Rsbo January-March 2014 by Univille

v. 11 – n. 1 – January/March 2014

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ISSN: Electronic version: 1984-5685

RSBO

Joinville – SC

v. 11

n. 1

106 p.

2014

Rector Sandra Aparecida Furlan Vice-Rector Alexandre Cidral Dean for Education Sirlei de Souza Dean for Research and Post-Graduation Denise Abatti Kasper Silva Dean for Extension and Community Affairs Claiton Emilio do Amaral Dean for Administration Cleiton Vaz Editorial Production Editora UNIVILLE Luciana Lourenço Ribeiro Vitor – Text revision and translation e-mail: llribeiro_3@hotmail.com Raphael Schmitz – Graphic design Claudio Alberto Lassance Rollin – Diagramming EDITORIAL BOARD Editor-in-chief Flares Baratto-Filho – Univille and UP, Brazil Administration Editors Fabricio Scaini – Univille, Brazil Luiz Carlos Machado Miguel – Univille, Brazil Carla Castiglia Gonzaga – UP, Brazil Tatiana Miranda Deliberador – UP, Brazil Associate Editors Edson Alves de Campos – Unesp, Brazil Sandra Rivera Fidel – Uerj, Brazil Gisele Maria Correr Nolasco – UP, Brazil Luiz Fernando Fariniuk – PUC/PR, Brazil Kathleen Neiva – University of Florida, USA Claudia Brizuela – University of Andes, Chile Johannes Ebert – University of Erlangen, Germany Nicolas Castrillon – University São Francisco of Quito, Ecuador

Editorial Board Alessandro Leite Cavalcanti – UEPB, Brazil Carlos Estrela – UFG, Brazil Christoph Kaaden – University of Munich, Germany Fernanda Pappen – UFPel, Brazil Fernando Branco Barletta – Ulbra, Brazil Fernando Goldberg – University of Salvador, Argentine Frank Lippert – Indiana University, USA Guilherme Carpena Lopes – UFSC, Brazil Jesus Djalma Pécora – Forp/USP, Brazil José Antônio Poli de Figueiredo – PUC/RS, Brazil José Carlos Laborde – Catholic University of Uruguay, Uruguay José Luiz Lage-Marques – USP, Brazil José Mondelli – FOB/USP, Brazil Juan Carlos Pontons-Melo – Sao Marcos University, Peru Lourenço Correr Sobrinho – FOP/Unicamp, Brazil Lúcia Helena Cevidanes – University of North Carolyn at Chapel Hill, USA Luciana Shaddox – University of Florida, USA Luis Sensi – University of Florida, USA Luiz Narciso Baratieri – UFSC, Brazil Manoel Damião Sousa-Neto – Forp/USP, Brazil Marco C. Bottino – Indiana University, USA M a r í a Merc e de s A z uer – Javer i a n a Un i versit y, Colombia Mário Tanomaru Filho – Unesp, Brazil Miguel González Rodríguez – Odonthos Institute, Dominican Republic Mu ha nad Hat a m leh – Un iversit y of Ma nchester, England Osmir Batista de Oliveira Júnior – Unesp, Brazil Pedro Bullon Fernandez – University of Sevilha, Spain Regina M. Puppin-Rontani – FOP/Unicamp, Brazil Richard L. Gregory – Indiana University, USA Rivail Antônio Sérgio Fidel – Uerj, Brazil Rodrigo Neiva – University of Florida, USA Sandra Milena Brinez Rodriguez – Javeriana University, Colombia Saulo Geraldeli – University of Florida, USA Ulrich Lohbauer – University of Erlangen, Germany Valentina Ulver de Beluatti – University of Maimonides, Argentine Valeria Gordan – University of Florida, USA Yara Teresinha Corrêa Silva Sousa – Unaerp, Brazil

The content of the articles is of sole responsibility of the authors.

Table of Contents

Guest editorial..................................................................................................................................... 5

Original Research Articles Bonding performance of a self-adhering flowable composite to indirect restorative materials............... 6 Rubens Nazareno Garcia, Caroline Silvestri Silva, Felipe de Oliveira Maisonnette, Giovana Gochinski Silva, Guilhermo Mocellin, José Ozelame, Laís Fracasso, Matheus Bernhardt Ozelame, Reinaldo Francisco do Nascimento, Ana Cristina Rocha Gomes

Endodontists perceptions of single and multiple visit root canal treatment: a survey in Florianópolis – Brazil ........................................................................................................13

Monica de Souza Netto, Flavia Saavedra, Jacy Simi Júnior, Ricardo Machado, Emmanuel João Nogueira Leal Silva, Luiz Pascoal Vansan

Repair on silorane-based composite . ................................................................................................ 19 Dilcele Silva Moreira Dziedzic, Shiffa al Sayd, Leonardo Fernandes da Cunha, Lino Oliveira Carvalho de Santana, Carla Castiglia Gonzaga, Adilson Yoshio Furuse

Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive – analysis by SEM and confocal laser scanning microscopy (CLSM)......................... 28 Aline de Oliveira Gonçalves, Cinthia Sawamura Kubo, Osmir Batista Oliveira Júnior, Edson Alves de Campos, Marcelo Ferrarezi Andrade

Comparative evaluation of pH and solubility of MTA Fillapex® endodontic sealer .............................. 41

Meiryelen Silva Finger, Gislaine Faraoni, Michel do Carmo Masson, Rogério Aparecido Minini dos Santos, Ana Claudia Baladelli Silva Cimardi, Fausto Rodrigo Victorino

Analysis of blood pressure during tooth extraction............................................................................ 47 Alessandro Hyczy Lisboa, Chigueyuki Jitumori, Evaldo Artur Hasselmann Júnior, Rafael Pes, Gibson Luis Pilatti

Immunoexpression of BMP-2 protein on bone repair of critical size defects treated with autogenous macerated adipose tissue................................................................................................ 52 Clayton Luiz Gorny Junior, Allan Fernando Giovanini, Juliana Vieira, João Cézar Zielak, Felipe Rychuv Santos, Carmen Lucia Mueller Storrer, Tatiana Miranda Deliberador

Analysis of tensile strength of poly(lactic-co-glycolic acid) (PLGA) membranes used for guided tissue regeneration................................................................................................... 59 Bruno Gasparini Betiatto de Sousa, Gabrielle Pedrotti, Ana Paula Sponchiado, Rafael Schlögel Cunali, Águedo Aragones, João Rodrigo Sarot, João Cézar Zielak, Bárbara Pick Ornaghi, Moira Pedroso Leão

Analysis of salivary pH, flow rate, buffering capacity, concentrations of calcium, urea and total proteins in 2-8 years-old children with Down’s syndrome............................................ 66 Gizele Franco, Rafaella Saab, Luciani Variani Pizzatto, Maria Fernanda Torres, Andréa Paula Fregoneze, João Armando Brancher

Adhesion and formation of tags from MTA Fillapex compared with AH Plus® cement.......................... 71 Marina Samara Baechtold, Ana Flávia Mazaro, Bruno Monguilott Crozeta, Denise Piotto Leonardi, Flávia Sens Fagundes Tomazinho, Flares Baratto-Filho, Gisele Aihara Haragushiku

Literature Review Article Cell adhesion in bone grafts associated to nanotechnology: a systematic review................................ 77 Haroldo Gurgel Mota-Filho, Amanda Alencar Cabral, Diego Moura Soares, Fernanda Ginani, Carlos Augusto Galvão Barboza

Case Report Articles Masseteric cysticercosis: an uncommon appearance diagnosed on ultrasound................................... 83 Vinod Vijay Chander, Sridevi Koduri, Atul Kaushik, Manpreet Kalra, Renu Tanwar, Sukeerat Mann

Unusual transmigration of canines – report of two cases in a family................................................... 88 Sulabha A. Narsapur, Sameer Choudhari, Shrishal Totad

Maxillary squamous cell carcinoma: diagnosis and evolution of an inoperable case .......................... 93 Luana Beber Yoshizumi, Mariah Scotti Alérico, José Miguel Amenábar Céspedes, Cleto Mariosvaldo Piazzetta, Cassius Carvalho Torres-Pereira

Oral rehabilitation of patient with severe early childhood caries: a case report................................. 100 Luciana Pedroso, Camila Zucuni, Letícia Westphalen Bento, Juliana Yassue Barbosa da Silva, Bianca Zimmermann Santos

Guest editorial Human tooth bank: importance and social role

The creation of the Human tooth bank (HTB) in the teaching institutions with graduation and post-graduation programs as well as technical secondary courses has great importance and relevant social role today. The quality of the teaching and consequently of the vocational training is directly and indirectly linked to the perspective of performing the clinical simulation in natural teeth and of conducting researches supporting the scientific advancement. Well-structured HTB has the prospect of capturing and providing teeth for academic activities. It has been emphasized that the same tooth can be employed at different stages of the courses, from the study of tooth morphology to prosthetic preparations after restorative and/or endodontic treatments. The processing of the teeth assures a safe handling for both the students and researchers maintaining the quality of the tooth and respecting the legal and environmental aspects. This RSBO issue publishes a literature review study which supports the processing of teeth at HTB and proposes the creation of a protocol/routine of procedures aiming at systematizing and ensuring the quality of this processing. Erica Lopes Ferreira Curitiba City Hall

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):6-12

Original Research Article

Bonding performance of a self-adhering flowable composite to indirect restorative materials Rubens Nazareno Garcia1, 2 Caroline Silvestri Silva1 Giovana Gochinski Silva1 Guilhermo Mocellin1 José Ozelame1 Laís Fracasso1 Matheus Bernhardt Ozelame1 Reinaldo Francisco do Nascimento4 Ana Cristina Rocha Gomes3 Corresponding author: Rubens Nazareno Garcia Universidade do Vale do Itajaí – Univali Curso de Odontologia – Grupo de Pesquisa Biomateriais em Odontologia Rua Uruguai, 458 – Centro CEP 88302-202 – Itajaí – SC – Brasil E-mail: rubensgarcia@univali.br School of Dentistry, Research Group on Biomaterials in Dentistry, University of Itajai Valley – Itajai – SC – Brazil. Department of Dentistry, University of Joinville Region – Joinville – SC – Brazil. 3 DDS and Dental Technician, RPD Dental Laboratory – Joinville – SC – Brazil. 4 Dental Technician, RPD Dental Laboratory – Joinville – SC – Brazil. 1 2

Received for publication: April 14, 2013. Accepted for publication: August 18, 2013.

