The preventive effect of grape seed extract on artificial enamel caries progression in a microbial biofilm-induced caries model.

JJOD 2295 1–9 journal of dentistry xxx (2014) xxx–xxx

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/jden 1 2 3

The preventive effect of grape seed extract on artificial enamel caries progression in a microbial biofilm-induced caries model

4 5

6 7 8 9 10 11 12

Wei Zhao a,b, Qian Xie a, Ana Karina Bedran-Russo c, Shuang Pang d, b a, Q2 Junqi Ling , Christine D. Wu * Q1

a

Department of Pediatric Dentistry, College of Dentistry, University of Illinois-Chicago, Chicago, IL, USA Guanghua School of Stomotology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China c Department of Restorative Dentistry, College of Dentistry, University of Illinois-Chicago, Chicago, IL, USA d Department of Oral Biology, College of Dentistry, University of Illinois-Chicago, Chicago, IL, USA b

article info

abstract

Article history:

Objectives: The aim of this study was to evaluate the effect of grape seed extract (GSE) on

Received 21 January 2014

enamel caries lesion formation in an in vitro Streptococcus mutans biofilm model.

Received in revised form

Methods: Enamel fragments were prepared from bovine incisors and divided into six

9 May 2014

treatment groups (n = 12): inoculated Brain Heart Infusion with 1% sucrose (BHIS), 1 mg/

Accepted 14 May 2014

mL GSE, 2 mg/mL GSE, 3 mg/mL GSE, 10 ppm fluoride as NaF, and uninoculated BHIS. For

Available online xxx

biofilm formation, tooth fragments were incubated anaerobically in polystyrene 6-well tissue culture plates containing BHIS, the respective agents, and S. mutans (1 105 CFU/

Keywords:

mL) for 24 h at 37 8C. Culture medium was replaced with fresh BHIS and respective agents

Grape seed extract

daily over a 7-day period. Following caries lesion formation, lesion depth (LD) and relative

Caries prevention

optical density (ROD) were determined by polarized light microscopy (PLM) and confocal

Microbial biofilm-induced caries

laser scanning microscopy (CLSM), respectively, to evaluate lesion progression.

model

Results: LDs of the 2 mg/mL GSE group (122.86 13.41 mm) and the 3 mg/mL GSE group

Enamel caries lesion

(111.92 11.39 mm)

Streptococcus mutans biofilm

(198.33 17.70 mm) and control groups (210.86 15.50 mm) (p < 0.05). Compared with the

were

significantly

smaller

than

those

of

the

1 mg/mL

GSE

2 mg/mL and 3 mg/mL groups, the control and 1 mg/mL GSE groups showed significantly lower ROD values when depth was less than 200 mm, indicating greater mineral loss. Conclusions: Dose-dependent GSE inhibits in vitro enamel caries formation due to its ability to suppress growth of S. mutans and the formation of biofilm and thus may be a promising Q4 agent for enamel caries prevention. # 2014 Published by Elsevier Ltd.

15 13 14 16 17 18

19 20

1.

Introduction

Although current preventive measures—including the administration of fluoride and broad-spectrum antimicrobials, along

with the reduction of sucrose intake and effective oral hygiene habits—have been demonstrated to decrease caries prevalence,1 dental caries remains one of the most prevalent diseases in humans.2 Dental caries is initiated by demineralization of the tooth surface through acid production from sugar

Q3 * Corresponding author. Tel.: +1 312 355 1990. E-mail address: [email protected] (C.D. WuQ2). Q2

http://dx.doi.org/10.1016/j.jdent.2014.05.006 0300-5712/# 2014 Published by Elsevier Ltd.

Please cite this article in press as: Zhao W, et al. The preventive effect of grape seed extract on artificial enamel caries progression in a microbial biofilm-induced caries model. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.05.006

21 22 23 24 25

JJOD 2295 1–9

2

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

journal of dentistry xxx (2014) xxx–xxx

by plaque biofilm. Streptococcus mutans is considered the principal cariogenic pathogen and plays a decisive role in the development of dental caries.3–5 Various measures have been developed to counteract the onset or progression of dental caries. One preventive method is the use of antimicrobial agents, such as chlorhexidine or NaF, to limit the growth and biofilm formation of cariogenic microorganisms, especially S. mutans, in the oral cavity.3–6 However, the conventional chemical antimicrobial agents have demonstrated some limitations and have not been recommended for regular caries prevention.7 In recent years, much attention has been focused on plant-derived natural antimicrobial compounds, with potential use as alternatives to the common chemicals used for caries prevention.8–17 Polyphenolic compounds (polyphenols) are secondary metabolites of plants and are commonly found in both edible and non-edible plants or plant-based foods and beverages. Their health benefits (e.g., antioxidant, anticancer, and anti-inflammatory) have been emphasized within the last decade.18 The consumption of polyphenol-rich foods or beverages has also been reported to benefit oral health, with antigingivitis and anticaries properties.14,18–22 In recent years, the antimicrobial and antiplaque activity of plant polyphenols has been demonstrated in many in vitro studies.16,17,23–25 Grape seed extract (GSE), derived from the seeds of Vitis vinifera, is rich in polyphenolic compounds. It consists mainly of free monomeric flavanols, i.e., the proanthocyanidins (PACs), as well as their dimeric, trimeric, tetrameric, and higher oligomeric forms, termed the oligomeric proanthocyanidins (OPACs). Examples of PACs contained in GSE are catechin, epicatechin, and epicatechin-3-O-gallate. These monomers are the structural building blocks of the OPCAs contained in GSE. GSE has attracted much attention in recent years due to its well-documented antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenic properties.26,27 PACs have remarkable dentine-specific protective effects by decreasing biodegradation rates and enhancing the mechanical properties of the organic matrix.28 The antimicrobial activity of GSE has been reported in the literature, but information on its effect on the growth of cariogenic bacteria, especially their resistant biofilm, is limited.29,30 Using an in vitro pH-cycling model, researchers have demonstrated the positive effect of GSE on artificial root caries by stabilizing the collagen-based tissues and promoting remineralization.31,32 However, there are no available data on the anticariogenic potential of GSE (in terms of its antibacterial and antibiofilm properties) in combating microbial biofilminduced artificial enamel caries. A more clinically relevant model is needed.33 It is hypothesized that GSE inhibits the growth and biofilm formation of S. mutans, thereby preventing the progression of artificial enamel caries. This study evaluated the preventive effect of GSE on caries lesion formation in an in vitro S. mutans biofilm model.

