A niobophosphate bioactive glass suspension for rewetting dentin: Effect on antibacterial activity, pH and resin-dentin bonding durability

Author’s Accepted Manuscript A niobophosphate bioactive glass suspension for rewetting dentin: effect on antibacterial activity, pH and resin-dentin bonding durability José Bauer, Allana Silva e Silva, Edilausson Moreno Carvalho, Ceci Nunes Carvalho, Ricardo Marins Carvalho, Adriana Pigozzo Manso www.elsevier.com/locate/ijadhadh

PII: DOI: Reference:

S0143-7496(18)30082-4 https://doi.org/10.1016/j.ijadhadh.2018.03.004 JAAD2166

To appear in: International Journal of Adhesion and Adhesives Received date: 5 June 2017 Accepted date: 22 February 2018 Cite this article as: José Bauer, Allana Silva e Silva, Edilausson Moreno Carvalho, Ceci Nunes Carvalho, Ricardo Marins Carvalho and Adriana Pigozzo Manso, A niobophosphate bioactive glass suspension for rewetting dentin: effect on antibacterial activity, pH and resin-dentin bonding durability, International Journal of Adhesion and Adhesives, https://doi.org/10.1016/j.ijadhadh.2018.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A niobophosphate bioactive glass suspension for rewetting dentin: effect on antibacterial activity, pH and resin-dentin bonding durability José Bauera Allana Silva e Silvaa Edilausson Moreno Carvalhoa Ceci Nunes Carvalhob Ricardo Marins Carvalhoc Adriana Pigozzo Mansoc a

Discipline of Dental Materials, School of Dentistry, University Federal of Maranhão (UFMA),

São Luis, Maranhão, Brazil. b

Department of Restorative Dentistry, School of Dentistry, University Ceuma, (UNICEUMA),

São Luis, Maranhão, Brazil. c

Department of Oral Biological and Medical Sciences, Division of Biomaterials, Faculty of

Dentistry, The University British of Columbia (UBC), Vancouver, BC, Canada.

Corresponding author: José Bauer, Universidade Federal do Maranhão (UFMA), Departamento de Odontologia I, Curso de Odontologia, Rua dos Portugueses, 1966, Campus Universitário do Bacanga, Phone: +55 (98) 32729541, CEP: 65080-805, São Luís – MA, e-mail: [email protected]

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A niobophosphate bioactive glass suspension for rewetting dentin: effect on antibacterial activity, pH and resin-dentin bonding durability Abstract Purpose: The aim of this study was evaluate the effect of rewetting suspensions of niobium phosphate bioactive glass (NbG) on the shear bond strength to dentin (SBS) and antibacterial activity. Materials and Methods: The NbG was prepared and characterized by XRD and SEM/EDS. Antibacterial activity (Streptococcus mutans ATCC 159) of suspensions NbG was evaluated (n=6). The pH of control and of suspensions NbG were monitored in different times. Dentin surface of sixty-five human third molars was etched with 35% phosphoric acid, rinsed with water and air-dried (n=13). The specimens were assigned into 5 groups of four different concentrations of NbG suspensions in distilled water (5%, 10%, 20%, 40% w/v) and one control group (distilled water). Dentin surface was dried and rewetted according to their respective groups and adhesive system (One Step) was applied. The specimens were assembled for SBS testing (Ultradent Jig) and two cylinders of resin composite (ÆLITE) were built on the bonded dentin surface. Specimens were tested after 24h and 3 months storage in PBS at 37°C. The data from each test were analyzed by appropriate statistical methods. Results: Suspensions containing the bioactive glass NbG can neutralize the pH. Significant reductions on SBS were observed after 3 months PBS storage for control and NbG 5% suspension groups. All the suspension groups showed significantly inhibition of S. mutans. Conclusion: Rewetting demineralized dentin with NbG 40% prevented bond strength reduction after 3 months storage and showed antibacterial effect against S. mutans. Keywords: Dentine (A), bioactive niobium phosphate glass, pH, adhesive, antibacterial properties.

