Materials Science and Engineering C 52 (2015) 267–272
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Designing dental composites with bioactive and bactericidal properties Xanthippi Chatzistavrou a,⁎, Saalini Velamakanni a, Kyle DiRenzo b, Anna Lefkelidou c, J. Christopher Fenno d, Toshihiro Kasuga e, Aldo R. Boccaccini f, Petros Papagerakis a,⁎ a
Orthodontics and Pediatric Dentistry, School of Dentistry, University of Michigan, Ann Arbor, MI, USA Materials Science and Engineering, University of Michigan, Ann Arbor, MI, USA School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece d Biologic & Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI, USA e Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Japan f Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Erlangen, Germany b c
a r t i c l e
i n f o
Article history: Received 10 August 2014 Received in revised form 25 February 2015 Accepted 26 March 2015 Available online 28 March 2015 Keywords: Ag-doped bioactive glass Dental composite Antibacterial properties Bioactivity Total bond strength
a b s t r a c t Objectives: The aim of this work was to fabricate and evaluate new antibacterial and bioactive composites capable of strictly controlling oral bacteria, enhancing apatite layer formation and retaining their mechanical properties. Methods: A new Ag-doped bioactive glass (Ag-BG) was incorporated into flowable dental composite (COMP) in different concentrations (1, 5, and 15 wt.%) in order to fabricate new combined bioactive and antibacterial composite materials (Ag-BGCOMPs). The antibacterial properties, bioactivity, and total bond strength of the Ag-BGCOMPs were evaluated. Results: The bioactivity of the Ag-BG was confirmed after its immersion in simulated body fluid (SBF). The total bond strength between the surrounding tooth tissue and the new composites or the control (dental composite alone) has not shown any statistically significant difference in the performed pilot study. Antibacterial activity was observed against Escherichia coli (E. coli) and Streptococcus mutans (S. mutans) for the Ag-BGCOMP 5 wt.% and 15 wt.% but not for the Ag-BGCOMP 1 wt.% or the control. Conclusions: This work contributes to our long term aim which is the fabrication of dental materials capable of reducing bacteria invasion and enhancing remineralization of the surrounding dental tissues. Significance: We anticipate that these new composites could ultimately prevent restoration failure by inhibiting the formation of secondary caries and by remineralizing the hard tissues surrounding tooth lesions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction It has been observed that two-thirds of all restorative dentistry involves the replacement of failed restorations [1,2], while most teeth that have compromised restorations develop symptoms requiring dental pulp treatment [3], with 1.5 million US restorations requiring root canal therapy [4] and millions of teeth finally extracted. High rates of treatment failure suggest that the current restorative approaches are not yet optimized and have a potential for improvement. There is a need for developing innovative restorative materials exhibiting antibacterial function to prevent the recurrence of caries and to repair and/or regenerate the defected dental tissue. Up-to-date, appropriate materials capable of exhibiting these features are not yet available. Towards this goal, several ion-releasing resin-based CaP cements have been proposed as potential cavity liner materials due to their ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Chatzistavrou), [email protected] (P. Papagerakis).
http://dx.doi.org/10.1016/j.msec.2015.03.062 0928-4931/© 2015 Elsevier B.V. All rights reserved.
ability to neutralize acid solution and to induce the remineralization of mineral-deficient carious dentine [5]. However, their relative weak antibacterial activity indicates the need for incorporation of other antibacterial agents in order to develop strong antibacterial composite materials for clinical use. Ag-containing resin-based materials have been also fabricated and used as adhesives in dental practice [6,7]. Although these materials presented antibacterial activity, their remineralization potential has not been confirmed. There is also much merit in the studies examining how current dental materials can be modified to maintain long lasting bioactive and anti-bacterial properties [8–10]. Many of these studies provide evidence for the potential beneficial use of bioactive glasses (BGs) as additives to dental materials to enhance their biological effect. Bioactive glasses have been reported to induce mineralization of dentin disc surfaces [11,12]. These results suggest that dental materials with incorporated BGs could be instrumental in the remineralization of affected human dental tissues [13]. Indeed, new materials that can remineralize the dental hard tissues [14] will find multiple applications in the minimal invasive
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dentistry field. The principals of minimal therapeutic restoration may be enhanced by using bioactive materials capable of releasing specific ions within the bonding interface, to evoke a positive response from the biological environment and to induce protection and/or remineralization of the mineral-depleted dental tissues [15–17]. The application of such materials can lead to a perfect bonding between the restorative material and the surrounding tooth tissues. However, bioactive dental materials with combined antibacterial properties that can eliminate future micro-penetration and secondary caries have not been developed. Due to their esthetics and direct-filling capability, resin composites have been the materials of choice in restorative dentistry [18,19]. One of the most common causes of replaced restorations is the secondary caries where active bacteria colonize the injury site and penetrate the dental pulp through material/dental tissues micro-gaps. Another major challenge with resin-based composites is bulk fracture [20]. Furthermore, it has been observed that composites allowed more accumulation of dental plaque on their surfaces than other restorative materials [21,22]. The aim of this work was to fabricate and evaluate new bioactive and antibacterial composite materials. Our hypothesis was that a silver containing bioactive composite could show enhanced antibacterial properties compared to a control composite material. A novel silver (Ag)-doped bioactive glass (Ag-BG) [23] was incorporated in different proportions in a flowable commercial composite to fabricate new bioactive and antibacterial composites (Ag-BGCOMPs). Moreover, we anticipate that Ca and P released ions from the incorporated BGs will enhance apatite layer formation, remineralizing the mineral-depleted dentin.
