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Effect of xylitol chewing gum enriched with propolis on dentin remineralization in vitro
Archives of Oral Biology 112 (2020) 104684
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Effect of xylitol chewing gum enriched with propolis on dentin remineralization in vitro
T
Wafa Gargouria,*, Rym Kammounb, Mazen Elleuchec, Mahdi Tlilib, Nabil Kechaoua, Sonia Ghoul-Mazgarb a
Research Group of Agri-Food Processing Engineering, National School of Engineers of Sfax, University of Sfax, Soukra road, BP 1173, 3038, Sfax, Tunisia Laboratory of Dento-Facial, Clinical and Biological Approach (ABCDF), Faculty of Dental Medicine, University of Monastir, Tunisia c La Confiserie Triki le Moulin, Sfax, Tunisia b
ARTICLE INFO
ABSTRACT
Keywords: Remineralization Propolis Dentin Chewing gum Hydroxyapatites Xylitol
Objectives: The aim of this study was to evaluate the efficiency of xylitol chewing gums enriched with propolis, remineralizing softly demineralized dentin in vitro. Design: Four groups of chewing gum were developed; Group1: xylitol (1.8 %), Group2: xylitol + casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) (3%), Group3: xylitol+Hydroxyapatite (3%) and Group4: xylitol + propolis (5%). A control group was designed without chewing gum, but with artificial saliva. Sections of embedded crowns and cleaned roots of twenty five bovine incisors were demineralized in carbonated drink. Crown specimens were half-varnished. Remineralization process was run for all the dental specimens in the 4 groups with gum extracts and in the control group with artificial saliva for 20 min at 37 °C three times a day during 7 days. Mineral contents were evaluated by scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX-SEM). Surface morphology and roughness were analyzed using atomic force microscopy (AFM). Micro-hardness was measured using Vickers micro-hardness tester among varnished and unvarnished sides. Results: Calcium/Phosphate mean ratio showed a significant decrease between the control group, group1, group2 and group4. Control group and group3 were not significantly different. Micro-hardness increased significantly for all treated groups. AFM showed obstruction of dentinal tubules in all the groups and roughness decreased in the treated side of the dentin compared to the untreated side for tested groups. Conclusion: Xylitol chewing gum enriched with propolis showed dentinal tubules occlusion, significant improvement of micro-hardness and slight decrease in roughness. Ca/P ratio analysis suggests that a mineral compound other than hydroxyapatite is responsible of tubules occlusion.
1. Introduction Remineralization of demineralized dentin is one of the target challenges in dentistry, intending not only to repair eroded dentin, but improving also the dentin bonding stability and treating dentin hypersensitivity (Niu et al., 2014). Dentin forms the bulkiest portion of dental hard tissues and is mainly constituted of tubules. These tubules are internally lined by a highly mineralized and non-collagenous intratubular dentin and externally surrounded by an intertubular dentin, consisting of a collagen matrix, non-collagenous proteins and hydroxyapatite crystals (Kinney, Marshall, & Marshall, 2003). Dentin demineralization under acidic challenges usually results in the dissolution of the peritubular dentin, which is detectable by the enlargement of the
⁎
tubule lumen, while the morphology of the intertubular dentin is preserved thanks to the persistence of the organic phase. Due to these structural particularities and changes, remineralization of demineralized dentin is a complex phenomenon. In fact, the remineralizing processes of demineralized dentin is supported by different strategies. The traditional ion-based strategy targets on the deposition of calcium and phosphate ions on the demineralized tissue. Among these agents, casein phosphopeptide-amorphouse calcium phosphate (CPP-ACP) is proposed to enhance remineralization and to reduce solubility of hydroxyapatite. Thereby, CPP-ACPcontaining products such as toothpaste, mouth rinse solutions and also chewing gum have been widely used (Wiegand & Attin, 2014). Otherwise, synthetic hydroxyapatite, that is similar to the biologically formed
Corresponding author. E-mail address: [email protected] (W. Gargouri).
https://doi.org/10.1016/j.archoralbio.2020.104684 Received 5 October 2019; Received in revised form 8 January 2020; Accepted 17 February 2020 0003-9969/ © 2020 Published by Elsevier Ltd.
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apatite in natural dentin, has also been suggested to be a promising biomimetic oral care ingredient. While hydroxyapatite has proved efficiency occluding open dentin tubules in clinical studies, in situ studies are still needed to further clarify the biomineralization process (Enax & Epple, 2018). Besides, considering that the current trend in the field of oral health stresses on the use of natural products for treating diseases, rather than allopathic medicine, new products emerged in the field of the remineralization of demineralized dentin. In this context, xylitol that is a natural herbal product that does not bring neither calcium nor phosphate and is frequently used in dental products for its antimicrobial properties, has been recently shown to have a remineralizing potential in vitro on eroded teeth by facilitating calcium movement and accessibility into the lesion pores (Gargouri, Zmantar, Kammoun, Kechaou, & Ghoul-Mazgar, 2018; Miake, Saeki, Takahashi, & Yanagisawa, 2003). In this context, the calcium and phosphate present in the saliva were sufficient to ensure the remineralisation. In another hand, Propolis, another natural product produced by bees, seems to present also a scientific importance in oral health regarding its bioactive properties (Gargouri, Osés, Fernández-Muiño, Sancho, & Kechaou, 2019). Several studies showed particularly the effect of propolis preserving connective elements in periodontium (Yuan, Wang, Shi, & Zhao, 2018) or enhancing healing, regeneration and biomineralization in bone (Meimandi-Parizi, Oryan, Sayahi, & BighamSadegh, 2018). However, there are scarcity and controversies in the literature regarding the dental remineralizing potential of propolis. In fact, while Chen and collaborators demonstrated occlusion of dentinal tubules using red propolis extracts (Chen, Parolia, Pau, & Celerino de Moraes Porto, 2015), Cardoso and collaborators suggested that the propolis ethanolic extract has no inhibitory action on the demineralization process of caries (Cardoso et al., 2016). Noticeably, dentin remineralization of demineralized dentin could not take place if crystallites are completely dissolved, or if the organic phase is collapsed hiding thus the damaged apatites (Niu et al., 2014). So, to backfill demineralized dentin, it is also important to preserve a slight amount of apatites in dentin and to act on a slightly and not a totally demineralized tissue managing the dentin connective phase. The connective phase of dentin contains particularly both collagen and noncollagenous proteins playing important roles in remineralization. In fact, while the collagen fibers constitute a basic template for mineral nucleation, the non-collagenous proteins or their biomimetic analogs seem to be essential for the non-classical crystallization pathway (Niu et al., 2014). Therefore, the aim of this study was to evaluate the efficiency of xylitol chewing gums enriched with propolis, remineralizing softly demineralized dentin in vitro, compared to xylitol chewing gums enriched in CPP-ACP and hydroxyapatite as conventional remineralizing agents bringing calcium and phosphate. The chemical, physical and morphological changes were investigated on dentin specimens after a demineralizing challenge.