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1. Introduction The bonding mechanism of adhesive systems involves the replacement of minerals removed from the hard dental tissue by resin monomers, in such a way that a polymer becomes micro-mechanically interlocked to the dental substrate. The etch-and-rinse bonding strategy involves the application of phosphoric acid gel to dental substrates to remove the minerals and expose the collagen fibrils in dentin [1]. The disadvantage is that there is a risk of collagen collapsing during the process, which leads to poor resin infiltration and a decrease in bond strength over time as a result of degradation of the expose and unprotected collagen fibrils by endogenous dentin matrix metalloproteinases [1,2]. Studies have shown that adhesive-dentin interfaces degrade after relatively short periods of time [2]. A preventive alternative would be to induce remineralization of the interfibrillar spaces not infiltrated by the adhesive resin, thus protecting the collagen fibrils. Some researchers have used bioactive glasses to induce deposition of hydroxyl-carbonate apatite for osseointegration [3], thus indicating that bioactive glasses are capable of inducing natural mineralization of demineralized tissue [4]. In adhesive dentistry, bioactive glasses have been used to promote therapeutic mineral deposition within the resindentin interfaces [5,6] and the biomimetic remineralization of denuded collagen fibrils appears to be an alternative strategy for the revival of resin-dentin bonds [7]. While most of the bioactive glasses are made of a mixture of calcium, phosphate and silica, the use of niobium pentoxide as new filler has been proposed in some dental materials [6,8]. The presence of niobium results in higher chemical durability of phosphate glasses [9], improved biocompatibility and increased radiopacity and microhardness of the experimental adhesive materials [8,10]. The use of bioactive glasses as an additive in dental materials has been gaining attention lately because of potential mechanical and biological benefits [11]. For instance, it has been 3

recently demonstrated that an experimental composite of gutta-percha and niobium phosphate glass (NbG] resulted in increased push-out dentin bond strength when used as filling material for endodontic therapy [12,13]. The inclusion of bioactive glass in the formulation of adhesive resins have been proposed as a way to provide a source of ions that would work in concert with the oral and dentinal fluids to induce mineralization at the bonded interface [14]. It is controversial, however, whether the ions entrapped in the cured adhesive would be able to leach out to the medium to react with the fluids. Some studies suggested that the direct application of bioactive glasses dispersed in water or water/acetone solutions on demineralized dentin could have potential therapeutics effects [6,15]. The fine glass particles dispersed in water could diffuse deep into the demineralized zone and provide a more intimate contact with the dentinal fluids in order to induce mineralization [16]. Additionally, if the glass can be dispersed in small-sized particles, this could facilitate diffusion to the bottom of the demineralized zone, thus being available in the vicinity of exposed collagen fibrils. Besides the remineralization capacity, the bioactive restorative material must also present an antibacterial activity. Secondary caries seems to be more associated with composite than with amalgam restorations in high-caries-risk patients [17]. An ideal restorative material should not only seal and prevent recontamination of the restorations, but also have antibacterial effects. Thus, the development of a bioactive restorative approach that is capable of preventing or reducing bacterial growth, and promoting a good seal in a moist environment would be ideal [18,19]. Although bioactive glasses have previously been used for dentine remineralization when dispersed in water solutions [16] there is no information about the antibacterial properties of NbG suspensions and its effect on dentin bond strength when used as rewetting suspensions prior to bonding. Thus, therapeutic benefit may therefore be gained by combining antibacterial properties. The aim of this study was to evaluate antibacterial properties of NbG suspensions and the 4