Keywords: ceramics; composite resins; metal ceramic alloys; shear strength.

Abstract Introduction: Simplified restorative materials may be a logical next step for dental manufacturers. Objective: The aim of this study was to evaluate the shear bond strength of a self-adhering flowable composite to four substrates used in indirect technique. Material and methods: Twenty-four samples (5 mm wide, 15 mm length and 2 mm thick / six blocks each substrate) were prepared in the dental prosthetic laboratory. The following materials were used: ceromer (SR Adoro/AD, Ivoclar Vivadent), leucite ceramic (IPS Empress Esthetic/EE, Ivoclar Vivadent), zirconia ceramic (ZirCAD/ZI, Ivoclar Vivadent); metal ceramic alloy (Fit Cast SB/ME, Talladium do Brasil). Samples of each substrate were divided into two groups

– RSBO. 2014 Jan-Mar;11(1):6-12 Garcia et al. – Bonding performance of a self-adhering flowable composite to indirect restorative materials

(n = 3). Two flowable composites (Control/FF – Filtek Z350 XT Flow/3M ESPE, and the self-adhering/DF – Dyad Flow/Kerr) were bonded to the four substrates. Four Tygon tubings were positioned over each sample, which were filled in with the composites FF and DF, and visible light-cured for 20 s. The tubings were removed to expose the specimens (12 per group) in format of cylinders and samples were stored in distilled water at 37±2°C for one week. After this period, each sample was attached to testing machine and the specimens were submitted to the shear bond strength test at speed of 1.0 mm/min, until failure. The results were analyzed by two-way ANOVA and Tukey test (p < 0.05). Results: The means (SD) were (in MPa): AD + FF = 34.4 ± 4.9; AD + DF = 28.2 ± 4.2; EE + FF = 29.7 ± 5.8; EE + DF = 32.3 ± 6.9; ZI + FF = 23.2 ± 5.4; ZI + DF = 8.5 ± 1.5; ME + FF = 28.9 ± 4.2; ME + DF = 31.7 ± 4.5. Conclusion: The efficacy of flowable composites is material-dependent. The self-adhering composite provided lower bond strength only to zirconia ceramic. Comparing with the control group, Dyad Flow showed lower bond strength to the ceromer and zirconia ceramic.

Introduction The demands from dentists and patients for tooth-colored posterior restorations such as inlays, onlays, and crowns have been increasing in recent years, also the luting techniques for these restorations. For larger restorations, indirect methods are superior alternatives to direct resin composite fillings [18]. The ceromers, leucitereinforced and zirconia-reinforced ceramics, and the metal-ceramic restorations can represent these materials, which can be luted by either conventional or adhesive technique, or other alternative of luting can be the new self-adhering flowable composite. Flowable composites were first introduced in 1995 to restore Class V lesions. They have excellent handling properties, low viscosity, and superior injectability. Easy handling is a highly desired characteristic because it reduces the working time of clinicians and chairside time of patients, according to Bayne et al. [1]. A new self-adhering flowable composite, Vertise Flow (Kerr, Orange, CA, USA – named Dyad Flow in Latin America), was recently introduced into the market, as well as the Fusio Dentin Liquid (Pentron Clinical, Orange, CA, USA). These adhesive-free composites are claimed to rely on chemical and micromechanical interaction between material and tooth structures or other substrates, achieved with incorporation of an acidic adhesive monomer into the flowable composites [2, 7, 11, 15, 22].

Owing to the novelty of this material, it seemed interesting to investigate further on the bonding performance of this new self-adhering flowable composite Dyad Flow. The aim of this study was to evaluate the shear bond strength of a self-adhering f lowable composite to four substrates used in indirect technique. The tested null hypothesis was that statistically similar bond strengths are achieved by the self-adhering flowable composite and the flowable composite of the control group.

Material and methods Twenty-four samples (5 mm wide, 15 mm length and 2 mm thick / six blocks to each substrate below) were obtained in the dental prosthetic laboratory, according to manufacturer’s instructions. The ceromer SR Adoro/AD blocks (Batch # R57506 / Exp: 07/2015) were prepared and light-cured in the Lumamat 100 Light Furnace; the leucite-reinforced ceramic IPS Empress Esthetic/EE blocks (Batch # KM0305 / Exp: 12/2030) were prepared in the hot pressing technique; the zirconia-reinforced ceramic IPS e.max ZirCAD/ZI blocks (Batch # L15418 / Exp: 12/2030) were prepared in the CAD/CAM (ComputerAided Design / Computer-Aided Manufacturing) technique – all materials from Ivoclar Vivadent, Schaan, Principality of Liechtenstein; and the metal ceramic alloy Fit Cast SB/ME blocks (Batch # 121165/Exp: undetermined / Talladium do Brasil,

– RSBO. 2014 Jan-Mar;11(1):6-12 Garcia et al. – Bonding performance of a self-adhering flowable composite to indirect restorative materials

Curitiba, PR, Brazil) were prepared in the lost wax technique. All the samples were sandblasted with aluminum oxide (90 µm / 2.5 Bar / 10 mm distance) and the samples of each substrate were divided into two groups – control and self-adhering flowable composites (n = 3). For AD and EE groups, after application of 10% hydrofluoric acid for 1 min (Condac Porcelana, FGM, Joinville, SC, Brazil – Batch # 031211 / Exp.: 12/2013), the samples were rinsed for 1 min, airdried for 1 min, followed by the application of the silane coupling agent (Monobond-S, monofunctional3-methacryloxypropyltrimethoxy sylane/3-MPS, Ivoclar Vivadent – Batch # N15219 / Exp.: 02/2013) for 1 min and then air-dried for 30 s. For ZI and ME groups, it was applied the metal & zirconia primer (Metal/Zirconia Primer, phosphonic acid acrylate in tert-Butyl alcohol, Ivoclar Vivadent – Batch # M68692 / Exp.: 02/2013) for 3 min and then air-dried for 30 s. Two flowable composites (Control/FF – Filtek Z350 XT Flow/3M ESPE, and the self-adhering/DF

– Dyad Flow/Kerr) were bonded to the four substrates (table I). According to Shimada et al. [18], for all groups and in each sample, four Tygon tubings (TYG-030, Saint-Gobain Performance Plastic, Maime Lakes, FL, USA) were positioned over the sample, which were filled in with the composites FF and DF, and visible light-cured (VLC) for 20 s (LED Bluephase – 1.200 mW/cm2 – Ivoclar Vivadent, Schaan, Principality of Liechtenstein). The tubings were removed to expose the specimens in format of cylinders (12 per group – area: 0.38 mm2 / by formula πR2) and samples were stored in distilled water at 37±2°C for one week. After this period, each sample was attached to the universal testing machine (Emic DL 1000, São José dos Pinhais, Pr, Brazil) and the specimens were submitted to shear bond strength test (SBS), applied at the base of the specimen/substrate cylinder with a thin wire/0.25 mm, at speed of 1.0 mm/min – until failure. The results were analyzed by two-way ANOVA (two flowable composites and four substrates) and Tukey test (p < 0.05).

Table I – Materials used

Material Adper Single Bond 2 Dental Adhesive pH ≈ 4.7 3M ESPE St. Paul, MN, USA Filtek Z350 XT Flow VLC Flowable Nanocomposite/A2 3M ESPE St. Paul, MN, USA Dyad Flow or Vertise Flow Self-Adhering Flowable Nanocomposite/A2 pH ≈ 1.9 before VLC pH ≈ 6.5-7 after Kerr, Orange, CA, USA

Batch # N2I1104BR Exp: 11/13

1211700713 Exp: 12/13

4398621 Item 34.384 Exp: 06/13

Composition Bis-GMA, HEMA, UDMA, dimethacrylates, ethanol, water, camphorquinone, photoinitiators, copolymer of polialcenoic acid, silica (5 nm) Bis-GMA, TEGDMA, Bis-EMA, silane-treated ceramic, silica, zirconium oxide - 55 vol% / 65 wt%

Protocol Apply the adhesive, gentle air 5 s, VLC 10 s

GPDM, prepolymerized filler, 1μm barium glass filler, nanosized colloidal silica, nanosized Ytterbium fluoride

Apply, brush a thin layer (< 0.5 mm) with pressure for 15–20 s, VLC 20 s

Apply and VLC 20 s

Composition as provided by respective manufacturer: Bis-GMA, bisphenol glycidyl dimethacrylate; HEMA, 2-hydroxyethyl methacrylate; UDMA, urethane dimethacrylate; TEGDMA, triethylene glycol dimethacrylate; Bis-EMA, bisphenol A polyethylene glycol dimethacrylate; GPDM, glycerol phosphate dimethacrylate. VLC: visible light-curing

Results ANOVA showed significant differences between flowable composites and among substrates (p < 0.001). Tukey test (p < 0.05) was applied to investigate the differences. The self-adhering composite provided lower bond strength just on zirconia ceramic. Comparing to the control group, Dyad Flow showed lower bond strength to the ceromer and zirconia ceramic (table II).

