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Wettability and surface morphology of eroded dentin treated with chitosan
Accepted Manuscript Title: Wettability of chitosan treated eroded dentin and analysis of surface morphology Author: Mirian Saavedra Ururahy Fabiana Almeida Curylofo-Zotti Rodrigo Galo Lucas Fabricio Bahia Nogueira Ana Paula Ramos Silmara Aparecida Milori Corona PII: DOI: Reference:
S0003-9969(16)30351-X http://dx.doi.org/doi:10.1016/j.archoralbio.2016.11.017 AOB 3759
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
1-12-2015 30-8-2016 29-11-2016
Please cite this article as: Ururahy Mirian Saavedra, Curylofo-Zotti Fabiana Almeida, Galo Rodrigo, Nogueira Lucas Fabricio Bahia, Ramos Ana Paula, Corona Silmara Aparecida Milori.Wettability of chitosan treated eroded dentin and analysis of surface morphology.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2016.11.017 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 proof before it is published in its final 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.
Wettability of chitosan treated eroded dentin and analysis of surface morphology Mirian Saavedra Ururahya, Fabiana Almeida Curylofo-Zottia Rodrigo Galob, Lucas Fabricio Bahia Nogueirac, Ana Paula Ramosc, Silmara Aparecida Milori Coronaa a
Department of Restorative Dentistry, Sao Paulo University, Ribeirao Preto, Café Avenue,
Monte Alegre, Ribeirão Preto, SP, Brazil. b
Department of Dentistry, Federal University of Jequitinhonha and Mucuri Valleys,
Diamantina, MG, Brazil. c
Department of Chemistry, Sao Paulo University, Bandeirantes Avenue, Ribeirao Preto, SP,
Brazil.
Corresponding author: Silmara Aparecida Milori Corona Restorative Dentistry Department, Dental Schoolof Ribeirão Preto, University of São Paulo, Café, s/n, Monte Alegre, Ribeirão Preto-SP, Brazil, 14040-904. Phone number: +55-16-33154075 E-mail: [email protected]
1
Highlights
The use of chitosan on dentin has shown favorable results in literature. However,
within the limit of our knowledge, there no studies that evaluated if the wettability of chitosan treated eroded dentin was influenced by the concentration of chitosan used.
It was concluded that chitosan, at concentrations of 2.5% and 5.0%, did not influence of the eroded dentin wettability.
Through SEM analysis, it was found particles of chitosan deposited on the surface and within the dentinal tubules.
Abstract Objective: To assess the effect of chitosan, at concentrations of 2.5% and 5.0%, on the eroded dentin wettability, followed by analysis of surface morphology by SEM. Methods: 104 bovine dentin slabs were ground, polished and then immersed in 20mL of citric acid (pH = 3.2) under continuous stirring for 2h. Specimens were randomly divided according to the dentin substrate: sound and eroded, and then, subdivided into 4 groups (n=10): without rewetting (control), 1% acetic acid, 2.5% chitosan and 5.0% chitosan. Then, a drop of the adhesive system Single Bond 2 (3M) was deposited onto surface of each specimen. The contact angle between dentin surface and the adhesive system was measured by using a goniometer. The other 24 specimens were subjected to analysis under SEM. Statistical analysis was performed using the normality test (Kolmogorov-Smirnov) and Analysis of Variance (ANOVA) (p> 0.05). Results: No differences were found between the angles produced on the eroded dentin rewetting with chitosan at the concentrations of 2.5% and 5%. Conclusion: The chitosan, regardless of the concentration used, did not influence of the eroded dentin wettability. Through SEM analysis, it was found particles of chitosan deposited on the surface and within the dentinal tubules.
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Key-words: eroded dentin, chitosan, wettability, contact angle
1. Introduction Dental erosion (more recently known as corrosion) (Grippo, 2012) occurs due to extended contact of dental tissues with acidic substances provided from intrinsic or extrinsic sources (Imfeld, 1996; Lussi & Hellwig, 2006), without the involvement of bacteria (Grippo, Simring,
& Schreiner,
2004).
Clinically,
this
demineralization
process
is
characterized by the progressive and irreversible loss of tooth structure (Addy & Shellis, 2006), which depending on the severity of the lesions may result in dental hypersensitivity (Barbour, Lussi, & Shellis, 2011), as well as functional and aesthetic damage. The erosion process provides an initial dissolution of peritubular dentin, increasing the diameter of the tubules. This can lead to demineralization of intertubular dentin, exposing the organic matrix which may result in a rough and porous surface (Meurman & ten Cate, 1991; Meurman & ten Cate, 1996; Ganss, Hardat, Blazek, Klimek, & Schlueter, 2009). In addition to the mineral dissolution, enzymatic collagen degradation (Ganss, Klimek, Brune, & Schürman, 2004; Schlueter, Neutard, von Hinckeldey, Klimek, & Ganss, 2010) can interfere with the quality of the collagen network (Sabatini & Pashley, 2014.). This fact can directly reflect on the success of composite restorations, which are preferable for being based on minimally invasive procedures (Atin & Wegehaupt, 2014), in addition to not being acid-soluble (Milosevic & Jones, 1996). In attempting to improve the quality and longevity of adhesive restorations, studies have evaluated the use of biopolymers, such as chitosan (Bedran-Russo, Pereira, Duarte, Drumond, & Yamauchi, 2007; Shrestha, Friedman, Kishen, 2011; Fawzy, Nitisusanta, Iqbal, Daood, Beng, & Neo, 2013), to increase the cross-linking between the collagen fibers, and neutralize matrix metalloproteinases (MMPs) (Xu, Neoh, Lin, & Kishen, 2011; Persadmehr, Torneck, Cvitkovitch, Pinto, Talior, Kazembe, Shrestha, McCulloch, & Kishen, 2014) Chitosan is a natural polysaccharide obtained through deacetylation of chitin (Muzzarelli & Rochetti, 1973) which is totally biocompatible, biodegradable and non-toxic 3
(Venter, Kotze, Auzely-Velty, & Rinaudo, 2006; Raafat & Sahl, 2009). This polymer has been used in order to strengthen (Fawzy, Nitisusanta, Iqbal, Daood, Beng, & Neo, 2013) and stabilize (Bedran-Russo, Pereira, Duarte, Drumond, & Yamauchi, 2007; Shrestha, Friedman, & Kishen, 2011) dentin collagen once its use is able to produce microfibrillar arrangements with superior mechanical properties (Dash, Chiellini, Ottenbrite, & Chiellini, 2011). Thus, due to the need for greater durability of restorative procedures on eroded dentin, its important evaluate if the wettability of chitosan treated eroded dentin will be influenced by the concentration of chitosan used. The null hypotheses tested were: 1) the type of substrate (sound or eroded dentin) does not influence the wettability of a total-etch adhesive system; 2) the rewetting with chitosan, at concentrations of 2.5% and 5.0%, both in sound or eroded dentin does not influence the wettability of a total-etch adhesive system.
