Abstract
Objective
The aim of the present study was to evaluate the effect of quercetin on the acid resistance of human dentin through both laboratory and clinical studies.
Methods
Two hundred and twelve dentin blocks (2 mm × 2 mm × 2 mm) were prepared and used. For the laboratory study, dentin specimens were randomly divided into 8 groups (n = 12): deionized water, ethanol, 1.23 × 10 4 μg/ml sodium fluoride (NaF), 120 μg/ml chlorhexidine, 183.2 μg/ml epigallocatechin gallate (EGCG), and 75 μg/ml, 150 μg/ml, and 300 μg/ml quercetin (Q75, Q150, and Q300). The specimens were treated with the respective solutions for 2 min and then subjected to in vitro erosion (4 cycles/d for 7 d). The surface microhardness loss (%SMH l ), erosive dentin wear, and surface morphology were evaluated and compared. For the impact on MMP inhibition, the release of crosslinked carboxyterminal telopeptide of type I collagen (ICTP) and the thickness of the demineralized organic matrix (DOM) were measured using additional dentin specimens. For the clinical study, the specimens were treated with NaF or Q300 for 2 min and then subjected to in vivo erosion (4 cycles/d for 7 d). The %SMH l and erosive dentin wear of the specimens were measured to determine whether quercetin similarly inhibits erosion in situ .
Results
The quercetin-treated group had a significantly lower %SMH l and erosive dentin wear than any other group, and the effect was concentration-dependent in vitro ( P < 0.05). Dentin treated with quercetin produced significantly less ICTP and had a thicker DOM than the control dentin ( P < 0.05). After in vivo erosion, the %SMH l and erosive dentin wear of the Q300 group were significantly lower than those of the control group ( P < 0.05).
Significance
The application of quercetin was shown, for the first time, to increase the acid resistance of human dentin, possibly through MMP inhibition and DOM preservation.
1
Introduction
Dental erosion is defined as the loss of tooth substance caused by acids without the involvement of microorganisms. According to a recent survey, the incidence of dental erosion has reached 57.1%, and the erosion reaches the dentin layer in 29.4% of these cases [ ]. The effective prevention of erosive tooth wear includes measures that can avoid or reduce direct contact with acidic products, reduce the erosive potential of acidic products, and increase the acid resistance of tooth substance [ , ].
Although fluoride is considered a well-established protective agent against dental caries, it is much less effective against erosive tooth wear [ , ]. It is noteworthy that the effect of fluoride against erosion seems to be more prominent for dentin than enamel [ ]. The enhanced protection conferred by fluoride to dentin could be attributed to the fact that fluoride inhibits matrix metalloproteinases (MMPs), slowing down erosion progression [ ]. The presence of a collagen matrix with a reduced mineral content in dentin makes it more susceptible than enamel to degradation by free- and collagen-bound proteases, such as MMPs [ ]. MMPs are involved in diverse physiological and pathological processes in the pulpo-dentin complex [ ]. In the oral environment, MMPs, including mainly MMP-2, MMP-8, and MMP-9, have been found to exist in saliva and dentin [ ]. It has been speculated that MMPs located within dentin could be exposed and activated during the erosion process and, hence, could contribute to tissue breakdown [ ]. Moreover, when dentin is exposed to acid attack, a superficial layer of demineralized organic matrix (DOM) is present on the dentin surface. DOM is considered an important barrier to protect the remaining dentin against erosion [ ] and abrasion [ ]. However, DOM is susceptible to breakdown by host-derived salivary or endogenous dentinal enzymes, such as MMPs [ , ].
Therefore, 2 strategies, including the application of MMP inhibitors and cross-linkers, have been proposed to challenge this enzymatic degradation. The evidence consists of the finding that MMPs increase the susceptibility to erosive dentin wear [ ] and that the application of MMP inhibitors [epigallocatechin gallate (EGCG) and chlorhexidine] reduces erosive dentin wear [ , ]. Moreover, crosslinking improves the mechanical properties of dentin and increases its resistance to enzymatic degradation [ ].
Flavonoids are a group of bioactive molecules that can perform various biological processes [ ]. Quercetin is a flavonoid found in citrus fruit and possesses multiple functions, including antioxidative [ ], anti-inflammatory [ ], and antiaggregatory effects [ ]. In adhesive dentistry, the pretreatment of the dentin surface with a quercetin/ethanol solution has been shown to effectively inhibit MMP activity in the hybrid layer, delay the collapse of the collagen fibrous network, and further improve dentin adhesion longevity [ , ]. It has also been shown that quercetin has a collagen crosslinking effect and can enhance the mechanical properties of demineralized dentin [ ]. Although the currently available evidence is indirect, theoretically, quercetin should facilitate the preservation of the dentin matrix and reduce the progression of dentin erosion. Thus, the aim of this study was to determine whether quercetin can inhibit erosive dentin wear in vitro and in situ / in vivo . The null hypothesis tested was that quercetin has no effect on the progression of dentin erosion.