2.

Materials and methods

2.1.

Test bacteria and grape seed extract (GSE)

79

80 81

The test bacterium used in this study was S. mutans UA159. Cultures were routinely grown in Brain Heart Infusion broth

(BHI; Difco, Detroit, MI, USA) under anaerobic conditions (Forma anaerobic chamber, 80% N2, 10% CO2, and 10% H 2) for 24 h at 37 8C overnight. For S. mutans biofilm formation, BHI with 1% sucrose (BHIS) was used. The grape seed extract (GSE) obtained from the seeds of Vitis vinifera was purchased from MegaNatural, Polyphenolics (Madera, CA, USA). It consists of 97.8% proanthocyanidins (PA) according to data provided by the manufacturer. For assays, GSE was dissolved in distilled water and filtered through an MFMillipore membrane (0.22 mm, Carrigtwohill Co., Cork, Ireland).

82 83 84 85 86 87 88 89 90 91 92

2.2. Determination of minimum inhibitory concentration (MIC) and minimum biofilm inhibition concentration (MBIC)

93 94

The MIC and MBIC of GSE of S. mutans were determined by a microdilution method in 96-well microliter plates (Corning, NY, USA). Each well contained S. mutans (final concentration 1 105 CFU/mL), BHI/BHIS medium, and serially diluted GSE (0.125–16 mg/mL). Control wells contained BHI/BHIS without GSE, uninoculated BHI/BHIS, or GSE alone. Chlorhexidine digluconate solution (CHX, Sigma, St. Louis, MO, USA) was used as a positive control. All plates were incubated in an anaerobic chamber (Forma anaerobic chamber, 80% N2, 10% CO2, and 10% H2) at 37 8C for 24 h, after which growth was determined spectophotometrically at 550 nm by means of a microplate reader (PowerWave 200, Bio-Tek Instruments, Winooski, VT, USA). The MIC was defined as the lowest concentration of GSE that inhibited the visible growth of S. mutans (OD550 < 0.05). MBIC was defined as the lowest GSE concentration that inhibited formation of S. mutans biofilms.17 The data were reported as the median of at least 3 independent tests.

95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112

2.3.

113

Enamel specimen preparation

Seventy-two extracted sound bovine incisors were used. The crowns were cut from the roots at the cemento-enamel junction with a cylindrical diamond bur (No. 557D, Brasseler, Savannah, GA, USA) in a high-speed handpiece. One enamel disc fragment (5 mm 4 mm 3 mm) was obtained per crown by means of a low-speed diamond blade (Isomet 1000, Buehler, Lake Bluff, IL, USA). Subsequently, enamel surfaces were ground flat and polished on a rotating polishing machine under water cooling (Phoenix Alpha; Buehler, Du¨sseldorf, Germany) with progressively finer grades of SiC grinding paper. Approximately 150–200 mm of surface enamel was removed. In total, 72 fragments were obtained and sealed with acid-resistant nail polish (Revlon Corp., New York, NY, USA), except for a 3 mm 2 mm window on each fragment.

114 115 116 117 118 119 120 121 122 123 124 125 126 127 128

2.4. In vitro caries lesion formation induced by S. mutans biofilm

129 130

Seventy-two bovine enamel fragments were randomly divided into six treatment groups ( n = 12) as follows: Group 1, Brain Heart Infusion with 1% sucrose (BHIS, as control); Group 2, BHIS + 1 mg/mL GSE; Group 3, BHIS + 2 mg/mL GSE; Group 4, BHIS + 3 mg/mL GSE; Group 5, BHIS + 10 ppm

131 132 133 134 135



136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161

NaF (positive control); and Group 6, uninoculated BHIS (as blank control). Based on pilot data, NaF at 10 ppm was the sub-MIC concentration which did not promote in vitro enamel demineralization and was chosen as the positive control. Twelve specimens in each group were assigned to 3 wells (4 in each well) of a polystyrene 6-well tissue culture plate (Corning, NY, USA). For biofilm formation, tooth fragments were pre-conditioned with sterile artificial saliva (AS)34 at 37 8C. After 2 h , AS was gently aspirated, and a 10-mL quantity of BHIS containing the respective agent and S. mutans (1 105 CFU/mL) was added, except for the uninoculated BHIS group (blank control). All plates were incubated under anaerobic conditions at 37 8C to allow for biofilm formation and growth. Culture media were replaced daily with fresh BHIS with respective agents and incubated over a 7-day period under anaerobic conditions. Following caries lesion formation, enamel fragments were rinsed with deionized water for 2 min to dislodge attached biofilm, and longitudinally sectioned through the lesions with a low-speed water-cooled diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA) into two halves. The two fragment halves were further examined with polarized light microscopy (PLM) and confocal laser scanning microscopy (CLSM), respectively, for caries lesions.

