MICROSCOPY RESEARCH AND TECHNIQUE 77:586–593 (2014)
In Situ Analysis of CO2 Laser Irradiation on Controlling Progression of Erosive Lesions on Dental Enamel TAISA PENAZZO LEPRI,1* RENATA SIQUEIRA SCATOLIN,1 VIVIAN COLUCCI,1 ADILIS KALINA DE ALEXANDRIA,2 LUCIANNE COPLE MAIA,2 CECILIA PEDROSO TURSSI,3 AND SILMARA APARECIDA MILORI CORONA1 1 Department of Restorative Dentistry, School of Dentistry of Ribeir~ ao Preto, University of S~ ao Paulo (USP) Avenida do Cafe, S/N Monte Alegre CEP: 14040-904 Ribeir~ ao Preto—SP, Brazil 2 Department of Pediatric and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro (UFRJ) Rua Prof., Rodolpho Paulo Rocco 325 CEP 21941-913 Cidade Universit aria—Rio de Janeiro—RJ, Brazil 3 Department of Restorative Dentistry, S~ ao Leopoldo Mandic Institute and Dental Research Center, Av. Jose Rocha Junqueira, 13 CEP 13045-755 Campinas—SP, Brazil
KEY WORDS
enamel; erosion; citric acid; CO2 laser
ABSTRACT The present study aimed to evaluate in situ the effect of CO2 laser irradiation to control the progression of enamel erosive lesions. Fifty-six slabs of bovine incisors enamel (5 3 3 3 2.5 mm3) were divided in four distinct areas: (1) sound (reference area), (2) initial erosion, (3) treatment (irradiated or nonirradiated with CO2 laser), (4) final erosion (after in situ phase). The initial erosive challenge was performed with 1% citric acid (pH 5 2.3), for 5 min, 23/day, for 2 days. The slabs were divided in two groups according to surface treatment: irradiated with CO2 laser (k 5 10.6 mm; 0.5 W) and nonirradiate. After a 2-day lead-in period, 14 volunteers wore an intraoral palatal appliance containing two slabs (irradiated and nonirradiated), in two intraoral phases of 5 days each. Following a cross-over design during the first intraoral phase, half of the volunteers immersed the appliance in 100 mL of citric acid for 5 min, 33/day, while other half of the volunteers used deionized water (control). The volunteers were crossed over in the second phase. Enamel wear was determined by an optical 3D profilometer. Three-way ANOVA for repeated measures revealed that there was no significant interaction between erosive challenge and CO2 laser irradiation (P 5 0.419). Erosive challenge significantly increased enamel wear (P 5 0.001), regardless whether or not CO2 laser irradiation was performed. There was no difference in enamel wear between specimens CO2-laser irradiated and non-irradiated (P 5 0.513). Under intraoral conditions, CO2 laser irradiation did not control the progression of erosive lesions in enamel caused by citric acid. Microsc. Res. Tech. 77:586–593, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION The new dietary habits of population, with frequent consumption of acidic beverages (Ehlen et al., 2008; Lussi et al., 2004) has led to a higher occurrence of pathological tooth wear (Johansson et al., 2002; Zero and Lussi, 2005), such as erosion (Bartlett et al., 2011; Lussi and Jaeggi, 2008). In dental erosion, the contact of the dental surfaces with acid of nonbacterial origin results in an irreversible tissue loss (Featherstone and Lussi, 2006). This pathology can be caused by intrinsic acids, as recurrent vomiting or regurgitation of gastric contents (Bartlett, 2006), or extrinsic factors (Lussi and Jaeggi, 2006), with the frequent intake of beverages identified as a major cause of dental erosion (Jager et al., 2012). In an attempt to prevent the occurrence of erosive lesions or to minimize their resulting damage, various strategies have been proposed and include reduction of acidic challenges and increase of acid resistance of dental hard tissues (Hjortsj€o et al., 2010; Magalh~ aes et al., 2008; Ramalho et al., 2013; Wiegand et al., 2010a). These strategies includes the lasers, among C V
2014 WILEY PERIODICALS, INC.
which the CO2 laser is widely used in dentistry for having wavelengths more effective in dental enamel (Esteves-Oliveira et al., 2008; Fried et al., 1997; Tsai el al., 2002), making it significantly less soluble to acids (Featherstone et al., 1998). It has been stated that the chemical and structural changes caused on dental surface by CO2 laser irradiation are responsible for the increase of acid resistance on enamel (Featherstone, 2000). Besides, the matrix of enamel is partially denatured and its permeability decreased after CO2 laser irradiation, which hampers the diffusion of acid into the pores of the substrate, reducing demineralization (Ramalho et al., 2013; Souza-Gabriel et al., 2010; Tepper et al., 2004).
*Correspondence to: Taısa Penazzo Lepri, Alameda Itu, 285 Ap 141 – S~ ao Paulo – SP, CEP 01421-000, Brazil. E-mail: [email protected] Received 26 March 2014; accepted in revised form 1 May 2014 REVIEW EDITOR: Prof. Alberto Diaspro Contract grant sponsor: State of S~ ao Paulo Research Foundation (FAPESP); Contract grant number: 2010/19247-8. DOI 10.1002/jemt.22377 Published online 13 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
CO2 LASER TO CONTROL ENAMEL EROSIVE LESIONS
In the context of dental erosion, it has already been demonstrated by in vitro (Esteves-Oliveira et al., 2012; Ramalho et al., 2013), and in situ studies associated with different fluorides (Wiegand et al., 2010b) that the CO2 laser can reduce enamel mineral loss. However, no study has investigated the influence of CO2 laser in preventing progression of dental erosion in enamel previously eroded. Thus, this study was designed in order to evaluate in situ the effect of CO2 laser in controlling the progression of erosive lesions on dental enamel. MATERIAL AND METHODS Experimental Design This study was a two-period crossover design. Each phase lasted for 5 days with a washout period of 15 days between them. The factors under evaluation were erosive challenge at two levels [(A) 1% citric acid pH 2.3 and (B) deionized water—control)] and CO2 laser irradiation at two levels [(I) present and (II) absent]. Volunteers wore slabs irradiated or not, and in alternate and independent phases, subjected them to erosive challenges or deionized water (control). Each group consisted of 14 slabs, randomly assigned to the 14 volunteers, which were considered as statistical blocks. The response variable was the measurement of wear in three distinct moments: after initial erosion, after treatment (irradiated or not with CO2 laser) and after in situ phase. The wear was quantitatively measured by optical profilometry and qualitatively by scanning electron microscopy (SEM) analysis. The experimental design is schematized in the flowchart (Fig. 1). Selection of Volunteers Fourteen volunteers aged 22–40 years who fulfilled the inclusion criteria (normal salivary flow, no evidence of active cavity and non-carious lesions and willing to follow the research schedule) without violating the exclusion criteria (use of any form of medication likely to interfere with salivary secretion, use of fixed or removable orthodontic appliances, pregnancy or breastfeeding, general/systemic illness) were enrolled. The experimental procedures used were performed with the informed consent of the subjects, following protocols reviewed and approved by the Ethics Committee of the Faculty of Dentistry of Ribeir~ ao Preto, University of S~ ao Paulo, Brazil (CAAE 02110512.2.0000.5419). Impression of maxillary arch was taken on each volunteer using a stock tray. Impression was poured in dental stone, and an upper removable appliance of acrylic resin (Jet, Artigos Odontologicos Cl assico, S~ ao Paulo, SP, Brazil) was constructed. Appliances had two retention slots (7 3 7 3 3 mm3), one on each side of the midline, which accommodated two slabs facing the oral cavity. Preparation of Enamel Slabs Freshly extracted bovine incisors stored in a 0.1% thymol solution at 4 C were cleaned with a scaler and water/pumice slurry in dental prophylactic cups Microscopy Research and Technique