2. Materials and methods 2.1 Experimental design The factors under study were dentin substrate (sound and eroded) and the rewetting of the surface (1% acetic acid, 2.5% chitosan and 5.0% chitosan). Specimens of the control group did not receive the rewetting with chitosan. Experimental units were 104 bovine incisors randomly assigned into groups. The response variable was the measurement of the contact angle (n=10) formed between dentin and the adhesive system and analysis of surface morphology by SEM (n=3). The design of the present study is presented in Figure 1. 2.2 Selection of teeth Bovine incisors stored in 0.1% thymol solution at 9°C were washed in running water for 24 hours to eliminate thymol residues. Then, teeth were examined under a 20x magnifier in
4
stereoscopic microscope (Leica Microsystems, Wetzlar, Germany). 104 incisors with no fractures lines or crown-deep cracks were selected.
2.3 Preparation of dentin slabs Roots were transversely sectioned in the cementoenamel junction using a doublefaced diamond disk (15HC 11-4244, Buehler, Illinois, USA) mounted in a low-speed handpiece (Isomet 1000; Buehler, Germany). Subsequently, crowns were sectioned in mesial-distal direction in order to obtain two hemi-sections (buccal and palatal) of each crown. From each buccal surface, one slab with the dimensions of 7.0 x 7.0 x 2.5 mm was obtained. The fragments were fixed in a teflon matrix using molten wax (Kota Ltd, SP, Brazil) with the enamel surface face up. The dentin surfaces was grounded with 320 grid silicon carbide paper under water cooling to remove lateral excess and set the size of the slabs, then, specimens were grounded with 1200 grid silicon carbide paper (Hermes Abrasives Ltd., VA, EUA) for 10 s. Polishing was performed with felt disk (ATM, Altenkirchen, Germany) and 0.3 mm alumina suspension (Arotec, SP, Brazil) for 5 s.
2.4 Erosion-like lesion formation To induce erosion lesions formation it was used a protocol previously established by Vanuspong, Eisenburger, & Addy, (2002). Each dentin slab was immersed in a beaker containing 20 mL of 0.3% citric acid (pH = 3.2) which was individually placed in a shaker under constant stirring, 50 rpm speed for a period of 2h. After this period, specimens were rinsed with distilled water and individually stored in vials containing artificial saliva for 24 hours at 37ºC. Artificial saliva was composed of methyl phydroxybenzoate (2.0 g), sodium carboxymethylcellulose (10.0 g), KCl (0.625 g), MgCl2.6H2O (0.059 g), CaCl2.2H2O (0.166 g), K2HPO4 (0.804 g ), and KH2PO4 (0.326 g) in 1000 mL of distilled water, according to the
5
protocol described by McKnight-Hanes & Whitford (1992) and modified by Amaechi, Higham, Edgar (1999).
2.5 Surface treatment All dentin specimens were conditioned with 37% phosphoric acid (Ultra-Etch Ultradent, SP, Brazil) for 15 sec, rinsed for 10s under running tap water, and dried with absorbent paper. For the specimens that received the rewetting, this procedure was performed with 2.5% and 5.0% chitosan (Arnaud, de Barros Neto, & Diniz, 2010). For the preparation of the chitosan, 2.5 g and 5.0 g of chitosan (Sigma-Aldrich, St. Louis, MO, USA) low molecular weight were weighed in an analytical balance (Bel analytical equipment Ltd, SP, Brazil). Then, chitosan was diluted in 100 ml of 1.0% of acetic acid solution, slowly added and under constant stirring (Marconi Equip. Lab., Piracicaba, Brazil) for 20 min (time enough to solubilise the polysaccharide). 1 mol/L of NaOH solution was added in order to avoid aggregation of the particles and raising the pH (Daood, Iqbal, Nitisusanta, Fawzy, & 2013; Fawzy, Nitisusanta, IIqbal, Daood, Beng, & Neo, 2013). A 0.5 mL glass syringe was employed for the application of chitosan which remained on the surface for 1 min (Arnaud, de Barros Neto, & Diniz, 2010), followed by water rinse for 5s.
2.6 Wettability analysis The wettability of dentine surface was determined measuring the contact angle (θ) by sessile drop method, using a goniometer (OCA 20-DataPhysics Instruments GmbH, Filderstadt, Germany). Each specimen was positioned on a mobile platform with adjustment screws. Then, a drop of adhesive (≈ 20L) (Single Bond Universal, 3M ESPE, St.Paul, EUA) was dispensed under the dentin surface. Through a lighting system with a tungesten lamp and a Charge-Coupled Device (CCD) camera, the drop image on the dentine surface was captured for a period of 2 minutes, at intervals of 1ms. The values of (θ) were analyzed by 6
the OCA-20 software. All procedures involving the measurement of (θ) were performed in a closed environment and at controlled room temperature of 25 °.
