Graphical abstract
Highlights
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Phenolic compounds from cashew nut shell liquid were investigated regarding the interaction with dentin collagen.
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Aroeira bark extract was also, for the first time, investigation on dentin biomodification.
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Major components of CNSL provide optimal collagen crosslinking significantly higher than PACs of grape-seed extract.
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Unlike aroeira and grape-seed extracts, treatment with CNSL does not stain dentin collagen in dark brown.
Abstract
Objectives
Several polyphenols from renewable sources were surveyed for dentin biomodification. However, phenols from cashew nut shell liquid (CNSL, Anacardium occidentale) and from Aroeira ( Myracrodruon urundeuva ) extract have never been evaluated. The present investigation aimed to compare the dentin collagen crosslinking (biomodification) effectiveness of polyphenols from Aroeira stem bark extract, proanthocyanidins (PACs) from grape-seed extract ( Vitis vinifera ), cardol and cardanol from CNSL after clinically relevant treatment for one minute.
Methods
Three-point bending test was used to obtain the elastic modulus of fully demineralized dentin beams before and after biomodification, whilst color change and mass variation were evaluated after four weeks water biodegradation. Color aspect was assessed by optical images after biodegradation whereas collagen cross-linking was investigated by micro-Raman spectroscopy. Statistical analysis was performed with repeated-measures two way ANOVA and Tukey’s test (p < 0.05).
Results
The increase in elastic modulus after biomodification was in the order cardol > cardanol > aroeira = PACs with cardol solution achieving mean 338.2% increase. The mass increase after biomodification followed the same order aforementioned. Nevertheless, after four weeks aging, more hydrophobic agent (cardanol) induced the highest resistance against water biodegradation. Aroeira and cardol attained intermediate outcomes whereas PACs provided the lower resistance. Tannin-based agents (Aroeira and PACs) stained the specimens in dark brown color. No color alteration was observed with cardol and cardanol treatments. All four agents achieved crosslinking in micro-Raman after one minute application.
Significance
In conclusion, major components of CNSL yield overall best dentin biomodification outcomes when applied for one minute without staining the dentin collagen.
1
Introduction
Major drawbacks in dentin bond are related to enzymatic and hydrolytic degradation of hybrid layer and resin-sparse collagen matrix . It is hypothesized that the quality and longevity of the dentin adhesion may be improved by increasing the dentin collagen mechanical properties . In this regard, dentin biomodification improves the stiffness of the adhesive interface and protects the collagen fibrils from biodegradation by promoting collagen crosslinking .
A variety of crosslinking (biomodification) agents have proved to effectively increase mechanical properties of dentin organic matrix . Nevertheless, condensed tannins, also known as proanthocyanidins (PACs), from grape seed extract ( Vitis vinifera ) are a group of plant-derived polyphenols with highlight and high crosslink potential to collagen thereby providing biostability . The positive influence of these substances on bond strength and dentin properties such as ultimate tensile strength , resistance to demineralization/biodegradation and elastic modulus was described in several investigations. Due to the dark color of PACs solution, the main shortcoming for its clinical use still is the risk to stain tooth substrates .
Further plant extracts from Anacardiaceae such as Anacardium occidentale (cashew) and Myracrodruon urundeuva (Aroeira) have potential significant capabilities for application in dentin biomodification. Cardol and cardanol are long carbon-chain phenols ( Fig. 1 ) obtained from the industrial extraction of cashew nut shell liquid (CNSL) during production of nuts. They have antioxidant capacity , enzyme inhibitory potential and two hydroxyls in p-position (in the case of cardol) similar to those of PACs ( Fig. 1 ). Extracts of Aroeira showed anti-inflammatory properties and antimicrobial activity . Moreover, major polyphenols of Aroeira are characterized by hydrolysable tannins with high chemical interaction with proteins producing tannin-protein complexes relatively insoluble and resistant to degradation .