2.3. Antibacterial properties The bactericidal properties of the extracts were tested against S. mutans ATCC 25175 basic cariogenic bacterium and E. coli ATCC 29522. A single colony of bacteria was inoculated in nutrient broth and grown overnight at 37 °C. After adjusting to an optical density equivalent to 108 cells per ml in PBS, sequential tenfold dilutions were added to tubes containing equal volumes of the extracts. The effect of the materials' extracts on bacterial growth was assayed by colony forming units (CFU) on nutrient agar plates after 24 h of growth. Cured specimens were sterilized by ethylene oxide gas. Sterilized samples were placed onto BHI (brain heart infusion broth) agar plates inoculated with 350 μl of 1 × 108 CFU/ml of each bacterium suspension, and the plates were incubated at 37 °C for 48 h. The inhibitory effect against E. coli and S. mutans was assayed, as it is indicated by the literature [25]. In particular, after incubation, samples were then removed, and bacterial growth under the specimens was observed by scanning electron microscopy (SEM). For SEM observation the specimens were rinsed with phosphate buffered saline (PBS), and then immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C. The specimens were rinsed with PBS and subjected to graded ethanol dehydrations. They were then rinsed twice with 100% hexamethyldisilazane. The specimens were then mounted on aluminium stubs with carbon cement and sputter coated with gold (Polaron E5100 Sputter Coater). Scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) capability was used for the characterization of the samples (AMRAY 1910 Field Emission Gun-type Scanning Electron Microscope (FEG-SEM) and JEOL JSM-840A). The sample size was triplicated for each sample case and for each tested bacterium. Qualitative analysis of samples' surface was performed and representative images are shown.
2. Materials and methods 2.4. Bioactive behavior 2.1. Ag-BG fabrication Ag-BG fabrication has been already presented in detail elsewhere [24]. Briefly, the fabrication of the new sol–gel derived Ag-doped bioactive glass is based on incorporating the sol–gel bioactive glass 58S (SiO2 58-CaO 33-P2O5 9 wt.%) being in the solution stage, into the solution stage of a new sol–gel glass (in the system SiO2 60-CaO 6-P2O5 3Al2O3 14-Na2O 5-K2O 5-Ag2O 7 wt.%). The resulting composite solution follows a specific heat treatment; initially aging process at 60 °C, then drying at 180 °C and finally stabilization at 700 °C. The final sol–gel derived Ag-doped bioactive glass (Ag-BG) is in the system SiO2 58.6-CaO 24.9-P2O5 7.2-Al2O3 4.2-Na2O 1.5-K2O 1.5-Ag2O 2.1 wt.% [24]. The novel Ag-BG is fabricated in powder form with particle size around ~25 μm.
The apatite-forming ability of the bioactive glass was examined through immersion in simulated body fluid (SBF), as this technique is commonly used to evaluate the bioactivity [26]. Three specimens (dimensions 10 mm diameter and 2 mm thickness) were immersed in SBF (30 ml) at 37 °C up to 20 days (with time points after 3, 7, 10, 15 and 20 days). The SBF solution was replaced every three days since there is a decrease in cation concentration due to the changes in the chemistry of the samples. At the end of each selected time period, samples were removed from the SBF solution, rinsed with 70% ethanol and distilled water, dried and stored in airtight containers for further investigation. The SBF treated samples were examined by SEM to assess the possible formation of a hydroxyapatite (HAp) layer on the material surface, as a marker of bioactive behavior [27]. 2.5. Mechanical properties
2.2. Fabrication of Ag-BGCOMP specimens Specimens with 0, 1, 5 and 15 wt.% of Ag-BG within the flowable composite (Ivoclar Vivadent, Tetric EvoFlow® Filling Material A1, United States and Canada) were formulated prior to the initiation of the test methods. Ag-BG in powder form with particle size of b 35 μm was incorporated manually. Samples without Ag-BG incorporation were used as controls (controls: Ag-BG 0 wt.%). Cured disc samples were prepared using Teflon molds (10 mm diameter, 2 mm thick, and weight 100mgr) applying the instructions of manufactures (two curing cycles of 10 s with halogen curing light (Valo, Ultradent (South Jordan, UT)) operating with a wavelength of 400–500 nm and an intensity of about 1000 mW/cm2. Both top and bottom surfaces were exposed to light, while there was almost no distance between the light-tip and the sample surface) and uncured samples (weight 100 mgr) both with Ag-BG incorporated at 0, 1, 5 and 15 wt.% immersed in PBS for 8 days. Moreover the changes at the pH values were followed for immersion period of a month.
Microtensile test method was used in a pilot study to measure total bond strength (μTBS) of both dentin and uncut enamel. It is considered as an initial means of characterizing the mechanical properties of AgBGCOMP material [28]. In particular, third molars were potted along the long access of the tooth and cut mid-coronally to expose the dentin (or enamel alone in case of the uncut enamel tests). The teeth were sanded with 320 grit paper, etched for 15 s using 35% phosphoric acid etchant, blotted dry until they were slightly moist, and then bonded with commercial bonding agent (AdheSE®, Ivoclar Vivadent) as well as ultimately, layered with the fabricated composite formulations. Specimens of 1 mm × 1 mm in transverse cross-section were cut using a hard tissue microtome along the long axis of the tooth, obtaining matchstick-shaped beams and creating microtensile samples. The specimens were immediately tested for the μTBS test. This testing procedure was executed using customized microtensile fixtures on a testing set-up comprising a LAC-1 (high speed controller single axis with built-in amplifier) and LAL300 linear actuator that had a stroke length of 50 mm