2.7 SEM analysis The slabs were placed in vials containing distilled water and were prepared according to the following protocol: cleaning in ultrasound (Ultrasonic Cleaner T-1449-D, Odontobrás, SP, Brazil) for 10min; drying with absorbent paper; application of EDTA gel for 30s and, then, slabs were immersed in glutaraldehyde 2.5% in 0.1M sodium cacodylate buffer, pH 7.4 (Merck KGaA, D-64293, Darmstadt, Germany), at 4ºC for 12 hours. After this period, slabs were rinsed and immersed in distilled water for 1 hour. Dehydration was performed using ascending grades of ethanol (Labsynth Ltd., Diadema, SP, Brazil.): 25% (20 min), 50% (20 min), 75% (20 min), 95% (30 min), 100% (60 min). Finally, slabs were immersed in HMDS solution (Merck Kgap, Damstadt, D64293, Germany) for 10 min. After drying, the slabs were fixed in stubs with double-sided adhesive carbon tape and were coated with gold in a vacuum-metalizing machine (SDC 050, Bal-Tec AG, FL9496, Balzers, Liechtenstein). Through a scanning electron microscope (JSM-6610LV, JEOL, Peabody, MA, USA) the most representative area of each group was photographed, at different magnifications.
2.8 Statistical analysis The values of (θ) are submitted to the normality test (Kolmogorov-Smirnov) and data presented normal distribution. Thus, data were analyzed using ANOVA. Multiple comparisons were performed using DUNCAN test at a 0.05 significance level. Statistical analysis was performed with the SPSS software for Windows, version 12.0 (SPSS Inc., Chicago, IL, USA).
3. Results
7
The mean values (standard deviation) and statistical comparisons of the contact angles after the dentin rewetting with chitosan, at different concentrations, are shown in table 1. The ANOVA showed no significant interaction between rewetting and the condition of the dentin substrate (p> 0.05). Rewetting with chitosan, independent of concentration, did not influence the contact angle between the dentin and the adhesive system (p>0.05). The average of the smallest contact angle ranged from 23.43 to 32.26°, being the smallest value to sound dentin rewett with 2.5% chitosan and the highest to the eroded dentin rewett with 1.0% acetic acid. The schematic representation of the contact angle measurement is shown in Figure 2.
In Figure 3, the scanning electron microscopy images represent the different groups of sound dentin. The image A shows demineralized surface after conditioning with 35% phosphoric acid. In the image B, it is possible to observe demineralization promoted by the application of 1.0% acetic acid (chitosan vehicle control), a similar pattern to that observed in image A. The use of 2.5% and 5.0% chitosan promoted the deposition of chitosan particles on the surface and within the dentinal tubules (Images C and D). In Figure 4, the scanning electron microscopy images represent the different groups of eroded dentin. The image A shows demineralized surface, after conditioning with 35% phosphoric acid, with no smear layer and open dentinal tubules. The image B, it is verified is a homogeneous surface with a demineralized organic matrix, with no smear layer and open dentinal tubules. The peritubular dentin presented completely demineralized and intertubular dentin exhibited a porous appearance with roughness and exposure of collagen fibrils, similar to the pattern observed in image A, however, with an increased of collagen fibers exposure. In the image C, it was found particles of chitosan deposited on the surface and within the dentinal tubules. In the image D, chitosan deposition predominantly occurred inside the dentinal tubules. 8
4. Discussion The use of chitosan on dentin has shown favorable results in regarding to remineralization (Del Carpio-Perochena, Bramante, Duarte, de Moura, Aouada, & Kishen, 2015), enabling the formation of a calcium phosphate layer on the demineralized dentin (Xu, Xin, Li, Huang, Zhou, & Liu, 2011). Furthermore, the use of chitosan promoted an increase the dentin bond strength (Sherestha & Kishen, 2012) making dentin structure more resist to hydrolytic degradation challenges, as well, collagen-degrading enzymes (Fawzy, Nitisusanta, Iqbal, Daood, Beng,
& Neo, 2013), which can favor an increase in the durability of
adhesive restorations (Xu, Giu, Wang, Yu , Xi, & Jial, 2010). Considering these favorable aspects of chitosan in dentin and in the face of difficulties to obtain optimum wetting of the adhesive system on the eroded dentin surface, it is important to assess the contact angle between adhesive system and the demineralized dentin, rewett with chitosan at 2.5% and 5.0%, and to correlate these findings with the dentin surface morphology. Based on the results of this study, the null hypothesis that the type of substrate (sound or eroded dentin) does not influence the wettability of a total-etch adhesive system was accepted. The application of phosphoric acid on eroded dentin (Figure 4A) propitiates a homogeneous surface with demineralized organic matrix, with no smear layer and with open dentinal tubules. This occurred in a less intense way when compared to acid-etched sound dentin (Figure 3A). However, there was no significant interaction between rewetting and the condition of the dentin substrate. Within the limits of our knowledge, comparison of these results with the literature becomes difficult, since there are no studies in which the eroded dentin rewetting with chitosan was evaluated. The null hypotesis that the rewetting with chitosan, independent of concentration, does not influence the wettability of a total-etch adhesive system was accepted. The fact that chitosan does not influence the contact angle, and thus the wettability, can ensure its use as an adjunct to promote a greater adhesion to eroded dentin. In addition, this 9