Although several plant-derived polyphenols were tested for dentin collagen crosslinking , cardol/cardanol from cashew nut shell liquid and Aroeira extract have never been evaluated. Therefore, the aim of the present study was to determine the effects of cardol, cardanol and Aroeira extract as crosslinkers on mechanical properties and degradation of demineralized dentin in comparison to PACs within clinically relevant application time. The hypothesis tested was that cardol, cardanol, PACs and Aroeira provide similar increase on elastic modulus and mass variation of demineralized dentin beams.
2
Materials and methods
2.1
Preparation of biomodification solutions
Cardol and cardanol were obtained from industrial cashew nut shell liquid (CNSL) supplied by Amendoas do Brasil LTDA (Fortaleza, Brazil) separated employing a methodology described by Lomonaco et al. . The products were also characterized by gas chromatography/mass spectrometry and 1 H nuclear magnetic resonance (NMR) to ensure their purity . The CNSL components were diluted in EtOH/H 2 O (1:1 volume ratio) at 2 wt% concentration. Aroeira extract was obtained from commercial mastic stem bark (M&A Natural Products, Fortaleza, Brazil) of M. urundeuva species through infusion in EtOH/H 2 O (1:1 volume ratio) with 15% bark (15 g bark in 100 mL hydroethanolic solution). After 5 min agitation in 25 °C, the mixture was filtered twice to obtain the solution of Aroeira polyphenols with approximately 2% concentration, as the total amount of polyphenols in Aroeira stem bark is approximately 13% . Proanthocyanidins (PACs) solution was prepared after dissolving 6.5% grape seed extract ( V. vinifera , Meganatural Gold, Madera, CA, USA) in EtOH/H 2 O (1:1 volume ratio) with 5 min agitation at 25 °C and final double filtering. The solutions were buffered to pH 7.2. Ethanol/water solution was employed to standardize the dissolution of agents, once cardanol has very low solubility in only distilled water. Chemical structures of major polyphenol from Aroeira, monomeric unit of proanthocyanidins from grape-seed extract, and structures of cardol and cardanol are depicted in Fig. 1 .
2.2
Structural characterization of cardanol and cardol
NMR spectra were recorded on Avance DRX-500 (500 MHz for 1 H, Bruker, Bremen, Germany) using CDCl 3 as solvent for cardanol, and acetone-d6 for cardol. The gas chromatography/mass spectrometry analyses were performed on GC/MS QP-2010 Ultra (Shimadzu, Tokyo, Japan), equipped with a (5%-phenyl)-methylpolysiloxane (DB-5) capillary column (30 m × 0.25 mm), using helium (He) as carrier gas and a flow rate of 1 mL/min in a splitless mode.
2.3
Sample preparation
Twenty five extracted caries-free human third molars were used in this study after approval by the Research Ethics Committee of Federal University of Ceará (protocol 1482602). Teeth were cut with slow-speed water-cooled diamond saw (Isomet 4000; Buehler, Lake Bluff, USA). One 0.5 mm-thick disk from middle coronal dentin was obtained from each tooth . Disks were then sectioned into dentin beams (1.7 mm × 0.5 mm × 6 mm). A total of 75 beams were obtained and were completely demineralized in 10% H 3 PO 4 solution for 5 h at 20 °C . Demineralization was confirmed with digital radiography.
2.4
Dentin biomodification
Demineralized dentin beams (n = 15/group) were randomly divided in five groups and treated with distilled water (control) or one of the four biomodification solutions tested (PACs from grape seed extract, cardol, cardanol and Aroeira bark extract). Before the treatment, the initial dry weight and initial flexural modulus (three-point bending test) were assessed as previously described . For biomodification, the demineralized beams were individually immersed in 1 mL of each solution for 1 min, to simulate a clinically relevant time. Afterwards, beams were vigorously rinsed with distilled water for 30 s to remove unbound crosslinkers. The flexural modulus was re-assessed immediately after immersion, and treated beams were individually stored in 1 mL artificial saliva containing 5 mM HEPES, 2.5 mM CaCl 2 , 0.05 mM ZnCl 2 and 120 mM NaCl (pH 7.4) at 37 °C for four weeks to undertake collagen degradation .