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and examined under 203 magnification using a stereomicroscope (Leica S6 D Stereozoom, Mycrosystems Leica AG, Switzerland). The teeth presenting cracks and hypoplasia were discarded. Roots were removed 2 mm below the cement-enamel junction using a low-speed water-cooled diamond saw (Isomet 1000; Buehler, Lake Bluff, IL). After, crowns were sectioned, resulting in two enamel slabs per tooth (5 3 3 3 2.5 mm3). The slabs were flattened and polished on a watercooled polishing machine (Phoenix b, Buehler, Lake Bluff, IL) with the aid of an apparatus that standardize the procedure. First, the enamel fragments were fixed in teflon matrix with the aid of a parallelometer (ElQuip, S~ ao Carlos, S~ ao Paulo, Brazil) to flatten the pulp wall, with 600-grit of Al2O3 papers (Norton Abrasivos, SP, Brazil) applied for 20 s with standardized load of 20 N. After flattening the pulp wall, slabs were removed from the matrix and again fixed for flatten and polishing the enamel surface, with 600 and 1200grit Al2O3 papers (Norton Abrasivos, SP, Brazil) with standardized load of 20N for 20 s and 0.3 mm alumina suspension (Buehler, Lake Bluff, IL) with standardized load of 20N for 60 s. Selection of Slabs Before initial erosive-like lesion formation, slabs were sterilized by microwave irradiation (Viana et al., 2010). After sterilization, slabs with cracks were discarded. Slabs were then pretested for Knoop microhardness (KHN) (HMV-2000, Shimadzu Corporation, Japan) by making three indentations at the center of the specimen, spaced 100 mm from each other, under 25-g load for 5 s (Magalh~ aes et al., 2007). The three readings were averaged and used as the microhardness values of each slab. Specimens with microhardness values 10% above or below the mean (300% 6 10%) were excluded, remaining 56 slabs. Specimens were coated with acid-resistant nail varnish (Colorama, Maybelline, Brazil) in three layers, except on the buccal surface, which remained uncoated. The slabs were divided in four distinct areas (1) sound, (2) initial erosion, (3) treatment (irradiated or not irradiated with CO2 laser), (4) final erosion (after the in situ phase), as presented in Figure 2. The sound region was considered as the reference area of each slab (1.25 3 3 mm2). The first area (1) was covered with resin composite, applied on non etched surface and in the absence of adhesive system. Erosion-like Lesion Formation To create initial erosive lesions, specimens were subjected to erosive challenges twice a day (at 9 a.m and 1 p.m) for 2 days. Each challenge consisted of individual immersion in 20 mL of citric acid 1% (pH 2.3) in an Erlenmeyer flask, which was then placed in an orbital shaker (CT155, Cientec, Piracicaba, SP, Brazil) with a stirring velocity of 100 rpm for 5 min (Amaechi et al., 1999). After each erosive challenge, specimens were rinsed for 10 s with deionized water and stored in 10 mL of
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Fig. 1. A. Removal of bovine incisor root 2 mm below the cementoenamel junction; B. Section crowns; C. Two enamel slabs (5 3 3 3 2.5 mm3); D. Planning and polishing; E. Initial microhardness measurements (baseline); F. Delimitation of the first reference area (not eroded) 1.5 3 3 mm2; G. Initial erosive challenge (formation of erosive lesion); H. Storage at 37 C; I. Delimitation of the second reference area (50% of the specimen covered); J. CO2 laser irradiation; K.
Delimitation of the third reference area (75% of the specimen covered); L. Mounting of slabs at the palatal devices; M. Ex vivo erosive challenges alternating; N. Removal of the delimited reference areas; O. Optical 3D profilometry analysis; P. Scanning electron microscopy analysis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
artificial saliva at 37 C for the 4-h interval between the erosive challenges as well as overnight. The artificial saliva (pH 5 7.0), which was changed before the first challenge of each day, was similar to that described by McKnight-Hanes and Whitford (1992) and modified by Amaechi et al.,1999. It was composed of methyl-p-hydroxybenzoate (2.0 g), sodium carboxymethylcellulose (10.0 g), KCl (0.625 g), MgCl2.6H2O (0.059 g), CaCl2.2H2O (0.166 g), K2HPO4
(0.804 g); and KH2PO4 (0.326 g) in 1000 mL of water solution. Surface Treatment After initial erosion-like lesion formation, the delimited area 2 (Fig. 2) of the specimen was also protected with a resin composite without etching or applying adhesive system. Eroded slabs were randomly Microscopy Research and Technique
CO2 LASER TO CONTROL ENAMEL EROSIVE LESIONS
Fig. 2. Schematic enamel specimens. A. Sound area (control); B. Initial erosion; C. Treatment (irradiated or not); D. after in situ phase. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
assigned into two groups according to the CO2 laser irradiation: (I) present and (II) absent. Specimens were irradiated with a PC015-A (Shanghai Jue Hua Laser Tech. Development, Shangai, China) in an ultrapulse and noncontact mode. The irradiation was performed unfocused 4-mm distant from the enamel surface (Tepper et al., 2004) with the aid of a device that sets the pen during the laser irradiation, which moves the second commands previously established through a computer connected to the scanning machine (MPC ElQuip, S~ ao Carlos, SP, Brazil), allowing the radiation to reach the entire uncovered area (delimited area 3 and 4) evenly. Each specimen was irradiated during 10 s, with duty time of 100 ms and idle time of 0.001 s. The set of parameters employed were: 10.6 lm, 0.5 W, average output 0.44 W, pulse energy of 0.05 mJ and energy density of 0.04 J cm22. After achieving the surface treatments, using the same protocol described above, a layer of composite resin was placed on area 3 to protect this surface from further erosive challenges to which the area 4 was exposed (Fig. 2). Mounting of the Slabs in Intra-Oral Devices Volunteers wore slabs treated with CO2 laser and nonirradiated in both experimental phases, changing the erosive challenges (citric acid or control) in each phase. Two enamel sections—one irradiated and one nonirradiated—were placed in each device. The slab fixation was performed with wax, 1-mm below the edge of the palatal appliance to prevent abrasion by contact with surface of the tongue. In Situ Erosive Challenges During a 2-day lead-in period, the volunteers were instructed to brush their teeth only with the toothpaste (Colgate Maximum Anticaries Protection, Colgate-Palmolive, Division of Kolynos from Brazil, Osasco, SP, Brazil) and toothbrush (Colgate Extra Clean, Colgate-Palmolive, Division of Kolynos from Brazil, Osasco, SP, Brazil) supplied by the researchers. These challenges began only on the second day of use of intraoral device to allow formation of acquired pellicle (Huysmans et al., 2011). A 15-day wash-out period was allowed between the two phases. Microscopy Research and Technique
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In the first phase, half of the volunteers immersed the oral appliance in 100 mL of citric acid solution (1%, pH 5 2.3), outside the mouth, during 5 min, three times/day, for a 4-day period. After the challenge, slabs were rinsed for 20 s under tap water and reinserted in the mouth. The other half of volunteers followed the same sequence, employing, however, deionized water instead of citric acid. In both experimental phases, the use of the devices was continuous, including during night time, with exception of meals time, beverage consumption and oral hygiene procedures (Scatolin et al., 2012). To avoid dehydration of specimens during these periods, the appliance was covered with humid cotton gauze. The control of the biofilm on the surface of the specimens was carried out at the end of each experimental day, dripping chlorhexidine 0.2% (Bioquanti Compounding Pharmacy, Ribeir~ ao Preto, SP, Brazil) on the fragments, for one minute, and rinsing with tap water, according to the protocol described by West et al. 1998. 3D Optical Noncontact Profilometry At the end of the in situ experiment, all the composite resin was removed, and the specimens had their four areas exposed (Fig. 2): sound (control), initial erosion, treatment (irradiated or nonirradiated), after in situ phase. The determination of topographic change was made with the aid of an optical 3D non-contact profilometer (PS50 Optical Profilometer, Nanovea, Irvine, CA). This device allowed a scanning over an area of 4 mm in length (x-axis) by 1.5 mm in width (y-axis). The measurements of capture were performed by a chromatic confocal sensor with white light axial source, scan velocity of 2 mm s21 and refraction index of 10.000. The 3D noncontact profilometry technique was used to determine tooth structure loss—erosion depth (ED). The ED was calculated from the step height (in lm). Three linear measurements were made involving the following areas: (1) between sound area and initial erosion area; (2) between sound area and treatment area; (3) between sound area and final in situ phase area. All measurements were done in triplicate and the mean values. SEM Analysis After profilometry analysis, three specimens were randomly selected from each group (n 5 3) to the imaged under SEM. Specimens were stored in Eppendorff flasks with deionized water and cleaned in an ultrasound apparatus (Ultrasonic Cleaner, Odontobras, Ribeir~ ao Preto, SP, Brazil) for 10 min. The specimens were then immersed in 2.5% glutaraldehyde solution buffered with 0.1 M sodium cacodylate (Merck KGaA, Darmstadt, D-64293, Germany) for 12 h at 4 C. After fixation, the specimens were washed in deionized water for 3 min, immersed in deionized water for 1 h, changing the water every 20 min. Subsequently, the specimens were dehydrated with increasing percentage of ethanol (Labsynth, Diadema, SP, Brazil), namely 25% (20 min), 50% (20 min), 75% (20 min), 95% (30 min), and 100% (60 min) and dried with absorbing paper. After that, specimens were fixed on stubs with double-faced carbon tape (3M, S~ ao
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TABLE 1. Means and standard deviations of wear values (mm) presented by each group CO2 laser irradiation 3 challenge Irradiated Non-irradiated Grand mean
Citric acid
Deionized water
36.04 (15.56) 35.99 (17.94) 36.01 (16.48) B
20.12 (11.81) 16.38 (4.80) 18.25 (9.05) A
Grand means followed by different letters indicate a significant difference.