compound presents proven benefits, being a biodegradable, biocompatible and non-toxic compound (Venter, Kotze, Auzely-Velty, & Rinaudo, 2006; Raafat & Sahl, 2009), as well as, chelating agent (Silva, Guedes, Pécora, & da Cruz-Filho, 2012). One of the possible reasons for the contact angle have not been influenced by chitosan treated eroded dentin may be related to the fact that chitosan was deposited predominantly within the dentinal tubules, as illustrated by the photomicrographs shown in Figures 4C and 4D. In our study the contact angle was analyzed exclusively at the x axis of the sample. Any change caused by the orientation of the tubules may occur only at the z-axis. In this way, using this technique, the orientation of the dentinal tubules did not influence the measurements, being the contact angle influenced, mainly, by the roughness and by the heterogeneity of the dentin substrate (Farge, Alderete, & Ramos, 2010). Studies have found that the chitosan, for having an acidic pH, shows a remarkable chelating ability for different metal ions (Kurita, Shimada, Nishuyama, Shimojoh, & Nishimura, 1988) being responsible for chelating of calcium ions in dentin, which resulted in the depletion of inorganic material from smear layer (Pimenta, Zaparolli, Pécora, & da CruzFilho, 2012). Silva, Guedes, Pécora, & da Cruz-Filho, (2012) studied the effect of chitosan on the dentin structure, in dependence on the time of application. It demonstrated that when 0.3% chitosan was used for 3 min, the smear was efficiently removed. The fact that the chitosan being a chelating agent (Kurita, Shimada, Nishuyama, Shimojoh, & Nishimura, 1988; Silva, Guedes, Pécora, & da Cruz-Filho, 2012) may explain the predominance of its particles within the dentinal tubules, and not on the surface. Chitosan is a highly charged molecule formed by the presence of bridges of hydrogen and hydroxyl groups in its polymer chain. Thus, due to loss of protons by the molecule, there was a covalently bond to the substrate (by chelating action) which promoted a greater accumulation of its particles in peritubular dentin, not influencing the measure of contact angle. Another aspect that can be considered, is the amount of protonated amino groups (NH3+) in the chitosan polymer chains being related to the solubility of the molecule. The 10
electrostatic repulsion of these groups occurs due of their greater or lesser amount. Thus, the fact that dentin rewetting with 5.0% chitosan (Figure 3D and 4D) exhibit
chitosan
particles, predominantly within the tubules, can be related to a greater repulsive electrostatic interaction of this groups. Factors such as surface tension of the liquid and 'free' energy of the solid can influence the wettability. To achieve optimum wettability, the 'free' energy of the solid has to be maximized, and the liquid must have low contact angle with solid (Tsujimoto, Takimoto, Ootsuka, Endo, Takamizawa, & Miyazaki, 2010). However, these factors did not influence on the contact angle between dentin surface and adhesive system, since, the wettability showed no differences both substrates tested. Both wettability as the infiltration depth of adhesives plays an important role in the quality and durability of the dentin-resin interface. The results of this study demonstrate that chitosan-impregnated eroded dentin did not Influence of substratum wettability. Thus, it may suggest that this biopolymer may be used to rehydrate exposed collagen matrix maintaining a more hydrophilic surface after acid etching.
5. Conclusion It was concluded that chitosan, at concentrations of 2.5% and 5.0%, did not influence of the eroded dentin wettability. Through SEM analysis, it was found particles of chitosan deposited on the surface and within the dentinal tubules. Author Disclosure Statement No competing financial interests exist.
Acknowledgments The authors are grateful to the agency CAPES for the financial support.
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Grippo, J. O., Simring, M., & Schreiner, S. (2004). Attrition, abrasion, corrosion and abfraction revisited a new perspective on tooth surface lesions. American Dental Association, 135, 1109-1118. Imfeld, T. (1996). Dental erosion. Definition, classification and links. European Journal of Oral Sciences, 104, 151–155. Kurita, K. Chemistry and application of chitin and chitosan. (1998). Polymer Degradation and Stability, 59, 117-120. Lussi & Hellwing E. (2006). Risk assessment and preventive measures. Monographs in Oral Science, 20, 190-191. McKnight-Hanes, C., & Whitford, GM (1992) Fluoride release from three glass ionomer materials and the effects of varnishing with or without finishing. Caries Research, 26, 345– 350. Meurman, J. H., Drysdale, T., & Frank, R. M. (1991). Experimental erosion of dentin. Scandinavian Journal of Dental Research, 99, 457-462. Meurman, J. H., & ten Cate, J. M. (1996). Pathogenesis and modifying factors of dental erosion. European Journal of Oral Sciences, 104, 199-206. Milosevic, A., & Jones, C. (1996). Use of resin-bonded ceramic crowns in a bulimic patient with severe tooth erosion. Quintessence International, 27, 123-127. Muzzarelli, R. A., & Rochetti, R. (1993). The determination of molybdenium in sea water by hot graphite atomic absorption spectrometry after concentration on p-aminobenzylcelulose or chitosan. Analytica Chimica Acta, 64, 371-379. Persadmehr, A., Torneck, C. D., Cvitkovitch, D. G., Pinto. V., Talior, I., Kazembe, M., Shrestha, S., McCulloch, C. A., Kishen, A. (2011), Bioactive chitosan nanoparticles and photodynamic therapy inhibit collagen degradation in vit ro. Journal of Endodontics, 40, 703-709. Pimenta, J. A., Zaparolli, D., Pécora, J. D., & da Cruz-Filho, A. M. (2012). Chitosan: effect of a new chelanting agent on the microhardness of root dentin. Brazilian Dental Journal, 23, 212-217. Raafat, D., & Sahl, H. G. (2009). Chitosan and its antimicrobial potential-a critical literature survey. Microbial Biotechnology, 2, 186-201. Sabatini, C., & Pashley, D. H. (2014). Mechanisms regulating the degradation of dentine matrices by endogenous dentin proteases and their role in dental adhesion. American Journal of Dentistry, 27, 203-214.
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Fig. 1. Experimental design.