effect when they are used to rewet demineralized dentin prior to bonding. The following hypotheses were tested: (i) NbG suspension had effect on inhibition of bacterial growth; (ii) NbG suspension in re-wetting condition avoids the reduction bond strength after 3 months of storage. 2. Material and Methods 2.1 Preparation of the niobium phosphate glass NbG powder was prepared in accordance with previous studies [10,12,13]. The glass was then crushed in a vibrating system (8000M, Mixer/Mill, SPEX SamplePrep, NJ, USA) with tungsten carbide grinding vial balls (SPEX SamplePrep, NJ, USA) for 30 minutes. After grinding, the resultant powder passed through a series of sieves of 150μm - 75μm - 53μm - 38μm - 20μm (Hogentogler & Co., Inc, Columbia, MD, USA). Only the powder that passed the 20μm sieve was further used to prepare the suspensions. Scanning electron microscopic/energy-dispersive (SEM/EDS, XL30 Philips, Eindhoven, Netherlands) was performed to observe the particles size and composition. The particle size distribution was determined using a CILAS laser diffraction particle size analyzer (Model 1064, CILAS, Orléans, France). X-ray diffraction analysis (XRD) was performed to verify the crystallinity of the bioactive glass powder. The XRD was operated with Cu Kα radiation (λ = 0.15418 nm). The diffraction patterns were obtained in the 2θ range from 10° to 90° in continuous mode at 2°/min (Bruker D8 Advance, Bruker Corp., Billerica, MA, USA). 2.2 Preparation of Suspensions Four suspensions and a control group (distilled water) were prepared by mixing different concentrations of NbG in 5 mL of distilled water: 1) Control Group (0% NbG, only distilled water); 2) 5% NbG (0.04g/mL); 3) 10% NbG (0.1g/mL); 4) 20% NbG (0.2g/mL) and 5) 40% NbG (0.4g/mL). The suspensions were stirred for 48 hours (Model 210T, Thermix Fisher, Waltham, MA, 5

USA) and then allowed to sediment in sealed glass vials. Just before use, the vial containing the suspension was stirred again for 10 seconds. 2.3 pH measurement The pH of distilled water was adjusted in 4 and 7. NBG glass particles were added at concentrations of 5, 10, 20 and 40% (the same used to rewetted the dentin). The pH was monitored in different times (1 minute, 1 hour, 24 hours, 48 hours and 7 days) by pH meter (Quimis, Model Q838-F Diadema, SP, Brazil). 2.4 Antibacterial activity The NbG powder was sterilized by autoclaving at 120°C for 15 minutes. Aliquots of frozen stocks of Streptococcus mutans (UA159) were placed on Brain Heart Infusion (BHI; Sigma-Aldrich, St Louis, MO, USA) agar plates and were incubated for 48h at 37°C. ColonyForming Units (CFU) were collected and transferred to tubes containing BHI broth supplemented with 1% sucrose and grown until late exponential phase [20]. In order to from a microbial inoculum, of the suspensions was adjusted to using as a control the standard solution paragraph 0.5 of the McFarland scale, resulting in a suspension with an approximate concentration of 108 CFU/mL24 [21]. Aliquots of 100 µl of BHI supplemented with 1% sucrose were added to each well of a sterile 96-well plate followed by 100 µl the NbG (5, 10, 20 and 40%). The plate was incubated for 24h at 37°C. After 24h, an aliquot of 100 µl from each well was transferred to tubes containing 1ml of sterile 0.9% NaCl and vortexed vigorously. Aliquots of these suspensions were serially diluted up to 10-8 and 2 drops of 10 µl of each dilution were inoculated on BHI agar (BD, Sparks, USA) to determine the number of CFU. The plates were incubated for 48h at 37°C, 10% CO2. After 48h, CFU were counted under stereomicroscope and the results were expressed as log10 CFU/mL. 2.5 Teeth Preparation for SBS testing 6