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with a peak force of 250 N, and a displacement resolution of 0.5 mm (SMAC Europe, Horsham, West Sussex, UK). 2.6. Statistical analysis The sample size for each case of Ag-BGCOMP composite was calculated based on prior data indicating that the difference in the μTBS of dentin is normally distributed with a SD no larger than 4.19 [29]. Assuming a hypothesized effect size of no less than 4.45 MPa [29], at a significance level of 5%, with a statistical power of 90%, a power analysis based on paired t-test indicates a sample size of eleven would be sufficient (power and sample size calculations, version 3.0.43, 2009). However, the measured specimens were more than that (18 for each group). The statistical analysis of the results from the mechanical tests was performed using the Student's t-tests (two-tailed) and ANOVA (one-way) using SigmaStat Software (level of significance p b 0.05). 3. Results 3.1. Antibacterial properties The extracts of the materials from both uncured and cured samples, after 8 days of immersion in PBS, show significant bacterial growth inhibition against S. mutans. The increase of the Ag-BG amount in the
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composites leads to increase of the bactericidal activity. Uncured samples of all Ag-BGCOMP present stronger bactericidal activity compared to the cured samples. Composites with Ag-BG 15 wt.% do not allow any bacterial growth at all (Fig. 1a). Composites with 5 wt.% Ag-BG also show significant inhibition of bacterial growth (p b 0.05) (Fig. 1a). In case of the Ag-BGCOMP with 1 wt.% Ag-BG there is no significant difference compared to the control (Fig. 1a). Because of that specimens with Ag-BG 1 wt.% were excluded from further studies. The pH value remains at neutral level for both cured and uncured samples even after a month of immersion in PBS (Fig. 1b). The surface morphology of the cured Ag-BGCOMPs was observed by SEM. The surface roughness can be qualitatively estimated by the images in Fig. 2. There is no obvious difference in the surface roughness between the control sample and Ag-BGCOMP with 5 wt.% Ag-BG, while it seems that there is increase of surface roughness by increasing the AgBG up to 15 wt.%. Bacteria inhibition on the surface of Ag-BGCOMPs was also observed by SEM. Bacteria were observed on the surface of all samples. In case of control samples (Ag-BG 0%) and Ag-BG 5% there is no significant difference on the number of bacteria observed on their surface, especially when they are tested against S. mutans. However, there is significant decrease in the number of bacteria observed on the surface of all samples with Ag-BG 15%. It was also observed that samples show increased antimicrobial action against E. coli compared to S. mutans (Fig. 3). 3.2. Bioactive behavior The ion release profile of Ag-BG has been studied in detail [23,24]. In particular, Ag, Ca, and P ions are released continuously from the first day of immersion in PBS up to more than a month. In case of Ag ions it should be mentioned that the released amount increases up to almost five days and then it shows a plateau for more than a month. Here, the capability of the bioactive glass to stimulate the formation of apatite layer when immersed in SBF was tested. As shown in Fig. 4b, a thick apatite layer was formed on the surface of the specimens after 3 days immersion in SBF. The apatite phase is also confirmed by the EDS spectrum. Ca and P ions are observed in Ca/P ratio around ~ 1.85, which is characteristic for non-stoichiometric hydroxyapatite phase. In contrast, no apatite formation was noted before immersion in SBF (Fig. 4a). 3.3. Mechanical properties Baseline studies on uncut enamel yielded mean μTBS values for control samples (Ag-BG 0 wt.%) and Ag-BGCOMPs with 15 wt.% (Fig. 5a), chosen as representative groups for an initial pilot study. Follow-up studies on dentin, including a midpoint value of Ag-BG 5 wt.% were also performed (Fig. 5b). The μTBS value of control samples was higher in both cases of uncut enamel and dentin compared to Ag-BG wt.15% and 5%. Ag-BG of 5 wt.% shows slightly higher values compared to AgBG of 15 wt.% (Fig. 5b). There is no significant difference (p = 0.2) between control and both Ag-BGCOMPs (Ag-BG 15, 5 wt.%) tested in case of dentin, while there is significant difference (p = 0.02) between control and Ag-BGCOMPS with 15 wt.% in case of uncut enamel. All samples show higher μTBS value in case of dentin compared to uncut enamel. 4. Discussion
Fig. 1. (a) Antibacterial activity of the extracts of cured and uncured samples, after 8 days of immersion in PBS, shows significant bacterial growth inhibition against S. mutans (error bars = ±SD; n = 3, *p b 0.05). (b) The pH value as a function of soaking time in PBS remains neutral for both cured and uncured Ag-BGCOMPs.
In this study we examined the possibility to generate new composite materials for dental applications that present bioactive and antibacterial properties. These new composites (AG-BGCOMP) were fabricated by addition of Ag-BG as a filler component of flowable composites. The increase concentration of Ag into the composites increases the amount of Ag ions released being the highest at 15% of Ag-BG. We, thus, postulate that the addition of Ag leads to improved antibacterial
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Fig. 2. SEM images showing the surface morphology of the control sample and the Ag-BGCOMP samples with 5 and 15 wt.% of Ag-BG.
properties while the pH remains at neutral levels even after a month of soaking period. This observation confirms that the bactericidal activity observed in our study is not due to changes at the pH value but it is exclusively due to the release of Ag ions. This behavior is explained considering that silver ions show antibacterial properties following several mechanisms, including interacting with thiol groups in proteins and interfering with DNA replication [30].
We have previously showed [23] that after 8 days the concentration of the released Ag from Ag-BG has reached a plateau with the released amount calculated around 0.7 ppm. Consistently, the extracts taken in this present study from Ag-BGCOMPS showed strong antibacterial properties after 8 days of immersion. However, photopolymerized specimens show lower antibacterial activity compared to the respective uncured samples. This trend indicates that the polymerization conversion
Fig. 3. Bacterial growth on the surface of cured specimens is observed by SEM. There is no significant difference between control samples and composites containing Ag-BG 5%. However, there is significant decrease in the number of bacteria observed on the surface of all samples with Ag-BG 15%. It was also observed that samples show increased antimicrobial action against E. coli compared to S. mutans.