2.5
Elastic modulus
Specimens were tested on three-point bending set-up in a universal testing machine (Instron 4484; Instron Inc., Canton, USA), with a 5 N load cell at 0.5 mm/min crosshead speed. Load–displacement curves were converted to stress–strain curves. Width and thickness of the specimens were measured and the apparent elastic modulus was calculated at 3% strain as previously described . Data were expressed in MPa, and the percentage variation in elastic modulus was calculated as the ratio of the final value (after biomodification) to the initial values (baseline).
2.6
Mass change
Demineralized dentin beams were weighed before (M 1 ) and after (M 2 ) dentin biomodification with an analytical balance (0.01 mg precision, AUX-220, Shimadzu, Tokyo, Japan). Further mass assessment was surveyed after 4 weeks degradation in artificial saliva (M 3 ). In each period, specimens were initially dried in a vacuum desiccator containing silica gel beads for 72 h at room temperature. Mass variation (W mc %) was determined as the percentage of gain or loss in mass for each specimen as previously described based on the following formula for biomodification:
where M 1 is the demineralized dentin beam mass before dentin biomodification and M 2 is the mass of biomodified dentin matrix. Moreover, to assess the biodegradation percentage mass variation, the following formula was used:
where M 1 is the demineralized dentin beam mass before dentin biomodification and M 3 is the mass of dentin matrix after four weeks artificial saliva immersion. After the final weighing, pictures of each specimen were obtained using a professional camera (Canon, Tokyo, Japan) with Macro lens (100 mm, Canon) in order to observe the final color and aspect of biomodified demineralized dentin beams.
2.7
Micro-Raman spectroscopy
Vibrational analysis of the demineralized dentin specimens before and after biomodification treatment as aforementioned was assessed using the Micro-Raman spectrophotometer (Xplora, Horiba JobinYvon, Paris, France) calibrated internally in zero using the silicon standard sample provided by the manufacturer. The configuration of the equipment were HeNe laser with 3.2 mW power, 633 nm laser wavelength, 10 s acquisition time, 5 accumulations, 1.5 μm spatial resolution, 2.5 cm −1 spectral resolution, 10× magnification lens (Olympus, London, UK) and 60 × 70 μm field area. For observation of dentin collagen cross-linking, the range was 700–1800 cm −1 to survey peaks/shoulders at 1117 cm −1 and 1235 cm −1 according to a previous investigation . Raman analyses were performed in triplicate per group.
2.8
Statistical analysis
Analysis of elastic modulus data was carried out via one-way repeated-measures ANOVA (biomodification agent), followed by Tukey’s post hoc test (p < 0.05), after passing normality (p = 0.657) and equal variance (p = 0.335) tests. The data of percentage modulus variation results, percentage mass change after biomodification, and percentage mass variation after 4 weeks degradation were separately analyzed by one-way ANOVA and Tukey’s test ( α = 5%).
2
Materials and methods
2.1
Preparation of biomodification solutions
Cardol and cardanol were obtained from industrial cashew nut shell liquid (CNSL) supplied by Amendoas do Brasil LTDA (Fortaleza, Brazil) separated employing a methodology described by Lomonaco et al. . The products were also characterized by gas chromatography/mass spectrometry and 1 H nuclear magnetic resonance (NMR) to ensure their purity . The CNSL components were diluted in EtOH/H 2 O (1:1 volume ratio) at 2 wt% concentration. Aroeira extract was obtained from commercial mastic stem bark (M&A Natural Products, Fortaleza, Brazil) of M. urundeuva species through infusion in EtOH/H 2 O (1:1 volume ratio) with 15% bark (15 g bark in 100 mL hydroethanolic solution). After 5 min agitation in 25 °C, the mixture was filtered twice to obtain the solution of Aroeira polyphenols with approximately 2% concentration, as the total amount of polyphenols in Aroeira stem bark is approximately 13% . Proanthocyanidins (PACs) solution was prepared after dissolving 6.5% grape seed extract ( V. vinifera , Meganatural Gold, Madera, CA, USA) in EtOH/H 2 O (1:1 volume ratio) with 5 min agitation at 25 °C and final double filtering. The solutions were buffered to pH 7.2. Ethanol/water solution was employed to standardize the dissolution of agents, once cardanol has very low solubility in only distilled water. Chemical structures of major polyphenol from Aroeira, monomeric unit of proanthocyanidins from grape-seed extract, and structures of cardol and cardanol are depicted in Fig. 1 .