162

2.5.

163 164 165 166 167 168 169 170 171 172 173

One half of each enamel fragment was embedded perpendicular to the demineralized surface in epoxy resin. The embedded samples were polished in a water-cooled polishing unit (EcoMet 3000, Buehler, Lake Bluff, IL, USA) with SiC grinding paper (400-, 600-, 1200-, and 2500-grit) and further polished with 4000-grit paper to obtain sections of approximately 80 mm thickness (EXAKT 400 CS & 420 CL Grinding Systems, EXAKT Technologies GmbH, Norderstedt, Germany). Lesion depths were quantified by polarized light microscopy (PLM, Leica Microsystems GmbH, Wetzlar, Germany).

174

2.6.

175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191

The remaining half of each enamel fragment was longitudinally sectioned through the lesions with a low-speed water-cooled diamond saw (Isomet 1000, Buehler, Lake Bluff, IL, USA) to obtain 300-mm-thick sections and further polished to 100-mm thickness. The samples were stained overnight with a 0.1 mM solution of rhodamine B (Aldrich Chemical Co., Milwaukee, WI, USA), washed in deionized water for 3 min, and observed with CLSM (LSM 510; Carl Zeiss, Jena, Germany) at an excitation wavelength of a 543nm HeNe laser, a 25-mm confocal slit, and a long-pass filter between 565 and 615 nm at a magnification of 200. Digital images were taken and analyzed (Image Pro-Plus version 5.1) for lesion progression. Lesion depths were measured at three defined points per lesion, and mean values were calculated. The optical density (OD), directly related to the porosity of the demineralized enamel, was quantitatively measured at the same depth .32,35 The formula

3

ODR = ODl/ODs 100% was used to calculate the relative optical density (ROD), with ODl the OD of the enamel caries lesion and OD s the OD of sound enamel of the same sample at the same level (depth from the surface).

192 193 194 195

2.7.

196

Statistical analysis

Statistical analysis was performed with SPSS software (SPSS 13.0 for Windows, SPSS, Chicago, IL, USA). Data were checked for normal distribution by the Shapiro–Wilk test. For each sample group, descriptive statistics included means, standard deviations, standard errors, and maximum and minimum values of lesion depths for all measured parameters. The data collected from PLM and CLSM were analyzed by one-way ANOVA and Scheffe´’s post hoc comparison tests. The level of significance was set at 5%.

197 198 199 200 201 202 203 204 205 206

3.

Results

3.1. GSE

Growth and biofilm inhibition of S. mutans UA159 by

207 208

Both growth and biofilm formation of S. mutans UA159 were inhibited by GSE at 4 mg/mL. Sub-MBIC concentrations of GSE, 1–3 mg/mL, were chosen as test concentrations in this study.

209 210 211 212

3.2.

213

Polarized light microscopy analysis

Confocal laser scanning microscopy (CLSM)

Polarized light microscopy (PLM)

Artificial caries lesions, very similar to natural enamel caries lesions, were noted in all specimens examined by polarized light microscopy. Representative PLM photomicrographs are shown in Fig. 1. It was apparent that the S. mutans biofilminduced lesion depths differed among the treatment and control groups (Fig. 2). Average lesion depths of the 2 mg/mL GSE-treated group (122.86 13.41 mm) and the 3 mg/mL GSEtreated group (111.92 11.39 mm) were significantly less than those of the 1 mg/mL GSE-treated (198.33 17.70 mm) and non-treated control group (210.86 15.50 mm) ( p < 0.05). However, no statistically significant differences were noted in lesion depths between the 1 mg/mL GSE and control groups, or between the 3 mg/mL and 2 mg/mL GSE groups. No obvious lesion formation was observed in the NaF group.

214 215 216 217 218 219 220 221 222 223 224 225 226 227 228

3.3.

229

Confocal laser scanning microscopy (CLSM)

When samples were stained with rhodamine B and examined by CLSM, a red fluorescent band was observed (Fig. 3). The demineralization of enamel caries lesions formed by S. mutans biofilm was evident from the increase in fluorescence (or less optical intensity). The lesion depth confirmed by CLSM was consistent with that by PLM. No artificial caries lesion was observed in the 10-ppm-NaF group. The relative optical density (ROD) values of different treatment groups at different sites of the lesion are presented in Table 1 and Fig. 4. Significant differences were found among different treatment groups. Compared with the 2 mg/mL and 3 mg/ mL groups, the control and 1 mg/mL GSE groups showed


230 231 232 233 234 235 236 237 238 239 240 241

JJOD 2295 1–9

4


Lesion Depth (μm)

Fig. 1 – Polarized light microscopy photomicrographs of artificial enamel lesions in each group after 7days of in vitro S. mutans biofilm induction: (a) control group; (b) 1 mg/mL GSE; (c) 2 mg/mL GSE; (d) 3 mg/mL GSE; (e) 10 ppm NaF; and (f) blank control (L, lesion; E, sound enamel). 250

*

*

200 150

#

#

100 50 0 Control

1 mg/ml GSE

2 mg/ml GSE

3 mg/lml GSE

10-ppm-NaF

Group Fig. 2 – Effect of grape seed extract (GSE) on artificial enamel lesion depth as determined by polarized light microscopy. Different symbols (*, #) indicate statistically significant differences between the treatment group and control group (p < 0.05). Please cite this article in press as: Zhao W, et al. The preventive effect of grape seed extract on artificial enamel caries progression in a microbial biofilm-induced caries model. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.05.006


5

Fig. 3 – Representative confocal laser scanning microscopic images of artificial enamel caries induced by S. mutans biofilm grown in the presence of GSE (L, lesion; E, sound enamel). (a) Control; (b) 1 mg/mL GSE; (c) 2 mg/mL GSE; (d) 3 mg/mL GSE; (e) 10 ppm F; and (f) blank control.