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Fig. 4. The capability of the bioactive glass to stimulate the formation of apatite layer when immersed in SBF was evaluated. The surface of bioactive glass before immersion in SBF did not show apatite formation (a). In contrast, formation of apatite layer is clearly detected after 3 days of immersion in SBF (b). The apatite phase is also confirmed by the EDS spectrum. Ca and P ions are observed in Ca/P ratio around ~1.85, which is characteristic for non-stoichiometric hydroxyapatite phase.
blocks some of the pathways for the ion release process, but it does not obstruct the process. Considering the difference on the antibacterial properties between the cured and uncured samples, it is essential to develop a material with sufficient ion release even in its cured state. In
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doing so, a long-term antibacterial activity would be ensured and pulp vitality would be protected against monomer elution. The increase of surface roughness with the incorporation of Ag-BG up to 15 wt.%, as it is observed from SEM images of surface morphology, may enhance bacteria attachment on their surface. Although Ag-BG is well incorporated, it seems that the amount of Ag-BG 5 wt.% is not sufficient to express a strong antibacterial action into the surface compared to controls. However, the increase of Ag-BG amount up to 15 wt.% leads to increase bactericidal activity compared to the controls, even though the surface roughness is expected higher. Limitation of our work was the study of antibacterial activity against two bacteria strains. Further studies to clarify the action of the different Ag-BGCOMPs concentrations into surface biofilm formation are needed to confirm relevant antibacterial properties of these composites. Nevertheless, this study highlights the potential benefits of Ag addition into flowable composites and provides foundation for future studies. Ag-BG is a silicate-based material with Ca/P ratio close to 4, expected of inducing apatite formation when immersed in SBF due to the dissolution and subsequent protonation of the Si–O groups to form silanols as seats for heterogeneous nucleation and crystallization [31]. The capability of the Ag-BG to form apatite layer after immersion in SBF is shown here. The incorporated bioactive and bactericidal glass into dental composite could then stimulate the formation of apatite layer in the same way via ion exchange process which takes place through the developed interfaces in the composite. However, a delay on the formation time of the apatite layer is expected due to lower amount of bioactive glass in the composites and the polymerization conversion which lead to decrease on the ion release rate. Moreover, it can be also suggested that the precipitation of a SiO2-rich layer and its reaction with Ca2 + and PO34 − species may further favor the formation of a high molecularweight complex (Ca/P–MMPs), which restricts the activities of enzymatic degradation of metallo-proteinases MMP-2 and MMP-9 within the hybrid layer [32]. Thus, the biomimetic remineralization of the new Ag-BGCOMP composites could eliminate hydrolysis of both collagen and resin components, while the release of calcium and phosphate ions could have therapeutic remineralizing effects for tooth lesion areas. The total bond strength values measured in our study were relatively low as it is expected for flowable composite [33,34]. The μTBS values of Ag-BGCOMPs do not show significant differences compared to the control, although lower degree of polymerization conversion is expected. This behavior can be assigned both to the incorporated amount and the specific particle size of the Ag-BG. Although esthetics issues due to silver oxidation may prevent the use of Ag-BGCOMPs into anterior teeth, its potential use will be a valuable asset in protecting posterior teeth. Furthermore, due to the bioactive properties of the new Ag-BGCOMPs, their interaction with the surrounding dental tissues could lead into establishing a strong bonding
Fig. 5. (a) μTBS values for control composites (Ag-BG 0%) and composites containing 15 wt.% Ag-BG on uncut enamel. (b) μTBS values were also measured on dentin for control composites (Ag-BG 0%) and for Ag-BG wt.5% and 15%.
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eliminating the need for bonding agents while providing a bacteria free environment. This could decrease time of treatment and simplify the procedure into one step Ag-BGCOMP system. Limitations of our study and future directions include the necessity to extend our studies on the antibacterial properties of the AgBGCOMPs against surface biofilms, to evaluate the hardness and the re-mineralization capability in in vitro models and to test the mechanical and re-mineralizing properties after long soaking period in medium, in order to observe the capability of Ag-BGCOMP to express long lasting properties. Nevertheless, this study provides foundation for generating a new line of anti-bacterial composites with strong bioactive features. These Ag-BGCOMPs could contribute in providing better treatment options for dental patients and finally could benefit the dental healthcare system. 5. Conclusions In this work new bactericidal and bioactive composites are fabricated by the incorporation of Ag-doped bioactive glass into a flowable composite. The antibacterial properties of the new Ag-BGCOMP composites are found to be significantly higher than the control while the mechanical properties are not significantly affected after Ag-BG addition into the flowable composite. Furthermore, Ag-BG provides apatite forming capability (bioactivity) into the flowable composite. The results of this work enhance the implementation of the ultimate goals which are to reduce the etiologic microbiota and contributing risk factors to halt the caries decay process and stimulate remineralization. Acknowledgments This work was supported by the University of Michigan, School of Dentistry, Department of Orthodontics & Pediatric Dentistry start-up Funds of Dr. Papagerakis. Authors wish to thank Ivoclar Vivadent for providing materials. References [1] F.J. Burke, S.W. Cheung, I.A. Mjör, N.H. Wilson, Reasons for the placement and replacement of restorations in vocational training practices, Prim. Dent. Care 6 (1999) 17–20. [2] G. Maupomé, A. Sheiham, Criteria for restoration replacement and restoration lifespan estimates in an educational environment, J. Oral Rehabil. 25 (1998) 896–901. [3] A. Zöllner, P. Gaengler, Pulp reactions to different preparation techniques on teeth exhibiting periodontal disease, J. Oral Rehabil. 27 (2000) 93–102. [4] Research NIoDaC, Biomimetics and Tissue Engineering, Bethesda, MD, National Institutes of Health, 2002. [5] M. Barounian, S. Hesaraki, A. Kazemzadeh, Development of strong and bioactive calcium phosphate cement as a light-cure organic–inorganic hybrid, J. Mater. Sci. Mater. Med. 23 (2012) 1569–1581. [6] F. Li, M.D. Weir, J. Chen, H.H. Xu, Comparison of quaternary ammonium-containing with nano-silver-containing adhesive in antibacterial properties and cytotoxicity, Dent. Mater. 29 (2013) 450–461. [7] M.A.S. Melo, L. Cheng, K. Zhang, M.D. Weir, L.K. Rodrigues, H.H. Xu, Novel dental adhesives containing nanoparticles of silver and amorphous calcium phosphate, Dent. Mater. 29 (2013) 199–210. [8] L. Graham, P.R. Cooper, N. Cassidy, J.E. Nor, A.J. Sloan, A.J. Smith, The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components, Biomaterials 27 (2006) 2865–2873. [9] A.J. Smith, Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators, J. Dent. Educ. 67 (2003) 678–689.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Designing dental composites with bioactive and bactericidal properties Xanthippi Chatzistavrou a,⁎, Saalini Velamakanni a, Kyle DiRenzo b, Anna Lefkelidou c, J. Christopher Fenno d, Toshihiro Kasuga e, Aldo R. Boccaccini f, Petros Papagerakis a,⁎ a