Fig. 2. Schematic representation of the contact angle measurement. (A) Sound and (B) eroded dentin. (1) No rewetting, (2) 1% acetic acid, (3) 2.5% chitosan, (4) and 5% chitosan. Fig. 3. Photomicrographs of sound dentin after different treatments performed: A) without rewetting (control); B. 1.0% Acetic acid 1%; C. 2.5% Chitosan; D. 5.0% Chitosan. Arrows indicate the presence of chitosan particles deposited at the opening of the tubules. High power magnification (5000X) images show detail of the region. Fig, 4. Photomicrographs of eroded dentin after different treatments performed: A) without rewetting (control); B. 1.0% Acetic acid 1%; C. 2.5% Chitosan; D. 5.0% Chitosan. Arrows indicate the presence of chitosan particles deposited at the opening of the tubules. High power magnification (5000X) images show detail of the region.
Fig 1
15
Fig 2
Fig 3 16
Fig 4
17
Table 1- Means and standard deviations of lower contact angle (ɵ°), presented by each group. REWETTING
DENTIN SUBSTRATE SOUND
ERODED
Without rewetting
29,51 ±7,70ab
30,26±6,0ab
1.0 % Ácetic acid
24,41±8,45ab
32,26±9,28b
2.5% Chitosan
23,34±4,56a
30,68±5,67ab
5.0% Chitosan
26,29±11,44ab
29,22±8,93ab
Similar letters indicate statistical similarity.
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S0003-9969(16)30351-X http://dx.doi.org/doi:10.1016/j.archoralbio.2016.11.017 AOB 3759
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
1-12-2015 30-8-2016 29-11-2016
Please cite this article as: Ururahy Mirian Saavedra, Curylofo-Zotti Fabiana Almeida, Galo Rodrigo, Nogueira Lucas Fabricio Bahia, Ramos Ana Paula, Corona Silmara Aparecida Milori.Wettability of chitosan treated eroded dentin and analysis of surface morphology.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2016.11.017 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 proof before it is published in its final 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.
Wettability of chitosan treated eroded dentin and analysis of surface morphology Mirian Saavedra Ururahya, Fabiana Almeida Curylofo-Zottia Rodrigo Galob, Lucas Fabricio Bahia Nogueirac, Ana Paula Ramosc, Silmara Aparecida Milori Coronaa a
Department of Restorative Dentistry, Sao Paulo University, Ribeirao Preto, Café Avenue,
Monte Alegre, Ribeirão Preto, SP, Brazil. b
Department of Dentistry, Federal University of Jequitinhonha and Mucuri Valleys,
Diamantina, MG, Brazil. c
Department of Chemistry, Sao Paulo University, Bandeirantes Avenue, Ribeirao Preto, SP,
Brazil.
Corresponding author: Silmara Aparecida Milori Corona Restorative Dentistry Department, Dental Schoolof Ribeirão Preto, University of São Paulo, Café, s/n, Monte Alegre, Ribeirão Preto-SP, Brazil, 14040-904. Phone number: +55-16-33154075 E-mail: [email protected]
1
Highlights
The use of chitosan on dentin has shown favorable results in literature. However,
within the limit of our knowledge, there no studies that evaluated if the wettability of chitosan treated eroded dentin was influenced by the concentration of chitosan used.
It was concluded that chitosan, at concentrations of 2.5% and 5.0%, did not influence of the eroded dentin wettability.
Through SEM analysis, it was found particles of chitosan deposited on the surface and within the dentinal tubules.
Abstract Objective: To assess the effect of chitosan, at concentrations of 2.5% and 5.0%, on the eroded dentin wettability, followed by analysis of surface morphology by SEM. Methods: 104 bovine dentin slabs were ground, polished and then immersed in 20mL of citric acid (pH = 3.2) under continuous stirring for 2h. Specimens were randomly divided according to the dentin substrate: sound and eroded, and then, subdivided into 4 groups (n=10): without rewetting (control), 1% acetic acid, 2.5% chitosan and 5.0% chitosan. Then, a drop of the adhesive system Single Bond 2 (3M) was deposited onto surface of each specimen. The contact angle between dentin surface and the adhesive system was measured by using a goniometer. The other 24 specimens were subjected to analysis under SEM. Statistical analysis was performed using the normality test (Kolmogorov-Smirnov) and Analysis of Variance (ANOVA) (p> 0.05). Results: No differences were found between the angles produced on the eroded dentin rewetting with chitosan at the concentrations of 2.5% and 5%. Conclusion: The chitosan, regardless of the concentration used, did not influence of the eroded dentin wettability. Through SEM analysis, it was found particles of chitosan deposited on the surface and within the dentinal tubules.
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Key-words: eroded dentin, chitosan, wettability, contact angle
1. Introduction Dental erosion (more recently known as corrosion) (Grippo, 2012) occurs due to extended contact of dental tissues with acidic substances provided from intrinsic or extrinsic sources (Imfeld, 1996; Lussi & Hellwig, 2006), without the involvement of bacteria (Grippo, Simring,
& Schreiner,
2004).