Paulo, SP, Brazil), covered with a 30 lm gold–platinum layer (Bal-Tec SCD 005, Zurich, Switzerland) in a vacuum apparatus (SDC 050, Balzers, Liechtenstein). Specimens were examined with a SEM (JEOL JSM model 5410; Jeol Technic, Tokyo, Japan). Initially, the specimens were analyzed in a panoramic vision, and latter, photomicrographs of the most representative area of each group were accomplished at a standardized magnification (3,0003) (Fig. 4). Statistical Analysis Three-way ANOVA for repeated measure was used to examine effects of the independent variables CO2 laser irradiation, Erosive Challenge and Volunteers and their interaction on surface loss (dependent variable). Significance level was set at 5% and statistical calculations were performed using SPSS 20 (SPSS, Chicago, IL). RESULTS Three-way ANOVA for repeated measures revealed that there was no significant interaction between ero-
sive challenge and CO2 laser irradiation (P 5 0.419). Erosive challenge significantly increased enamel wear (P 5 0.001), regardless whether or not CO2 laser irradiation was performed. There was no difference in enamel wear between specimens CO2-laser irradiated and those left non-irradiated (P 5 0.513). Volunteer factor showed no significant effect (P 5 0.544) (Table 1). By paired t tests, applied to the initial erosion 3 post treatment conditions, regardless of the challenge that would be further applied, it was noted that the irradiated substrates (P 5 0.109), and nonirradiated specimens (P 5 0.631), were not significantly worn. Using this same test, when compared the samples submitted to the challenge with deionized water, it was noted that neither irradiated (P 5 0.062), nor the nonirradiated (P 5 0.170) specimens presented significant increased wear in relation to the initial erosion. Figure 3 shows the 3D profilometry images of experimental groups, in which it can be observed the enamel wear between the different analyzed areas: sound area and initial erosion area; sound area and treatment area; sound area and in situ phase area. In Figure 4, SEM images of a representative sample from each group show different phases of the experiment. Image A shows the sound enamel surface (control). The image of uncovered enamel prisms after in vitro erosive challenges can be seen at Image B. Irradiated enamel surface (Image C) shows some areas where the CO2 laser did not present an interaction with the enamel and areas where the laser irradiation provided a protective layer (circled area). The images
Fig. 3. The 3D noncontact optical profilometry images: A. Irradiated with CO2 laser1 citric acid; Birradiated with CO2 laser1 deionized water; C. Nonirradiated 1 citric acid; D. Nonirradiated 1 deionized water. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Microscopy Research and Technique
CO2 LASER TO CONTROL ENAMEL EROSIVE LESIONS
Fig. 4. Scanning electron microscopy images. A. Sound surface (control); B. Initially eroded surface; C. Surface irradiated with CO2 laser; D. Nonirradiated surface; E. Surface irradiated with CO2 laser and eroded at the in situ phase; F. Nonirradiated surface and eroded at in situ phase; G. Surface irradiated and noneroded at in situ phase; H. Nonirradiated surface and noneroded at the in situ phase.
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that presents the enamel slightly overcast by a protective layer as exclusive of irradiated surfaces, as shown at Image E (irradiated and eroded enamel). Image F, which represents the eroded enamel, presents a marked etched-like aspect, with huge exposition of the enamel prisms, representing a high degree of enamel dissolution. The irradiated but not eroded enamel, presented at Image G, shows small enamel dissolution, preserving the enamel prisms. Image H, representative of a specimen that was neither irradiated nor eroded in situ, shows moderate enamel demineralization, due exclusively to the in vitro erosive challenges. DISCUSSION Given the increased incidence of dental erosion (Jaeggi and Lussi, 2006) it is necessary, besides the search for preventive measures, treatments to minimize progression and damage already established on eroded enamel. The CO2 laser produces radiation at the infrared spectrum region very close to the apatite absorbing bands (Featherstone and Nelson, 1987; Featherstone et al., 1998; Nelson et al., 1987) leading to greater light absorption on the enamel and conversion into heat. When hydroxyapatite is heated at a temperature higher than 400 C, it’s possible to obtain a decrease or complete elimination of carbonate from the enamel surface, thus increasing the acid resistance (McCormack et al., 1995; Zuerlein et al., 1999). The results from the present study showed that, by means of the methodology employed, it was found no significant interaction of erosive challenge and CO2 laser irradiation, in other words, the treatment with CO2 laser at a wavelength 10.6 mm was not able to control the enamel wear caused by citric acid when evaluate by 3D optical profilometry. At the SEM images, the surface of non-irradiated specimens after the erosive challenges was similar to the non-irradiated and non-eroded with the enamel prisms slightly exposed. The irradiated specimens preserved its most superficial enamel layer. After the in situ phase, the specimens that were irradiated and eroded presented a more homogeneous surface when compared to the nonirradiated and eroded. Aiming an increase of enamel acid resistance, studies have suggested that, when irradiated, the enamel temperature must range between 600 and 900 C, thus leading to changes like increase of hydroxyapatite crystals (Ferreira et al., 1989; McCormack et al., 1995), melting (Ferreira et al., 1989; Kuroda and Fowler 1984; McCormack et al., 1995; Meurman et al., 1992; Nelson et al., 1987; Tsai et al., 2002), fusion and recristalization (Kantola, 1972). In the present study, the employ of lowest pulse duration and lowest energy density (0.04 J cm22), which cause less morphological changes, may not have been adequate to produce the expected effect. These parameters were employed aiming to provide lower heat propagation to the inner tooth layers, thus decreasing the risk of pulpal damage (Fried et al., 1996). Esteves Oliveira et al., 2011 and Ramalho et al., 2013, employing higher energy densities (0.3 J cm22), found a reduction on the enamel wear after erosion, and decrease of mineral loss and a rehardening of the eroded enamel, respectively. In an attempt to simulate the treatment of previously eroded enamel, in the present study we performed erosive challenges in vitro (two times a day, 2