Orthodontics and Pediatric Dentistry, School of Dentistry, University of Michigan, Ann Arbor, MI, USA Materials Science and Engineering, University of Michigan, Ann Arbor, MI, USA School of Dentistry, Aristotle University of Thessaloniki, Thessaloniki, Greece d Biologic & Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor, MI, USA e Department of Frontier Materials, Nagoya Institute of Technology, Nagoya, Japan f Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Erlangen, Germany b c
a r t i c l e
i n f o
Article history: Received 10 August 2014 Received in revised form 25 February 2015 Accepted 26 March 2015 Available online 28 March 2015 Keywords: Ag-doped bioactive glass Dental composite Antibacterial properties Bioactivity Total bond strength
a b s t r a c t Objectives: The aim of this work was to fabricate and evaluate new antibacterial and bioactive composites capable of strictly controlling oral bacteria, enhancing apatite layer formation and retaining their mechanical properties. Methods: A new Ag-doped bioactive glass (Ag-BG) was incorporated into flowable dental composite (COMP) in different concentrations (1, 5, and 15 wt.%) in order to fabricate new combined bioactive and antibacterial composite materials (Ag-BGCOMPs). The antibacterial properties, bioactivity, and total bond strength of the Ag-BGCOMPs were evaluated. Results: The bioactivity of the Ag-BG was confirmed after its immersion in simulated body fluid (SBF). The total bond strength between the surrounding tooth tissue and the new composites or the control (dental composite alone) has not shown any statistically significant difference in the performed pilot study. Antibacterial activity was observed against Escherichia coli (E. coli) and Streptococcus mutans (S. mutans) for the Ag-BGCOMP 5 wt.% and 15 wt.% but not for the Ag-BGCOMP 1 wt.% or the control. Conclusions: This work contributes to our long term aim which is the fabrication of dental materials capable of reducing bacteria invasion and enhancing remineralization of the surrounding dental tissues. Significance: We anticipate that these new composites could ultimately prevent restoration failure by inhibiting the formation of secondary caries and by remineralizing the hard tissues surrounding tooth lesions. © 2015 Elsevier B.V. All rights reserved.
1. Introduction It has been observed that two-thirds of all restorative dentistry involves the replacement of failed restorations [1,2], while most teeth that have compromised restorations develop symptoms requiring dental pulp treatment [3], with 1.5 million US restorations requiring root canal therapy [4] and millions of teeth finally extracted. High rates of treatment failure suggest that the current restorative approaches are not yet optimized and have a potential for improvement. There is a need for developing innovative restorative materials exhibiting antibacterial function to prevent the recurrence of caries and to repair and/or regenerate the defected dental tissue. Up-to-date, appropriate materials capable of exhibiting these features are not yet available. Towards this goal, several ion-releasing resin-based CaP cements have been proposed as potential cavity liner materials due to their ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Chatzistavrou), [email protected] (P. Papagerakis).
http://dx.doi.org/10.1016/j.msec.2015.03.062 0928-4931/© 2015 Elsevier B.V. All rights reserved.
ability to neutralize acid solution and to induce the remineralization of mineral-deficient carious dentine [5]. However, their relative weak antibacterial activity indicates the need for incorporation of other antibacterial agents in order to develop strong antibacterial composite materials for clinical use. Ag-containing resin-based materials have been also fabricated and used as adhesives in dental practice [6,7]. Although these materials presented antibacterial activity, their remineralization potential has not been confirmed. There is also much merit in the studies examining how current dental materials can be modified to maintain long lasting bioactive and anti-bacterial properties [8–10]. Many of these studies provide evidence for the potential beneficial use of bioactive glasses (BGs) as additives to dental materials to enhance their biological effect. Bioactive glasses have been reported to induce mineralization of dentin disc surfaces [11,12]. These results suggest that dental materials with incorporated BGs could be instrumental in the remineralization of affected human dental tissues [13]. Indeed, new materials that can remineralize the dental hard tissues [14] will find multiple applications in the minimal invasive
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dentistry field. The principals of minimal therapeutic restoration may be enhanced by using bioactive materials capable of releasing specific ions within the bonding interface, to evoke a positive response from the biological environment and to induce protection and/or remineralization of the mineral-depleted dental tissues [15–17]. The application of such materials can lead to a perfect bonding between the restorative material and the surrounding tooth tissues. However, bioactive dental materials with combined antibacterial properties that can eliminate future micro-penetration and secondary caries have not been developed. Due to their esthetics and direct-filling capability, resin composites have been the materials of choice in restorative dentistry [18,19]. One of the most common causes of replaced restorations is the secondary caries where active bacteria colonize the injury site and penetrate the dental pulp through material/dental tissues micro-gaps. Another major challenge with resin-based composites is bulk fracture [20]. Furthermore, it has been observed that composites allowed more accumulation of dental plaque on their surfaces than other restorative materials [21,22]. The aim of this work was to fabricate and evaluate new bioactive and antibacterial composite materials. Our hypothesis was that a silver containing bioactive composite could show enhanced antibacterial properties compared to a control composite material. A novel silver (Ag)-doped bioactive glass (Ag-BG) [23] was incorporated in different proportions in a flowable commercial composite to fabricate new bioactive and antibacterial composites (Ag-BGCOMPs). Moreover, we anticipate that Ca and P released ions from the incorporated BGs will enhance apatite layer formation, remineralizing the mineral-depleted dentin.