242 243 244 245 246 247 248

lower ROD values when depths were less than 200 mm, indicating higher mineral loss ( p < 0.05). However, no significant difference in ROD values was detected between the control and 1 mg/mL GSE groups, or between the 2 mg/ mL and 3 mg/mL groups ( p > 0.05). At any lesion depth, the highest ROD value was always observed in the 10-ppmNaF group, revealing no obvious mineral loss. For other

groups, the highest values of ROD were observed at the lesion surface (depth = 10 mm), indicating the least mineral loss, which corresponded to the intact surface of the caries lesion. The lowest ROD values were observed at the body of the lesion (depth = 50 mm) in all GSE-treated groups and the control group, demonstrating the greatest mineral loss.


249 250 251 252 253 254 255

JJOD 2295 1–9

6


Table 1 – Relative optical densities (ROD) of artificial enamel lesions induced by S. mutans biofilm treated by GSE at different depths. Depth from the surface (mm)

Control (%)

Mean SD

Mean SD

10 20 50 80 110 140 170 200 230 250

65.45 12.28a 32.82 9.12a 20.54 4.59a 32.17 3.94a 40.93 10.27a 53.11 12.07a 86.94 4.85a 94.16 6.87a 96.19 5.87 96.59 5.72

58.61 13.63 a 31.33 6.65 a 23.70 10.40 a 27.36 9.47 a 44.28 9.70 a 62.60 15.09 a 87.79 6.80 a 93.71 6.10 a 96.18 3.94 96.47 3.63

1 mg/mL GSE (%)

2 mg/mL GSE (%)

3 mg/mL GSE (%)

10 ppm F (%)

Mean SD

Mean SD

Mean SD

72.08 7.99 b 58.48 7.68 b 33.62 8.78 b 60.05 3.39 b 80.55 6.18 b 97.31 3.30 b 98.94 1.64 b 99.88 1.25 b 99.58 1.38 99.49 1.43

76.97 9.47 b 71.68 10.77 c 38.66 6.48 b 72.34 3.04 c 90.34 3.79 bc 100 2.09 b 100 1.70 b 99.47 2.26 b 98.71 3.00 99.10 3.00

96.86 3.28 c 98.53 3.01 d 99.27 1.53 c 9943 1.42 d 100.65 1.44 c 100.28 1.00 b 99.72 0.88 b 100.13 1.05 b 100.06 1.18 99.68 0.96

ROD was calculated as ODR = ODl/ODs 100%; ODl was the OD of the lesion, and ODs was the OD of sound enamel tissue as a blank control at the same level. Different letters (a–d) indicate statistically significant differences in each row.

256

4.

In caries research, the chemical induction of caries in in vitro pH-cycling models is a common approach to investigation of the mechanisms involved in the de- and remineralization of enamel.9,36–40 However, direct acid exposure lacks the bacterial biofilm interactions that characterize caries formation in vivo. Many of the recent studies on de- and remineralization have demonstrated increased preferences in the microcosm biofilm models by subjecting enamel to microbial biofilminduced acid challenges that better simulate the oral environment.16,41,42 Since it is widely accepted that S. mutans plays an important role in the development of dental caries, the approach with in vitro S. mutans biofilm models to produce caries-like lesions has been accepted for testing the protective effects of various antimicrobial agents on the demineralization of caries lesions.43–46 To the best of our knowledge, fewer attempts have been made to study the effects of natural antimicrobials on caries progression in bacterial biofilm models. In the present study, enamel caries lesions with depths of 120–200 mm were observed in the 7-day S. mutans biofilm model, comparable with those obtained from previous pH-cycling and bacterial models.32,41 Although it is impossible for in vitro models to simulate completely the complex intraoral conditions associated with natural caries, the caries-like enamel lesions formed in this S. mutans biofilm model showed all of the principal histological features of natural caries and

may be used as a pre-clinical model for study of the protective properties of caries-preventive agents in enamel demineralization. Fluoride, delivered systemically or topically, has been used for decades successfully as an effective caries preventing agent by enhancing the remineralization of and inhibiting demineralization of the dental hard tissue.47 However, studies have shown that dental fluorosis may be associated with inappropriate levels of fluoride in infant formula or the drinking water.48,49 With the root hard tissues (dentine) being more prone to acid dissolution, fluoride use is often insufficient to prevent decay.50 To render caries prevention less dependent on fluoride, antimicrobial therapy has been considered as an alternative. In this regard, much attention has been given to chlorhexidine (CHX). CHX inhibits growth of specific cariogenic bacteria, acid production in plaque, and the demineralization of enamel in in situ model.51,52 It has been incorporated into mouthrinses, gels, toothpastes and varnishes. However, due to the current lack of evidence on long-term clinical outcomes and the reported side effects (e.g., teeth stating, altered taste sensation and transient oral microflora), CHX has not been the agent of choice for caries prevention.7,50 In recent years, there has been a popular demand for ‘‘natural alternatives’’ to the use of synthetic antimicrobial chemicals to provide oral health benefits. Effort has been expended to develop natural products as caries-preventive agents due to the increased incidence of resistant organisms