days) prior to CO2 laser irradiation, differently from what happens in most studies of enamel erosion (Esteves-Oliveira et al., 2011; Ramalho et al., 2013; Steiner-Oliveira et al., 2010; Wiegand et al., 2010a), which search for the prevention of a non-existent erosive lesion instead of the control of a already established lesion. Besides, the exposure time of specimens to erosive challenges may have been too long (5 min of exposure, 33 a day, during 4 days), so that the possible positive effects of laser not persisted for this long. Most of the acidic beverages ingested by population contain citric acid, so its use is already validated in studies of dental erosion (West, 1998). Some of these beverages appear to be more erosive than other, with the same pH. As the citric acid comes in contact with the tooth surface, through its chelating properties it is capable to increase the erosive process in vivo by interaction with saliva, lowering the pH of the oral cavity (West, 2001), increasing the softening and dissolving the mineral content of the enamel (Meurman, 1996). In the present study, using the topographic profile to evaluate the reduction on the progression of erosive lesions, only structural changes were evaluated, and these changes, such as irregularities, may have masked a possible positive action of the CO2 laser irradiation related to chemical changes, which could increase the enamel resistance to further erosive challenges. Among the available analyses to quantify the dental hard tissue loss due to erosion, particular attention is dedicated to the profilometry, since it’s easily performed, capable to assess initial changes on the surface and also the tissue loss at advanced stages. This analyses is indicate to evaluated the enamel while the changes on the subsurface mineral contents are relatively small (Ganss et al., 2009), and the noncontact profilometer minimizes possible errors on the results when analyzing softened surfaces (Barbour and Rees, 2004). The employ of profilometry was validated for erosion (Ganss et al., 2005), being considered a “gold standard” (Hall et al., 1997). In the present study, to prepare the specimens for quantifying wear by profilometry, the enamel surface was planed and polished perpendicular (at 90 ) to enamel prisms in order to achieve evenly surfaces, as recommended by Hjorstj€o (2010). Nevertheless, due to the curvature of the teeth, this may not always occur, and specimens may have differently exposed enamel prisms, thus affecting their acid susceptibility (Ganss, 2000; Rios, 2006). Thus, considering the methodology and parameters employed on this study, it can be concluded that CO2 laser irradiation was unable to reduce in situ the progression of erosive lesions on enamel caused by citric acid. ACKNOWLEDGEMENT The authors thank the Federal University of Rio de Janeiro, for authorization to use the 3D optical profilometer. REFERENCES Amaechi BT, Higham SM, Edgar WM, Milosevic A. 1999. Thickness of acquired salivary pellicle as a determinant of the sites of dental erosion. J Dent Res 78:1821–1828. Barbour ME, Rees JS. 2004. The laboratory assessment of enamel erosion: A review. J Dent 32:591–602.
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CO2 LASER TO CONTROL ENAMEL EROSIVE LESIONS Bartlett D. 2006. Intrinsic causes of erosion. Monogr Oral Sci. Basel Karger 20:119–139. Bartlett DW, Fares J, Shirodaria S, Chiu K, Ahmad N, Sherriff M. 2011. The association of tooth wear, diet and dietary habits in adults aged 18–30 years old. J Dent 39:811–816. Ehlen LA, Marshall TA, Qian F, Wefel JS, Warren JJ. 2008. Acidic beverages increase the risk of in vitro tooth erosion. Nutr Res 28: 299–303. Esteves-Oliveira M, Apel C, Gutknecht N, Velloso WF, Jr, Cotrim MEB, Eduardo PC, Zezell DM. 2008. Low-fluence CO2 laser irradiation decreases enamel solubility. Laser Phys 18:478–485. Esteves-Oliveira M, Pasaporti C, Heussen N, Eduardo CP, Lampert F, Apel C. 2011. Rehardening of acid-softened enamel and prevention of enamel softening through CO2 laser irradiation. J Dent 39: 414–421. Esteves-Oliveira M, Yu H, de Paula Eduardo C, Meister J, Lampert F, Attin T, Wiegand A. 2012. Screening of CO2 laser (10.6 lm) parameters for prevention of enamel erosion. Photomed Laser Surg 30:331–338. Featherstone JDB. 2000. Caries detection and prevention with laser energy. Dent Clin North Am 44:955–969. Featherstone JDB, Lussi A. 2006. Understanding the chemistry of dental erosion. Monogr Oral Sci Basel Karger 20:66–76. Featherstone JD, Nelson DG. 1987. Laser effects on dental hard tissues. Adv Dent Res 1:21–26. Featherstone JD, Barrett-Vespone NA, Fried D, Kantorowitz, Seka W. 1998. CO2 laser inhibitor of artificial caries-like lesions progression in dental enamel. J Dent Res 77:1397–1403. Ferreira JM, Palamara J, Phakey PP, Rachinger WA, Orams HJ. 1989. Effects of the continuous-wave CO2 laser on the ultrastructure of human dental enamel. Arch Oral Biol 34:551–561. Fried D, Seka W, Glena RE, Featherston JDB. 1996. Thermal response of hard dental tissues to 9–11 mm CO2 laser irradiation. Opt Eng 35:1976–1984. Fried D, Glena RE, Featherstone JDB, Seka W. 1997. Permanent and transient changes in the reflectance of CO2 laser-irradiated dental hard tissues at lambda 1 9.3, 9.6, 10.3, and 10.6 microns and at fluences of 1–20 J/cm2. Lasers Surg Med 20:22–31. Ganss C, Klimek J, Schwarz N. 2000. A comparative profilometric in vitro study of the susceptibility of polished and natural human enamel and dentine surfaces to erosive demineralization. Arch Oral Biol 45:897–902. Ganss C, Lussi A, Klimek J. 2005. Comparison of calcium/phosphorus analysis, longitudinal microradiography and profilometry for the quantitative assessment of erosive demineralization. Caries Res 39:178–184. Ganss C, Lussi A, Scharmann I, Weigelt T, Hardt M, Klimek J, Schlueter N. 2009. Comparison of calcium analysis, longitudinal microradiography and profilometry for quantitative assessment of erosion in dentine. Caries Res 43:422–429. Hall AF, Sadler JP, Strang R, De Josselin DJ, Foye RH, Creanor SL. 1997. Application of transverse microradiography for measurement of mineral loss by acid erosion. Adv Dent Res 11:420–425. Hjortsj€o C, Jonski G, Young A, Saxegaard E. 2010. Effect of acidic fluoride treatments on early enamel erosion lesions—A comparison of calcium and profilometric analyses. Arch Oral Biol 55:229–234. Huysmans MC, Jager DH, Ruben JL, Unk DE, Klijn CP, Vieira AM. 2011. Reduction of erosive wear in situ by stannous fluoridecontaining toothpaste. Caries Res 45:518–523. Jaeggi T, Lussi A. 2006. Prevalence, incidence and distribution of erosion. Monogr Oral Sci Basel Karger 20:44–65. Jager DHJ, Vieira AM, Ruben JL, Huysmans MCDNJM. 2012. Estimated erosive potential depends on exposure time. J Dent 40:1103–1108. Johansson AK, Lingstrom P, Birkhed D. 2002. Comparison of factors potentially related to the occurrence of dental erosion in high- and low-erosion groups. Eur J Oral Sci 110:204–211. Kantola S. 1972. Laser induced effects on tooth structure. A study of changes in the calcium and phosphorus contents in dentine by electron probe microanalysis. Acta Odontol Scand 30:463–474. Kuroda S, Fowler BO. 1984. Compositional, structural and phase changes in vitro laser-irradiated human tooth enamel. Calcif Tissue Int 36:361–369. Lussi A. Jaeggi T. 2006. Chemical factors of erosion. Monogr Oral Sci Basel Karger 20:77–87. Lussi A, Jaeggi T. 2008. Erosion-diagnosis and risk factors. Clin Oral Investig 12:5–13.
