dental pulpal cells (hDPCs) was measured by monitoring their metabolic activity using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Ten human third molars were collected with written informed consent from patients (20–34 years of age) who have been planned to have those teeth extracted for orthodontic or therapeutic reasons. The teeth were mechanically fractured with surgical chisels longitudinally. Dental pulp tissues were obtained using forceps. The pulp tissues were stored in alpha Minimal Essential Medium (GIBCO Invitrogen Corp., Paisley, Scotland, UK), which had previously been supplemented with foetal-calf serum (FCS), penicillin (100 U/mL), streptomycin (100 mg/mL) and glutamine (2 mmol/L). The pulpal tissues were minced and cultured in 6-well plates for two weeks at 37 °C in a humidified atmosphere containing 5% CO 2 and 95% atmospheric air. The medium was changed twice a week.

The cultured cells were expanded to 4× dilution after achieving 80% confluency. Cells sub-cultured to the sixth or seventh passages were utilised for the experiment. The cells (3 × 10 4 ) were seeded on the pulpal side of the dentine discs (0.28 cm 2 ) in 24-well plates in an incubator with 5% CO 2 and 95% air at 37 °C. The discs were transferred back to the same wells to receive the QAS and CHX treatments.

Application of QAS and CHX

The occlusal side of the dentine disc was etched using 35% phosphoric acid (Scotchbond etchant, 3M ESPE) for 15 s, rinsed with deionised water and blot-dried using lint-free tissues. Two percent QAS was applied onto the occlusal side of the dentine discs using a sterile microbrush for 20 s, followed by blot-drying. Likewise, 2% CHX was applied, while no pre-treatment was performed in the control group. After treatment with the cavity disinfectants, the dentine discs were returned to the CO 2 incubator for an additional 24 h.

MTT assay

The hDPCs cultured on dentine discs were retrieved by washing with phosphate buffered saline and incubating with 500 μg/mL MTT for 4 h at 37 °C. The insoluble formazan produced by the cells was dissolved in 400 μL acidified isopropanol (0.04 N HCl). Three 100 μL aliquots of the dissolved formazan supernatants were transferred to a 96-well plate and cell viability was evaluated by spectrophotometry at 570 nm using a microplate reader (Thermo Plate, Nanshan District, Shenzhen, Guangdong, China). Twelve replicates were performed for each experimental group and the results were expressed as percentages of the MTT activity of hDPSCs grown on culture plates, which was normalised to present 100% cell viability.

Statistical analysis

The bond strength data was expressed as means ± standard deviations and analysed using a statistical package (SigmaStat Version 20, SPSS, Chicago, IL, USA). Because the bond strength values were normally distributed (Shapiro–Wilk test) and homoscedastic (modified Levene test), the data were analysed by three-way analysis of variance (ANOVA) to examine the effects of “disinfectants”, “adhesives” and “time”, and the interaction of those factors on microtensile bond strength. Post hoc multiple comparisons were performed using Tukey’s test. For nanoleakage evaluation, the inter-observer reproducibility and the intra-observer reproducibility were evaluated using weighted Kappa ( κ w ) statistic. The nanoleakage scores were treated as ordinal data. The Cochran-Mantel-Haenszel (CMH) method was used to test significant differences of the five treatment groups (control, 2% CHX, 2% QAS, 5% QAS, 10% QAS) at the two time periods (24 h vs. 12 months). A significant level of α = 0.05 was used. Data for cell viability assay were analysed using one-way ANOVA followed by Tukey’s multiple comparison procedures.

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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Effect of a novel quaternary ammonium silane cavity disinfectant on durability of resin–dentine bond
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