120

Control

100

ROD ( %)

257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281

Discussion

1 mg/ml GSE

80

2 mg/ml GSE

60

3 mg/ml GSE

40

10-ppm-NaF

20 0 0

50

100

150

200

250

300

Depth from the surface (μm) Fig. 4 – CLSM profile of each group and the schematic relative optical density vs. depth (mm) for the six experimental groups: control, inoculated BHIS; GSE, grape seed extract. Please cite this article in press as: Zhao W, et al. The preventive effect of grape seed extract on artificial enamel caries progression in a microbial biofilm-induced caries model. Journal of Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.05.006

282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309


310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369

and the adverse effects of existing therapies.13,21,33,53–55 Various plant-derived natural products have demonstrated the ability to inhibit the growth or biofilm formation of cariogenic organisms, especially S. mutans.8–13 However, the exact mechanisms and effectiveness of these antimicrobial phytochemicals on caries prevention are still unclear. Recently, the tea polyphenol epigallocatechin gallate (EGCG) was reported to inhibit the cariogenic virulence factors and biofilm formation of S. mutans by suppressing gtf genes.8,9,16,17 As a natural product, grape seed extract (GSE), high in proanthocyanidins, has demonstrated its potential oral health benefits.14,56–58 Bedran-Russo et al.28 used GSE to biomodify dentine matrices and reported the increased mechanical properties and stability of the tensile properties of dentine matrices. Their study suggested that GSE, by affecting collagen biochemistry and interacting with proteoglycans, enhanced the preventive and restorative/reparative abilities of dentine. Additional studies have also demonstrated that GSE positively affected the demineralization/remineralization processes of artificial root caries lesions and strengthened root dentine, suggesting its potential use in the prevention of and therapy for root caries.31,32 The antimicrobial properties of GSE against Gram-negative and Gram-positive bacteria have been reported ,29,30,59 and the high concentrations of flavonoids and their derivatives, and stilbenes, were thought to be responsible for the activity .60 At a concentration of 2 mg/mL, GSE inhibited oral pathogens including anaerobic periodontal bacteria, S. mutans, and other oral bacteria .13,61 In this study, the GSE at 4 mg/mL inhibited the growth and biofilm formation of S. mutans, in agreement with the results of previous studies. Since it is well-documented that bacterial biofilm cells are significantly more resistant to antimicrobials than are planktonic cells, a clinically more relevant microbial biofilm model should be utilized to assess the antimicrobial effect of GSE and its potential as a cariespreventive agent .33 At present, limited data are available regarding the effect of GSE on enamel caries progression in a bacterial biofilm model. Analysis of our data indicated that the antibiofilm activity of GSE against S. mutans, at sub-MIC levels, was reflected in the reduction of artificial enamel lesions in a dose-dependent manner (Fig. 2). In the S. mutans biofilm model, the caries-protective effect of GSE on early enamel caries lesions was demonstrated by the significant decreases in lesion depths and changes in ROD values in all treatment groups. The lesion depths and ROD vs. depth were related to the concentration of GSE. GSE at 2 mg/mL showed protective efficacy similar to that of the 3 mg/mL GSE-treated group. However, the depths of the lesions treated with 1 mg/ mL GSE were not statistically significantly different from those of the non-treated control, indicating that a concentration of 1 mg/mL is insufficient for enamel caries prevention. Analysis of our data supports the hypothesis that GSE inhibited S. mutans biofilm formation and growth, thereby reducing acid production and demineralization of enamel caries lesions. Different methods have been developed to measure de-/ re-mineralization in enamel .35,62 Transverse microradiography (TMR), considered to be the gold standard, is traditionally used to quantify mineral content, mineral changes, and

7

mineral distribution .63 In recent years, confocal laser scanning microscopy (CLSM) has been successfully used to evaluate artificial caries lesions .32,35,64,65 Several studies have compared TMR and CLSM for the measurement of mineral losses and concluded that the parameters measured by CLSM agreed well with those quantified by TMR, and that there are statistically significant correlations between them in enamel studies .62,66,67 CLSM is a new, non-destructive, 3dimensional technique for microscopic tomography, and samples can be morphologically observed after a single cutting-and-polishing procedure, thus avoiding the introduction of technical artefacts. In this investigation, the mineral changes in the S. mutans -induced artificial enamel caries lesions formed in the presence of different concentrations of GSE were determined by CLSM to measure the area of fluorescence. Lesion fluorescence depends upon the amount of fluorescent dye penetrating porosities of demineralized enamel, so the OD value is directly related to the porosity of the demineralized enamel in CLSM analysis. When mineral loss occurs, OD decreases as the porosity of enamel increases. Accordingly, relative optical density (ROD), calculated as ROD = ODl/ODs, should decrease with further demineralization. ROD values obtained in this study successfully demonstrated the different quantities of mineral lost from artificial bovine enamel caries treated by different concentrations of GSE at each depth, compatible with depth measures of PLM and the histological appearance of the lesion. On the basis of its precise data, we confirmed that CLSM is a valid method for the rapid quantification of mineral loss from the artificial enamel caries formed in a biofilm model, and then for comparison of the protective effects of dose-dependent anticaries agents on enamel demineralization. As a promising cariostatic agent, GSE may be incorporated into toothpaste, gel, varnish or mouthrinse, as with the topical application of fluoride and CHX. For example, a major polyphenol present in green tea, the epigallocatechin-3gallate (EGCG), has been added to a glass ionomer cement (GIC) which demonstrated improved antibacterial and physical properties.68 A recent study69 reported that topical use of grape seed extract before bonding procedures neutralized the effects of enamel bleaching, indicating that staining of the enamel may be a possible side effect which needs to be considered. For topical application of GSE, additional in vitro and in vivo data are needed to verify the outcomes. Based on results of this in vitro study, it can be concluded that GSE inhibits in vitro enamel caries formation due to its ability to suppress the growth and biofilm formation of S. mutans, thus reducing the biofilm mass and acid production. GSE at a concentration of 2 mg/mL significantly inhibits caries lesion depth and mineral loss, which may indicate its promising potential as a natural agent for enamel caries prevention. Future studies in a multi-species or microcosm biofilm model are warranted.