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Lussi A, Jaeggi T, Zero D. 2004. The role of diet in the aetiology of dental erosion. Caries Res 38:34–44. Magalh~ aes AC, Rios D, Delbem ACB, Buzalaf MAR, Machado MAAM. 2007. Influence of fluoride dentifrice on brushing abrasion of eroded human enamel: an in situ/ex vivo study. Caries Res 41:77–79. Magalh~ aes AC, Kato MT, Rios D, Wiegand A, Attin A, Buzalaf MAR. 2008. The effect of an experimental 4% TiF4 varnish compared to NaF varnishes and 4% TiF4 solution on dental erosion in vitro. Caries Res 42:269–274. McCormack SM, Fried D, Featherstone JDB, Glena RE, Seka W. 1995. Scanning electron microscope observations of CO2 laser effects on dental enamel. J Dent Res 74:1702–1708. McKnight-Hanes C, Whitford GM. 1992. Fluoride release from three glass ionomer materials and the effects of varnishing with or without finishing. Caries Res 26:345–350. Meurman JH, ten Cate JM. 1996. Pathogenesis and modifying factors of dental erosion. Eur J Oral Sci 104:199–206. Meurman JH, Voegel JC, Rauhamaa-M€ akinen R, Gasser P, Thomann JM, Hemmerle J, Luomanen M, Paunio I, Frank RM. 1992. Effects of carbon dioxide, Nd:YAG and carbon-dioxide-Nd:YAG combination lasers at high energy densities on synthetic hydroxyapatite. Caries Res 26:77–83. Nelson DG, Wefel JS, Jongebloed WL, Featherstone JD. 1987. Morphology, histology and crystallography of human dental enamel treated with pulsed low energy infrared laser radiation. Caries Res 21:411–426. Ramalho KM, de Paula Eduardo C, Heussen N, Rocha RG, Lampert F, Apel C, Esteves-Oliveira M. 2013. Protective effect of CO2 laser (10.6 lm) and fluoride on enamel erosion in vitro. Lasers Med Sci 28:71–78. Rios D, Hon orio HM, Magalh~ aes AC, Machado MAAM, Delbem ACB, Silva SMB. 2006. Effect of salivary stimulation on erosion subjected or not to abrasion in human and bovine enamel: An in situ/ ex vivo study. Caries Res 40:218–223. Scatolin RS, Galo R, Corona AS. In situ effect of dentifrices associated to CO2 laser in the permeability of eroded root dentin. Photomed Laser Surg 30:573–578. Souza-Gabriel AE, Colucci V, Turssi CP, Serra MC, Corona SA. 2010. Microhardness and SEM after CO2 laser irradiation or fluoride treatment in human and bovine enamel. Microsc Res Tech 73: 1030–1035. Steiner-Oliveira C, Rodrigues LKA, Soares LES, Martin AA, Zazell DM, Nobre dos Santos M. 2006. Chemical, morphological and thermal effects of 10.6 lm CO2 laser on the inhibition of enamel demineralization. Dent Mater J 25:455–462. Steiner-Oliveira C, Nobre-dos-Santos M, Zero DT, Eckert G, Hara AT. 2010. Effect of a pulsed CO2 laser and fluoride on the prevention of enamel and dentine erosion. Arch Oral Biol 55:127–133. Tepper SA, Zehnder M, Pajarola GF, Schmidlin PR. 2004. Increased fluoride uptake and acid resistance by CO2 laser-irradiation through topically applied fluoride on human enamel in vitro. J Dent 32:635–641. Tsai CL, Lin YT, Huang ST, Chang HW. 2002. In vitro acid resistance of CO2 and Nd:YAG laser treated human tooth enamel. Caries Res 36:423–429. Viana PS, Machado AL, Giampaolo ET, Pavarina AC, Vergani CE. 2010. Disinfection of bovine enamel by microwave irradiation: Effect on the surface microhardness and demineralization/remineralization processes. Caries Res 44:349–357. West NX, Maxwell A, Hughes JA, Parker DM, Newcombe RG, Addy M. 1998. A method to measure clinical erosion: The effect of orange juice consumption on erosion of enamel. J Dent 26:329–335. West NX, Hughes JA, Addy M. 2001. The effect of pH on the erosion of dentine and enamel by dietary acids in vitro. J Oral Rehabil 28: 860–864. Wiegand A, Hiestand B, Sener B, Magalh~ aes AC, Roos M, Attin T. 2010a. Effect of TiF4,ZrF4, Hf4F and AmF on erosion and erosion/ abrasion of enamel and dentin in situ. Arch Oral Biol 55:223–228. Wiegand A, Magalh~ aes AC, Navarro RS, Schmidlin PR, Rios D, Buzalaf MA, Attin T. 2010b. Effect of titanium tetrafluoride and amine fluoride treatment combined with carbon dioxide laser irradiation on enamel and dentin erosion. Photomed Laser Surg 28:219–226. Zero DT, Lussi A. 2005. Erosion—Chemical and biological factors of importance to the dental practitioner. Int Dent J 55:285–290. Zuerlein MJ, Fried D, Featherstone JDB. 1999. Modeling the modification depth of carbon dioxide laser-treated dental enamel. Lasers Surg Med 25:335–347.
In Situ Analysis of CO2 Laser Irradiation on Controlling Progression of Erosive Lesions on Dental Enamel TAISA PENAZZO LEPRI,1* RENATA SIQUEIRA SCATOLIN,1 VIVIAN COLUCCI,1 ADILIS KALINA DE ALEXANDRIA,2 LUCIANNE COPLE MAIA,2 CECILIA PEDROSO TURSSI,3 AND SILMARA APARECIDA MILORI CORONA1 1 Department of Restorative Dentistry, School of Dentistry of Ribeir~ ao Preto, University of S~ ao Paulo (USP) Avenida do Cafe, S/N Monte Alegre CEP: 14040-904 Ribeir~ ao Preto—SP, Brazil 2 Department of Pediatric and Orthodontics, School of Dentistry, Federal University of Rio de Janeiro (UFRJ) Rua Prof., Rodolpho Paulo Rocco 325 CEP 21941-913 Cidade Universit aria—Rio de Janeiro—RJ, Brazil 3 Department of Restorative Dentistry, S~ ao Leopoldo Mandic Institute and Dental Research Center, Av. Jose Rocha Junqueira, 13 CEP 13045-755 Campinas—SP, Brazil
KEY WORDS
enamel; erosion; citric acid; CO2 laser
ABSTRACT The present study aimed to evaluate in situ the effect of CO2 laser irradiation to control the progression of enamel erosive lesions. Fifty-six slabs of bovine incisors enamel (5 3 3 3 2.5 mm3) were divided in four distinct areas: (1) sound (reference area), (2) initial erosion, (3) treatment (irradiated or nonirradiated with CO2 laser), (4) final erosion (after in situ phase). The initial erosive challenge was performed with 1% citric acid (pH 5 2.3), for 5 min, 23/day, for 2 days. The slabs were divided in two groups according to surface treatment: irradiated with CO2 laser (k 5 10.6 mm; 0.5 W) and nonirradiate. After a 2-day lead-in period, 14 volunteers wore an intraoral palatal appliance containing two slabs (irradiated and nonirradiated), in two intraoral phases of 5 days each. Following a cross-over design during the first intraoral phase, half of the volunteers immersed the appliance in 100 mL of citric acid for 5 min, 33/day, while other half of the volunteers used deionized water (control). The volunteers were crossed over in the second phase. Enamel wear was determined by an optical 3D profilometer. Three-way ANOVA for repeated measures revealed that there was no significant interaction between erosive challenge and CO2 laser irradiation (P 5 0.419). Erosive challenge significantly increased enamel wear (P 5 0.001), regardless whether or not CO2 laser irradiation was performed. There was no difference in enamel wear between specimens CO2-laser irradiated and non-irradiated (P 5 0.513). Under intraoral conditions, CO2 laser irradiation did not control the progression of erosive lesions in enamel caused by citric acid. Microsc. Res. Tech. 77:586–593, 2014. V 2014 Wiley Periodicals, Inc. C
INTRODUCTION The new dietary habits of population, with frequent consumption of acidic beverages (Ehlen et al., 2008; Lussi et al., 2004) has led to a higher occurrence of pathological tooth wear (Johansson et al., 2002; Zero and Lussi, 2005), such as erosion (Bartlett et al., 2011; Lussi and Jaeggi, 2008). In dental erosion, the contact of the dental surfaces with acid of nonbacterial origin results in an irreversible tissue loss (Featherstone and Lussi, 2006). This pathology can be caused by intrinsic acids, as recurrent vomiting or regurgitation of gastric contents (Bartlett, 2006), or extrinsic factors (Lussi and Jaeggi, 2006), with the frequent intake of beverages identified as a major cause of dental erosion (Jager et al., 2012). In an attempt to prevent the occurrence of erosive lesions or to minimize their resulting damage, various strategies have been proposed and include reduction of acidic challenges and increase of acid resistance of dental hard tissues (Hjortsj€o et al., 2010; Magalh~ aes et al., 2008; Ramalho et al., 2013; Wiegand et al., 2010a). These strategies includes the lasers, among C V