2
Materials and methods
The study protocol was reviewed and approved by the Institutional Review Board, School and Hospital of Stomatology, Fujian Medical University, China (FMUSS-18-021). All volunteers participated after providing signed informed consent.
2.1
Specimen preparation
One hundred and eight extracted human third molars that were stored in 0.1% thymol solution at 4 °C were used within 1 month after extraction. Two hundred and twelve dentin blocks (2 mm × 2 mm × 2 mm) were obtained from mid-crown portions of the teeth using a low-speed saw (Isomet, Buehler, Lake Bluff, IL, USA) with water cooling. The blocks were further embedded with acrylic resin (Paladur, Heraeus Kulzer, Germany) using a custom-made silicone mold, exposing the erosion surface. The final dimensions of the specimens were as follows: top surface 3 mm × 3 mm, bottom surface 4 mm × 4 mm, and thickness 3 mm. The specimens were polished with a series of water-cooled carborundum discs (#320, #600 and #1200; Buehler) and cleaned in an ultrasound bath for 2 min. Finally, the specimens were allocated to groups according to the surface microhardness (Vickers diamond, 100 g, 10 s; HXD 1000 TMC, Shanghai Taiming Optics Ltd., China) and stratified randomization. To provide reference surfaces for profilometric analysis, two layers of nail varnish (Revlon Corp., NY, USA) were applied to both sides of each sample, leaving 1 mm of the middle exposed to acid challenge. All the samples were kept moistened to avoid shrinkage of the dentin organic matrix during the experiment.
2.2
Effects of quercetin on erosive dentin wear (laboratory study)
The specimens were randomly divided into 8 groups according to the different solutions applied (n = 12): 120 μg/ml chlorhexidine, 183.2 μg/ml EGCG, and 75 μg/ml, 150 μg/ml, and 300 μg/ml quercetin (Q75, Q150, and Q300). Sodium fluoride (NaF, 1.23 × 10 4 μg/ml) was used as a positive control, while deionized water (solvents for chlorhexidine, EGCG, and NaF) and ethanol (solvent for quercetin) were used as negative controls.
Over the experimental period, specimens were subjected to a 7-day erosion cycling regimen. The specimens were immersed in artificial saliva for 1 h to allow for the formation of an acquired salivary pellicle on the dentin surfaces [ ]. The artificial saliva was mixed according to the formulation described by Klimek et al. [ ]. After being dried briefly, the specimens were treated by topical application of the respective solutions for 2 min using a microbrush. The specimens were then stored in artificial saliva for 2 h and subsequently subjected to daily erosion cycles. For each cycle, the specimens were immersed in 5 ml of citric acid (pH = 2.45) for 5 min at room temperature, rinsed with tap water for 10 s, and exposed to 5 ml of artificial saliva for 1 h. After being subjected to 4 daily cycles, the specimens were stored in artificial saliva overnight.
The nail varnish was then carefully removed with a scalpel blade without touching the dentin surface [ ]. The surface profile of each specimen was measured at baseline and at the end of the experiment using a contact profilometer (SEF 680, Kosaka Laboratory, Japan). Erosive dentin wear (μm) was calculated based on the profiles from the reference surfaces to the eroded surfaces.
The surface microhardness (SMH) values were measured at baseline and at the end of the experiment. The mean SMH values were then used to calculate the percentage of SMH loss (%SMH l ) [ ].
After the 7-day experiment concluded, 2 specimens from each group were randomly selected for scanning electron microscopy (SEM). The specimens were fixed in 2.5% glutaraldehyde for 4 h and then washed in PBS buffer for 30 min at room temperature. The specimens were dehydrated in an ascending ethanol concentration series (30%, 50%, 70%, 80%, 90%, and 100%, 10 min/concentration) at room temperature. The specimens were fractured into 2 halves to allow for the observation of the sample surface and a transverse section of the demineralized dentin and DOM [ ]. They were then subjected to critical point drying and gold sputtering. The specimens were observed using a scanning electron microscope (Quanta 250, FEI, USA) with an acceleration voltage of 25 kV.