propolis, reduced into powder was introduced to the gum base in a concentration of 5% during the manufacturing process. In fact, propolis powder was incorporated in chewing gum due to the high amount of phenolic compounds and the strong potential of biological activities, recently reported for Tunisian propolis from Béja region (Gargouri et al., 2019). It has been reduced into powder using spray drying technique B-290 (BÜCHI Labortechnik AG, Flawil, Suisse), in order to represent a promising ingredient in food industries and in chewing gum in our case. 2.2. Dental samples preparation A total of 25 fresh bovine incisors free of caries and defects were collected for this study. Teeth were disinfected in 5% sodium hypochlorite for two hours, then the crowns were separated from the roots using a diamond disc mounted on low speed headpiece and all the specimens were stored in formol solution at 4 °C until required. 2.2.1. Crown treatment Crowns were embedded in acrylic resin (BMS 017 Trayplast kit. Methyl Methacrylate co-polymer) and placed into Teflon moulds measuring 10 * 10 mm. Embedded blocks were cut under water irrigation using a low-speed diamond saw (Isomet, Buehler, Lake Bluff, ILL, USA) to expose a flat dentin surface. The surfaces were then polished using 200, 400 and 600 grit silicon carbide papers (Buehler, Lake Bluff, IL) to obtain a flat and highly polished resin block measuring 10 mm * 10 mm * 2 mm with the dentin surface exposed. Samples were randomly divided into five groups of five crowns each, a control group CTRL without using any chewing gum extract (artificial saliva), Group1 (xylitol chewing gum without remineralizing agents), Group2 (xylitol chewing gum with CPP-ACP), Group3 (xylitol chewing gum with hydroxyapatite) and Group4 (xylitol chewing gum with propolis). 2.2.2. Root treatment Dental roots were cleaned using scalers, then cementum was removed using a cylindrical-shaped bur and silicon carbide paper (600, 800 and 1000). Dental root slides of 2 mm thick were cut using a diamond disc mounted on low speed headpiece and stored in dei-onized water until use. Dental root slides were randomly distributed among the 5 groups as described previously with a total mean weight of 2.3 g for each group. 2.3. Demineralization challenge Crown specimens and roots of all groups were immersed in 10 ml of a fresh carbonated drink (Coca-Cola, pH = 2.7, Tunisia) for 5 min under continuous agitation at room temperature before rinsing with distilled water. Four consecutive erosive cycles were carried out. 2.4. Remineralization process Concerning the crown specimens, a layer of acid-resistant nail varnish was applied on the half of the exposed dentin in order to create an unexposed area (eroded side) and an exposed one (treated side). For the gum extracts, three pieces of each chewing-gum group were grinded in a mortar with 30 ml of artificial saliva (1.5 mM CaCl2, 1.0 mM KH2PO4 and 50 mM NaCl, pH 7.0) (Esteves-Oliveira et al., 2011), during 5 min, then the final solution was collected in a clean container for the remineralization process. The pH of the final solutions was recorded as follow; Group1, pH = 6.85 ± 0.34; Group2, pH = 6.79 ± 0.39; Group3, pH = 6.76 ± 0.17; Group4, pH = 7.15 ± 0.16. Remineralization was conducted by immersing each group of acid-eroded dentin specimen in 10 ml of the gum extract for 20 min at 37 °C three times a day (10 am, 1 pm and 3 pm) during 7 days. In-between the treatments, specimens were stored in freshly prepared artificial saliva. Varnish was removed from crown samples before following analysis. For root
2. Material and methods 2.1. Sugar-free chewing gum preparation Four groups of chewing gum were developed in the research and development laboratory of a Tunisian confectioner, TRIKI-Le Moulin, Sfax; Group1: xylitol (1.8 %), Group2: xylitol + CPP-ACP (3%), Group3: xylitol + hydroxyapatite (3%) and Group4: xylitol + propolis (5%). For the manufacturing process, gum base was melted in a mixer under heating then flavorings, sweeteners, colorants, humectants, and emulsifying agents were added as described previously (Gargouri et al., 2018). The remineralizing agents CPP-ACP and hydroxyapatite were purchased and directly added in the preparation process of chewing gums of groups 2 and 3, respectively. For the group4, Tunisian brown 2
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Fig. 1. Schematic representation of experimental study design.
specimens, remineralization process was directly performed without applying acid-resistant nail varnish. After finishing treatments, all the specimens were kept in de-ionized water (pH = 7,38) until analysis.
RP % =
2.5. EDX/SEM analysis
2.7. Surface micro-hardness
Chemical elements were evaluated on root slides using energy dispersive X-ray microanalysis system (EDX) coupled to a ThermoScientific™ Q250 scanning electron microscope (SEM) under ×60 magnification at 10 Kv (Fig. 1). Eighteen point measurements were checked on the surface of each slide. Analyses of the elements were assessed using the Pathfinder Software (ThermoScientific™ Q250). The elements quantified were Ca and P net counts and the Ca/P stoichiometric ratios were evaluated using the following formula: Ca (mol)/P (mol)% = [Ca (weight%)/40.08 (g/mol)]/ [P (weight %)/30.97 (g/mol)], considering that the molecular masses of Ca and P being 40.08 g and 30.97 g respectively.
The Vickers hardness number (VHN) was measured on the crown specimens eroded and treated with chewing gum using a Micro Hardness Tester [HMV 2 T] applying a load of 2 N for 15 s. Three indentations 100 μm apart were made on the eroded as well as on the treated sides of each dentin surface of the crown specimens. The Vickers Micro hardness mean of the dentin specimens were evaluated among three specimens of each group. Mean and standard deviation of the hardness values were statistically analyzed with ANOVA followed by Tukey’s HSD test. The hardness progression (VHN%) was calculated by the following formula:
VHN % =
2.6. Atomic force microscopy (AFM) To analyze the surface morphology and roughness of crown specimens, a non-contact mode atomic force microscopy (AFM, XE-70, Park System) was used (Fig. 1). AFM was equipped with a PPP-NCHR cantilever at 249 kHz resonant frequency and images were analyzed using XEI Software (Park Systems, Korea); All images contained 256 × 256 data points. The surface roughness (Rrms) of the analyzed surface was also measured by this microscope and the roughness progression (RP%) was calculated by the following formula:
Rafter treatment
Rbefore treatment
Rbefore treatment
VHNafter treatment
× 100
VHNbefore treatment
VHNbefore treatment
× 100
2.8. Statistical analysis Data were analysed using the Statistical Package for Social Science (SPSS, version 18.0, SPSS, Chicago, IL, USA) through the one-way ANOVA, Means-Test-Tukey’s and the Student’s t-test The level of significance was defined as 0.05. All results were reproduced in at least three independent experiments. 3
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Fig. 2. Calcium, phosphate and calcium/phosphate ratio evaluation on dentinal surfaces of the roots and expressed in weight percentages. Data are mean ± standard deviation (SD) of at least three independent experiments. Asteriks denote significant differences (P < 0.05).
3. Results
3.2. Physical and morphological analysis of the crown dentinal surfaces
3.1. Chemical analysis of calcium and phosphate values on the root dentinal surfaces
3.2.1. Surface micro-hardness The surface micro-hardness of the intact dentin was recorded as 57.13 ± 0.71. The VHN analysis revealed a significant increase of dentin micro-hardness of each surface for each group, compared to control group (Fig. 3). The mean values of micro-hardness were 38.4 ± 2.46, 35.90 ± 1.13, 43.43 ± 2.75 and 34.57 ± 0.95 for the eroded sides respectively in groups 1, 2, 3 and 4. The mean values of micro-hardness were 41.06 ± 2.25, 49.7 ± 1.90, 50.43 ± 3.56 and 41.07 ± 1.86, for the treated side respectively in groups 1, 2, 3 and 4. The micro-hardness was significantly higher in the surfaces treated with chewing-gum extracts compared to the untreated surfaces of each group, except for the control group (P < 0.05). The improvement of micro-hardness, comparing the treated and eroded sides of each group, was evaluated at 5.96 %, 7%, 38.51 %, 16.18 % and 18.83 % respectively for the control group and groups 1, 2, 3 and 4. Mean values and standard deviations for each group were displayed in Table 1.