2.2
Structural characterization of cardanol and cardol
NMR spectra were recorded on Avance DRX-500 (500 MHz for 1 H, Bruker, Bremen, Germany) using CDCl 3 as solvent for cardanol, and acetone-d6 for cardol. The gas chromatography/mass spectrometry analyses were performed on GC/MS QP-2010 Ultra (Shimadzu, Tokyo, Japan), equipped with a (5%-phenyl)-methylpolysiloxane (DB-5) capillary column (30 m × 0.25 mm), using helium (He) as carrier gas and a flow rate of 1 mL/min in a splitless mode.
2.3
Sample preparation
Twenty five extracted caries-free human third molars were used in this study after approval by the Research Ethics Committee of Federal University of Ceará (protocol 1482602). Teeth were cut with slow-speed water-cooled diamond saw (Isomet 4000; Buehler, Lake Bluff, USA). One 0.5 mm-thick disk from middle coronal dentin was obtained from each tooth . Disks were then sectioned into dentin beams (1.7 mm × 0.5 mm × 6 mm). A total of 75 beams were obtained and were completely demineralized in 10% H 3 PO 4 solution for 5 h at 20 °C . Demineralization was confirmed with digital radiography.
2.4
Dentin biomodification
Demineralized dentin beams (n = 15/group) were randomly divided in five groups and treated with distilled water (control) or one of the four biomodification solutions tested (PACs from grape seed extract, cardol, cardanol and Aroeira bark extract). Before the treatment, the initial dry weight and initial flexural modulus (three-point bending test) were assessed as previously described . For biomodification, the demineralized beams were individually immersed in 1 mL of each solution for 1 min, to simulate a clinically relevant time. Afterwards, beams were vigorously rinsed with distilled water for 30 s to remove unbound crosslinkers. The flexural modulus was re-assessed immediately after immersion, and treated beams were individually stored in 1 mL artificial saliva containing 5 mM HEPES, 2.5 mM CaCl 2 , 0.05 mM ZnCl 2 and 120 mM NaCl (pH 7.4) at 37 °C for four weeks to undertake collagen degradation .
2.5
Elastic modulus
Specimens were tested on three-point bending set-up in a universal testing machine (Instron 4484; Instron Inc., Canton, USA), with a 5 N load cell at 0.5 mm/min crosshead speed. Load–displacement curves were converted to stress–strain curves. Width and thickness of the specimens were measured and the apparent elastic modulus was calculated at 3% strain as previously described . Data were expressed in MPa, and the percentage variation in elastic modulus was calculated as the ratio of the final value (after biomodification) to the initial values (baseline).
2.6
Mass change
Demineralized dentin beams were weighed before (M 1 ) and after (M 2 ) dentin biomodification with an analytical balance (0.01 mg precision, AUX-220, Shimadzu, Tokyo, Japan). Further mass assessment was surveyed after 4 weeks degradation in artificial saliva (M 3 ). In each period, specimens were initially dried in a vacuum desiccator containing silica gel beads for 72 h at room temperature. Mass variation (W mc %) was determined as the percentage of gain or loss in mass for each specimen as previously described based on the following formula for biomodification:
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