– RSBO. 2014 Jan-Mar;11(1):6-12 Garcia et al. – Bonding performance of a self-adhering flowable composite to indirect restorative materials

Table II – SBS means (±SD) in MPa and Tukey test (p < 0.05)

Ceromer Leucite-reinforced ceramic Zirconia-reinforced ceramic Metal ceramic alloy

Filtek Flow 34.4 ± 29.7 ± 23.2 ± 28.9 ±

Flowable composite (FF/control) Dyad Flow (DF/self-adhering) 4.9 A a 28.2 ± 4.2 B a 5.8 A b 32.3 ± 6.9 A a 5.4 A c 08.5 ± 1.5 B b 4.2 A b 31.7 ± 4.5 A a

Means followed by the same lower case within columns and capital letters within rows did not differ significantly by Tukey test (p < 0.05)

Discussion Based on the findings of the present study, the formulated null hypothesis was accepted for leucitereinforced ceramic and metal ceramic alloy. However, it was rejected for ceromer and zirconia-reinforced ceramic, because the results differ significantly in shear bond strength to these substrates. Laboratory tests are still useful at promptly yielding first-hand information. Specifically, bond strength tests have been considered to provide a quantitative assessment of materials adhesion, based on the concept that the stronger the bond, the better it will resist against contraction and functional stresses [20]. This study focused on the evaluation of the shear bond strength (SBS) of self-adhering flowable composite Dyad Flow to four substrates used in indirect technique, using microshear methodology proposed by Shimada et al. [18]. This type of mechanical test solves problems related to tension propagations at the bonded interface in larger areas. It presents the advantage that several specimens can be obtained from one sample without cutting it, being easier and cheaper than the microtensile test [17]. Indirect restorations have been used to reduce or minimize polymerization shrinkage of the resin composite direct restorations. One possible reason for this is the small amount of resin cement used in luting procedures. This technique provides better sealing than that of direct composites. Moreover, it is used to facilitate the reproduction of the dental anatomy, in order to improve control of the marginal fit, proximal and occlusal contacts. Regarding to ceromers, when compared to other indirect restorations, as ceramics, they present a more simple fabrication technique, less wear on the antagonist teeth, the possibility of intra-oral repair and lower cost [6]. With regards to ceramics, they are used to achieve esthetic dental restorations because of their superior color, and their clinical success is determined by the bond strength and bonding durability of the resin cement to tooth and ceramic [14].

The application of resin cements for toothcolored indirect restorations have increased considerably because of their ability to set completely and their greater resistance to occlusal loading when compared to the conventional cementation. This luting usually requires several steps to secure optimal adhesion. However, recently some selfetching primers and self-adhesive resin cements have been introduced, yielding major improvements in bonding to tooth structures. The use of these products is a result of attempts to improve the bonding quality while reducing the number of necessary procedures [18]. To this study, it was speculated that the use of one newly self-adhering composite could result in the similar shear bond strength, although the control group had other flowable composite instead of resin cement. First of all, the samples of ceromers and leucite-reinforced ceramics were sandblasted with aluminum oxide. Sandblasting of the interior surface of these materials is a common practice in laminates or crown restorations because the roughened surface enables a strong mechanical bond with resin-based dental materials [24]. After that, according to the manufacturer’s instructions, the ceromer and leucite-reinforced ceramic was etched with 10% hydrofluoric acid and silanation. The preferred manner of conditioning these surfaces is by etching with hydrofluoric acid, followed by the application of a silane coupling agent to achieve high bond strength. The acid works by creating surface pits via preferential dissolution of the glassy phase from the ceramic matrix and the dissolution of the resin matrix of the ceromer [5]. Treatment of the etched surface with silane increases the wettability and forms a covalent bond [16]. The 3-MPS (silane) is known to promote the adhesion through chemical and physical coupling between metal-composites, ceramic-composites, and composites containing methacrylate groups. Also, three chemical interaction mechanisms are possible for the bond strength of the flowable composites to other composite, the ceromer,

10 – RSBO. 2014 Jan-Mar;11(1):6-12 Garcia et al. – Bonding performance of a self-adhering flowable composite to indirect restorative materials

according to the findings of Teixeira et al. [19]: 1) the adhesion between the polymer matrices, from both f lowable composites and ceromer; 2) the adhesion between the fillers particles exposed of both composites; and 3) the formation of a micro-network of the polymer chains and the fillers particles of both composites. This latter mechanism would likely dominate and produce the greatest contribution with regards to acceptable bond strength, as it was possible to observe inside both the control and self-adhering groups. However, by comparing the groups, the DF group showed lower bond strength, with statistical difference between them. It is speculated that the adhesive system Adper Single Bond 2 used prior to flowable composite of the control group helped the wettability of the ceromer substrate, resulting in higher bond strength to ceromer in control group. As the aforementioned author information about the filler particles, it is interesting to report some information on Dyad Flow filler system. According to the Technical Bulletin Kerr/35104 (2010), the type, proportion, and size of each filler particles were carefully chosen for optimized wetting, mechanical strength, and polishability. Dyad Flow consists of 4 filler types: 1) a prepolymerized filler, 2) a 1micron barium glass filler, 3) a nanosized colloidal silica, and 4) a nanosized Ytterbium fluoride. The average particle size of Dyad Flow is 1 micron. The pre-polymerized filler (PPF) enhances the handling characteristics of the material, making it smooth and easy to manipulate. Nanoparticles enhance the polishability of the material and achieve special rheological property; also nanoytterbium fluoride particles give to Dyad Flow a superb radiopacity index for easy detection with X-rays. The Technical Bulletin Kerr/35104 (2010) also shows that the Dyad Flow has one common element in all Kerr bonding agents, that is, the GPDM adhesive monomer, a phosphate functional group that creates a chemical bond with the calcium ions of the tooth. GPDM monomers ensure a tenacious bond to both enamel and dentin, evidenced by the strength known to all generations of the OptiBond adhesive family. A GPDM adhesive monomer acts like a coupling agent. On one hand, it has an acidic phosphate group for etching the tooth structure and also for chemically bonding to the calcium ions within the tooth structure. On the other hand, it has two methacrylate functional groups for copolymerization with other methacrylate monomers to provide increased crosslinking density and enhanced mechanical strength for the polymerized adhesive.

As can be seen, the resin matrix of the Dyad Flow consists of multifunctional acidic methacrylates. If a high content of acidic functional monomers can react with the substrate like the leucite-reinforced ceramic used in this study, and achieve enough micromechanical and chemical bond strength, it is possible to hypothesize that this self-adhering composite can be used to bond successfully to this type of ceramics surface. Using microshear methodology and Dyad Flow, there is no comparison to the literature until this moment, regarding to bond strength to ceromers and ceramics – just the internal data of the manufacturer Kerr, reported at the Technical Bulletin Kerr/35104 (2010). In this technical report, the porcelain showed 33.9 MPa (without using hydrofluoric acid and silane), while in this study the leucite-reinforced ceramics exhibited 32.3 MPa. Moreover, the composite showed 34.2 MPa in the technical report, while in this study the ceromer presented 28.2 MPa, but they did not inform which was the type of the composite: direct or indirect. The findings of Garcia et al. [9] showed similar bond strength to leucite-reinforced ceramics, but the authors used only resin cements – RelyX ARC and RelyX U100, both from 3M ESPE. Metallic restorations have a long-standing history of clinical use in dentistry. However, increase in patients’ aesthetic expectations and demands caused the metallic restorations to be sidelined and led to the development of porcelain-fusedto-metal (PFM) restorations. Despite the brittle nature of porcelain, PFM crowns are widely used because the metallic frameworks afford superior mechanical durability. On the other hand, the use of metals in PFM restorations gives rise to gingival discolorations and metal-related allergies, according to Kuriyama et al. [12]. IPS e.maz ZirCAD is an yttrium-stabilized zirconia oxide block. It is suitable for indications that require high strength, including posterior bridges. The composition and physical properties of zirconia oxide-based ceramics differ substantially from silicabased ceramics, such that conventional acid-etching has no positive effects on the resin bond to zirconia oxide ceramics [3]. As resin bond to high-strength ceramics is less predictable, alternative bonding techniques are required to achieve a strong and long-term durable resin bond. Primers containing phosphonic acid compound as the active ingredient, could establish a chemical bond to oxidic surfaces, such as Metal/Zirconia Primer. Hence, they can be an alternative to promote adhesion to oxide ceramics such as zirconia, except for DF. In this study, it was not enough to obtain a reliable bond strength

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between DF and zirconia. However, the control group showed statistically higher bond strength. Also, it is speculated that the adhesive system Adper Single Bond 2 used prior to flowable composite FF of the control group helped the wettability of the substrate zirconia-reinforced ceramic. The opposite situation occurred for the metal ceramic alloy Fit Cast SB. In an attempt to improve the bonding of composite to alloys, and according to Di Francescantonio et al. [4], there are many methods like electrolytic etching, chemical etchings, adhesive primer application, and silica coating methods for surface treatment of metal alloys. This study also employed Metal/Zirconia Primer to metal ceramic alloy, which showed similar bond strength between the control group FF and the self-adhering composite DF, without statistically significant difference. Sandblasting with aluminum oxide particles removes contaminated layers and creates a roughened surface which provides mechanical interlocking for the composites as well as providing a great surface area for bonding. It has been reported that the sandblasting process could form a passive film made of Ni, Cr and Co oxides, according to the findings of Yoshida et al. [23]. Metal/Zirconia Primer, as already described, con si st s of pho sphon ic acid ac r yl ate. T he ma nufacturer claimed t hat it is suitable for oxide ceramic and all type of metal alloy. In this study, the results showed the efficiency of Metal/ Zirconia Primer in enhancing shear bond strength between Ni-Cr alloy and the self-adhering flowable composite Dyad Flow. The results were similar to Di Francescantonio et al. [4], however they used others primers and others metal ceramic alloys. Finally, it has been observed that shear bond testing tends to produce cohesive failures of the substrate [8, 10], usually to dental substrates. It is observed that a bigger piece of cohesive fracture in the substrate is pulled out after the transition from adhesive to cohesive fracture [21]. The improvement of the bonding properties of restorative materials have increased the bond strength and changed the failure pattern [13, 17]. This transition is most likely related to the changing stress pattern as the crack progresses across the interface. By using optical microscopy in this study and according to the authors above, it was observed mixed failures within AD and EE groups, followed by cohesive failures (in the ceromer and in the leucite-reinforced ceramic) and adhesive failures. With regards to zirconiareinforced ceramic (ZI) and metal ceramic alloy (ME), mainly adhesive failures were observed.