Calcium and phosphate presence was proved in all samples (Fig. 2). Calcium weight percentage quantified at the surface of the slides showed an increased level in the treated groups with a significant difference (P < 0.05) evaluated by Student’s t-test between the control group (24.39 % ± 0.02) and the groups2 (25.74 % ± 0.06) and 3 (29.88 % ± 0.33). A significant decreased level was noticed between the control group (24.39 % ± 0.02) and the group1 (24.40 % ± 0.01) and group4 (24.18 % ± 0.02). Phosphate weight percentage quantified at the surface of the slides and evaluated by Student’s t-test showed an increased level in the treated groups with a significant difference (P < 0.05) between the control group (12.61 % ± 0.05) and the groups1 (12.98 % ± 0.06), 2 (13.57 % ± 0.04), 3 (14.77 % ± 0.09) and 4 (12.86 % ± 0.02). The Calcium/Phosphate ratio (Ca/P mean ratio), evaluated for the weight percentage in each group showed a significant difference (P < 0.05) between the control group (1.54 ± 0.05), group1 (1.44 ± 0.04), group2 (1.46 ± 0.02) or group4 (1.45 ± 0.03). The control group and group3 (1.50 ± 0.04) were not significantly different (P > 0.05).
3.2.2. Morphology Qualitative analysis of the three dimensional topographic maps obtained with the AFM revealed dentinal tubules cut either transversely or longitudinally on the eroded sides of the specimens. On the treated sides, the tubule sections were evidenced only in the control group,
Fig. 3. Micro-hardness mean on dentinal surfaces of the crowns. Data are mean ± SD of at least three independent experiments. Asterisks denote significant differences (P < 0.05).
4
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Table 1 Micro-hardness improvement (VHN%) among the sides. Groups
VHN before treatment (HV)
VHN after treatment (HV)
VHN%
Control Group1 Group2 Group3 Group4
29.50 ± 0.72 38.40 ± 2.46 35.90 ± 1.13 43.43 ± 2.75 34.57 ± 0.95
31.27 ± 1.46 41.06 ± 2.25 49.70 ± 1.90 50.43 ± 3.56 41.07 ± 1.86
5.96 ± 3.27a 7.00 ± 2.71a 38.51 ± 6.26c 16.11 ± 3.30b 18.83 ± 5.18b
Each measurement was performed in triplicate and the results are mean ± SD. Means followed by distinct letters are statically different (P < 0.05).
while they were hidden on the other treated specimens with irregular surfaces for group1 and 2 and flat surfaces for group3 and 4 (Fig. 4).
4. Discussion Our study clearly demonstrated the capacity of xylitol chewing gums enriched with propolis, remineralizing softly demineralized dentin by enhancing its mineral content, recovering its physical properties and occluding its dentinal tubules. During the process of chewing gum preparation, the percentages of remineralizing agents incorporated were based on the manufacturer preference and taste tests. In fact, as a sweetener, 1.8 % was the percentage of the usual incorporation of xylitol into sugar-free chewing gums. With regard to the propolis powder, several preliminary tests have been carried out in order to determine the percentage of adequate incorporation into chewing gum. Three percentages of incoporation of
3.2.3. Roughness Quantitative roughness measures evaluated with the AFM decreased in the treated side of the dentin compared to the untreated side for groups 1, 2, 3 and 4 respectively at a rate of 22.73, 36.33, 4.24 and 2.94 %. In the control group, roughness progression increased at a rate of 5.84 % between the sides. The mean dentin roughness of intact group was 119 nm. Dentin roughness and their progression for each group were displayed in Table 2.
Fig. 4. Morphology of the dentinal surfaces observed under an atomic force microscope. 5
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those enriched with CPP-ACP. The micro-hardness improvement of demineralized dentin after Propolis treatment suggests an intrafibrillar mineralization process. This data also suggests using this extract improving dentin bonding properties. It is noticeably that the microhardness improvement of demineralized dentin after CPP-ACP treatment evaluated in our study matches with data recorded in the literature (Khamverdi, Kordestani, & Soltanian, 2017) suggesting also an intrafibrillar remineralization. However, the morphological analysis described in our study showed not only an occlusion of tubules but also mineral precipitations in group1 and 2, suggesting rather an extrafibrillar remineralization for CPP-ACP. Concomitant intrafibrillar and interfibrillar remineralization processes are suggested here for CPPACP. Besides, smooth and homogeneous surfaces without mineral precipitates were observed for groups 3 and 4, suggesting a good interaction with the organic network of collagen for hydroxyapatite and propolis groups. Due to its biocompatibility and similarity to biologically formed hydroxyapatite in natural dentin, the efficiency of hydroxyapatite occluding open dentin tubules has been frequently confirmed (Baglar, Erdem, Dogan, & Turkoz, 2018; Enax & Epple, 2018). The effectiveness of chewing gum enriched with propolis occluding dentinal tubules was evidenced in our study using AFM approach and was matching what was described with SEM approaches using ethanolic extracts of propolis by Hongal, Torwane, Goel, & Chandrashekar (2014). This point suggests considering chewing gums enriched with propolis for the treatment of dentin hypersensitivity. Apart from the 2-D data, AFM provides also dimensional topographic information about a sample by probing its surface with a sharp tip. The vertical movements of the tip are recorded and used to construct a quantitative 3 dimensional topographic map. Roughness as evaluated considering the z-axial displacement of the tip, showed a decrease in the treated side of the dentin compared to the untreated one for all the groups except the control one. For xylitol chewing gum enriched with CPP-ACP, our results describing a roughness decrease matched with those described by Lechner, Röper, Messerschmidt, Blume, & Magerle (2015), using GC-Tooth Mousse after coca cola etching, but not with those of Poggio, Lombardini, Vigorelli, and Ceci (2013), describing an increasing roughness using the same products. Such controversies may be attributed as suggested by Marshall, Balooch, Tench, Kinney, and Marshall (1993) to the partial collapse of the collagen matrix under acid effect. In fact, during dentin demineralization, the preferential attack on the peritubular dentin is evidenced, while modifications occur at a slower rate on the collapsed intertubular dentin (Marshall et al., 1993). In our study, freshly extracted teeth were fixed in formol solution in order to preserve the organic matrix during the experiment and soft drink was used for demineralization instead of classical acids to simulate daily oral conditions. Concerning hydroxyapatite, our results were also in concordance with those published by Poggio, Lombardini, Vigorelli, Colombo, and Chiesa (2014), using Biorepair Plus-Total Protection, a toothpaste containing zinc hydroxyapatite. While progression of dentin roughness due to the action of chewing gum enriched with propolis was evidenced in our study, it was as low (2.94 %) as that of the group treated with hydroxyapatite (4.24 %). This point could be an advantage, considering that surface roughness of the dentin could lead to biofilm accumulation, increasing the risk of caries. This point suggests considering chewing-gums enriched with propolis preventing dental caries. Considering that dentin remineralization involves the stabilization of both organic and inorganic components, propolis seems to have an advantage over the other remineralizing agents that only bring ions for remineralization. In fact, we think that the remineralizing effect observed with propolis is not only attributed to the calcium phosphate precipitation derived from the artificial saliva solution as suggested by Toledano, Osorio, Cabello, and Osorio (2014) or from the propolis, but is also due to the organic matrix stabilization provided by this product. In fact, the calcium and phosphate amounts evaluated in the chewing gums of our study were weak (data non shown) and couldn’t be