370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423

Conflict of interest

424

There were no conflicts of interest on the part of any authors or investigators.

425 427 426


JJOD 2295 1–9

8

428

429 430 431


Acknowledgement

20.

Q5 This study was supported by the Department of Pediatric Q6 Dentistry, University of Illinois at Chicago (UIC), College of

21.

Dentistry. 22.

432

references

433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488

1. Moynihan P, Petersen PE. Diet, nutrition and the prevention of dental diseases. Public Health Nutrition 2004;7:201–26. 2. Selwitz RH, Ismail AI, Pitts NB. Dental caries. Lancet 2007;369:51–9. 3. Beighton D. The complex oral microflora of high-risk individuals and groups and its role in the caries process. Community Dentistry and Oral Epidemiology 2005;33:248–55. 4. Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiological Reviews 1986;50:353–80. 5. van Houte J. Role of micro-organisms in caries etiology. Journal of Dental Research 1994;73:672–81. 6. Marsh PD. Antimicrobial strategies in the prevention of dental caries. Caries Research 1993;27(Suppl. 1):72–6. 7. Autio-Gold J. The role of chlorhexidine in caries prevention. Operative Dentistry 2008;33:710–6. 8. Cheng L, Exterkate RA, Zhou X, Li J, ten Cate JM. Effect of Galla chinensis on growth and metabolism of microcosm biofilms. Caries Research 2011;45:87–92. 9. Cheng L, ten Cate JM. Effect of Galla chinensis on the in vitro remineralization of advanced enamel lesions. International Journal of Oral Science 2010;2:15–20. 10. Nassar HM, Li M, Gregory RL. Effect of honey on Streptococcus mutans growth and biofilm formation. Applied and Environment Microbiology 2012;78:536–40. 11. Percival RS, Devine DA, Duggal MS, Chartron S, Marsh PD. The effect of cocoa polyphenols on the growth, metabolism, and biofilm formation by Streptococcus mutans and Streptococcus sanguinis. European Journal of Oral Sciences 2006;114:343–8. 12. Sasaki H, Matsumoto M, Tanaka T, Maeda M, Nakai M, Hamada S, et al. Antibacterial activity of polyphenol components in oolong tea extract against Streptococcus mutans. Caries Research 2004;38:2–8. 13. Smullen J, Koutsou GA, Foster HA, Zumbe A, Storey DM. The antibacterial activity of plant extracts containing polyphenols against Streptococcus mutans. Caries Research 2007;41:342–9. 14. Koo H, Duarte S, Murata RM, Scott-Anne K, Gregoire S, Watson GE, et al. Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on salivacoated apatitic surface and on dental caries development in vivo. Caries Research 2010;44:116–26. 15. Wu CD. Grape products and oral health. Journal of Nutrition 2009;139:1818S–23S. 16. Xu X, Zhou XD, Wu CD. The tea catechin epigallocatechin gallate suppresses cariogenic virulence factors of Streptococcus mutans. Antimicrobial Agents and Chemotherapy 2011;55:1229–36. 17. Xu X, Zhou XD, Wu CD. Tea catechin epigallocatechin gallate inhibits Streptococcus mutans biofilm formation by suppressing gtf genes. Archives of Oral Biology 2012;57:678–83. 18. Ferrazzano GF, Amato I, Ingenito A, De Natale A, Pollio A. Anti-cariogenic effects of polyphenols from plant stimulant beverages (cocoa, coffee, tea). Fitoterapia 2009;80:255–62. 19. Rivero-Cruz JZM, Kinghorn A, Wu CD. Antimicrobial constituents in Thompson seedless raisins (Vitis vinifera)