Conclusion Within the limitation of this study, it was observed that the efficacy of flowable composites is material-dependent. The self-adhering composite provided lower bond strength only to zirconia ceramic. Comparing to the control group, Dyad Flow showed lower bond strength to the ceromer and zirconia ceramic. The Dyad Flow can provide acceptable bond strength; however further studies on the properties and action mechanism of this material are necessary.

References 1. Bayne SC, Thompson JY, Swift Jr. EJ, Stamatiades P, Wilkerson M. A characterization of first-generation flowable composites. J Am Dent Assoc. 1998;129:567-77. 2. Bektas OO, Eren D, Akin EG, Akin H. Evaluation of a self-adhering flowable composite in terms of micro-shear bond strength and microleakage. Acta Odontol Scand. 2013 May-Jul;71(3-4):541-6. 3. Blatz MB, Sadan A, Kern M. Resin-ceramic bonding: a review of the literature. J Prosthet Dent. 2003 Mar;89(3):268-74. 4. Di Francescantonio M, Oliveira MT, Garcia RN, Romanini JC, Silva NR, Giannini M. Bond strength of resin cements to Co-Cr and Ni-Cr metal alloys using adhesive primers. J Prosthodont. 2010 Feb;19(2):125-9. 5. Edris A, Al Jabr A, Cooley RL, Barghi N. SEM evaluation of etch patterns by three etchants on three porcelains. J Prosthet Dent. 1990 Dec;64(6):734-9. 6. Elias RV, Osório AB, Sarmento HR, Camacho GB, Demarco FF. Type of resin cement influences microleakage in ceromer inlays? Rev Gaúcha Odontol. 2013;61:13-9. 7. Fu J, Kakuda S, Pan F, Hoshika S, Ting S, Fukuoka A et al. Bonding performance of a newly developed step-less all-in-one system on dentin. Dent Mater J. 2013;32(2):203-11. 8. Garcia RN, de Goes MF, Giannini M. Effect of water storage on bond strength of self-etching adhesives to dentin. J Contemp Dent Pract. 2007 Nov 1;8(7):46-53.

12 – RSBO. 2014 Jan-Mar;11(1):6-12 Garcia et al. – Bonding performance of a self-adhering flowable composite to indirect restorative materials

9. Garcia RN, Nascimento RF, Gomes ACR, Giannini M, Miguel LCM, Moon PC. Bond strength of resin cements to leucite-reinforced ceramics. RSBO. 2012 Apr-Jun;9(2):183-9. 10. Garcia RN, Reis AF, Giannini M. Effect of activation mode of dual-cured resin cements and low-viscosity composite liners on bond strength to dentin. J Dent. 2007 Jul;35(7):564-9. 11. Juloski J, Goracci C, Rengo C, Giovannetti A, Vichi A, Vulicevic ZR et al. Enamel and dentin bond strength of new simplified adhesive materials with and without preliminary phosphoric acid-etching. Am J Dent. 2012 Aug;25(4):239-43. 12. Kuriyama S, Terui Y, Higuchi D, Goto D, Hotta Y, Manabe A et al. Novel fabrication method for zirconia restorations: bonding strength of machinable ceramic to zirconia with resin cements. Dent Mater J. 2011;30(3):419-24. 13. Menezes FC, Silva SB, Valentino TA, Oliveira MA, Rastelli AN, Gonçalves LS. Evaluation of bond strength and thickness of adhesive layer according to the techniques of applying adhesives in composite resin restorations. Quintessence Int. 2013 Jan;44(1):9-15.

17. Sano H, Shono T, Sonoda H, Takatsu T, Ciucchi B, Carvalho RM. Relation between surface area for adhesion and tensile bond strength – evaluation of a microtensile bond test. Dent Mater. 1994 Jul;10(4):236-40. 18. Shimada Y, Yamaguchi S, Tagami J. Microshear bond strength of dual-cured resin cement to glass ceramics. Dent Mater. 2002 Jul;18(5):380-8. 19. Teixeira EC, Bayne SC, Thompson JY, Ritter AV, Swift EJ. Shear bond strength of self-etching bonding systems in combination with various composites used for repairing aged composites. J Adhes Dent. 2005 Summer;7(2):159-64. 20. Van Meerbeek B, De Munck J, Yoshida Y, Inoue S, Vargas M, Vijay P et al. Buonocore memorial lecture. Adhesion to enamel and dentin: current status and future challenges. Oper Dent. 2003;28:215-35. 21. Versluis A, Tantbirojn D, Douglas WH. Why do shear bond tests pull out dentin? J Dent Res. 1997 Jun;76(6):1298-307.

14. Peumans M, Van Meerbeek B, Lambrechts P, Vanherle G. Porcelain veneers: a review of the literature. J Dent. 2000 Mar;28(3):163-77.

22. Vichi A, Margvelashvili M, Goracci C, Papacchini F, Ferrari M. Bonding and sealing ability of a new self-adhering flowable composite resin in class I restorations. Clin Oral Investig. 2013 Jul;17(6):1497-506.

15. Poitevin A, De Munck J, Van Ende A, Suyama Y, Mine A, Peumans M et al. Bonding effectiveness of self-adhesive composites to dentin and enamel. Dent Mater. 2013 Feb;29(2):221-30.

23. Yoshida Y, Kamada K, Atsuta M. Adhesive primers for bonding cobalt-chromium alloy to resin. J Oral Rehabil. 1999;26:475-8.

16. Roulet JF, Söderholm KJ, Longmate J. Effects of treatment and storage conditions on ceramic/composite bond strength. J Dent Res. 1995;74(1):381-7.

24. Zhang Y, Lawn BR, Rekow ED, Thompson VP. Effect of sandblasting on the long-term performance of dental ceramics. J Biomed Mater Res B Appl Biomater. 2004 Nov 15;71(2):381-6.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):14-9

Original Research Article

Endodontists perceptions of single and multiple visit root canal treatment: a survey in Florianópolis – Brazil Monica de Souza Netto1 Flavia Saavedra2 Jacy Simi Júnior1 Ricardo Machado1 Emmanuel João Nogueira Leal Silva3 Luiz Pascoal Vansan4 Corresponding author: Ricardo Machado Faculdades Unidas do Norte de Minas (Funorte/Sobrás) Rua Trajano, n. 265 – Centro CEP 88010-010 – Florianópolis – SC - Brasil E-mail: ricardo.machado.endo@gmail.com Integrated College from North of Minas Gerais – Florianópolis – SC – Brazil. State University of Rio de Janeiro – Rio de Janeiro – RJ – Brazil. 3 Grande Rio University – Duque de Caxias – RJ – Brazil. 4 Ribeirão Preto Dental School, University of São Paulo – Ribeirão Preto – SP – Brazil. 1 2

Received for publication: October 12, 2013. Accepted for publication: November 18, 2013.

Keywords: Endodontics; single visit; multiple visit.

Abstract Introduction: One of the main debates that have occurred in Endodontics is about the amount of sessions required to complete an appropriate treatment. There are very different philosophies regarding this matter. Objective: to the aim of this study was to investigate the Endodontists’ point of view regarding single and multiple visit root canal treatment, identifying the basis on which the choice is made and how the information necessary for the choice is acquired. Material and methods: Endodontists registered in the dental practice board of Florianópolis/SC were contacted, and if they agreed to participate, they were interviewed using a questionnaire. The following topics were addressed: demographics, current clinical procedures, treatment rationales and preferences. Forty-three endodontists agreed to participate in the study. Results: Single visit endodontic treatment is carried out in 59.5% of biopulpectomy cases, 31.0% of necropulpectomy cases without lesion and only 11.9% in necropulpectomy cases with periapical lesion. The presence of vital pulp (81.4%) and a canal

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without exudate (93.0%) are the most important criteria for carrying out single visit endodontic treatment. The most used intracanal medication was calcium hydroxide. The lack of studies comparing long-term success on single visit endodontic treatment is considered an important issue to determine this therapy. Conclusion: It can be concluded that Florianopolis-Brazil endodontists prefer multiple visit over single visit root canal treatment in pulp necrosis cases. When pulp vitality is not compromised there is an increase in the number of endodontists who choose single visit root canal treatment.