Table 2 Dentin roughness (Rrms) and its progression (RP%). Groups
Rrms before treatment (nm)
Rrms after treatment (nm)
Roughness progression (RP%)
Control Group1 Group2 Group3 Group4
219.20 136.20 204.21 181.71 170.26
232.00 105.24 130.01 174.00 165.25
5.84 % −22.73% −36.33% −4.24% −2.94%
propolis powder in chewing gum were tried; 2.5 %, 5% and 7.5 %. At 2.5 %, the new taste of propolis was not detected while the incorporation at 7.5 % led a strong and pronounced taste of propolis that was not supported by tasters. For these reasons, the concentration of 5% was chosen for propolis. Chemically, our study demonstrated an increase of Calcium and Phosphate values on treated surfaces, suggesting the adsorption of these ions on the treated surfaces of demineralized dentin. The adsorption of calcium was supported by the decreased amount of this ion in the extract solution as explored every day (data not shown). However, the Ca/P ratio was different among the groups, suggesting the formation of different mineral compounds in xylitol, CPP-ACP and propolis groups compared to the apatite described in the control and hydroxyapatite groups. In fact, several calcium phosphate arrangements could occur during remineralisation leading to several products of variable formulas (Huang & Best, 2014). Our study showed a ratio of 1.5 for control group and group3, suggesting an apatite configuration of the precipitate, while the ratio was inferior for the other groups suggesting rather an octacalcium phosphate phase. Generally, the higher is the Ca/P ratio, the lower is the solubility of the precipitate. Though the remineralized product formed in our study could be more vulnerable in acidic environment. Besides, if the simple precipitation of minerals into the demineralized dentin matrix provides an increased mineral content, it may not necessarily provide an optimal interaction with the organic components of the dentin matrix. In fact, the organic matrix is mainly composed of collagen acting as a template for mineral deposition in the presence of non-collagenous proteins (NCP). Preserving the collagen structure is a key factor for intra and extrafibrillar remineralization. While the intrafibrillar mineralization occurs within the pores of collagen fibrils transforming there amorphous calcium phosphate to nanoparticles to crystalline apatites, the interfibrillar mineralization occurs in the interstitial spaces between the fibrils and involves larger size and more randomly oriented cristallites. It is the intrafibrillar remineralization that is determinant for the dentin’s physical properties (Zhong et al., 2015). So, the physical appreciation of the dentin remineralization seemed to be necessary and the exploration of our samples revealed that the quality of the remineralisation was not also equivalent between the groups. In fact, according to the results of this study, the mean baseline micro-hardness values of dentin samples ranged from 34.43 to 43.43 ± VHN (Table 1). These Vicker’s micro-hardness values were in accordance with those published in the literature (Fuentes, Toledano, Osorio, & Carvalho, 2003; Mollica, Torres, Gonçalves, & Mancini, 2012). However, and at our knowledge, this is the first study exploring the micro-hardness of eroded dentin following propolis treatment. While it is proved that propolis is able to enhance micro-hardness of enamel (Giamalia, Steinberg, Grobler, & Gedalia, 1999) or glass ionomere cement (Altunsoy, Tanrıver, Türkan, Uslu, & Silici, 2016), nothing is mentioned in the literature concerning its action on dentin. Besides, our study showed an improvement of the micro-hardness of eroded dentin treated with xylitol chewing gum enriched with propolis that was more important than those observed with xylitol chewing gums alone or those enriched with hydroxyapatite, but less important than 6
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involved alone to explain the remineralizing process. In the same perspective, previous studies have proved that combining an organic stabilizer as green tea with CPP-ACP for example enhances the remineralizing properties of the product (Jose, Sanjeev, & Sekar, 2016) while green tea alone is involved in the tooth protection against dental erosion (Jaâfoura et al., 2014). The efficiency of propolis combined with a remineralizing agent providing calcium and phosphate such as CPPACP should be further explored for dentin remineralization.
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Critical Reviews in Oral Biology and Medicine: An Official Publication of the American Association of Oral Biologists, 14(1), 13–29. Lechner, B.-D., Röper, S., Messerschmidt, J., Blume, A., & Magerle, R. (2015). Monitoring demineralization and subsequent remineralization of human teeth at the DentinEnamel Junction with atomic force microscopy. ACS Applied Materials & Interfaces, 7(34), 18937–18943. https://doi.org/10.1021/acsami.5b04790. Marshall, G. W., Balooch, M., Tench, R. J., Kinney, J. H., & Marshall, S. J. (1993). Atomic force microscopy of acid effects on dentin. Dental Materials, 9(4), 265–268. https:// doi.org/10.1016/0109-5641(93)90072-X. Meimandi-Parizi, A., Oryan, A., Sayahi, E., & Bigham-Sadegh, A. (2018). Propolis extract a new reinforcement material in improving bone healing: An in vivo study. International Journal of Surgery, 56, 94–101. https://doi.org/10.1016/j.ijsu.2018.06. 006. Miake, Y., Saeki, Y., Takahashi, M., & Yanagisawa, T. (2003). Remineralization effects of xylitol on demineralized enamel. Journal of Electron Microscopy, 52(5), 471–476. Mollica, F. B., Torres, C. R. G., Gonçalves, S. E. P., & Mancini, M. N. G. (2012). Dentine microhardness after different methods for detection and removal of carious dentine tissue. Journal of Applied Oral Science, 20(4), 449–454. https://doi.org/10.1590/ S1678-77572012000400010. Niu, L., Zhang, W., Pashley, D. H., Breschi, L., Mao, J., Chen, J., et al. (2014). Biomimetic remineralization of dentin. Dental Materials: Official Publication of the Academy of Dental Materials, 30(1), https://doi.org/10.1016/j.dental.2013.07.013. Poggio, C., Lombardini, M., Vigorelli, P., & Ceci, M. (2013). Analysis of dentin/enamel remineralization by a CPP-ACP paste: AFM and SEM study. Scanning, 35(6), 366–374. https://doi.org/10.1002/sca.21077. Poggio, C., Lombardini, M., Vigorelli, P., Colombo, M., & Chiesa, M. (2014). The role of different toothpastes on preventing dentin erosion: An SEM and AFM study®. Scanning, 36(3), 301–310. https://doi.org/10.1002/sca.21105. Toledano, M., Osorio, E., Cabello, I., & Osorio, R. (2014). Early dentine remineralisation: Morpho-mechanical assessment. Journal of Dentistry, 42(4), 384–394. https://doi. org/10.1016/j.jdent.2014.01.012. Wiegand, A., & Attin, T. (2014). Randomised in situ trial on the effect of milk and CPPACP on dental erosion. Journal of Dentistry, 42(9), 1210–1215. https://doi.org/10. 1016/j.jdent.2014.07.009. Yuan, X., Wang, Y., Shi, B., & Zhao, Y. (2018). Effect of propolis on preserving human periodontal ligament cells and regulating pro-inflammatory cytokines. Dental Traumatology, 34(4), 245–253. https://doi.org/10.1111/edt.12411. Zhong, B., Peng, C., Wang, G., Tian, L., Cai, Q., & Cui, F. (2015). Contemporary research findings on dentine remineralization. Journal of Tissue Engineering and Regenerative Medicine, 9(9), 1004–1016. https://doi.org/10.1002/term.1814.