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

against selected oral pathogens. Phytochemistry Letters 2008;1:151–4. Signoretto C, Canepari P, Stauder M, Vezzulli L, Pruzzo C. Functional foods and strategies contrasting bacterial adhesion. Current Opinion in Biotechnology 2012;23:160–7. Wu CD. The impact of food components and dietary factors on oral health. Journal of Food and Drug Analysis 2012;20(Suppl. 1):5. Wu CD, Wei GX. Tea as a functional food for oral health. Nutrition 2002;18:443–4. Furiga A, Lonvaud-Funel A, Dorignac G, Badet C. In vitro anti-bacterial and anti-adherence effects of natural polyphenolic compounds on oral bacteria. Journal of Applied Microbiology 2008;105:1470–6. Ferrazzano GF, Amato I, Ingenito A, Zarrelli A, Pinto G, Pollio A. Plant polyphenols and their anti-cariogenic properties: a review. Molecules 2011;16:1486–507. Ferrazzano GF, Roberto L, Amato I, Cantile T, Sangianantoni G, Ingenito A. Antimicrobial properties of green tea extract against cariogenic microflora: an in vivo study. Journal of Medicinal Food 2011;14:907–11. Perumalla AVS, Hettiarachchy NS. Green tea and grape seed extracts—potential applications in food safety and quality. Food Research International 2011;44:827–39. Xia EQ, Deng GF, Guo YJ, Li HB. Biological activities of polyphenols from grapes. International Journal of Molecular Sciences 2010;11:622–46. Bedran-Russo AK, Castellan CS, Shinohara MS, Hassan L, Antunes A. Characterization of biomodified dentin matrices for potential preventive and reparative therapies. Acta Biomaterialia 2011;7:1735–41. Ahn J, Grun IU, Mustapha A. Effects of plant extracts on microbial growth, color change, and lipid oxidation in cooked beef. Food Microbiology 2007;24:7–14. Jayaprakasha GK, Selvi T, Sakariah KK. Antibacterial and antioxidant activities of grape (Vitis vinifera) seed extracts. Food Research International 2003;36:117–22. Pavan S, Xie Q, Hara AT, Bedran-Russo AK. Biomimetic approach for root caries prevention using a proanthocyanidin-rich agent. Caries Research 2011;45: 443–7. Xie Q, Bedran-Russo AK, Wu CD. In vitro remineralization effects of grape seed extract on artificial root caries. Journal of Dentistry 2008;36:900–6. Jeon JG, Rosalen PL, Falsetta ML, Koo H. Natural products in caries research: current (limited) knowledge, challenges and future perspective. Caries Research 2011;45:243–63. Wei GX, Xu X, Wu CD. In vitro synergism between berberine and miconazole against planktonic and biofilm Candida cultures. Archives of Oral Biology 2011;56:565–72. Gonzalez-Cabezas C, Fontana M, Dunipace AJ, Li Y, Fischer GM, Proskin HM, et al. Measurement of enamel remineralization using microradiography and confocal microscopy. A correlational study. Caries Research 1998;32:385–92. Alves KM, Franco KS, Sassaki KT, Buzalaf MA, Delbem AC. Effect of iron on enamel demineralization and remineralization in vitro. Archives of Oral Biology 2011;56:1192–8. Amaechi BT, Porteous N, Ramalingam K, Mensinkai PK, Ccahuana Vasquez RA, Sadeghpour A, et al. Remineralization of artificial enamel lesions by theobromine. Caries Research 2013;47:399–405. Huang S, Gao S, Cheng L, Yu H. Remineralization potential of nano-hydroxyapatite on initial enamel lesions: an in vitro study. Caries Research 2011;45:460–8. Ten Cate JM, Duijsters PP. Alternating demineralization and remineralization of artificial enamel lesions. Caries Research 1982;16:201–10.


489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557


558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613

40. White DJ. The application of in vitro models to research on demineralization and remineralization of the teeth. Advances in Dental Research 1995;9:175–93. discussion 94–7. 41. Shu M, Wong L, Miller JH, Sissons CH. Development of multi-species consortia biofilms of oral bacteria as an enamel and root caries model system. Archives of Oral Biology 2000;45:27–40. 42. Thneibat A, Fontana M, Cochran MA, Gonzalez-Cabezas C, Moore BK, Matis BA, et al. Anticariogenic and antibacterial properties of a copper varnish using an in vitro microbial caries model. Operative Dentistry 2008;33:142–8. 43. Azevedo MS, van de Sande FH, Romano AR, Cenci MS. Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges. Caries Research 2011;45: 510–7. 44. Seemann R, Bizhang M, Kluck I, Loth J, Roulet JF. A novel in vitro microbial-based model for studying caries formation–development and initial testing. Caries Research 2005;39:185–90. 45. Silva TC, Pereira AF, Exterkate RA, Bagnato VS, Buzalaf MA, Machado MA, et al. Application of an active attachment model as a high-throughput demineralization biofilm model. Journal of Dentistry 2012;40:41–7. 46. van de Sande FH, Azevedo MS, Lund RG, Huysmans MC, Cenci MS. An in vitro biofilm model for enamel demineralization and antimicrobial dose-response studies. Biofouling 2011;27:1057–63. 47. ten Cate JM. Contemporary perspective on the use of fluoride products in caries prevention. British Dental Journal 2013;214:161–7. 48. Yeung CA. A systematic review of the efficacy and safety of fluoridation. Evidence-Based Dentistry 2008;9:39–43. 49. Santos AP, Oliveira BH, Nadanovsky P. Effects of low and standard fluoride toothpastes on caries and fluorosis: systematic review and meta-analysis. Caries Research 2013;47:382–90. 50. ten Cate JM. The need for antibacterial approaches to improve caries control. Advances in Dental Research 2009;21:8–12. 51. Kim SS, Kim S, Kim E, Hyun B, Kim KK, Lee BJ. Synergistic inhibitory effect of cationic peptides and antimicrobial agents on the growth of oral streptococci. Caries Research 2003;37:425–30. 52. Ribeiro CC, Ccahuana-Vasquez RA, Carmo CD, Alves CM, Leitao TJ, Vidotti LR, et al. The effect of iron on Streptococcus mutans biofilm and on enamel demineralization. Brazilian Oral Research 2012;26:300–5. 53. Otake S, Makimura M, Kuroki T, Nishihara Y, Hirasawa M. Anticaries effects of polyphenolic compounds from Japanese green tea. Caries Research 1991;25:438–43. 54. Koo H, Pearson SK, Scott-Anne K, Abranches J, Cury JA, Rosalen PL, et al. Effects of apigenin and tt-farnesol on glucosyltransferase activity, biofilm viability and caries development in rats. Oral Microbiology and Immunology 2002;17:337–43.