Introduction Single and multiple visit root canal treatment has been the subject of long-standing debate in the endodontic community, not only on the biological and efficiency point of view, but also on the operator and patient‘s comfort, satisfaction and preferences [29]. Both options of treatment, single and multiple visits, are based on solid studies, but the number of different opinions is still significant [12, 14, 16, 18, 24, 26, 30, 33, 39]. Single visit endodontic therapy has many advantage, e.g. (a) it reduces the number of patient appointments; (b) it eliminates the chance for interappointment microbial contamination; (c) it allows for the immediate use of the canal space retention of a post; and (d) it allows the endodontists perform the root canal filling when they are more familiar with the canal anatomy [4, 22, 32, 40]. However, two concerns regarding single visit root canal treatment still make many endodontists do not use this therapy: (a) the incidence of flare-ups and (b) the long-term success [9, 36]. Recent studies demonstrated no differences between single and multiple visit treatment regarding to postoperative complications [1, 5, 31]; however, there is a lack of conclusive studies demonstrating the long-term success of single visit treatment in necrotic pulp teeth [14, 26, 39]. The aim of the present study was to investigate endodontists’ point of view regarding single and multiple visit root canal treatment, identifying the basis on which the choice is made and how the information necessary for the choice is acquired.

Material and methods A questionnaire was sent to 103 endodontists, resident in Florianópolis/SC, Brazil and listed in the Brazilian Federal Board of Dentists. It was e mailed with wording briefly explaining the purpose of the study. The questionnaires were divided in

two sections: (i) The first one was about age, gender, place of work, time since graduation and level of academic degree; (ii) The second one consisted in questions about current clinical procedures, treatment options, preferences and opinion about single and multiple visit canal treatment. The answers were recorded and entered into spread sheet software for analysis. Descriptive statistics were used.

Results Profile of the Endodontists and clinical preferences and procedures Forty-three endodontists agreed to participate in the study. 53.5% were male and 46.5% were female. Of these, 88.4% had neither a master nor doctorate degree. Most of them have more than ten years of specialist practice (60.5%). Almost 80% of all participants used hand files for canal preparation, mostly in a crown-down technique. The sodium hypochlorite is the preferential irrigation substance for 86% endodontists.

Opinion about single and multiple visit canal treatment Concerning to the time to complete a single visit treatment, most part of the professionals rep or ted t a k i n g more t h a n 9 0 m i nute s to complete the treatment of a molar both for vital and necrotic pulp (44% and 60%, respectively). Single visit therapy is usually practiced by almost 60.0% of all endodontists interviewed in cases of vital pulp, but when the biological status of the pulp becomes necrotic very few still choose this treatment type (table I). However, most of participants were willing to provide single visit t reat ment in cases of necrot ic pulp w it hout periapical lesion (53.4%) rather than in cases with periapical lesion (79.0%).

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Table I – Number of sessions until root canal obturation

Status of the pulp/ Number of sessions

1 (%)

2 (%)

3 or + (%)

Vital

25 (59.5%)

15 (35.7%)

2 (4.8%)

Necrotic with lesion

13 (31.0%)

19 (45.2%)

10 (23.8%)

Necrotic without lesion

5 (11.9%)

19 (45.2%)

18 (42.9%)

When questioned about what were the most commonly volunteered reasons for not performing a single visit therapy in teeth with pulp necrosis with or without periapical lesion, 60.5% of the participants answered the “importance of the intracanal medication”. On the other hand, when asked about the main reason to perform a single visit treatment, 81.4% of the professionals answered that vital pulp is the most important aspect (table II). The most important reason to allow this treatment type was the absence of exudate for 93.0% of them. Other reasons are shown in table III. Table II – Reasons to not perform and to perform single visit treatments

Reasons to not perform single visit treatments

n (%)

Reasons to perform single visit treatments

n (%)

Intracanal medication

26 (60.5%)

Vital pulp

35 (81.4%)

Post-operative pain

14 (32.5%)

Inter-appointment contamination

23 (53.5%)

Doubts about biological healing

13 (30.2%)

Absence of lesion

14 (32.5%)

Flare-up

11 (25.6%)

Patient preference

13 (30.2%)

Others

3 (7.0%)

Others

10 (23.3%)

Table III – Most important signs to enable single visit treatments

Most important sings to enable single visit treatments Absence of exudate Absence of pain Absence of edema Time Absence of sinus track Absence of severe pain Others

n (%) 40 28 27 26 19 17 3

(93.0%) (65.1%) (63.0%) (60.5%) (44.2%) (39.5%) (7.0%)

Discussion The aim of the present study was to investigate the endodontists’ point of view regarding to single and multiple visit root canal treatment. When the answers of the questionnaire were analyzed, we realized that the single visit therapy is wellaccepted in cases of vital pulp. The concern about the chance of inter-appointment contamination, especially in vital pulp teeth, was also named as one of the important causes to perform a single visit treatment. These results are in agreement with the endodontic literature. Several studies have

recognized the great importance of coronal seal for the long-term success of endodontic therapy [19, 20, 32, 35]. With regard to the endodontic treatment in teeth with necrotic pulp, the most of specialists still prefer to perform multiple visit therapy. Only a small amount of then prioritize a single visit therapy in this cases. These results are similar to previous studies, such as Araujo Filho et al. [3] who also found that single visits are widely accepted by the Endodontists from Rio de Janeiro, mainly in teeth with vital pulp. In contrast, the percentage of participants that performed single visit treatments decreases broadly in cases of necrosis with and without periapical lesion, which can probably be justified by another data from this research that shows the value of intracanal medication on the biological healing for these Brazilian endodontists, even though the current best available evidence does not support such notion [28]. A lt houg h si ng le v isit t reat ment was not performed by most of t he endodont ists, t he treatment of necrotic teeth with periapical lesion has been done successfully by many authors who justify the results by the elimination of bacterial contamination in the root canal through adequate instrumentation, irrigation and filling [2, 7, 41].

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Despite the large refusal, the treatment of teeth with pulp necrosis, with or without periapical lesion, has been successfully established and approved by many authors. Field et al. [11] retrospectively assessed the success rate of single visit root canal therapy. Both vital and necrotic cases, as well as those with and without periradicular disease were included. They found that 199 teeth had clinical and radiographic success over 223 available cases. The major listed problem to not perform root canal treatment in single visit is the importance of using an intracanal medication to promote a better disinfection process. The most reported intracanal medication was calcium hydroxide for the time of seven days. Several studies highlighted the benefits of the use of calcium hydroxide during endodontic therapy [33, 34]. Trope [36] demonstrated that the use of intracanal medication with calcium hydroxide can improve healing when compared to single visit therapy. Despite of the well-known disinfection properties of this medication, several studies demonstrate its inability to completely eliminate microorganisms from the root canal system [18, 25, 26]. Vivacqua-Gomes et al. [37] demonstrated that calcium hydroxide was unable to eliminate Enterococcus faecalis completely from dentinal tubules after seven or fourteen days. In this present study, dry root canal without exudates, was pointed out as one of the important factors during the decision-making to perform a single visit appointment. These results are in agreement with previous studies [38, 41] which confirm that the moisture condition of root canal is an important issue in the decision to perform this approach. Moisture and liquids can negatively affect the sealing ability. It may inhibit, prolong or accelerate the setting process of root canal sealers, which may result in higher leakage [27]. In t his present st udy, most i nter v iewed endodontists reported the use of hand files and the Crown-Down technique. It has been shown that this technique can reduce the chance of accumulation of smear-layer in the apical area, improving the prognosis of immediate obturation [10]. Also, some studies demonstrated that shaping the canal by using Crown-Down philosophy provides a cleaner apical third of the root canals [6]. The use of hand files instead of mechanical Ni-Ti files could be explained by the late popularization of these instruments and the discontinuous in the education of the endodontists in Brazil. 60.5% of all specialists have more than 10 years of experience, and at the time they were studying, Ni-Ti mechanical files were not so popular. Also, almost 90% of

them have neither master’s degree nor doctorate choosing to keep on a private practice career, which among Brazilian’s endodontists almost always results in lack of updating on clinical and scientific knowledge. Concerning to the irrigation solution, sodium hypochlorite and the EDTA were the most used ones. These results are in agreement with the literature, which confirms the bactericidal, organic tissue dissolution potential and low surface tension of the former [17, 23], associate to the ability to remove smear-layer of EDTA [15]. Although there is a paradigm about single visit on the Brazilian endodontic community, it is important to highlight that regarding single versus multiple visit therapy, there is very few or no difference between its quality, post operative complication incidence, success and failure clinical indexes, which reinforce the practical of single session therapy [8, 13, 21].

Conclusion Within the results of this study, it can be concluded that most of Florianopolis’ Endodontists perform single-visit root canal treatment in cases of vital pulp endodontic therapy, but in cases of necrotic pulp, most of the participants perform multiple-visit endodontic treatment. The main and most important reason to perform a single-visit endodontic treatment is pulp vitality.

References 1. Albashaireh ZS, Alnegrish AS. Post-obturation pain after single and multiple visit endodontic therapy. A prospective study. J Dent. 1998;26:222-32. 2. Araújo Filho WR, Sendra MC. Tratamento endodôntico em sessão única. Avaliação clínicoradiográfica. Rev Cient OCEx. 1998;2:6-8. 3. Araújo Filho WR, Cabreira MS, Costa FF. Tratamento endodôntico em sessão única: levantamento da opinião de endodontistas da cidade do Rio de Janeiro. Rev Bras Odontol. 2003;60:103-5. 4. Ashkenaz, PJ. One-visit endodontics. Dent Clin North Am. 1984;28:853-63. 5. Bhagwat S, Mehta D. Incidence of post-operative pain following single visit endodontics in vital and non-vital teeth: An in vivo study. Contemp Clin Dent. 2013;4:295-302.