5. Conclusion Our study evidenced that xylitol chewing gum enriched with propolis is able to enhance the biomineralization of demineralized dentin in an intrafibrillar manner, to form a mineral compound different to hydroxyapatite closing opened tubules. These points suggest considering chewing gums enriched with propolis as a dentin conditioner improving dentin bonding biomaterials and as a remineralizing agent occluding dentinal tubules for the treatment of dentin hypersensitivity. Other studies are needed to clarify the mechanism of action of propolis during dentin remineralization exploring the specific propolis role either preserving dentin collagen, acting as NCP analogues or inhibiting matrix enzymes. CRediT authorship contribution statement Wafa Gargouri: Conceptualization, Data curation, Formal analysis, Investigation, Software, Writing - review & editing. Rym Kammoun: Data curation, Formal analysis, Investigation, Software, Writing - review & editing. Mazen Elleuche: Funding acquisition, Resources. Mahdi Tlili: Investigation. Nabil Kechaou: Conceptualization, Project administration, Validation. Sonia Ghoul-Mazgar: Conceptualization, Formal analysis, Methodology, Project administration, Supervision, Validation, Writing - review & editing. Declaration of Competing Interest Wafa Gargouri benefited from a scholarship granted by the confectioner TRIKI-Le Moulin (Sfax, Tunisia). Mazen Elleuche works at Triki le Moulin. Acknowledgment The authors are grateful to the Food Technology laboratory in the University of Burgos (Spain), for their help in preparing propolis powder incorporated in the chewing gum. Pr Tarek Bouraoui from the National Engineering School of Monastir is thanked for his help with the microhardness analysis. References Altunsoy, M., Tanrıver, M., Türkan, U., Uslu, M. E., & Silici, S. (2016). In vitro evaluation of microleakage and microhardness of ethanolic extracts of propolis in different proportions added to glass ionomer cement. The Journal of Clinical Pediatric Dentistry, 40(2), 136–140. https://doi.org/10.17796/1053-4628-40.2.136. Baglar, S., Erdem, U., Dogan, M., & Turkoz, M. (2018). Dentinal tubule occluding capability of nano-hydroxyapatite; the in-vitro evaluation. Microscopy Research and Technique, 81(8), 843–854. https://doi.org/10.1002/jemt.23046. Cardoso, J. G., Iorio, N. L. P., Rodrigues, L. F., Couri, M. L. B., Farah, A., Maia, L. C., et al. (2016). Influence of a Brazilian wild green propolis on the enamel mineral loss and Streptococcus mutans’ count in dental biofilm. Archives of Oral Biology, 65, 77–81. https://doi.org/10.1016/j.archoralbio.2016.02.001. Chen, C. L., Parolia, A., Pau, A., & Celerino de Moraes Porto, I. C. (2015). Comparative evaluation of the effectiveness of desensitizing agents in dentine tubule occlusion using scanning electron microscopy. Australian Dental Journal, 60(1), 65–72. https:// doi.org/10.1111/adj.12275. Enax, J., & Epple, M. (2018). Synthetic hydroxyapatite as a biomimetic oral care agent.
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Effect of xylitol chewing gum enriched with propolis on dentin remineralization in vitro
T
Wafa Gargouria,*, Rym Kammounb, Mazen Elleuchec, Mahdi Tlilib, Nabil Kechaoua, Sonia Ghoul-Mazgarb a
Research Group of Agri-Food Processing Engineering, National School of Engineers of Sfax, University of Sfax, Soukra road, BP 1173, 3038, Sfax, Tunisia Laboratory of Dento-Facial, Clinical and Biological Approach (ABCDF), Faculty of Dental Medicine, University of Monastir, Tunisia c La Confiserie Triki le Moulin, Sfax, Tunisia b
ARTICLE INFO
ABSTRACT
Keywords: Remineralization Propolis Dentin Chewing gum Hydroxyapatites Xylitol
Objectives: The aim of this study was to evaluate the efficiency of xylitol chewing gums enriched with propolis, remineralizing softly demineralized dentin in vitro. Design: Four groups of chewing gum were developed; Group1: xylitol (1.8 %), Group2: xylitol + casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) (3%), Group3: xylitol+Hydroxyapatite (3%) and Group4: xylitol + propolis (5%). A control group was designed without chewing gum, but with artificial saliva. Sections of embedded crowns and cleaned roots of twenty five bovine incisors were demineralized in carbonated drink. Crown specimens were half-varnished. Remineralization process was run for all the dental specimens in the 4 groups with gum extracts and in the control group with artificial saliva for 20 min at 37 °C three times a day during 7 days. Mineral contents were evaluated by scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX-SEM). Surface morphology and roughness were analyzed using atomic force microscopy (AFM). Micro-hardness was measured using Vickers micro-hardness tester among varnished and unvarnished sides. Results: Calcium/Phosphate mean ratio showed a significant decrease between the control group, group1, group2 and group4. Control group and group3 were not significantly different. Micro-hardness increased significantly for all treated groups. AFM showed obstruction of dentinal tubules in all the groups and roughness decreased in the treated side of the dentin compared to the untreated side for tested groups. Conclusion: Xylitol chewing gum enriched with propolis showed dentinal tubules occlusion, significant improvement of micro-hardness and slight decrease in roughness. Ca/P ratio analysis suggests that a mineral compound other than hydroxyapatite is responsible of tubules occlusion.
1. Introduction Remineralization of demineralized dentin is one of the target challenges in dentistry, intending not only to repair eroded dentin, but improving also the dentin bonding stability and treating dentin hypersensitivity (Niu et al., 2014). Dentin forms the bulkiest portion of dental hard tissues and is mainly constituted of tubules. These tubules are internally lined by a highly mineralized and non-collagenous intratubular dentin and externally surrounded by an intertubular dentin, consisting of a collagen matrix, non-collagenous proteins and hydroxyapatite crystals (Kinney, Marshall, & Marshall, 2003). Dentin demineralization under acidic challenges usually results in the dissolution of the peritubular dentin, which is detectable by the enlargement of the
⁎
tubule lumen, while the morphology of the intertubular dentin is preserved thanks to the persistence of the organic phase. Due to these structural particularities and changes, remineralization of demineralized dentin is a complex phenomenon. In fact, the remineralizing processes of demineralized dentin is supported by different strategies. The traditional ion-based strategy targets on the deposition of calcium and phosphate ions on the demineralized tissue. Among these agents, casein phosphopeptide-amorphouse calcium phosphate (CPP-ACP) is proposed to enhance remineralization and to reduce solubility of hydroxyapatite. Thereby, CPP-ACPcontaining products such as toothpaste, mouth rinse solutions and also chewing gum have been widely used (Wiegand & Attin, 2014). Otherwise, synthetic hydroxyapatite, that is similar to the biologically formed
Corresponding author. E-mail address: [email protected] (W. Gargouri).
https://doi.org/10.1016/j.archoralbio.2020.104684 Received 5 October 2019; Received in revised form 8 January 2020; Accepted 17 February 2020 0003-9969/ © 2020 Published by Elsevier Ltd.
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apatite in natural dentin, has also been suggested to be a promising biomimetic oral care ingredient. While hydroxyapatite has proved efficiency occluding open dentin tubules in clinical studies, in situ studies are still needed to further clarify the biomineralization process (Enax & Epple, 2018). Besides, considering that the current trend in the field of oral health stresses on the use of natural products for treating diseases, rather than allopathic medicine, new products emerged in the field of the remineralization of demineralized dentin. In this context, xylitol that is a natural herbal product that does not bring neither calcium nor phosphate and is frequently used in dental products for its antimicrobial properties, has been recently shown to have a remineralizing potential in vitro on eroded teeth by facilitating calcium movement and accessibility into the lesion pores (Gargouri, Zmantar, Kammoun, Kechaou, & Ghoul-Mazgar, 2018; Miake, Saeki, Takahashi, & Yanagisawa, 2003). In this context, the calcium and phosphate present in the saliva were sufficient to ensure the remineralisation. In another hand, Propolis, another natural product produced by bees, seems to present also a scientific importance in oral health regarding its bioactive properties (Gargouri, Osés, Fernández-Muiño, Sancho, & Kechaou, 2019). Several studies showed particularly the effect of propolis preserving connective elements in periodontium (Yuan, Wang, Shi, & Zhao, 2018) or enhancing healing, regeneration and biomineralization in bone (Meimandi-Parizi, Oryan, Sayahi, & BighamSadegh, 2018). However, there are scarcity and controversies in the literature regarding the dental remineralizing potential of propolis. In fact, while Chen and collaborators demonstrated occlusion of dentinal tubules using red propolis extracts (Chen, Parolia, Pau, & Celerino de Moraes Porto, 2015), Cardoso and collaborators suggested that the propolis ethanolic extract has no inhibitory action on the demineralization process of caries (Cardoso et al., 2016). Noticeably, dentin remineralization of demineralized dentin could not take place if crystallites are completely dissolved, or if the organic phase is collapsed hiding thus the damaged apatites (Niu et al., 2014). So, to backfill demineralized dentin, it is also important to preserve a slight amount of apatites in dentin and to act on a slightly and not a totally demineralized tissue managing the dentin connective phase. The connective phase of dentin contains particularly both collagen and noncollagenous proteins playing important roles in remineralization. In fact, while the collagen fibers constitute a basic template for mineral nucleation, the non-collagenous proteins or their biomimetic analogs seem to be essential for the non-classical crystallization pathway (Niu et al., 2014). Therefore, the aim of this study was to evaluate the efficiency of xylitol chewing gums enriched with propolis, remineralizing softly demineralized dentin in vitro, compared to xylitol chewing gums enriched in CPP-ACP and hydroxyapatite as conventional remineralizing agents bringing calcium and phosphate. The chemical, physical and morphological changes were investigated on dentin specimens after a demineralizing challenge.