55. Wu CD, Wei GX. Tea as a functional food for oral health. In: Michael Wilson, editor. Food constituents and oral health: current status and future prospect. Cambridge: Woodheading Publishing; 2009. p. 396–417. 56. Castellan CS, Bedran-Russo AK, Karol S, Pereira PN. Longterm stability of dentin matrix following treatment with various natural collagen cross-linkers. Journal of the Mechanical Behavior of Biomedical Materials 2011;4:1343–50. 57. Castellan CS, Pereira PN, Grande RH, Bedran-Russo AK. Mechanical characterization of proanthocyanidin-dentin matrix interaction. Dental Materials 2010;26:968–73. 58. Dos Santos PH, Karol S, Bedran-Russo AK. Long-term nanomechanical properties of biomodified dentin-resin interface components. Journal of Biomechanics 2011;44:1691–4. 59. Cueva C, Mingo S, Munoz-Gonzalez I, Bustos I, Requena T, del Campo R, et al. Antibacterial activity of wine phenolic compounds and oenological extracts against potential respiratory pathogens. Letters in Applied Microbiology 2012;54:557–63. 60. Anastasiadi M, Chorianopoulos NG, Nychas GJ, Haroutounian SA. Antilisterial activities of polyphenol-rich extracts of grapes and vinification byproducts. Journal of Agricultural and Food Chemistry 2009;57:457–63. 61. Furiga A, Lonvaud-Funel A, Badet C. In vitro study of antioxidant capacity and antibacterial activity on oral anaerobes of a grape seed extract. Food Chemistry 2009;113:1037–40. 62. Fontana M, Li Y, Dunipace AJ, Noblitt TW, Fischer G, Katz BP, et al. Measurement of enamel demineralization using microradiography and confocal microscopy. A correlation study. Caries Research 1996;30:317–25. 63. Arends J, Dijkman T, Christoffersen J. Average mineral loss in dental enamel during demineralization. Caries Research 1987;21:249–54. 64. de Carvalho FG, Puppin-Rontani RM, Soares LE, Santo AM, Martin AA, Nociti-Junior FH. Mineral distribution and CLSM analysis of secondary caries inhibition by fluoride/MDPBcontaining adhesive system after cariogenic challenges. Journal of Dentistry 2009;37:307–14. 65. Min JH, Kwon HK, Kim BI. The addition of nano-sized hydroxyapatite to a sports drink to inhibit dental erosion: in vitro study using bovine enamel. Journal of Dentistry 2011;39:629–35. 66. Meyer-Lueckel H, Paris S. Progression of artificial enamel caries lesions after infiltration with experimental light curing resins. Caries Research 2008;42:117–24. 67. Ogaard B, Duschner H, Ruben J, Arends J. Microradiography and confocal laser scanning microscopy applied to enamel lesions formed in vivo with and without fluoride varnish treatment. European Journal of Oral Sciences 1996;104:378–83. 68. Hu J, Du X, Huang C, Fu D, Ouyang X, Wang Y. Antibacterial and physical properties of EGCG-containing glass ionomer cements. Journal of Dentistry 2013;41:927–34. 69. Vidhya S, Srinivasulu S, Sujatha M, Mahalaxmi S. Effect of grape seed extract on the bond strength of bleached enamel. Operative Dentistry 2011;36:433–8.

9 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670


The effect of ozone on progression or regression of artificial caries-like enamel lesions in vitro.

Remineralizing Effect of Child Formula Dentifrices on Artificial Enamel Caries Using a pH Cycling Model.

Effect of xylitol varnishes on remineralization of artificial enamel caries lesions in vitro.

Caries-preventive fluoride mouthrinses.

Caries-preventive salt fluoridation.

Caries-preventive fluoride dentifrices.

Fatty acid effect on sucrose-induced enamel demineralization and cariogenicity of an experimental biofilm-caries model.

Influence of posteruptive age of enamel on its susceptibility to artificial caries.

Caries-preventive effect of two concentrations of stannous fluoride mouthrinse.

Effect of intensified caries prevention on approximal caries in adolescents with high caries risk.

Beneficial clinical effects of grape seed proanthocyanidin extract on the progression of carotid atherosclerotic plaques.

fluoride complexes on fluoride uptake and caries-like lesion formation in enamel.

Preventive dentistry: Early childhood caries in infants.

Comparison of the initial streptococcal microflora on dental enamel in caries-active and in caries-inactive individuals.

Remineralization Potential of Three Tooth Pastes on Enamel Caries.

The effect of grape seed extract on estrogen levels of postmenopausal women: a pilot study.

The effect of Medicaid primary care provider reimbursement on access to early childhood caries preventive services.

Effect of Enamel Caries Lesion Baseline Severity on Fluoride Dose-Response.

Inhibitory effect of liposomal solutions of grape seed extract on the formation of heterocyclic aromatic amines.

Effects of fluoride on caries development and progression in vivo.

The effect of phenytoin medication on dentin apposition, root length, and caries progression in rat molars.

Effect of Grape Seed Extract on Lipid Profile and Expression of Interleukin-6 in Polycystic Ovarian Syndrome Wistar Rat Model.

May caries-preventive fluoride regimes have an effect on dental erosive wear? An in situ study.

Clinical testing of a topical fluoride caries preventive programme.