Sixty-five extracted human third molars were collected and stored in 0.5% chloramine solution until use. The study protocol was reviewed by the local Human Ethic Review Board (UBC CREB: H13-01451). Flat superficial dentin surfaces were created after removal of the occlusal enamel with a slow speed diamond saw (Isomet 5000 – Buehler, Lake Bluff, Illinois, USA) under water-cooling. The roots were then removed by transversally sectioning the teeth slightly below the CEJ level resulting in crown segments that were embedded in transparent acrylic resin (Orthodontic Resin, Dentsply Caulk, Milford, DE, USA). The exposed flat dentin surfaces were further polished in a polishing machine (Unipol 1210, MTI Corporation, CA, USA) under water-cooling with 180-grit and 320-grit silicon carbide abrasive papers for 20 seconds each. The specimens were randomly assigned into 5 different groups as previously described (n=13). The dentin surface was etched for 15s with 35% phosphoric acid gel (Select HV Etch without BAC, Bisco, Schaumburg, IL, USA), rinsed with water for 20s and air-dried with an oil free air syringe for 30s. The surface was then rewetted with either one of the four different concentrations of NbG suspension or distilled water for 10s (Microbrush, Aplicator regular – Ref X-80250P, Bisco, USA). A mild air blow was applied to remove excess water and leave the surface moist for bonding. Two consecutive coats of adhesive system (One Step, Bisco, Inc., Schaumburg, IL, USA) were applied to dentin for 10s with a microbrush, air-dried for 10s at 20 cm distance and light polymerized for 10s (Bluephase 2.0i, Ivoclar Vivadent AG, Schaan, Liechtenstein) at 1500 mW/cm² (LED Radiometer, SDS, Kerr, USA). Next, a dental composite resin (ÆLITE, Bisco, Inc., Schaumburg, IL, USA) was incrementally built on the bonded dentin surface using a cylindrical (2.38mm in diameter x 3 mm in height) plastic mould (Ultradent JIG, South Jordan, Utah, USA) designed for shear bond testing and light polymerized for 40s (Bluephase 2.0i, 7

Ivoclar Vivadent AG, Schaan, Liechtenstein). Two cylinders of resin composite were bonded to the same dentin surface for shear bond strength testing after 24 hours and 3 months. Details of the materials used are shown in Table 1. Table 1 – Materials composition and information supplied in the respective MSDS. Name Composition Bach Number (Manufacture) Select HV Etch 35% H3PO4 (without benzalkonium chloride) Y095 (Bisco) Bis-phenol A diglycidylmethacrylate
 (BIS-GMA), One Step Biphenyl dimethacrylate (BPDM), Hydroxyethyl 1200013326 (Bisco) methacrylate (HEMA) and Acetone. Ethoxylated Bis-GMA, Triethyleneglycol Ælite Body A3 Dimethacrylate (TEGDMA), Glass filler, amorphous 1200013317 (Bisco) silica. The bonded samples were stored in a phosphate-calcium buffer solution (PBS, Dulbecco’s Phosphate Buffered Saline, Sigma Adrich, St Louis, MO, USA) in an oven at 37 °C. The composition of the PBS (in g/L) was CaCl2.2H2O (0.133), MgCl2.6H2O (0.1), KH2PO4 (0.2), KCl (0.2) and Na2HPO4 (1.15). Storage solution was refreshed every month. At the appropriate testing period, 24 hours or 3 months, the resin-dentin bonded specimens were tested with Bisco Shear Bond Tester (Bisco, Schaumburg, IL, USA) at a crosshead speed of 0.5 mm/min until fracture. Shear bond strength was expressed in MPa. 2.6 Failure Pattern analysis Failure modes were examined using a digital microscope at 40X (Nikon Eclipse 80i microscope; Nikon Instruments Inc, Tokyo, Japan) and classified as cohesive (failure exclusively within resin composite), adhesive (failure at adhesive/substrate interface) and mixed (adhesive failure mixed with partially cohesive failure of the neighboring substrates). 2.7 Statistical analysis Statistical analysis was performed using the SigmaPlot 13 software (SigmaPlot v. 13.0,

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Systat Software Inc., San Jose, USA). Data were analyzed for normality of distribution by the Shapiro-Wilk test (=0.05). Comparison of antibacterial activity data was performed with oneway ANOVA and SBS data were submitted to two-way ANOVA and Holm-Sidak tests (=0.05). 3. Results 3.1 Analyses of niobium phosphate glass powder The SEM/EDS spectra of the NbG powder confirmed the composition of sodium, magnesium, aluminum, phosphorus, calcium and niobium. The glass powder used to produce the suspensions exhibited particles with irregular aspect (Figure 1). The particle size analysis showed a distribution of particles with a mean diameter of 5.92 μm (Figure 2). Figure 1 - (1A) Analysis of composition of the particles of the NbG by EDS confirming the presence of the main elements such as calcium, phosphorus, niobium, sodium and magnesium. (1B) SEM of NbG glass shows particles with sizes smaller than 20μm (2.500x - bar = 20μm).