propolis, reduced into powder was introduced to the gum base in a concentration of 5% during the manufacturing process. In fact, propolis powder was incorporated in chewing gum due to the high amount of phenolic compounds and the strong potential of biological activities, recently reported for Tunisian propolis from Béja region (Gargouri et al., 2019). It has been reduced into powder using spray drying technique B-290 (BÜCHI Labortechnik AG, Flawil, Suisse), in order to represent a promising ingredient in food industries and in chewing gum in our case. 2.2. Dental samples preparation A total of 25 fresh bovine incisors free of caries and defects were collected for this study. Teeth were disinfected in 5% sodium hypochlorite for two hours, then the crowns were separated from the roots using a diamond disc mounted on low speed headpiece and all the specimens were stored in formol solution at 4 °C until required. 2.2.1. Crown treatment Crowns were embedded in acrylic resin (BMS 017 Trayplast kit. Methyl Methacrylate co-polymer) and placed into Teflon moulds measuring 10 * 10 mm. Embedded blocks were cut under water irrigation using a low-speed diamond saw (Isomet, Buehler, Lake Bluff, ILL, USA) to expose a flat dentin surface. The surfaces were then polished using 200, 400 and 600 grit silicon carbide papers (Buehler, Lake Bluff, IL) to obtain a flat and highly polished resin block measuring 10 mm * 10 mm * 2 mm with the dentin surface exposed. Samples were randomly divided into five groups of five crowns each, a control group CTRL without using any chewing gum extract (artificial saliva), Group1 (xylitol chewing gum without remineralizing agents), Group2 (xylitol chewing gum with CPP-ACP), Group3 (xylitol chewing gum with hydroxyapatite) and Group4 (xylitol chewing gum with propolis). 2.2.2. Root treatment Dental roots were cleaned using scalers, then cementum was removed using a cylindrical-shaped bur and silicon carbide paper (600, 800 and 1000). Dental root slides of 2 mm thick were cut using a diamond disc mounted on low speed headpiece and stored in dei-onized water until use. Dental root slides were randomly distributed among the 5 groups as described previously with a total mean weight of 2.3 g for each group. 2.3. Demineralization challenge Crown specimens and roots of all groups were immersed in 10 ml of a fresh carbonated drink (Coca-Cola, pH = 2.7, Tunisia) for 5 min under continuous agitation at room temperature before rinsing with distilled water. Four consecutive erosive cycles were carried out. 2.4. Remineralization process Concerning the crown specimens, a layer of acid-resistant nail varnish was applied on the half of the exposed dentin in order to create an unexposed area (eroded side) and an exposed one (treated side). For the gum extracts, three pieces of each chewing-gum group were grinded in a mortar with 30 ml of artificial saliva (1.5 mM CaCl2, 1.0 mM KH2PO4 and 50 mM NaCl, pH 7.0) (Esteves-Oliveira et al., 2011), during 5 min, then the final solution was collected in a clean container for the remineralization process. The pH of the final solutions was recorded as follow; Group1, pH = 6.85 ± 0.34; Group2, pH = 6.79 ± 0.39; Group3, pH = 6.76 ± 0.17; Group4, pH = 7.15 ± 0.16. Remineralization was conducted by immersing each group of acid-eroded dentin specimen in 10 ml of the gum extract for 20 min at 37 °C three times a day (10 am, 1 pm and 3 pm) during 7 days. In-between the treatments, specimens were stored in freshly prepared artificial saliva. Varnish was removed from crown samples before following analysis. For root
2. Material and methods 2.1. Sugar-free chewing gum preparation Four groups of chewing gum were developed in the research and development laboratory of a Tunisian confectioner, TRIKI-Le Moulin, Sfax; Group1: xylitol (1.8 %), Group2: xylitol + CPP-ACP (3%), Group3: xylitol + hydroxyapatite (3%) and Group4: xylitol + propolis (5%). For the manufacturing process, gum base was melted in a mixer under heating then flavorings, sweeteners, colorants, humectants, and emulsifying agents were added as described previously (Gargouri et al., 2018). The remineralizing agents CPP-ACP and hydroxyapatite were purchased and directly added in the preparation process of chewing gums of groups 2 and 3, respectively. For the group4, Tunisian brown 2
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Fig. 1. Schematic representation of experimental study design.
specimens, remineralization process was directly performed without applying acid-resistant nail varnish. After finishing treatments, all the specimens were kept in de-ionized water (pH = 7,38) until analysis.
RP % =
2.5. EDX/SEM analysis
2.7. Surface micro-hardness
Chemical elements were evaluated on root slides using energy dispersive X-ray microanalysis system (EDX) coupled to a ThermoScientific™ Q250 scanning electron microscope (SEM) under ×60 magnification at 10 Kv (Fig. 1). Eighteen point measurements were checked on the surface of each slide. Analyses of the elements were assessed using the Pathfinder Software (ThermoScientific™ Q250). The elements quantified were Ca and P net counts and the Ca/P stoichiometric ratios were evaluated using the following formula: Ca (mol)/P (mol)% = [Ca (weight%)/40.08 (g/mol)]/ [P (weight %)/30.97 (g/mol)], considering that the molecular masses of Ca and P being 40.08 g and 30.97 g respectively.
The Vickers hardness number (VHN) was measured on the crown specimens eroded and treated with chewing gum using a Micro Hardness Tester [HMV 2 T] applying a load of 2 N for 15 s. Three indentations 100 μm apart were made on the eroded as well as on the treated sides of each dentin surface of the crown specimens. The Vickers Micro hardness mean of the dentin specimens were evaluated among three specimens of each group. Mean and standard deviation of the hardness values were statistically analyzed with ANOVA followed by Tukey’s HSD test. The hardness progression (VHN%) was calculated by the following formula:
VHN % =
2.6. Atomic force microscopy (AFM) To analyze the surface morphology and roughness of crown specimens, a non-contact mode atomic force microscopy (AFM, XE-70, Park System) was used (Fig. 1). AFM was equipped with a PPP-NCHR cantilever at 249 kHz resonant frequency and images were analyzed using XEI Software (Park Systems, Korea); All images contained 256 × 256 data points. The surface roughness (Rrms) of the analyzed surface was also measured by this microscope and the roughness progression (RP%) was calculated by the following formula:
Rafter treatment
Rbefore treatment
Rbefore treatment
VHNafter treatment
× 100
VHNbefore treatment
VHNbefore treatment
× 100
2.8. Statistical analysis Data were analysed using the Statistical Package for Social Science (SPSS, version 18.0, SPSS, Chicago, IL, USA) through the one-way ANOVA, Means-Test-Tukey’s and the Student’s t-test The level of significance was defined as 0.05. All results were reproduced in at least three independent experiments. 3
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Fig. 2. Calcium, phosphate and calcium/phosphate ratio evaluation on dentinal surfaces of the roots and expressed in weight percentages. Data are mean ± standard deviation (SD) of at least three independent experiments. Asteriks denote significant differences (P < 0.05).