Clinically,
this
demineralization
process
is
characterized by the progressive and irreversible loss of tooth structure (Addy & Shellis, 2006), which depending on the severity of the lesions may result in dental hypersensitivity (Barbour, Lussi, & Shellis, 2011), as well as functional and aesthetic damage. The erosion process provides an initial dissolution of peritubular dentin, increasing the diameter of the tubules. This can lead to demineralization of intertubular dentin, exposing the organic matrix which may result in a rough and porous surface (Meurman & ten Cate, 1991; Meurman & ten Cate, 1996; Ganss, Hardat, Blazek, Klimek, & Schlueter, 2009). In addition to the mineral dissolution, enzymatic collagen degradation (Ganss, Klimek, Brune, & Schürman, 2004; Schlueter, Neutard, von Hinckeldey, Klimek, & Ganss, 2010) can interfere with the quality of the collagen network (Sabatini & Pashley, 2014.). This fact can directly reflect on the success of composite restorations, which are preferable for being based on minimally invasive procedures (Atin & Wegehaupt, 2014), in addition to not being acid-soluble (Milosevic & Jones, 1996). In attempting to improve the quality and longevity of adhesive restorations, studies have evaluated the use of biopolymers, such as chitosan (Bedran-Russo, Pereira, Duarte, Drumond, & Yamauchi, 2007; Shrestha, Friedman, Kishen, 2011; Fawzy, Nitisusanta, Iqbal, Daood, Beng, & Neo, 2013), to increase the cross-linking between the collagen fibers, and neutralize matrix metalloproteinases (MMPs) (Xu, Neoh, Lin, & Kishen, 2011; Persadmehr, Torneck, Cvitkovitch, Pinto, Talior, Kazembe, Shrestha, McCulloch, & Kishen, 2014) Chitosan is a natural polysaccharide obtained through deacetylation of chitin (Muzzarelli & Rochetti, 1973) which is totally biocompatible, biodegradable and non-toxic 3
(Venter, Kotze, Auzely-Velty, & Rinaudo, 2006; Raafat & Sahl, 2009). This polymer has been used in order to strengthen (Fawzy, Nitisusanta, Iqbal, Daood, Beng, & Neo, 2013) and stabilize (Bedran-Russo, Pereira, Duarte, Drumond, & Yamauchi, 2007; Shrestha, Friedman, & Kishen, 2011) dentin collagen once its use is able to produce microfibrillar arrangements with superior mechanical properties (Dash, Chiellini, Ottenbrite, & Chiellini, 2011). Thus, due to the need for greater durability of restorative procedures on eroded dentin, its important evaluate if the wettability of chitosan treated eroded dentin will be influenced by the concentration of chitosan used. The null hypotheses tested were: 1) the type of substrate (sound or eroded dentin) does not influence the wettability of a total-etch adhesive system; 2) the rewetting with chitosan, at concentrations of 2.5% and 5.0%, both in sound or eroded dentin does not influence the wettability of a total-etch adhesive system.
2. Materials and methods 2.1 Experimental design The factors under study were dentin substrate (sound and eroded) and the rewetting of the surface (1% acetic acid, 2.5% chitosan and 5.0% chitosan). Specimens of the control group did not receive the rewetting with chitosan. Experimental units were 104 bovine incisors randomly assigned into groups. The response variable was the measurement of the contact angle (n=10) formed between dentin and the adhesive system and analysis of surface morphology by SEM (n=3). The design of the present study is presented in Figure 1. 2.2 Selection of teeth Bovine incisors stored in 0.1% thymol solution at 9°C were washed in running water for 24 hours to eliminate thymol residues. Then, teeth were examined under a 20x magnifier in
4
stereoscopic microscope (Leica Microsystems, Wetzlar, Germany). 104 incisors with no fractures lines or crown-deep cracks were selected.
2.3 Preparation of dentin slabs Roots were transversely sectioned in the cementoenamel junction using a doublefaced diamond disk (15HC 11-4244, Buehler, Illinois, USA) mounted in a low-speed handpiece (Isomet 1000; Buehler, Germany). Subsequently, crowns were sectioned in mesial-distal direction in order to obtain two hemi-sections (buccal and palatal) of each crown. From each buccal surface, one slab with the dimensions of 7.0 x 7.0 x 2.5 mm was obtained. The fragments were fixed in a teflon matrix using molten wax (Kota Ltd, SP, Brazil) with the enamel surface face up. The dentin surfaces was grounded with 320 grid silicon carbide paper under water cooling to remove lateral excess and set the size of the slabs, then, specimens were grounded with 1200 grid silicon carbide paper (Hermes Abrasives Ltd., VA, EUA) for 10 s. Polishing was performed with felt disk (ATM, Altenkirchen, Germany) and 0.3 mm alumina suspension (Arotec, SP, Brazil) for 5 s.
2.4 Erosion-like lesion formation To induce erosion lesions formation it was used a protocol previously established by Vanuspong, Eisenburger, & Addy, (2002). Each dentin slab was immersed in a beaker containing 20 mL of 0.3% citric acid (pH = 3.2) which was individually placed in a shaker under constant stirring, 50 rpm speed for a period of 2h. After this period, specimens were rinsed with distilled water and individually stored in vials containing artificial saliva for 24 hours at 37ºC. Artificial saliva was composed of methyl phydroxybenzoate (2.0 g), sodium carboxymethylcellulose (10.0 g), KCl (0.625 g), MgCl2.6H2O (0.059 g), CaCl2.2H2O (0.166 g), K2HPO4 (0.804 g ), and KH2PO4 (0.326 g) in 1000 mL of distilled water, according to the
5
protocol described by McKnight-Hanes & Whitford (1992) and modified by Amaechi, Higham, Edgar (1999).
2.5 Surface treatment All dentin specimens were conditioned with 37% phosphoric acid (Ultra-Etch Ultradent, SP, Brazil) for 15 sec, rinsed for 10s under running tap water, and dried with absorbent paper. For the specimens that received the rewetting, this procedure was performed with 2.5% and 5.0% chitosan (Arnaud, de Barros Neto, & Diniz, 2010). For the preparation of the chitosan, 2.5 g and 5.0 g of chitosan (Sigma-Aldrich, St. Louis, MO, USA) low molecular weight were weighed in an analytical balance (Bel analytical equipment Ltd, SP, Brazil). Then, chitosan was diluted in 100 ml of 1.0% of acetic acid solution, slowly added and under constant stirring (Marconi Equip. Lab., Piracicaba, Brazil) for 20 min (time enough to solubilise the polysaccharide). 1 mol/L of NaOH solution was added in order to avoid aggregation of the particles and raising the pH (Daood, Iqbal, Nitisusanta, Fawzy, & 2013; Fawzy, Nitisusanta, IIqbal, Daood, Beng, & Neo, 2013). A 0.5 mL glass syringe was employed for the application of chitosan which remained on the surface for 1 min (Arnaud, de Barros Neto, & Diniz, 2010), followed by water rinse for 5s.