2014 WILEY PERIODICALS, INC.
which the CO2 laser is widely used in dentistry for having wavelengths more effective in dental enamel (Esteves-Oliveira et al., 2008; Fried et al., 1997; Tsai el al., 2002), making it significantly less soluble to acids (Featherstone et al., 1998). It has been stated that the chemical and structural changes caused on dental surface by CO2 laser irradiation are responsible for the increase of acid resistance on enamel (Featherstone, 2000). Besides, the matrix of enamel is partially denatured and its permeability decreased after CO2 laser irradiation, which hampers the diffusion of acid into the pores of the substrate, reducing demineralization (Ramalho et al., 2013; Souza-Gabriel et al., 2010; Tepper et al., 2004).
*Correspondence to: Taısa Penazzo Lepri, Alameda Itu, 285 Ap 141 – S~ ao Paulo – SP, CEP 01421-000, Brazil. E-mail: [email protected] Received 26 March 2014; accepted in revised form 1 May 2014 REVIEW EDITOR: Prof. Alberto Diaspro Contract grant sponsor: State of S~ ao Paulo Research Foundation (FAPESP); Contract grant number: 2010/19247-8. DOI 10.1002/jemt.22377 Published online 13 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
CO2 LASER TO CONTROL ENAMEL EROSIVE LESIONS
In the context of dental erosion, it has already been demonstrated by in vitro (Esteves-Oliveira et al., 2012; Ramalho et al., 2013), and in situ studies associated with different fluorides (Wiegand et al., 2010b) that the CO2 laser can reduce enamel mineral loss. However, no study has investigated the influence of CO2 laser in preventing progression of dental erosion in enamel previously eroded. Thus, this study was designed in order to evaluate in situ the effect of CO2 laser in controlling the progression of erosive lesions on dental enamel. MATERIAL AND METHODS Experimental Design This study was a two-period crossover design. Each phase lasted for 5 days with a washout period of 15 days between them. The factors under evaluation were erosive challenge at two levels [(A) 1% citric acid pH 2.3 and (B) deionized water—control)] and CO2 laser irradiation at two levels [(I) present and (II) absent]. Volunteers wore slabs irradiated or not, and in alternate and independent phases, subjected them to erosive challenges or deionized water (control). Each group consisted of 14 slabs, randomly assigned to the 14 volunteers, which were considered as statistical blocks. The response variable was the measurement of wear in three distinct moments: after initial erosion, after treatment (irradiated or not with CO2 laser) and after in situ phase. The wear was quantitatively measured by optical profilometry and qualitatively by scanning electron microscopy (SEM) analysis. The experimental design is schematized in the flowchart (Fig. 1). Selection of Volunteers Fourteen volunteers aged 22–40 years who fulfilled the inclusion criteria (normal salivary flow, no evidence of active cavity and non-carious lesions and willing to follow the research schedule) without violating the exclusion criteria (use of any form of medication likely to interfere with salivary secretion, use of fixed or removable orthodontic appliances, pregnancy or breastfeeding, general/systemic illness) were enrolled. The experimental procedures used were performed with the informed consent of the subjects, following protocols reviewed and approved by the Ethics Committee of the Faculty of Dentistry of Ribeir~ ao Preto, University of S~ ao Paulo, Brazil (CAAE 02110512.2.0000.5419). Impression of maxillary arch was taken on each volunteer using a stock tray. Impression was poured in dental stone, and an upper removable appliance of acrylic resin (Jet, Artigos Odontologicos Cl assico, S~ ao Paulo, SP, Brazil) was constructed. Appliances had two retention slots (7 3 7 3 3 mm3), one on each side of the midline, which accommodated two slabs facing the oral cavity. Preparation of Enamel Slabs Freshly extracted bovine incisors stored in a 0.1% thymol solution at 4 C were cleaned with a scaler and water/pumice slurry in dental prophylactic cups Microscopy Research and Technique
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and examined under 203 magnification using a stereomicroscope (Leica S6 D Stereozoom, Mycrosystems Leica AG, Switzerland). The teeth presenting cracks and hypoplasia were discarded. Roots were removed 2 mm below the cement-enamel junction using a low-speed water-cooled diamond saw (Isomet 1000; Buehler, Lake Bluff, IL). After, crowns were sectioned, resulting in two enamel slabs per tooth (5 3 3 3 2.5 mm3). The slabs were flattened and polished on a watercooled polishing machine (Phoenix b, Buehler, Lake Bluff, IL) with the aid of an apparatus that standardize the procedure. First, the enamel fragments were fixed in teflon matrix with the aid of a parallelometer (ElQuip, S~ ao Carlos, S~ ao Paulo, Brazil) to flatten the pulp wall, with 600-grit of Al2O3 papers (Norton Abrasivos, SP, Brazil) applied for 20 s with standardized load of 20 N. After flattening the pulp wall, slabs were removed from the matrix and again fixed for flatten and polishing the enamel surface, with 600 and 1200grit Al2O3 papers (Norton Abrasivos, SP, Brazil) with standardized load of 20N for 20 s and 0.3 mm alumina suspension (Buehler, Lake Bluff, IL) with standardized load of 20N for 60 s. Selection of Slabs Before initial erosive-like lesion formation, slabs were sterilized by microwave irradiation (Viana et al., 2010). After sterilization, slabs with cracks were discarded. Slabs were then pretested for Knoop microhardness (KHN) (HMV-2000, Shimadzu Corporation, Japan) by making three indentations at the center of the specimen, spaced 100 mm from each other, under 25-g load for 5 s (Magalh~ aes et al., 2007). The three readings were averaged and used as the microhardness values of each slab. Specimens with microhardness values 10% above or below the mean (300% 6 10%) were excluded, remaining 56 slabs. Specimens were coated with acid-resistant nail varnish (Colorama, Maybelline, Brazil) in three layers, except on the buccal surface, which remained uncoated. The slabs were divided in four distinct areas (1) sound, (2) initial erosion, (3) treatment (irradiated or not irradiated with CO2 laser), (4) final erosion (after the in situ phase), as presented in Figure 2. The sound region was considered as the reference area of each slab (1.25 3 3 mm2). The first area (1) was covered with resin composite, applied on non etched surface and in the absence of adhesive system. Erosion-like Lesion Formation To create initial erosive lesions, specimens were subjected to erosive challenges twice a day (at 9 a.m and 1 p.m) for 2 days. Each challenge consisted of individual immersion in 20 mL of citric acid 1% (pH 2.3) in an Erlenmeyer flask, which was then placed in an orbital shaker (CT155, Cientec, Piracicaba, SP, Brazil) with a stirring velocity of 100 rpm for 5 min (Amaechi et al., 1999). After each erosive challenge, specimens were rinsed for 10 s with deionized water and stored in 10 mL of
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Fig. 1. A. Removal of bovine incisor root 2 mm below the cementoenamel junction; B. Section crowns; C. Two enamel slabs (5 3 3 3 2.5 mm3); D. Planning and polishing; E. Initial microhardness measurements (baseline); F. Delimitation of the first reference area (not eroded) 1.5 3 3 mm2; G. Initial erosive challenge (formation of erosive lesion); H. Storage at 37 C; I. Delimitation of the second reference area (50% of the specimen covered); J. CO2 laser irradiation; K.