3. Results
3.2. Physical and morphological analysis of the crown dentinal surfaces
3.1. Chemical analysis of calcium and phosphate values on the root dentinal surfaces
3.2.1. Surface micro-hardness The surface micro-hardness of the intact dentin was recorded as 57.13 ± 0.71. The VHN analysis revealed a significant increase of dentin micro-hardness of each surface for each group, compared to control group (Fig. 3). The mean values of micro-hardness were 38.4 ± 2.46, 35.90 ± 1.13, 43.43 ± 2.75 and 34.57 ± 0.95 for the eroded sides respectively in groups 1, 2, 3 and 4. The mean values of micro-hardness were 41.06 ± 2.25, 49.7 ± 1.90, 50.43 ± 3.56 and 41.07 ± 1.86, for the treated side respectively in groups 1, 2, 3 and 4. The micro-hardness was significantly higher in the surfaces treated with chewing-gum extracts compared to the untreated surfaces of each group, except for the control group (P < 0.05). The improvement of micro-hardness, comparing the treated and eroded sides of each group, was evaluated at 5.96 %, 7%, 38.51 %, 16.18 % and 18.83 % respectively for the control group and groups 1, 2, 3 and 4. Mean values and standard deviations for each group were displayed in Table 1.
Calcium and phosphate presence was proved in all samples (Fig. 2). Calcium weight percentage quantified at the surface of the slides showed an increased level in the treated groups with a significant difference (P < 0.05) evaluated by Student’s t-test between the control group (24.39 % ± 0.02) and the groups2 (25.74 % ± 0.06) and 3 (29.88 % ± 0.33). A significant decreased level was noticed between the control group (24.39 % ± 0.02) and the group1 (24.40 % ± 0.01) and group4 (24.18 % ± 0.02). Phosphate weight percentage quantified at the surface of the slides and evaluated by Student’s t-test showed an increased level in the treated groups with a significant difference (P < 0.05) between the control group (12.61 % ± 0.05) and the groups1 (12.98 % ± 0.06), 2 (13.57 % ± 0.04), 3 (14.77 % ± 0.09) and 4 (12.86 % ± 0.02). The Calcium/Phosphate ratio (Ca/P mean ratio), evaluated for the weight percentage in each group showed a significant difference (P < 0.05) between the control group (1.54 ± 0.05), group1 (1.44 ± 0.04), group2 (1.46 ± 0.02) or group4 (1.45 ± 0.03). The control group and group3 (1.50 ± 0.04) were not significantly different (P > 0.05).
3.2.2. Morphology Qualitative analysis of the three dimensional topographic maps obtained with the AFM revealed dentinal tubules cut either transversely or longitudinally on the eroded sides of the specimens. On the treated sides, the tubule sections were evidenced only in the control group,
Fig. 3. Micro-hardness mean on dentinal surfaces of the crowns. Data are mean ± SD of at least three independent experiments. Asterisks denote significant differences (P < 0.05).
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Table 1 Micro-hardness improvement (VHN%) among the sides. Groups
VHN before treatment (HV)
VHN after treatment (HV)
VHN%
Control Group1 Group2 Group3 Group4
29.50 ± 0.72 38.40 ± 2.46 35.90 ± 1.13 43.43 ± 2.75 34.57 ± 0.95
31.27 ± 1.46 41.06 ± 2.25 49.70 ± 1.90 50.43 ± 3.56 41.07 ± 1.86
5.96 ± 3.27a 7.00 ± 2.71a 38.51 ± 6.26c 16.11 ± 3.30b 18.83 ± 5.18b
Each measurement was performed in triplicate and the results are mean ± SD. Means followed by distinct letters are statically different (P < 0.05).
while they were hidden on the other treated specimens with irregular surfaces for group1 and 2 and flat surfaces for group3 and 4 (Fig. 4).
4. Discussion Our study clearly demonstrated the capacity of xylitol chewing gums enriched with propolis, remineralizing softly demineralized dentin by enhancing its mineral content, recovering its physical properties and occluding its dentinal tubules. During the process of chewing gum preparation, the percentages of remineralizing agents incorporated were based on the manufacturer preference and taste tests. In fact, as a sweetener, 1.8 % was the percentage of the usual incorporation of xylitol into sugar-free chewing gums. With regard to the propolis powder, several preliminary tests have been carried out in order to determine the percentage of adequate incorporation into chewing gum. Three percentages of incoporation of
3.2.3. Roughness Quantitative roughness measures evaluated with the AFM decreased in the treated side of the dentin compared to the untreated side for groups 1, 2, 3 and 4 respectively at a rate of 22.73, 36.33, 4.24 and 2.94 %. In the control group, roughness progression increased at a rate of 5.84 % between the sides. The mean dentin roughness of intact group was 119 nm. Dentin roughness and their progression for each group were displayed in Table 2.
Fig. 4. Morphology of the dentinal surfaces observed under an atomic force microscope. 5
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those enriched with CPP-ACP. The micro-hardness improvement of demineralized dentin after Propolis treatment suggests an intrafibrillar mineralization process. This data also suggests using this extract improving dentin bonding properties. It is noticeably that the microhardness improvement of demineralized dentin after CPP-ACP treatment evaluated in our study matches with data recorded in the literature (Khamverdi, Kordestani, & Soltanian, 2017) suggesting also an intrafibrillar remineralization. However, the morphological analysis described in our study showed not only an occlusion of tubules but also mineral precipitations in group1 and 2, suggesting rather an extrafibrillar remineralization for CPP-ACP. Concomitant intrafibrillar and interfibrillar remineralization processes are suggested here for CPPACP. Besides, smooth and homogeneous surfaces without mineral precipitates were observed for groups 3 and 4, suggesting a good interaction with the organic network of collagen for hydroxyapatite and propolis groups. Due to its biocompatibility and similarity to biologically formed hydroxyapatite in natural dentin, the efficiency of hydroxyapatite occluding open dentin tubules has been frequently confirmed (Baglar, Erdem, Dogan, & Turkoz, 2018; Enax & Epple, 2018). The effectiveness of chewing gum enriched with propolis occluding dentinal tubules was evidenced in our study using AFM approach and was matching what was described with SEM approaches using ethanolic extracts of propolis by Hongal, Torwane, Goel, & Chandrashekar (2014). This point suggests considering chewing gums enriched with propolis for the treatment of dentin hypersensitivity. Apart from the 2-D data, AFM provides also dimensional topographic information about a sample by probing its surface with a sharp tip. The vertical movements of the tip are recorded and used to construct a quantitative 3 dimensional topographic map. Roughness as evaluated considering the z-axial displacement of the tip, showed a decrease in the treated side of the dentin compared to the untreated one for all the groups except the control one. For xylitol chewing gum enriched with CPP-ACP, our results describing a roughness decrease matched with those described by Lechner, Röper, Messerschmidt, Blume, & Magerle (2015), using GC-Tooth Mousse after coca cola etching, but not with those of Poggio, Lombardini, Vigorelli, and Ceci (2013), describing an increasing roughness using the same products. Such controversies may be attributed as suggested by Marshall, Balooch, Tench, Kinney, and Marshall (1993) to the partial collapse of the collagen matrix under acid effect. In fact, during dentin demineralization, the preferential attack on the peritubular dentin is evidenced, while modifications occur at a slower rate on the collapsed intertubular dentin (Marshall et al., 1993). In our study, freshly extracted teeth were fixed in formol solution in order to preserve the organic matrix during the experiment and soft drink was used for demineralization instead of classical acids to simulate daily oral conditions. Concerning hydroxyapatite, our results were also in concordance with those published by Poggio, Lombardini, Vigorelli, Colombo, and Chiesa (2014), using Biorepair Plus-Total Protection, a toothpaste containing zinc hydroxyapatite. While progression of dentin roughness due to the action of chewing gum enriched with propolis was evidenced in our study, it was as low (2.94 %) as that of the group treated with hydroxyapatite (4.24 %). This point could be an advantage, considering that surface roughness of the dentin could lead to biofilm accumulation, increasing the risk of caries. This point suggests considering chewing-gums enriched with propolis preventing dental caries. Considering that dentin remineralization involves the stabilization of both organic and inorganic components, propolis seems to have an advantage over the other remineralizing agents that only bring ions for remineralization. In fact, we think that the remineralizing effect observed with propolis is not only attributed to the calcium phosphate precipitation derived from the artificial saliva solution as suggested by Toledano, Osorio, Cabello, and Osorio (2014) or from the propolis, but is also due to the organic matrix stabilization provided by this product. In fact, the calcium and phosphate amounts evaluated in the chewing gums of our study were weak (data non shown) and couldn’t be