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6. Bhuyan A, Boruah L, Katak R. Histological evaluation of the effectiveness of four instrumentation techniques for cleaning apical third of root canals – an in vitro study. J Conserv Dent. 2006;9:134-9. 7. Buchanan SL. The art of endodontics: selected case histories, part 2. Dent Today. 1997;16:50-1. 8. Davini F, Cunha RS, Fontana CE, Silveira CFM, Bueno CES. Radix entomolaris – a case report. RSBO. 2012;9:340-4. 9. Eleazer PD, Eleazer KR. Flare-up rate in pulpally necrotic molars in one-visit versus two-visit endodontic treatment. J Endod. 1998;24:614-6. 10. Fava LRG. A comparison of one versus tab appointment endodontic therapy in teeth with nonvital pulps. Int Endod J. 1989;22:179-83. 11. Field JM, Gutmann JL, Solomon ES, Rakusin H. A clinical radiographic retrospective assessment of the success rate of single-visit root canal treatment. Int Endod J. 2004;37:70-82. 12. Figini L, Lodi G, Gorni F, Gagliani M. Single versus multiple visits for endodontic treatment of permanent teeth. Cochrane Database Syst Rev. 2007;34:1041-7.

20. Madison S, Wilcox LR. An evaluation of coronal microleakage in endodontically treated teeth. Part Ill. In vivo study. J Endod. 1988;14:455-8. 21. Miranda TC. O tratamento endodôntico em sessão única depende do estado pulpar e/ou perirradicular? APCD. 1998 Jan-Feb. 22. Morse DR. One-visit endodontics. Hawaii Dent J. 1987;18:12-4. 23. Murad C, Sassone L, Souza M, Fidel R, Fidel S, Hirata R. Antimicrobial activity of sodium hypoclorite, chlorexidine and MTAD against enterococcus faecalis biofilm on human dentin matrix in vitro. RSBO. 2012;9:143-50. 24. Ng YL, Mann V, Rahbaran S, Lewsey J, Gulabivala K. Outcome of primary root canal treatment: systematic review of the literature – part 2. Influence of clinical factors. Int Endod J. 2008;41:6-31. 25. Peters LB, Van Winkelhoff AJ, Buijs JF, Wesselink PR. Effects of instrumentation, irrigation and dressing with calcium hydroxide on infection in pulpless teeth with periapical bone lesions. Int Endod J. 2002;35:13-21.

13. Fontana CE, Ibanéz CDM, Davini F, De Martin AS, Silveira CFM, Rocha DGP et al. Endodontic therapy of maxillary second molar showing an unusual internal anatomy. RSBO. 2012;9:213-7.

26. Peters LB, Wesselink PR. Periapical healing of endodontically treated teeth in one and two visists obturated in the presence or absence of detectable microorganisms. Int Endod J. 2002;35:660-7.

14. Friedman S. Success and failure of initial endodontic therapy. Ont Dent. 1997;74:35-8.

2 7 . R o g g e n d o r f M J, E b e r t J , P e t s c h e l t A , Frankenberger R. Influence of moisture on the apical seal of root canal fillings with five different types of sealer. Int Endod J. 2007;33:31-3.

15. Hulsmann M, Heckendorff M, Lennon A. Chelating agents in root canal treatment: mode of action and indications for their use. Int Endod J. 2003;36:810-30. 16. Inamoto K, Kojima K, Nagamatsu K, Hamaguch A, Nakata K, Nakamura H. A survey of the incidence of single-visit endodontics. J Endod. 2002;28:371-4. 17. Leonardo MR, Leal JM. Endodontia: tratamento de canais radiculares. 3. ed. São Paulo: Panamericana; 1998. 901 p.

28. Sathorn C, Parashos P, Messer H. Antibacterial efficacy of calcium hydroxide intracanal dressing: a systematic review and meta-analysis. Int Endod J. 2007;40:2-10. 29. Sathorn C, Parashos P, Messer H. Australian endodontists’ perceptions of single and multiple visit root canal treatment. Int Endod J. 2009;42:811-8.

18. Machado MEL. Endodontia: da biologia à técnica. 1. ed. São Paulo: Santos; 2007. 484 p.

30. Sathorn C, Parashos P, Messer H. Effectiveness of single versus multiple visit endodontic treatment of teeth with apical periodontitis: a systematic review and metaanalysis. Int Endod J. 2005;38:347-55.

19. Madison S, Swanson KL, Chiles SA. An evaluation of coronal microleakage in endodontically treated teeth. Part II. Sealer types. J Endod. 1987;13:109-12.

31. Singh S, Garg A. Incidence of post-operative pain after single visit and multiple visit root canal treatment: a randomized controlled trial. J Conserv Dent. 2012;15:323-7.

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32. Siqueira Jr. JF, Rôças IN, Lopes HP, Uzeda M. Coronal leakage of two root canal sealers containing calcium hidroxide after exposure in human saliva. J Endod. 1999;25:14-6. 33. Sjogren U, Figdor D, Peerson S, Sundqvist G. Influence of infection at the time of root filing on the outcome of endodontic treatment of teeth apical periodontitis. Int Endod J. 1997;30:297-306. 34. Sjogren U, Hagglund B, Sundqvist G, Wing K. Factors affecting the long term results of endodontic treatment. J Endod. 1990;16:498-504. 35. Swanson KS, Madison S. An evaluation of coronal microleakage in endodontically treated teeth. Part I. Time periods. J Endod. 1987;13:56-9. 36. Trope M. Flare-up rate of single-visits endodontics. Int Endod J. 1991;24:24-7.

37. Vivacqua-Gomes N, Gurgel-Filho ED, Gomes BPFA, Ferraz CCR, Zaia AA, Souza-Filho FJ. Recovery of Enterococcus faecalis after single or multiple visit root canal treatments carried out in infected teeth ex vivo. Int Endod J. 2005;38:697-704. 38. Walton R, Fouad A. Endodontic interappointment flare-ups: a prospective study of incidence and related factors. J Endod. 1992;18:172-7. 39. Weiger R, Rosendahl R, Lost C. Influence of calcium hydroxide intracanal dressings on the prognosis of teeth with endodontically induced periapical lesions. Int Endod J. 2000;33:219-26. 40. Whal MJ. Myths of single visit endodontics. Gen Dent. 1996;44:126-31. 41. Wolch I. One appointment endodontic treatment. Dent J. 1975;41:613-5.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):19-27

Original Research Article

Repair on silorane-based composite Dilcele Silva Moreira Dziedzic1 Shiffa al Sayd1 Leonardo Fernandes da Cunha2 Lino Oliveira Carvalho de Santana1 Carla Castiglia Gonzaga1 Adilson Yoshio Furuse1 Corresponding author: Dilcele Silva Moreira Dziedzic Universidade Positivo – Graduação em Odontologia Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: dilcele@up.edu.br 1 2

Department of Dentistry, Positivo University – Curitiba – PR – Brazil. Private practice – Brasília – DF – Brazil.

Received for publication: March 18, 2013. Accepted for publication: July 20, 2013.

Keywords: silorane resins; composite resins; shear strength.

Abstract Introduction: Silorane-based composites have low polymerization shrinkage and good color stability. However, the effectiveness and the best surface treatment to carry out repairs to this type of restoration is unclear. Objective: To evaluate the effect of different types of repair made on a silorane-based composite. Material and methods: 80 disks of silorane-based composite were prepared (Filtek P90, 3M ESPE) and divided into eight groups (n = 10), according to the surface treatment being carried out before repairs of either the same silorane composite or a dimethacrylate material (Filtek Z350, 3M ESPE) were conducted. In two groups the immediate adhesion without repair (positive control) was evaluated. In other two groups repairs without any surface treatment (negative control) were evaluated. Surface treatments before the repair of the four remaining groups included the application of adhesive systems specific to silorane (Silorane System Adhesive, 3M ESPE) or to dimethacrylate (Adper Single Bond 2, 3M ESPE) and roughening followed by application of adhesive system. All groups were stored into distilled water at 37°C for 1 week prior to the microshear bond strength evaluation. Results: The group immediate adhesion silorane-dimethacrylate and group repair silorane-dimethacrylate

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without surface treatment showed lower microshear bond strength values and were statistically different from groups with surface treatment and immediate adhesion silorane-silorane (p < 0.05). Conclusion: Surface treatments with application of adhesive systems compatible with the repair material or roughening prior to the application of these adhesive systems are suitable for repairing silorane-based composites.

Introduction Both the resin composites and the adhesive systems are very versatile and their use have favored the greatest advancements in aesthetic Dentistry leading to the preservation of tooth tissues and obtaining nearly invisible restorations by little wearing of the sound tooth structure. Thus, Dentistry has been currently experiencing the perspectives predicted for over one decade [19], by emphasizing restorative direct techniques and conservative preparations. However, the polymerization shrinkage of dimethacrylate-based composites has been considered as their major shortcoming resulting in marginal failures and leakage. The current development of composites whose polymerization occurs by the opening of the rings rather than by free radical polymerization and approximation of dimethacrylate monomers has evidenced a reduction in polymerization shrinkage. Silorane-based resin composites have their organic matri x formed by siloxane and oxirane with cationic polymerization and opening of the rings during polymerization reaction [14, 24]. This has resulted in low polymerization shrinkage, insolubility to simulated biological fluids, and good color stability when compared with dimethacrylatebased composites s [5, 7, 10, 24]. Despite these advantages, further studies are necessary to evaluate the behavior of silorane-based resin composites in cases requiring repair. During t he clinica l life of a restorat ion, failures can occur, which leads to the indication of repair instead of its total replacement. Clinical researches on repairing, reshaping and sealing of direct restorations have demonstrated that these minimal intervention procedures are conservative alternatives indicated in cases of failure in marginal adaptation and staining [13, 21]. Therefore, when properly indicated, the repair procedure has been considered as an alternative to total replacement of a restoration [6] decreasing the weariness of the sound tooth structure and the time amount required to remove the restorative material completely [16].