2.3. Antibacterial properties The bactericidal properties of the extracts were tested against S. mutans ATCC 25175 basic cariogenic bacterium and E. coli ATCC 29522. A single colony of bacteria was inoculated in nutrient broth and grown overnight at 37 °C. After adjusting to an optical density equivalent to 108 cells per ml in PBS, sequential tenfold dilutions were added to tubes containing equal volumes of the extracts. The effect of the materials' extracts on bacterial growth was assayed by colony forming units (CFU) on nutrient agar plates after 24 h of growth. Cured specimens were sterilized by ethylene oxide gas. Sterilized samples were placed onto BHI (brain heart infusion broth) agar plates inoculated with 350 μl of 1 × 108 CFU/ml of each bacterium suspension, and the plates were incubated at 37 °C for 48 h. The inhibitory effect against E. coli and S. mutans was assayed, as it is indicated by the literature [25]. In particular, after incubation, samples were then removed, and bacterial growth under the specimens was observed by scanning electron microscopy (SEM). For SEM observation the specimens were rinsed with phosphate buffered saline (PBS), and then immersed in 1% glutaraldehyde in PBS for 4 h at 4 °C. The specimens were rinsed with PBS and subjected to graded ethanol dehydrations. They were then rinsed twice with 100% hexamethyldisilazane. The specimens were then mounted on aluminium stubs with carbon cement and sputter coated with gold (Polaron E5100 Sputter Coater). Scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) capability was used for the characterization of the samples (AMRAY 1910 Field Emission Gun-type Scanning Electron Microscope (FEG-SEM) and JEOL JSM-840A). The sample size was triplicated for each sample case and for each tested bacterium. Qualitative analysis of samples' surface was performed and representative images are shown.
2. Materials and methods 2.4. Bioactive behavior 2.1. Ag-BG fabrication Ag-BG fabrication has been already presented in detail elsewhere [24]. Briefly, the fabrication of the new sol–gel derived Ag-doped bioactive glass is based on incorporating the sol–gel bioactive glass 58S (SiO2 58-CaO 33-P2O5 9 wt.%) being in the solution stage, into the solution stage of a new sol–gel glass (in the system SiO2 60-CaO 6-P2O5 3Al2O3 14-Na2O 5-K2O 5-Ag2O 7 wt.%). The resulting composite solution follows a specific heat treatment; initially aging process at 60 °C, then drying at 180 °C and finally stabilization at 700 °C. The final sol–gel derived Ag-doped bioactive glass (Ag-BG) is in the system SiO2 58.6-CaO 24.9-P2O5 7.2-Al2O3 4.2-Na2O 1.5-K2O 1.5-Ag2O 2.1 wt.% [24]. The novel Ag-BG is fabricated in powder form with particle size around ~25 μm.
The apatite-forming ability of the bioactive glass was examined through immersion in simulated body fluid (SBF), as this technique is commonly used to evaluate the bioactivity [26]. Three specimens (dimensions 10 mm diameter and 2 mm thickness) were immersed in SBF (30 ml) at 37 °C up to 20 days (with time points after 3, 7, 10, 15 and 20 days). The SBF solution was replaced every three days since there is a decrease in cation concentration due to the changes in the chemistry of the samples. At the end of each selected time period, samples were removed from the SBF solution, rinsed with 70% ethanol and distilled water, dried and stored in airtight containers for further investigation. The SBF treated samples were examined by SEM to assess the possible formation of a hydroxyapatite (HAp) layer on the material surface, as a marker of bioactive behavior [27]. 2.5. Mechanical properties
2.2. Fabrication of Ag-BGCOMP specimens Specimens with 0, 1, 5 and 15 wt.% of Ag-BG within the flowable composite (Ivoclar Vivadent, Tetric EvoFlow® Filling Material A1, United States and Canada) were formulated prior to the initiation of the test methods. Ag-BG in powder form with particle size of b 35 μm was incorporated manually. Samples without Ag-BG incorporation were used as controls (controls: Ag-BG 0 wt.%). Cured disc samples were prepared using Teflon molds (10 mm diameter, 2 mm thick, and weight 100mgr) applying the instructions of manufactures (two curing cycles of 10 s with halogen curing light (Valo, Ultradent (South Jordan, UT)) operating with a wavelength of 400–500 nm and an intensity of about 1000 mW/cm2. Both top and bottom surfaces were exposed to light, while there was almost no distance between the light-tip and the sample surface) and uncured samples (weight 100 mgr) both with Ag-BG incorporated at 0, 1, 5 and 15 wt.% immersed in PBS for 8 days. Moreover the changes at the pH values were followed for immersion period of a month.
Microtensile test method was used in a pilot study to measure total bond strength (μTBS) of both dentin and uncut enamel. It is considered as an initial means of characterizing the mechanical properties of AgBGCOMP material [28]. In particular, third molars were potted along the long access of the tooth and cut mid-coronally to expose the dentin (or enamel alone in case of the uncut enamel tests). The teeth were sanded with 320 grit paper, etched for 15 s using 35% phosphoric acid etchant, blotted dry until they were slightly moist, and then bonded with commercial bonding agent (AdheSE®, Ivoclar Vivadent) as well as ultimately, layered with the fabricated composite formulations. Specimens of 1 mm × 1 mm in transverse cross-section were cut using a hard tissue microtome along the long axis of the tooth, obtaining matchstick-shaped beams and creating microtensile samples. The specimens were immediately tested for the μTBS test. This testing procedure was executed using customized microtensile fixtures on a testing set-up comprising a LAC-1 (high speed controller single axis with built-in amplifier) and LAL300 linear actuator that had a stroke length of 50 mm