The XRD analysis of NbG powder is presented in Figure 3. The curve, with smooth inclination and no sharp diffraction peaks, shows the absence of crystals, typical of materials with high amorphicity. 3.2 pH measurement Figure 4 (A and B) shows the pH values of the different suspensions and control at the different initial pH (4 and 7) and times. At pH 4 (Fig. 4A): it is possible to observe a

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neutralization of pH after the period of 1 h in the groups where NbG glass was added, on the other hand the control group remains at the acid pH independent of the evaluation interval. At pH 7 (Fig. 4B): shows that pH is close to neutrality independent of the evaluated group, but in the NbG 40% group a more basic pH is observed at the end of 7 days. Figure 2 – Particle size distribution of the NbG powder.

Figure 3 - XRD analysis of the synthesized particles of bioactive niobium phosphate glasses.

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Figure 4 (A and B) - Measurement of pH values over time in glass suspensions NBG different initial pH (4 and 7).

3.3 Antibacterial activity One-way ANOVA revealed that there were significant differences in the antibacterial activity among the different NbG concentration solutions (p<0.0001). Antibacterial activity was observed in the groups of suspensions with glass NbG compared with NaCl (negative control). Chlorhexidine (positive control) demonstrated a higher antibacterial activity among the tested groups (Figure 5). Figure 5 - Results of antibacterial activity glass suspensions and the respective controls are expressed as mean log10 CFU.

Bars with dissimilar letters indicate values that are significantly different from each other (p < 0.05).

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3.4 Shear Bonding Strength testing Two-way ANOVA revealed significant interactions between main factors (p=0.037). Holm-Sidak post-hoc tests showed significant decrease of SBS after 3 months storage only for control (distilled water) and 5% NbG suspension groups. No significant reductions in bond strength were observed for higher concentrations (10, 20 and 40%) of NbG suspensions (Figure 6). Figure 6 –Shear bond strength (MPa ± SD) to dentin according to suspensions and storage time.

Identical letters indicate no significant differences between the values (immediate and 3 months) in the same group (p > 0.05).

The distribution (%) of fracture patterns for each experimental condition is shown in Table 2. All groups showed a predominance of adhesive failure mode and some mixed fractures. Table 2 – Distribution of the fracture pattern according to groups (%). Storage Groups 24 hs Adhesive Mixed Cohesive Adhesive Control 61.50 38.50 0 61.50 5 % NbG 53.85 46.15 0 76.90 10 % NbG 69.25 30.75 0 61.50 20 % NbG 84.61 7.69 7.69 84.60 40 % NbG 61.50 38.50 0 53.80

3 months Mixed 38.50 23.10 38.50 15.40 46.20

Cohesive 0 0 0 0 0

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4. Discussion This study found that suspensions of NbG presented antibacterial activity and also influenced the stability of SBS after 3 months of storage in PBS. The suspension with NbG particles showed bacterial inhibition values that were significantly higher than the control group. These findings support a previous study in which experimental gutta-percha containing NbG also resulted in significant inhibition of biofilm formation [13]. Although the antibacterial mechanism of bioactive glasses is not completely understood, it may be related to an increase in the pH of the surrounding media [22]. In this study, the pH changes were measured in the solutions for a period of up to 7 days. These findings indicate that NbG particles are capable of rapidly neutralizing solutions with low pH (pH 4) and maintain the pH in neutral conditions (pH 7). The pH environment un important role on antibacterial activity, some materials that had a high alkaline capable of releasing highly alkali ions (Na+, Ca+2), which damage bacterial membranes, proteins, and nucleic acids, leading to cell death [23,24]. The antibacterial activity is potentially of great importance as the infiltration of microorganisms may cause secondary caries, which jeopardize the longevity of resin–dentin interface [6]. Therefore, this technique could prevent the bacteria growth in the tooth cavity and inhibit the invading bacteria along the toothrestoration interface. Storage time had a significant effect on the bond strength, causing reduction of the values for 2 groups (control and NbG 5%) of the five solutions tested. The NbG powder included particles that were smaller than previously reported in other studies using bioactive glasses [14,15,22,25]. Some studies showed that a nanoparticulated bioactive glass release [22] could facilitate the dissolution of ions (Ca+2 and PO4-3) from the glass and thereby accelerate the remineralization of the collagen matrix [16]. In addition to remineralization capacity, it has been reported that bioactive materials may 13