Notwithstanding, the repairing requires that the restoration surface is adequately treated [1, 9]. Among these treatments, silanization has shown controversial results, with either effectiveness in [22] or unable of increasing the bond strength [3]. On the other hand, the surface roughness has exhibited good results [9], probably because of promoting a micromechanical linking between the old resin composite and the new restorative system. The superficial treatments for repairing most taught in dental schools have been the roughening of the exposed surface, etching with phosphoric acid, cleaning with pumice and abrading with aluminum oxide [1]. The materials most used in repairing have been the adhesive agents, resin composite, silane agent, and flowable resin [1]. However, most of the studies were conducted with resins containing organic matrixes with similar chemical characteristics [9, 11]. The concern with the compatibility between resins with different organic matrix, such as silorane-based and conventional resins, is clinically relevant for eventual repairs because not even the dentist knows the composite type employed firstly [4]. The surface weariness followed by siloranebased bonding agent has been recommended to repair silorane-based resins with similar resins while the application of the primer agent has been contraindicated [17]. On the other hand, when a dimethacrylate-based resin is used in the repair, an application of an intermediary layer of siloranebased bonding agent has been advised [12, 23]. Dimethacrylate-based material bond strength to silorane-base materials has been little studied probably because the latter is recently on the market. Consequently, both the repairing effectiveness and which would be the best surface treatment has not been clarified yet. Moreover, the behavior of the interaction between the different layers should be evaluated at long-term, which can be simulated by laboratorial studies. The aim of this study was to evaluate the microshear bond strength of the repair interface between a silorane-/silorane-based resin as well as a silorane-/dimethacrylate-based resin.

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Material and methods Eighty matrixes made from acrylic resin (Clássico, São Paulo, SP, Brazil) within PVC tubes (3/4” in diameter, 1 cm in height), with a center cylindrical orifice with 5 mm in diameter and 3 mm in mean depth. The restorative materials and the adhesive agents employed are described in table I. Table I – Composition of the materials employed

Material, batch, shade

Manufacturer

Filtek P90 80284930218 A3

3M ESPE

Filtek Z350 XT 80284930218 A3B

3M ESPE

Silorane adhesive system – Self-Etch 3M ESPE Primer Silorane adhesive system – Bond

3M ESPE

Single Bond

3M ESPE

Composition Particles of quartz and silica/silane, yttrium fluoride, Di-34-epoxy cyclohexyl dimethyl silane, 3-4 poly methyl siloxaneepoxy-cycle, functional di- and oligo-siloxane, initiator system: camphorquinone and iodonium salt (donator of eletrons), stabilizers and pigments. Bis-GMA (bisphenol A glycidyl methacrylate), UDMA (urethane dimethacrylate), TEGDMA (triethylene glycol dimethacrylate), bis-EMA (bisphenol A ethoxylated dimethacrylate), particles of silica and zirconia/silane, BHT (Butyl hydroxy toluene), photoinitiator system and pigments. Phosphate methacrylates, copolymer of Vitrebond, BisGMA, HEMA (hydroxy-ethylene glycol dimethacrylate), water, ethanol, particles of silica treated with silane, initiators, and stabilizers. Hydrophobic dimethacrylate, phosphate methacrylates, TEGDMA, particles of silica treated with silane, initiators, and stabilizers. BisGMA, HEMA, dimethacrylates, ethanol, water, photoinitiator system, functional copolymer of methacrylate of polyacrylic and polyalkenoic acids.

The silorane-based resin composite (Filtek P90, 3M ESPE, St. Paul, USA) was inserted within the orifice of acrylic resin matrix with the aid of a spatula, at two increments, in order to obtain a base disc as substrate for the silorane-based resin composite. Each increment (1.5 mm in thickness) was individually light-cured for 40 s with the aid of a light-curing unit (Coltolux LED, Coltène/Whaledent, Altstätte, Switzerland), at intensity of 1,000 mW/cm2. After the insertion of the last increment of resin composite P90, a polyester strip (TDV Dental Ltda., Pomerode, SC, Brazil) was placed onto the surface of the resin not polymerized and pushed with the aid of a glass lamina with 1 mm in thickness to achieve a flat surface prior to photoactivation, which was carried out through the polyester strip. To obtain the aging of the resin composite specimens prior to the repairing procedures, the discs were stored into distilled water at 37°C for one week. This was executed to mimic the condition of a restoration exposed to the humidity within oral cavity. The discs were divided into six groups (G1 to G6). The specimens from G1, G3 and G5 were repaired with a silorane-based material (Filtek P90), while those from G2, G4 and G6 were repaired

with a dimethacrylate-based material (Filtek Z350 XT, 3M ESPE, St. Paul, USA). The repairs were executed after the washing of the discs with air jet for 15 s and drying with air jet for 20 s at 10 cm of distance. G5 and G6 specimens were roughened with the aid of 600-grit sandpaper, at a single direction with water lubrication (Ecomet 250, Lake Buff, IL, USA), prior to the treatment with the adhesive systems and repairing procedures. The specimens of G4 and G6 underwent 37% phosphoric acid etching (Dentak Villevie, Joinville, SC, Brazil) for 30 s. G6 specimens received silane application (ProSil, FGM, Joinville, SC, Brazil), while the adhesive system Single Bond 2 (3M ESPE, St. Paul, USA) was applied onto the specimens of G4 and G6 prior to the repairing procedure with dimethacrylate-based resin (Z350 XT). The adhesive system Single Bond 2 application was performed with the aid of a microbrush, twice, followed by gentle air drying for 5 s and light-curing for 10 s. The specimens of G3 and G5 were washed and dried before the treatment with the adhesive system P90 (3M ESPE, St. Paul, USA) and the repairing procedure was executed with silorane-

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based material (P90). The application of the two-flask adhesive system P90 (Self-Etch Primer and Bond agent) was executed according to the manufacturer’s recommendation. The self-etch primer agent was applied with the aid of a microbrush for 15 s, followed by gentle air drying and light-curing for 10 s. Table II exhibited the description of the experimental groups. Table II – Groups and treatments prior to either repairing procedure (G1 to G6) or immediate adhesion (G7 and G8) Groups G1 G2 G3 G4 G5 G6 G7 G8

Aging 1 week water at 1 week water at 1 week water at 1 week water at 1 week water at 1 week water at No No

into 37°C into 37°C into 37°C into 37°C into 37°C into 37°C

600-grit sandpaper roughness

Washing for 37% 15 s, drying phosphoric for 20 s acid etching

Silane

Adhesive

Repairing resin

Yes

P90

Yes

Z350

Yes

P90

Yes

Single Bond

Z350

Yes

P90

Yes

Single Bond

Z350

No No

P90 Z350

Cylindrical silicone tubes (Perfitécnica, Salto, SP, Brazil) with 0.95 mm in internal diameter and 1.5 mm in height were used as matrixes for either the immediate adhesion or repairing procedure after the surface treatment of each silorane disc, according to the group assigned (n = 10). One portion of resin composite was inserted into the cylindrical silicone tube with the aid of a spatula. After the cleaning of the tube extremities with the same spatula, the set was put in contact with the center of the silorane disc with the aid of dental tweezers. The photoactivation of the resin within the cylindrical tube in contact with the disc enabled their adhesion therefore simulating the execution of a repairing procedure, but with standardized characteristics. Elapsing 10 minutes, the silicone tube was cut with the aid of a size 15 blade to expose the resin cylinder and the repairing interface. Following, the specimens were stored into distilled water at 37°C. C yl i nders of si lora ne- (F i ltek P 90) a nd dimethacrylate-based (Z350 XT) materials were constructed directly onto the discs of G7 and G8, respectively, without previous aging, to analyze the immediate adhesion between the materials. Thus, 80 specimens were submitted to micro shear bond strength test through a universal testing machine (EMIC, model DL3000, São José dos Pinhais, PR, Brazil) and observed regarding to failure types. The acrylic resin matrixes were mounted into a round device and fixed to the testing

machine with load applied with the aid of a steel wire (0.2 mm in diameter) and crosshead speed of 0.5 mm/min until rupture. The bonding strength was calculated with the aid of software linked to the testing machine by dividing the maximum force recorded (in Newton) by the bonding area (in mm2), and expressed in MPa. After the shear bond strength test, the specimens were exa mined w it h t he a id of stereoscopic magnifying glass at x30 magnification (SZX9, Olympus, Tokyo, Japan), to determine the failure mode, which were classified as follows: adhesive (at the bonding interface), cohesive (at the base resin or cylinder resin) and mixed. The data was submitted to statistical analysis through Student’s t test with level of confidence of 95%.

Results Micro shear bond strength values indicated an increasing in the bonding strength of the repairing procedures with roughening and application of the adhesive system onto the base disc (P90). Moreover, the results showed in table III and figure 1 demonstrated that the differences among G1, G3, G4, G5, G6 and G7 were not statistically significant (p < 0.05). The greatest micro shear bond strength mean values were observed in the specimens of G7 with immediate adhesion between silorane-/siloranebased resins without previous aging.