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with a peak force of 250 N, and a displacement resolution of 0.5 mm (SMAC Europe, Horsham, West Sussex, UK). 2.6. Statistical analysis The sample size for each case of Ag-BGCOMP composite was calculated based on prior data indicating that the difference in the μTBS of dentin is normally distributed with a SD no larger than 4.19 [29]. Assuming a hypothesized effect size of no less than 4.45 MPa [29], at a significance level of 5%, with a statistical power of 90%, a power analysis based on paired t-test indicates a sample size of eleven would be sufficient (power and sample size calculations, version 3.0.43, 2009). However, the measured specimens were more than that (18 for each group). The statistical analysis of the results from the mechanical tests was performed using the Student's t-tests (two-tailed) and ANOVA (one-way) using SigmaStat Software (level of significance p b 0.05). 3. Results 3.1. Antibacterial properties The extracts of the materials from both uncured and cured samples, after 8 days of immersion in PBS, show significant bacterial growth inhibition against S. mutans. The increase of the Ag-BG amount in the
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composites leads to increase of the bactericidal activity. Uncured samples of all Ag-BGCOMP present stronger bactericidal activity compared to the cured samples. Composites with Ag-BG 15 wt.% do not allow any bacterial growth at all (Fig. 1a). Composites with 5 wt.% Ag-BG also show significant inhibition of bacterial growth (p b 0.05) (Fig. 1a). In case of the Ag-BGCOMP with 1 wt.% Ag-BG there is no significant difference compared to the control (Fig. 1a). Because of that specimens with Ag-BG 1 wt.% were excluded from further studies. The pH value remains at neutral level for both cured and uncured samples even after a month of immersion in PBS (Fig. 1b). The surface morphology of the cured Ag-BGCOMPs was observed by SEM. The surface roughness can be qualitatively estimated by the images in Fig. 2. There is no obvious difference in the surface roughness between the control sample and Ag-BGCOMP with 5 wt.% Ag-BG, while it seems that there is increase of surface roughness by increasing the AgBG up to 15 wt.%. Bacteria inhibition on the surface of Ag-BGCOMPs was also observed by SEM. Bacteria were observed on the surface of all samples. In case of control samples (Ag-BG 0%) and Ag-BG 5% there is no significant difference on the number of bacteria observed on their surface, especially when they are tested against S. mutans. However, there is significant decrease in the number of bacteria observed on the surface of all samples with Ag-BG 15%. It was also observed that samples show increased antimicrobial action against E. coli compared to S. mutans (Fig. 3). 3.2. Bioactive behavior The ion release profile of Ag-BG has been studied in detail [23,24]. In particular, Ag, Ca, and P ions are released continuously from the first day of immersion in PBS up to more than a month. In case of Ag ions it should be mentioned that the released amount increases up to almost five days and then it shows a plateau for more than a month. Here, the capability of the bioactive glass to stimulate the formation of apatite layer when immersed in SBF was tested. As shown in Fig. 4b, a thick apatite layer was formed on the surface of the specimens after 3 days immersion in SBF. The apatite phase is also confirmed by the EDS spectrum. Ca and P ions are observed in Ca/P ratio around ~ 1.85, which is characteristic for non-stoichiometric hydroxyapatite phase. In contrast, no apatite formation was noted before immersion in SBF (Fig. 4a). 3.3. Mechanical properties Baseline studies on uncut enamel yielded mean μTBS values for control samples (Ag-BG 0 wt.%) and Ag-BGCOMPs with 15 wt.% (Fig. 5a), chosen as representative groups for an initial pilot study. Follow-up studies on dentin, including a midpoint value of Ag-BG 5 wt.% were also performed (Fig. 5b). The μTBS value of control samples was higher in both cases of uncut enamel and dentin compared to Ag-BG wt.15% and 5%. Ag-BG of 5 wt.% shows slightly higher values compared to AgBG of 15 wt.% (Fig. 5b). There is no significant difference (p = 0.2) between control and both Ag-BGCOMPs (Ag-BG 15, 5 wt.%) tested in case of dentin, while there is significant difference (p = 0.02) between control and Ag-BGCOMPS with 15 wt.% in case of uncut enamel. All samples show higher μTBS value in case of dentin compared to uncut enamel. 4. Discussion
Fig. 1. (a) Antibacterial activity of the extracts of cured and uncured samples, after 8 days of immersion in PBS, shows significant bacterial growth inhibition against S. mutans (error bars = ±SD; n = 3, *p b 0.05). (b) The pH value as a function of soaking time in PBS remains neutral for both cured and uncured Ag-BGCOMPs.
In this study we examined the possibility to generate new composite materials for dental applications that present bioactive and antibacterial properties. These new composites (AG-BGCOMP) were fabricated by addition of Ag-BG as a filler component of flowable composites. The increase concentration of Ag into the composites increases the amount of Ag ions released being the highest at 15% of Ag-BG. We, thus, postulate that the addition of Ag leads to improved antibacterial
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Fig. 2. SEM images showing the surface morphology of the control sample and the Ag-BGCOMP samples with 5 and 15 wt.% of Ag-BG.
properties while the pH remains at neutral levels even after a month of soaking period. This observation confirms that the bactericidal activity observed in our study is not due to changes at the pH value but it is exclusively due to the release of Ag ions. This behavior is explained considering that silver ions show antibacterial properties following several mechanisms, including interacting with thiol groups in proteins and interfering with DNA replication [30].
We have previously showed [23] that after 8 days the concentration of the released Ag from Ag-BG has reached a plateau with the released amount calculated around 0.7 ppm. Consistently, the extracts taken in this present study from Ag-BGCOMPS showed strong antibacterial properties after 8 days of immersion. However, photopolymerized specimens show lower antibacterial activity compared to the respective uncured samples. This trend indicates that the polymerization conversion
Fig. 3. Bacterial growth on the surface of cured specimens is observed by SEM. There is no significant difference between control samples and composites containing Ag-BG 5%. However, there is significant decrease in the number of bacteria observed on the surface of all samples with Ag-BG 15%. It was also observed that samples show increased antimicrobial action against E. coli compared to S. mutans.