2.6 Wettability analysis The wettability of dentine surface was determined measuring the contact angle (θ) by sessile drop method, using a goniometer (OCA 20-DataPhysics Instruments GmbH, Filderstadt, Germany). Each specimen was positioned on a mobile platform with adjustment screws. Then, a drop of adhesive (≈ 20L) (Single Bond Universal, 3M ESPE, St.Paul, EUA) was dispensed under the dentin surface. Through a lighting system with a tungesten lamp and a Charge-Coupled Device (CCD) camera, the drop image on the dentine surface was captured for a period of 2 minutes, at intervals of 1ms. The values of (θ) were analyzed by 6
the OCA-20 software. All procedures involving the measurement of (θ) were performed in a closed environment and at controlled room temperature of 25 °.
2.7 SEM analysis The slabs were placed in vials containing distilled water and were prepared according to the following protocol: cleaning in ultrasound (Ultrasonic Cleaner T-1449-D, Odontobrás, SP, Brazil) for 10min; drying with absorbent paper; application of EDTA gel for 30s and, then, slabs were immersed in glutaraldehyde 2.5% in 0.1M sodium cacodylate buffer, pH 7.4 (Merck KGaA, D-64293, Darmstadt, Germany), at 4ºC for 12 hours. After this period, slabs were rinsed and immersed in distilled water for 1 hour. Dehydration was performed using ascending grades of ethanol (Labsynth Ltd., Diadema, SP, Brazil.): 25% (20 min), 50% (20 min), 75% (20 min), 95% (30 min), 100% (60 min). Finally, slabs were immersed in HMDS solution (Merck Kgap, Damstadt, D64293, Germany) for 10 min. After drying, the slabs were fixed in stubs with double-sided adhesive carbon tape and were coated with gold in a vacuum-metalizing machine (SDC 050, Bal-Tec AG, FL9496, Balzers, Liechtenstein). Through a scanning electron microscope (JSM-6610LV, JEOL, Peabody, MA, USA) the most representative area of each group was photographed, at different magnifications.
2.8 Statistical analysis The values of (θ) are submitted to the normality test (Kolmogorov-Smirnov) and data presented normal distribution. Thus, data were analyzed using ANOVA. Multiple comparisons were performed using DUNCAN test at a 0.05 significance level. Statistical analysis was performed with the SPSS software for Windows, version 12.0 (SPSS Inc., Chicago, IL, USA).
3. Results
7
The mean values (standard deviation) and statistical comparisons of the contact angles after the dentin rewetting with chitosan, at different concentrations, are shown in table 1. The ANOVA showed no significant interaction between rewetting and the condition of the dentin substrate (p> 0.05). Rewetting with chitosan, independent of concentration, did not influence the contact angle between the dentin and the adhesive system (p>0.05). The average of the smallest contact angle ranged from 23.43 to 32.26°, being the smallest value to sound dentin rewett with 2.5% chitosan and the highest to the eroded dentin rewett with 1.0% acetic acid. The schematic representation of the contact angle measurement is shown in Figure 2.
In Figure 3, the scanning electron microscopy images represent the different groups of sound dentin. The image A shows demineralized surface after conditioning with 35% phosphoric acid. In the image B, it is possible to observe demineralization promoted by the application of 1.0% acetic acid (chitosan vehicle control), a similar pattern to that observed in image A. The use of 2.5% and 5.0% chitosan promoted the deposition of chitosan particles on the surface and within the dentinal tubules (Images C and D). In Figure 4, the scanning electron microscopy images represent the different groups of eroded dentin. The image A shows demineralized surface, after conditioning with 35% phosphoric acid, with no smear layer and open dentinal tubules. The image B, it is verified is a homogeneous surface with a demineralized organic matrix, with no smear layer and open dentinal tubules. The peritubular dentin presented completely demineralized and intertubular dentin exhibited a porous appearance with roughness and exposure of collagen fibrils, similar to the pattern observed in image A, however, with an increased of collagen fibers exposure. In the image C, it was found particles of chitosan deposited on the surface and within the dentinal tubules. In the image D, chitosan deposition predominantly occurred inside the dentinal tubules. 8
4. Discussion The use of chitosan on dentin has shown favorable results in regarding to remineralization (Del Carpio-Perochena, Bramante, Duarte, de Moura, Aouada, & Kishen, 2015), enabling the formation of a calcium phosphate layer on the demineralized dentin (Xu, Xin, Li, Huang, Zhou, & Liu, 2011). Furthermore, the use of chitosan promoted an increase the dentin bond strength (Sherestha & Kishen, 2012) making dentin structure more resist to hydrolytic degradation challenges, as well, collagen-degrading enzymes (Fawzy, Nitisusanta, Iqbal, Daood, Beng,
& Neo, 2013), which can favor an increase in the durability of
adhesive restorations (Xu, Giu, Wang, Yu , Xi, & Jial, 2010). Considering these favorable aspects of chitosan in dentin and in the face of difficulties to obtain optimum wetting of the adhesive system on the eroded dentin surface, it is important to assess the contact angle between adhesive system and the demineralized dentin, rewett with chitosan at 2.5% and 5.0%, and to correlate these findings with the dentin surface morphology. Based on the results of this study, the null hypothesis that the type of substrate (sound or eroded dentin) does not influence the wettability of a total-etch adhesive system was accepted. The application of phosphoric acid on eroded dentin (Figure 4A) propitiates a homogeneous surface with demineralized organic matrix, with no smear layer and with open dentinal tubules. This occurred in a less intense way when compared to acid-etched sound dentin (Figure 3A). However, there was no significant interaction between rewetting and the condition of the dentin substrate. Within the limits of our knowledge, comparison of these results with the literature becomes difficult, since there are no studies in which the eroded dentin rewetting with chitosan was evaluated. The null hypotesis that the rewetting with chitosan, independent of concentration, does not influence the wettability of a total-etch adhesive system was accepted. The fact that chitosan does not influence the contact angle, and thus the wettability, can ensure its use as an adjunct to promote a greater adhesion to eroded dentin. In addition, this 9