Delimitation of the third reference area (75% of the specimen covered); L. Mounting of slabs at the palatal devices; M. Ex vivo erosive challenges alternating; N. Removal of the delimited reference areas; O. Optical 3D profilometry analysis; P. Scanning electron microscopy analysis. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
artificial saliva at 37 C for the 4-h interval between the erosive challenges as well as overnight. The artificial saliva (pH 5 7.0), which was changed before the first challenge of each day, was similar to that described by McKnight-Hanes and Whitford (1992) and modified by Amaechi et al.,1999. It was composed of methyl-p-hydroxybenzoate (2.0 g), sodium carboxymethylcellulose (10.0 g), KCl (0.625 g), MgCl2.6H2O (0.059 g), CaCl2.2H2O (0.166 g), K2HPO4
(0.804 g); and KH2PO4 (0.326 g) in 1000 mL of water solution. Surface Treatment After initial erosion-like lesion formation, the delimited area 2 (Fig. 2) of the specimen was also protected with a resin composite without etching or applying adhesive system. Eroded slabs were randomly Microscopy Research and Technique
CO2 LASER TO CONTROL ENAMEL EROSIVE LESIONS
Fig. 2. Schematic enamel specimens. A. Sound area (control); B. Initial erosion; C. Treatment (irradiated or not); D. after in situ phase. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
assigned into two groups according to the CO2 laser irradiation: (I) present and (II) absent. Specimens were irradiated with a PC015-A (Shanghai Jue Hua Laser Tech. Development, Shangai, China) in an ultrapulse and noncontact mode. The irradiation was performed unfocused 4-mm distant from the enamel surface (Tepper et al., 2004) with the aid of a device that sets the pen during the laser irradiation, which moves the second commands previously established through a computer connected to the scanning machine (MPC ElQuip, S~ ao Carlos, SP, Brazil), allowing the radiation to reach the entire uncovered area (delimited area 3 and 4) evenly. Each specimen was irradiated during 10 s, with duty time of 100 ms and idle time of 0.001 s. The set of parameters employed were: 10.6 lm, 0.5 W, average output 0.44 W, pulse energy of 0.05 mJ and energy density of 0.04 J cm22. After achieving the surface treatments, using the same protocol described above, a layer of composite resin was placed on area 3 to protect this surface from further erosive challenges to which the area 4 was exposed (Fig. 2). Mounting of the Slabs in Intra-Oral Devices Volunteers wore slabs treated with CO2 laser and nonirradiated in both experimental phases, changing the erosive challenges (citric acid or control) in each phase. Two enamel sections—one irradiated and one nonirradiated—were placed in each device. The slab fixation was performed with wax, 1-mm below the edge of the palatal appliance to prevent abrasion by contact with surface of the tongue. In Situ Erosive Challenges During a 2-day lead-in period, the volunteers were instructed to brush their teeth only with the toothpaste (Colgate Maximum Anticaries Protection, Colgate-Palmolive, Division of Kolynos from Brazil, Osasco, SP, Brazil) and toothbrush (Colgate Extra Clean, Colgate-Palmolive, Division of Kolynos from Brazil, Osasco, SP, Brazil) supplied by the researchers. These challenges began only on the second day of use of intraoral device to allow formation of acquired pellicle (Huysmans et al., 2011). A 15-day wash-out period was allowed between the two phases. Microscopy Research and Technique
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In the first phase, half of the volunteers immersed the oral appliance in 100 mL of citric acid solution (1%, pH 5 2.3), outside the mouth, during 5 min, three times/day, for a 4-day period. After the challenge, slabs were rinsed for 20 s under tap water and reinserted in the mouth. The other half of volunteers followed the same sequence, employing, however, deionized water instead of citric acid. In both experimental phases, the use of the devices was continuous, including during night time, with exception of meals time, beverage consumption and oral hygiene procedures (Scatolin et al., 2012). To avoid dehydration of specimens during these periods, the appliance was covered with humid cotton gauze. The control of the biofilm on the surface of the specimens was carried out at the end of each experimental day, dripping chlorhexidine 0.2% (Bioquanti Compounding Pharmacy, Ribeir~ ao Preto, SP, Brazil) on the fragments, for one minute, and rinsing with tap water, according to the protocol described by West et al. 1998. 3D Optical Noncontact Profilometry At the end of the in situ experiment, all the composite resin was removed, and the specimens had their four areas exposed (Fig. 2): sound (control), initial erosion, treatment (irradiated or nonirradiated), after in situ phase. The determination of topographic change was made with the aid of an optical 3D non-contact profilometer (PS50 Optical Profilometer, Nanovea, Irvine, CA). This device allowed a scanning over an area of 4 mm in length (x-axis) by 1.5 mm in width (y-axis). The measurements of capture were performed by a chromatic confocal sensor with white light axial source, scan velocity of 2 mm s21 and refraction index of 10.000. The 3D noncontact profilometry technique was used to determine tooth structure loss—erosion depth (ED). The ED was calculated from the step height (in lm). Three linear measurements were made involving the following areas: (1) between sound area and initial erosion area; (2) between sound area and treatment area; (3) between sound area and final in situ phase area. All measurements were done in triplicate and the mean values. SEM Analysis After profilometry analysis, three specimens were randomly selected from each group (n 5 3) to the imaged under SEM. Specimens were stored in Eppendorff flasks with deionized water and cleaned in an ultrasound apparatus (Ultrasonic Cleaner, Odontobras, Ribeir~ ao Preto, SP, Brazil) for 10 min. The specimens were then immersed in 2.5% glutaraldehyde solution buffered with 0.1 M sodium cacodylate (Merck KGaA, Darmstadt, D-64293, Germany) for 12 h at 4 C. After fixation, the specimens were washed in deionized water for 3 min, immersed in deionized water for 1 h, changing the water every 20 min. Subsequently, the specimens were dehydrated with increasing percentage of ethanol (Labsynth, Diadema, SP, Brazil), namely 25% (20 min), 50% (20 min), 75% (20 min), 95% (30 min), and 100% (60 min) and dried with absorbing paper. After that, specimens were fixed on stubs with double-faced carbon tape (3M, S~ ao
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TABLE 1. Means and standard deviations of wear values (mm) presented by each group CO2 laser irradiation 3 challenge Irradiated Non-irradiated Grand mean
Citric acid
Deionized water
36.04 (15.56) 35.99 (17.94) 36.01 (16.48) B
20.12 (11.81) 16.38 (4.80) 18.25 (9.05) A
Grand means followed by different letters indicate a significant difference.