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Fig. 4. The capability of the bioactive glass to stimulate the formation of apatite layer when immersed in SBF was evaluated. The surface of bioactive glass before immersion in SBF did not show apatite formation (a). In contrast, formation of apatite layer is clearly detected after 3 days of immersion in SBF (b). The apatite phase is also confirmed by the EDS spectrum. Ca and P ions are observed in Ca/P ratio around ~1.85, which is characteristic for non-stoichiometric hydroxyapatite phase.
blocks some of the pathways for the ion release process, but it does not obstruct the process. Considering the difference on the antibacterial properties between the cured and uncured samples, it is essential to develop a material with sufficient ion release even in its cured state. In
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doing so, a long-term antibacterial activity would be ensured and pulp vitality would be protected against monomer elution. The increase of surface roughness with the incorporation of Ag-BG up to 15 wt.%, as it is observed from SEM images of surface morphology, may enhance bacteria attachment on their surface. Although Ag-BG is well incorporated, it seems that the amount of Ag-BG 5 wt.% is not sufficient to express a strong antibacterial action into the surface compared to controls. However, the increase of Ag-BG amount up to 15 wt.% leads to increase bactericidal activity compared to the controls, even though the surface roughness is expected higher. Limitation of our work was the study of antibacterial activity against two bacteria strains. Further studies to clarify the action of the different Ag-BGCOMPs concentrations into surface biofilm formation are needed to confirm relevant antibacterial properties of these composites. Nevertheless, this study highlights the potential benefits of Ag addition into flowable composites and provides foundation for future studies. Ag-BG is a silicate-based material with Ca/P ratio close to 4, expected of inducing apatite formation when immersed in SBF due to the dissolution and subsequent protonation of the Si–O groups to form silanols as seats for heterogeneous nucleation and crystallization [31]. The capability of the Ag-BG to form apatite layer after immersion in SBF is shown here. The incorporated bioactive and bactericidal glass into dental composite could then stimulate the formation of apatite layer in the same way via ion exchange process which takes place through the developed interfaces in the composite. However, a delay on the formation time of the apatite layer is expected due to lower amount of bioactive glass in the composites and the polymerization conversion which lead to decrease on the ion release rate. Moreover, it can be also suggested that the precipitation of a SiO2-rich layer and its reaction with Ca2 + and PO34 − species may further favor the formation of a high molecularweight complex (Ca/P–MMPs), which restricts the activities of enzymatic degradation of metallo-proteinases MMP-2 and MMP-9 within the hybrid layer [32]. Thus, the biomimetic remineralization of the new Ag-BGCOMP composites could eliminate hydrolysis of both collagen and resin components, while the release of calcium and phosphate ions could have therapeutic remineralizing effects for tooth lesion areas. The total bond strength values measured in our study were relatively low as it is expected for flowable composite [33,34]. The μTBS values of Ag-BGCOMPs do not show significant differences compared to the control, although lower degree of polymerization conversion is expected. This behavior can be assigned both to the incorporated amount and the specific particle size of the Ag-BG. Although esthetics issues due to silver oxidation may prevent the use of Ag-BGCOMPs into anterior teeth, its potential use will be a valuable asset in protecting posterior teeth. Furthermore, due to the bioactive properties of the new Ag-BGCOMPs, their interaction with the surrounding dental tissues could lead into establishing a strong bonding
Fig. 5. (a) μTBS values for control composites (Ag-BG 0%) and composites containing 15 wt.% Ag-BG on uncut enamel. (b) μTBS values were also measured on dentin for control composites (Ag-BG 0%) and for Ag-BG wt.5% and 15%.
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eliminating the need for bonding agents while providing a bacteria free environment. This could decrease time of treatment and simplify the procedure into one step Ag-BGCOMP system. Limitations of our study and future directions include the necessity to extend our studies on the antibacterial properties of the AgBGCOMPs against surface biofilms, to evaluate the hardness and the re-mineralization capability in in vitro models and to test the mechanical and re-mineralizing properties after long soaking period in medium, in order to observe the capability of Ag-BGCOMP to express long lasting properties. Nevertheless, this study provides foundation for generating a new line of anti-bacterial composites with strong bioactive features. These Ag-BGCOMPs could contribute in providing better treatment options for dental patients and finally could benefit the dental healthcare system. 5. Conclusions In this work new bactericidal and bioactive composites are fabricated by the incorporation of Ag-doped bioactive glass into a flowable composite. The antibacterial properties of the new Ag-BGCOMP composites are found to be significantly higher than the control while the mechanical properties are not significantly affected after Ag-BG addition into the flowable composite. Furthermore, Ag-BG provides apatite forming capability (bioactivity) into the flowable composite. The results of this work enhance the implementation of the ultimate goals which are to reduce the etiologic microbiota and contributing risk factors to halt the caries decay process and stimulate remineralization. Acknowledgments This work was supported by the University of Michigan, School of Dentistry, Department of Orthodontics & Pediatric Dentistry start-up Funds of Dr. Papagerakis. Authors wish to thank Ivoclar Vivadent for providing materials. References [1] F.J. Burke, S.W. Cheung, I.A. Mjör, N.H. Wilson, Reasons for the placement and replacement of restorations in vocational training practices, Prim. Dent. Care 6 (1999) 17–20. [2] G. Maupomé, A. Sheiham, Criteria for restoration replacement and restoration lifespan estimates in an educational environment, J. Oral Rehabil. 25 (1998) 896–901. [3] A. Zöllner, P. Gaengler, Pulp reactions to different preparation techniques on teeth exhibiting periodontal disease, J. Oral Rehabil. 27 (2000) 93–102. [4] Research NIoDaC, Biomimetics and Tissue Engineering, Bethesda, MD, National Institutes of Health, 2002. [5] M. Barounian, S. Hesaraki, A. Kazemzadeh, Development of strong and bioactive calcium phosphate cement as a light-cure organic–inorganic hybrid, J. Mater. Sci. Mater. Med. 23 (2012) 1569–1581. [6] F. Li, M.D. Weir, J. Chen, H.H. Xu, Comparison of quaternary ammonium-containing with nano-silver-containing adhesive in antibacterial properties and cytotoxicity, Dent. Mater. 29 (2013) 450–461. [7] M.A.S. Melo, L. Cheng, K. Zhang, M.D. Weir, L.K. Rodrigues, H.H. Xu, Novel dental adhesives containing nanoparticles of silver and amorphous calcium phosphate, Dent. Mater. 29 (2013) 199–210. [8] L. Graham, P.R. Cooper, N. Cassidy, J.E. Nor, A.J. Sloan, A.J. Smith, The effect of calcium hydroxide on solubilisation of bio-active dentine matrix components, Biomaterials 27 (2006) 2865–2873. [9] A.J. Smith, Vitality of the dentin-pulp complex in health and disease: growth factors as key mediators, J. Dent. Educ. 67 (2003) 678–689.
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