inhibit endogenous enzymes that promote collagen degradation [25]. This could explain why bond strengths remained stable when higher concentrations (10, 20 and 40%) of NbG were used for the re-wetting suspensions. The creation of a moderate alkaline environment within the bonded-dentine interface due to the ion exchange between Na+ and Ca+2 ions from the glass and the H3O+ from the physiological medium [9] could have contributed to the inhibition of pH-sensitive enzymes [25]. Perhaps this is one reason for the bond strength results remained stable after three months of storage for the higher concentrated suspensions. Currently, the addition of bioactive glass to resinous materials appears to result in controversial outcomes in terms of bond strengths [6,14,15,26,27] as it not clear through which mechanisms bioactive glasses could benefit the interface. However, the move to continued investigation is worthwhile since it has been demonstrated that the incorporation of 45S5, NbG or another bioactive particles in many dental materials might contribute to mineral deposition, antibacterial activity and MMP activity [12,27-32]. A major difference between the composition of NbG and a commercially available version of bioactive glass (45S5) is merely the presence of niobium. It has been shown that the presence of niobium resulted in improved chemical durability of phosphate glasses [9]. Contrary to silica, niobium would effectively participate in the reaction of osseointegration [33] due to its high biocompatibility [34]. Tamai et al. [35] has shown that bioactive glasses with niobium increased the alkaline phosphatase activity and calcification in osseointegration, with the amount of calcified tissue being directly proportional to the concentration of dissolved niobium ions. Interestingly, the best results of antibacterial activity and bond strength were also found in the highest concentrated 40% group. This can also be attributed to the increase in the pH of the suspensions after mixing with NbG. Independent of starting pH (4 and 7), a neutral or alkaline 14

pH was sustained for up to seven days in suspension, and such pH could have contributed to the antibacterial effect observed (Figure 4). It is possible that this bonding approach may also render free particles of the NbG glass on the dentinal surface to release ions of Ca2+ and PO43- to induce remineralization of the collagen layer. Remineralization of the dentinal tissue, prevention of collagen degradation [25], and inhibition of bacterial growth [16] are among the desirable benefits of the incorporation of bioactive glass to the bonding procedure [18]. With these two advantages (bioactivity and antibacterial activity), re-wetting technique becomes of great interest in cases of partial removal of carious tissue [19]. A bioactive suspension could be beneficial, because the fine particles from the suspension directly contacts the dentin and infiltrates into dentinal tubules to help disinfect the prepared tooth cavity and promote the remineralization of the dentinal tissue affected. This study demonstrated that suspensions of NbG applied to dentin after H3PO4 etching did not compromise the bond strength of One Step adhesive to dentin. More importantly, it showed that SBS can remain stable after 3 months, depending on the % of NbG in the suspension. The combination of antibacterial properties with the ability to preserve resin-dentin bonds make the effort to further investigate NbG as potential addition to bonding procedures. 5. Conclusion Rewetting demineralized dentin with NbG 10-40% bioactive glass prevented bond strength reduction after 3-month storage in PBS and NbG 40% had a best performance in antibacterial properties when compared with negative control.

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6. Acknowledgements This study was supported by the Foundation for the Support of Scientific and Technological Research of Maranhão (FAPEMA BEPP-05585/15) and by the National Council for Scientific and Technological Development grant (CNPq - 237066/2012-2).

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