Paulo, SP, Brazil), covered with a 30 lm gold–platinum layer (Bal-Tec SCD 005, Zurich, Switzerland) in a vacuum apparatus (SDC 050, Balzers, Liechtenstein). Specimens were examined with a SEM (JEOL JSM model 5410; Jeol Technic, Tokyo, Japan). Initially, the specimens were analyzed in a panoramic vision, and latter, photomicrographs of the most representative area of each group were accomplished at a standardized magnification (3,0003) (Fig. 4). Statistical Analysis Three-way ANOVA for repeated measure was used to examine effects of the independent variables CO2 laser irradiation, Erosive Challenge and Volunteers and their interaction on surface loss (dependent variable). Significance level was set at 5% and statistical calculations were performed using SPSS 20 (SPSS, Chicago, IL). RESULTS Three-way ANOVA for repeated measures revealed that there was no significant interaction between ero-
sive challenge and CO2 laser irradiation (P 5 0.419). Erosive challenge significantly increased enamel wear (P 5 0.001), regardless whether or not CO2 laser irradiation was performed. There was no difference in enamel wear between specimens CO2-laser irradiated and those left non-irradiated (P 5 0.513). Volunteer factor showed no significant effect (P 5 0.544) (Table 1). By paired t tests, applied to the initial erosion 3 post treatment conditions, regardless of the challenge that would be further applied, it was noted that the irradiated substrates (P 5 0.109), and nonirradiated specimens (P 5 0.631), were not significantly worn. Using this same test, when compared the samples submitted to the challenge with deionized water, it was noted that neither irradiated (P 5 0.062), nor the nonirradiated (P 5 0.170) specimens presented significant increased wear in relation to the initial erosion. Figure 3 shows the 3D profilometry images of experimental groups, in which it can be observed the enamel wear between the different analyzed areas: sound area and initial erosion area; sound area and treatment area; sound area and in situ phase area. In Figure 4, SEM images of a representative sample from each group show different phases of the experiment. Image A shows the sound enamel surface (control). The image of uncovered enamel prisms after in vitro erosive challenges can be seen at Image B. Irradiated enamel surface (Image C) shows some areas where the CO2 laser did not present an interaction with the enamel and areas where the laser irradiation provided a protective layer (circled area). The images
Fig. 3. The 3D noncontact optical profilometry images: A. Irradiated with CO2 laser1 citric acid; Birradiated with CO2 laser1 deionized water; C. Nonirradiated 1 citric acid; D. Nonirradiated 1 deionized water. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Microscopy Research and Technique
CO2 LASER TO CONTROL ENAMEL EROSIVE LESIONS
Fig. 4. Scanning electron microscopy images. A. Sound surface (control); B. Initially eroded surface; C. Surface irradiated with CO2 laser; D. Nonirradiated surface; E. Surface irradiated with CO2 laser and eroded at the in situ phase; F. Nonirradiated surface and eroded at in situ phase; G. Surface irradiated and noneroded at in situ phase; H. Nonirradiated surface and noneroded at the in situ phase.
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that presents the enamel slightly overcast by a protective layer as exclusive of irradiated surfaces, as shown at Image E (irradiated and eroded enamel). Image F, which represents the eroded enamel, presents a marked etched-like aspect, with huge exposition of the enamel prisms, representing a high degree of enamel dissolution. The irradiated but not eroded enamel, presented at Image G, shows small enamel dissolution, preserving the enamel prisms. Image H, representative of a specimen that was neither irradiated nor eroded in situ, shows moderate enamel demineralization, due exclusively to the in vitro erosive challenges. DISCUSSION Given the increased incidence of dental erosion (Jaeggi and Lussi, 2006) it is necessary, besides the search for preventive measures, treatments to minimize progression and damage already established on eroded enamel. The CO2 laser produces radiation at the infrared spectrum region very close to the apatite absorbing bands (Featherstone and Nelson, 1987; Featherstone et al., 1998; Nelson et al., 1987) leading to greater light absorption on the enamel and conversion into heat. When hydroxyapatite is heated at a temperature higher than 400 C, it’s possible to obtain a decrease or complete elimination of carbonate from the enamel surface, thus increasing the acid resistance (McCormack et al., 1995; Zuerlein et al., 1999). The results from the present study showed that, by means of the methodology employed, it was found no significant interaction of erosive challenge and CO2 laser irradiation, in other words, the treatment with CO2 laser at a wavelength 10.6 mm was not able to control the enamel wear caused by citric acid when evaluate by 3D optical profilometry. At the SEM images, the surface of non-irradiated specimens after the erosive challenges was similar to the non-irradiated and non-eroded with the enamel prisms slightly exposed. The irradiated specimens preserved its most superficial enamel layer. After the in situ phase, the specimens that were irradiated and eroded presented a more homogeneous surface when compared to the nonirradiated and eroded. Aiming an increase of enamel acid resistance, studies have suggested that, when irradiated, the enamel temperature must range between 600 and 900 C, thus leading to changes like increase of hydroxyapatite crystals (Ferreira et al., 1989; McCormack et al., 1995), melting (Ferreira et al., 1989; Kuroda and Fowler 1984; McCormack et al., 1995; Meurman et al., 1992; Nelson et al., 1987; Tsai et al., 2002), fusion and recristalization (Kantola, 1972). In the present study, the employ of lowest pulse duration and lowest energy density (0.04 J cm22), which cause less morphological changes, may not have been adequate to produce the expected effect. These parameters were employed aiming to provide lower heat propagation to the inner tooth layers, thus decreasing the risk of pulpal damage (Fried et al., 1996). Esteves Oliveira et al., 2011 and Ramalho et al., 2013, employing higher energy densities (0.3 J cm22), found a reduction on the enamel wear after erosion, and decrease of mineral loss and a rehardening of the eroded enamel, respectively. In an attempt to simulate the treatment of previously eroded enamel, in the present study we performed erosive challenges in vitro (two times a day, 2
days) prior to CO2 laser irradiation, differently from what happens in most studies of enamel erosion (Esteves-Oliveira et al., 2011; Ramalho et al., 2013; Steiner-Oliveira et al., 2010; Wiegand et al., 2010a), which search for the prevention of a non-existent erosive lesion instead of the control of a already established lesion. Besides, the exposure time of specimens to erosive challenges may have been too long (5 min of exposure, 33 a day, during 4 days), so that the possible positive effects of laser not persisted for this long. Most of the acidic beverages ingested by population contain citric acid, so its use is already validated in studies of dental erosion (West, 1998). Some of these beverages appear to be more erosive than other, with the same pH. As the citric acid comes in contact with the tooth surface, through its chelating properties it is capable to increase the erosive process in vivo by interaction with saliva, lowering the pH of the oral cavity (West, 2001), increasing the softening and dissolving the mineral content of the enamel (Meurman, 1996). In the present study, using the topographic profile to evaluate the reduction on the progression of erosive lesions, only structural changes were evaluated, and these changes, such as irregularities, may have masked a possible positive action of the CO2 laser irradiation related to chemical changes, which could increase the enamel resistance to further erosive challenges. Among the available analyses to quantify the dental hard tissue loss due to erosion, particular attention is dedicated to the profilometry, since it’s easily performed, capable to assess initial changes on the surface and also the tissue loss at advanced stages. This analyses is indicate to evaluated the enamel while the changes on the subsurface mineral contents are relatively small (Ganss et al., 2009), and the noncontact profilometer minimizes possible errors on the results when analyzing softened surfaces (Barbour and Rees, 2004). The employ of profilometry was validated for erosion (Ganss et al., 2005), being considered a “gold standard” (Hall et al., 1997). In the present study, to prepare the specimens for quantifying wear by profilometry, the enamel surface was planed and polished perpendicular (at 90 ) to enamel prisms in order to achieve evenly surfaces, as recommended by Hjorstj€o (2010). Nevertheless, due to the curvature of the teeth, this may not always occur, and specimens may have differently exposed enamel prisms, thus affecting their acid susceptibility (Ganss, 2000; Rios, 2006). Thus, considering the methodology and parameters employed on this study, it can be concluded that CO2 laser irradiation was unable to reduce in situ the progression of erosive lesions on enamel caused by citric acid. ACKNOWLEDGEMENT The authors thank the Federal University of Rio de Janeiro, for authorization to use the 3D optical profilometer. REFERENCES Amaechi BT, Higham SM, Edgar WM, Milosevic A. 1999. Thickness of acquired salivary pellicle as a determinant of the sites of dental erosion. J Dent Res 78:1821–1828. Barbour ME, Rees JS. 2004. The laboratory assessment of enamel erosion: A review. J Dent 32:591–602.
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