compound presents proven benefits, being a biodegradable, biocompatible and non-toxic compound (Venter, Kotze, Auzely-Velty, & Rinaudo, 2006; Raafat & Sahl, 2009), as well as, chelating agent (Silva, Guedes, Pécora, & da Cruz-Filho, 2012). One of the possible reasons for the contact angle have not been influenced by chitosan treated eroded dentin may be related to the fact that chitosan was deposited predominantly within the dentinal tubules, as illustrated by the photomicrographs shown in Figures 4C and 4D. In our study the contact angle was analyzed exclusively at the x axis of the sample. Any change caused by the orientation of the tubules may occur only at the z-axis. In this way, using this technique, the orientation of the dentinal tubules did not influence the measurements, being the contact angle influenced, mainly, by the roughness and by the heterogeneity of the dentin substrate (Farge, Alderete, & Ramos, 2010). Studies have found that the chitosan, for having an acidic pH, shows a remarkable chelating ability for different metal ions (Kurita, Shimada, Nishuyama, Shimojoh, & Nishimura, 1988) being responsible for chelating of calcium ions in dentin, which resulted in the depletion of inorganic material from smear layer (Pimenta, Zaparolli, Pécora, & da CruzFilho, 2012). Silva, Guedes, Pécora, & da Cruz-Filho, (2012) studied the effect of chitosan on the dentin structure, in dependence on the time of application. It demonstrated that when 0.3% chitosan was used for 3 min, the smear was efficiently removed. The fact that the chitosan being a chelating agent (Kurita, Shimada, Nishuyama, Shimojoh, & Nishimura, 1988; Silva, Guedes, Pécora, & da Cruz-Filho, 2012) may explain the predominance of its particles within the dentinal tubules, and not on the surface. Chitosan is a highly charged molecule formed by the presence of bridges of hydrogen and hydroxyl groups in its polymer chain. Thus, due to loss of protons by the molecule, there was a covalently bond to the substrate (by chelating action) which promoted a greater accumulation of its particles in peritubular dentin, not influencing the measure of contact angle. Another aspect that can be considered, is the amount of protonated amino groups (NH3+) in the chitosan polymer chains being related to the solubility of the molecule. The 10
electrostatic repulsion of these groups occurs due of their greater or lesser amount. Thus, the fact that dentin rewetting with 5.0% chitosan (Figure 3D and 4D) exhibit
chitosan
particles, predominantly within the tubules, can be related to a greater repulsive electrostatic interaction of this groups. Factors such as surface tension of the liquid and 'free' energy of the solid can influence the wettability. To achieve optimum wettability, the 'free' energy of the solid has to be maximized, and the liquid must have low contact angle with solid (Tsujimoto, Takimoto, Ootsuka, Endo, Takamizawa, & Miyazaki, 2010). However, these factors did not influence on the contact angle between dentin surface and adhesive system, since, the wettability showed no differences both substrates tested. Both wettability as the infiltration depth of adhesives plays an important role in the quality and durability of the dentin-resin interface. The results of this study demonstrate that chitosan-impregnated eroded dentin did not Influence of substratum wettability. Thus, it may suggest that this biopolymer may be used to rehydrate exposed collagen matrix maintaining a more hydrophilic surface after acid etching.
5. Conclusion It was concluded that chitosan, at concentrations of 2.5% and 5.0%, did not influence of the eroded dentin wettability. Through SEM analysis, it was found particles of chitosan deposited on the surface and within the dentinal tubules. Author Disclosure Statement No competing financial interests exist.
Acknowledgments The authors are grateful to the agency CAPES for the financial support.
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Fig. 1. Experimental design.
Fig. 2. Schematic representation of the contact angle measurement. (A) Sound and (B) eroded dentin. (1) No rewetting, (2) 1% acetic acid, (3) 2.5% chitosan, (4) and 5% chitosan. Fig. 3. Photomicrographs of sound dentin after different treatments performed: A) without rewetting (control); B. 1.0% Acetic acid 1%; C. 2.5% Chitosan; D. 5.0% Chitosan. Arrows indicate the presence of chitosan particles deposited at the opening of the tubules. High power magnification (5000X) images show detail of the region. Fig, 4. Photomicrographs of eroded dentin after different treatments performed: A) without rewetting (control); B. 1.0% Acetic acid 1%; C. 2.5% Chitosan; D. 5.0% Chitosan. Arrows indicate the presence of chitosan particles deposited at the opening of the tubules. High power magnification (5000X) images show detail of the region.
Fig 1
15
Fig 2
Fig 3 16
Fig 4
17
Table 1- Means and standard deviations of lower contact angle (ɵ°), presented by each group. REWETTING
DENTIN SUBSTRATE SOUND
ERODED
Without rewetting
29,51 ±7,70ab
30,26±6,0ab
1.0 % Ácetic acid
24,41±8,45ab
32,26±9,28b
2.5% Chitosan
23,34±4,56a
30,68±5,67ab
5.0% Chitosan
26,29±11,44ab
29,22±8,93ab
Similar letters indicate statistical similarity.
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