2.3
Effect of quercetin on DOM and the release of crosslinked carboxyterminal telopeptide of type I collagen (ICTP)
Seventy-two dentin specimens were prepared and divided into 6 groups (n = 12): ethanol, deionized water, NaF, Q75, Q150, and Q300. The specimens were treated with the respective solutions and subjected to in vitro erosion cycles, as mentioned above. Surface profilometry was performed before and after the treatment with 100 U/ml type I collagen enzyme at 37 °C for 5 d to remove the DOM [ ]. The thickness of the DOM (μm) was calculated based on the differences between pre- and post-treatment profiles [ ].
Twenty-four dentin blocks were demineralized with 10% phosphoric acid (pH = 1.0) (Panreac Química, Barcelona, Spain) for 18 h, rinsed with deionized water with shaking at 4 °C for 72 h to remove the acid, and then dried over anhydrous calcium sulfate for 8 h [ ]. The dentin blocks were randomly divided into 6 groups (n = 4): ethanol, deionized water, NaF, Q75, Q150, and Q300. They were dipped for 2 min in the respective solutions and then incubated separately in 1 ml of artificial saliva in Eppendorf tubes at 37 °C for 7 d [ ].
After the specimens were removed, tubes were centrifuged (3000 rpm for 10 min at 4 °C) and 50 μl of the medium supernatant in each tube was collected for ICTP telopeptides analysis using an ELISA kit (ICTP ELISA, MBbio, Jiangsu, China). The telopeptide results were expressed in μg/l.
2.4
Effects of quercetin on erosive dentin wear (clinical study)
The in situ / in vivo experimental protocol has been described in detail previously [ ]. Ten healthy volunteers (6 males and 4 females, mean age: 25.3 years) who fulfilled the inclusion criteria were examined and recruited for this clinical study. Each volunteer wore an intraoral appliance containing 2 slots (located on the buccal surfaces of the central incisors) to allow for the storage of the dentin specimens. An opening of 3 × 3 mm was made on each slot to allow for the acid exposure.
At the beginning of each experimental day, the intraoral appliances with samples were worn for 1 h. The specimens were then treated in vivo with 300 μg/ml quercetin or 1.23 × 10 4 μg/ml NaF for 2 min using a microbrush by a study coordinator. After rinsing with 20 ml of tap water for 10 s, the appliances were worn for 2 h, followed by a daily in vivo erosive challenge. The erosive challenge consisted of rinsing with citric acid (pH = 2.45) for 5 min at short intervals and then expectorating the rinse, 4 times per day for 7 d. The appliances were worn for 1 h between each erosive challenge. The %SMH l and erosive dentin wear were measured as mentioned above.
2.5
Statistical analysis
The assumptions regarding the equality of the variances and normal distribution of errors were confirmed using the Levene test and the Kolmogorov–Smirnov test, respectively. For erosive dentin wear, DOM, and release of ICTP, the data were statistically analyzed using two-way analysis of variance (ANOVA) and Tukey’s test. For the %SMHl, the data were statistically analyzed using one-way analysis of covariance (ANCOVA) and Tukey’s test. The data were analyzed using the SPSS statistical software package (SPSS 19.0 for MAC, SPSS, Chicago, IL, USA) at a level of P < 0.05. All statistical analyses were performed at a significance level of 0.05.
3
Results
3.1
Effects of quercetin on erosive dentin wear (laboratory study)
After the samples were subjected to in vitro erosion cycles, the %SMH l of specimens in different groups ranged from 8.75 to 37.95 ( Table 1 ). One-way ANCOVA revealed that statistically significant differences were found among different groups ( Table 2 ). The Q300 group exhibited the least %SMH l , which was significantly lower than those of the positive (NaF) and negative (deionized water and ethanol) controls (all P < 0.001).
| Group | SMH 1 (HV) | SMH 2 (HV) | %SMH l |
|---|---|---|---|
| Deionized water | 64.23 ± 2.19 | 39.83 ± 4.93 | 37.95 ± 7.86 a |
| Ethanol | 64.42 ± 1.68 | 39.14 ± 3.22 | 39.24 ± 4.69 a |
| NaF | 65.36 ± 1.82 | 47.67 ± 3.61 | 27.08 ± 4.90 b |
| Chlorhexidine | 65.03 ± 1.79 | 52.36 ± 3.68 | 19.46 ± 5.64 c |
| EGCG | 65.10 ± 2.16 | 52.28 ± 2.83 | 19.58 ± 5.51 c |
| Q75 | 65.22 ± 2.16 | 53.25 ± 4.21 | 18.31 ± 6.40 c |
| Q150 | 65.02 ± 2.19 | 54.89 ± 2.95 | 15.48 ± 5.41 c |
| Q300 | 65.11 ± 1.66 | 59.42 ± 3.68 | 8.75 ± 4.95 d |
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