Table 2 Dentin roughness (Rrms) and its progression (RP%). Groups
Rrms before treatment (nm)
Rrms after treatment (nm)
Roughness progression (RP%)
Control Group1 Group2 Group3 Group4
219.20 136.20 204.21 181.71 170.26
232.00 105.24 130.01 174.00 165.25
5.84 % −22.73% −36.33% −4.24% −2.94%
propolis powder in chewing gum were tried; 2.5 %, 5% and 7.5 %. At 2.5 %, the new taste of propolis was not detected while the incorporation at 7.5 % led a strong and pronounced taste of propolis that was not supported by tasters. For these reasons, the concentration of 5% was chosen for propolis. Chemically, our study demonstrated an increase of Calcium and Phosphate values on treated surfaces, suggesting the adsorption of these ions on the treated surfaces of demineralized dentin. The adsorption of calcium was supported by the decreased amount of this ion in the extract solution as explored every day (data not shown). However, the Ca/P ratio was different among the groups, suggesting the formation of different mineral compounds in xylitol, CPP-ACP and propolis groups compared to the apatite described in the control and hydroxyapatite groups. In fact, several calcium phosphate arrangements could occur during remineralisation leading to several products of variable formulas (Huang & Best, 2014). Our study showed a ratio of 1.5 for control group and group3, suggesting an apatite configuration of the precipitate, while the ratio was inferior for the other groups suggesting rather an octacalcium phosphate phase. Generally, the higher is the Ca/P ratio, the lower is the solubility of the precipitate. Though the remineralized product formed in our study could be more vulnerable in acidic environment. Besides, if the simple precipitation of minerals into the demineralized dentin matrix provides an increased mineral content, it may not necessarily provide an optimal interaction with the organic components of the dentin matrix. In fact, the organic matrix is mainly composed of collagen acting as a template for mineral deposition in the presence of non-collagenous proteins (NCP). Preserving the collagen structure is a key factor for intra and extrafibrillar remineralization. While the intrafibrillar mineralization occurs within the pores of collagen fibrils transforming there amorphous calcium phosphate to nanoparticles to crystalline apatites, the interfibrillar mineralization occurs in the interstitial spaces between the fibrils and involves larger size and more randomly oriented cristallites. It is the intrafibrillar remineralization that is determinant for the dentin’s physical properties (Zhong et al., 2015). So, the physical appreciation of the dentin remineralization seemed to be necessary and the exploration of our samples revealed that the quality of the remineralisation was not also equivalent between the groups. In fact, according to the results of this study, the mean baseline micro-hardness values of dentin samples ranged from 34.43 to 43.43 ± VHN (Table 1). These Vicker’s micro-hardness values were in accordance with those published in the literature (Fuentes, Toledano, Osorio, & Carvalho, 2003; Mollica, Torres, Gonçalves, & Mancini, 2012). However, and at our knowledge, this is the first study exploring the micro-hardness of eroded dentin following propolis treatment. While it is proved that propolis is able to enhance micro-hardness of enamel (Giamalia, Steinberg, Grobler, & Gedalia, 1999) or glass ionomere cement (Altunsoy, Tanrıver, Türkan, Uslu, & Silici, 2016), nothing is mentioned in the literature concerning its action on dentin. Besides, our study showed an improvement of the micro-hardness of eroded dentin treated with xylitol chewing gum enriched with propolis that was more important than those observed with xylitol chewing gums alone or those enriched with hydroxyapatite, but less important than 6
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involved alone to explain the remineralizing process. In the same perspective, previous studies have proved that combining an organic stabilizer as green tea with CPP-ACP for example enhances the remineralizing properties of the product (Jose, Sanjeev, & Sekar, 2016) while green tea alone is involved in the tooth protection against dental erosion (Jaâfoura et al., 2014). The efficiency of propolis combined with a remineralizing agent providing calcium and phosphate such as CPPACP should be further explored for dentin remineralization.
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5. Conclusion Our study evidenced that xylitol chewing gum enriched with propolis is able to enhance the biomineralization of demineralized dentin in an intrafibrillar manner, to form a mineral compound different to hydroxyapatite closing opened tubules. These points suggest considering chewing gums enriched with propolis as a dentin conditioner improving dentin bonding biomaterials and as a remineralizing agent occluding dentinal tubules for the treatment of dentin hypersensitivity. Other studies are needed to clarify the mechanism of action of propolis during dentin remineralization exploring the specific propolis role either preserving dentin collagen, acting as NCP analogues or inhibiting matrix enzymes. CRediT authorship contribution statement Wafa Gargouri: Conceptualization, Data curation, Formal analysis, Investigation, Software, Writing - review & editing. Rym Kammoun: Data curation, Formal analysis, Investigation, Software, Writing - review & editing. Mazen Elleuche: Funding acquisition, Resources. Mahdi Tlili: Investigation. Nabil Kechaou: Conceptualization, Project administration, Validation. Sonia Ghoul-Mazgar: Conceptualization, Formal analysis, Methodology, Project administration, Supervision, Validation, Writing - review & editing. Declaration of Competing Interest Wafa Gargouri benefited from a scholarship granted by the confectioner TRIKI-Le Moulin (Sfax, Tunisia). Mazen Elleuche works at Triki le Moulin. Acknowledgment The authors are grateful to the Food Technology laboratory in the University of Burgos (Spain), for their help in preparing propolis powder incorporated in the chewing gum. Pr Tarek Bouraoui from the National Engineering School of Monastir is thanked for his help with the microhardness analysis. References Altunsoy, M., Tanrıver, M., Türkan, U., Uslu, M. E., & Silici, S. (2016). In vitro evaluation of microleakage and microhardness of ethanolic extracts of propolis in different proportions added to glass ionomer cement. The Journal of Clinical Pediatric Dentistry, 40(2), 136–140. https://doi.org/10.17796/1053-4628-40.2.136. Baglar, S., Erdem, U., Dogan, M., & Turkoz, M. (2018). Dentinal tubule occluding capability of nano-hydroxyapatite; the in-vitro evaluation. Microscopy Research and Technique, 81(8), 843–854. https://doi.org/10.1002/jemt.23046. Cardoso, J. G., Iorio, N. L. P., Rodrigues, L. F., Couri, M. L. B., Farah, A., Maia, L. C., et al. (2016). Influence of a Brazilian wild green propolis on the enamel mineral loss and Streptococcus mutans’ count in dental biofilm. Archives of Oral Biology, 65, 77–81. https://doi.org/10.1016/j.archoralbio.2016.02.001. Chen, C. L., Parolia, A., Pau, A., & Celerino de Moraes Porto, I. C. (2015). Comparative evaluation of the effectiveness of desensitizing agents in dentine tubule occlusion using scanning electron microscopy. Australian Dental Journal, 60(1), 65–72. https:// doi.org/10.1111/adj.12275. Enax, J., & Epple, M. (2018). Synthetic hydroxyapatite as a biomimetic oral care agent.
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