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Table III – Micro shear bond strength means and standard deviations (MPa) of the different groups Groups

Bond strength (MPa)

13.71 ± 8.67

4.09 ± 3.87

15.41 ± 8.91

11.41 ± 6.82

19.33 ± 5.29

14.97 ± 3.69

21.28 ± 5.90

1.52 ± 1.86

a b

Superscript letters mean statistically significant differences (p < 0.05)

Figure 1 – Micro shear bond strength of the different groups (MPa, means and standard deviations). G1 to G6 received repairing procedures (G1, G3 and G5 with P90; G2, G4 and G6 with Z350). G1 and G2 did not receive surface treatment, G3 and G4 received adhesive system; G5 and G6 received roughening procedure and adhesive system. G7 and G8 received immediate adhesion without repairing with either P90 or Z350, respectively

The smallest micro shear bond strength mean values were observed in the specimens of G8 and G2, that is, in the repairing procedure between silorane-/dimethacrylate-based material without surface treatment and in the immediate silorane/dimet hacr ylate-based materia l respect ively. Significantly highest micro shear bond strength values were observed in groups in which the surfaces had been treated with adhesive systems (G5 and G6), both for repairing procedures with silorane (G3 and G5) and dimethacrylate (G4 and G6), but without statistically significant differences

when compared with G7 (immediate adhesion between silorane-/silorane-based materials). The decreasing of the adhesivity after the aging period of the material surface was observed by comparing G7 w it h G1, w it hout statistica lly significa nt differences. Fig ures 2 a nd 3 exempli f ied t he fa ilure t y pes seen w it h t he a id of t he stereoscopic magnifying glass at x30 magnification. Cohesive failures were observed when part of the material within the cylinder was retained onto the base resin (figure 3B). The highest micro shear bond strength mean values (table III), exhibited by the immediate adhesion between silorane-/siloranebased materia ls (G7), a nd corresponded to cohesive failures. All failures of the immediate adhesion between silorane-/dimethacrylate-based materials (G8) and of the repairing procedure between silorane-/dimethacrylate-based materials without surface material (G2) were adhesive type (figure 2). In the groups submitted to the surface treatment with adhesive system and roughening, both for silorane-/silorane-based materials and silorane-/dimethacrylate-based materials, showing bond strength mean values significantly higher than those of G2 and G8, exhibited mixed and cohesive failures. In mixed failures, fragments of the cylinder resin were retained into the base resin (figure 3C). All failures within G2 and G8 were adhesive failures (figure 2), with the smallest micro shear bond strength mean values (figure 1). Adhesive failures where the base resin (substrate of resin composite P90) were clean and smooth, without resin remnants of the cylinder resin (figure 3D).

Figure 2 – Percentage (%) of the failure types during the micro shear bond strength testing (n = 10, per group)

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Figure 3 – Silicone cylinder and disc surfaces after the micro shear bond strength testing (x30 magnification): (a) empty cylinder to show its internal area to be filled with the resins to simulate the repairing procedure (0.95 mm in internal diameter); (b) cohesive failure; (c) mixed failure; (d) adhesive failure

Discussion B y c on s ide r i n g t he d i f fe re nt c he m i c a l characteristics between the dimethacrylate- and silorane-based resin composite, the aim of this study was to evaluate the bond strength between them. In the dimethacrylate-based composites, the oxygen-inhibited layer formed onto the surface after the polymerization of the material increments in contact with the environmental air enables a better adhesion between the layers of these materials [8]. On the other hand, the cationic polymerization of silorane-based materials do not lead to the formation of oxygen-inhibited layer and the decreasing of adhesion among successive increments of silorane over time [23] would cause the decreasing of micro shear bond strength. For this reason, in this present study, the preparation of each specimen of

G7 (control group – immediate adhesion between silorane/silorane) was executed with less than 5 min, to assure the greatest reactivity among successive increments, as suggested by Tezvergil-Mutluay et al. [23], who observed the greastet immediate adhesion values of silorane increments when those were inserted and light-cured in few seconds [23]. The greatest micro shear bond strength values were observed in the specimens of G7, as cylinders of silorane-based composites were made immediately onto discs of the same resin, therefore enabling cohesive failures within all specimens. As discussed above, the chemical reactivity of the components of silorane-based composites accounting for the adhesion of successive increments decreases over time and could affect the adhesion of repairing procedures [23]. Notwithstanding, in this present study, the reduction of the adhesivity

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after the aging of the material surface was not stat ist ica lly significa nt when t he specimens submitted to immediate adhesion between silorane/silorane-based material (G7) were compared with those underwent repairing without surface treatment (G1). These results demonstrated that despite of the aging period and conditions, the material still has enough reactivity to provide adhesion with chemically-compatible repairing material. The smallest micro shear bond strength mean values were seen in G8 specimens submitted to i m me d i ate ad he sion b et we en si lora ne -/ dimethacrylate-based materials. Other authors still verified a smaller immediate bond strength between silorane-/dimethacrylate-based materials, without either aging period or surface treatment [23]. The result of the lack of adhesion between the dimethacrylate-based and freshly-light-cured silorane-based material when this exhibits the greatest chemical reactivity confirms the lack of chemical affinity between the materials. This present study used a one-week period into distilled water at 37°C as aging method. Different authors studying repairing procedures on siloranebased resins have employed different techniques to mimic the conditions to which the restoration are submitted within oral cavity: 72 hours into saline solution at 37°C [12], 24 hours into water at 100ºC [15], one month into water at 60°C [18], and 24 hours into water at 37°C [20]. These methods have been used to promote the water sorption and leaching of components not participating in the polymerization reaction, prior to the repairing procedure. Micro shear bond strength testing was chosen in this study because it does not require additional cuts after the bonding of the segments during the preparation of the specimens, taking into consideration the low bond strength observed by other authors in both the immediate adhesion and the repairing between silorane- and dimethacrylatebased resins [4, 12, 23]. The evaluation method of micro shear bond strength through orthodontic wire has been sug gested as an alternative to microtensile bond strength because of its easier execution and good distribution of stresses in the adhesive interfaces [2]. According to Tezvergil-Mutluay et al. [23], the interposition of silorane-based adhesive system as intermediary layer would provide a better immediate adhesion between silorane-/dimethacrylate-based materials than that provided by dimethacrylatebased adhesive systems. The initial adhesion between silorane-/dimethacrylate-based materials thorugh silorane adhesive system, composed by

dimethacrylate with carboxylic and phosphoric acid groups, enables the reaction of the phosphate group with oxirane and that of acrylate with dimethacrylate promoting chemical adhesion [12, 23]. The effect of the lack of chemical affinity between silorane and dimethacrylate impairing the initial adhesion (G8) was not decreased by the surface aging of G2, but it occurred with the application of dimethacrylate-based adhesive system (G4). In the treatment with the exclusive application of the dimethacrylate-based adhesive system (G4) prior to repairing procedure, the micro shear bond strength values were not statistically different from those of the initial silorane-silorane adhesion (G7). The effect of dimethacrylate-based adhesive system application was more substantial than the decreasing of silorane chemical reactivity during the aging period, with statistically significant difference between G3 and G8. In G5 and G6, where the repaired surfaces were treated with roughening prior to the application of the adhesive systems, bond strength values were not statistically different from those of the immediate silorane/silorane adhesion (G7), both repaired with silorane- (G5) and dimethacrylate-based resins (G6). The roughening of material surface to be repaired promoted the weariness and development of micro rugosities. The intermediary interfaces created by roughening and the filling with low viscosity material provide micro retentions and allow a better adaptation with the repairing material of high viscosity. The roughening obtained with the aid of 600-grit sandpaper, employed in this present study, was also used by other authors [12]. Other roughening methods have been used in silorane repairing: 80-grit sandpaper [18], 320-grit sandpaper [15], abrasion with diamond drill and aluminum oxide blasting [25]. The surface treatment for the repairing between silorane-/dimethacrylate-based materials with roughening but without silane application prior to the application of the dimethacrylate adhesive system was not analyzed by this present study. The silane application executed in the repairing procedure of G6 could have contributed to increase the micro shear bond strength values as observed by other authors who used silane-based adhesive system [18] and silane prior to the adhesive system [25], which suggested that the silane promoted a greater superficial leakage. The greatest micro shear bond strength mean values observed in G7 (table III) corresponded to cohesive failures (figure 2). In mixed failures, fragments of the cylinder resin were retained into

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the base (figure 3C). The failures in G2 and G8 were of adhesive type (figure 2), with the smallest micro shear bond strength mean values (table III). In adhesive failures, the silorane discs were clean and smooth, without fragments of the cylinder resin (figure 3D). These results suggested that there would be little or none adhesion between the two resins. In this present study, adhesive systems indicated for either P90 and dimethacrylate, considering the two possible clinical situations: the composition of the original resin to be repaired is either known or unknown. Although this present study did not verify significant differences between the roughening treatment followed by the adhesive system and the single application of the adhesive system, roughening seems to be a more reliable surface treatment for repairing procedures because it increases the adhesion area.

3. Brosh T, Pilo R, Bichacho N, Blutstein R. Effect of combinations of surface treatments and bonding agents on the bond strength of repaired composites. J Prosth Dent. 1997 Feb;77(2):122-6.

Conclusion

7. Eick JD, Smith RE, Pinzino CS, Kostoryz EL. Stability of silorane dental monomers in aqueous systems. J Dent. 2006 Jul;34(6):405-10.

Surface treatments with the application of either adhesive systems or roughening enabled the repairing procedures in silorane-based resin composites when an adhesive system compatible with the repairing material was used. These results suggested that repairing procedures onto silorane-based resin composites can be executed successfully, regardless of the surface roughening and the composition of the repairing material.

Acknowledgments Th is st udy wa s c a r r ied out du r i n g t he Undergraduate Research Program of Positivo University, Curitiba, Paraná, Brazil (2012/2013). The restorative materials employed were kindly supplied by 3M ESPE.

References 1. Blum IR, Lynch CD, Wilson NH. Teaching of direct composite restoration repair in undergraduate dental schools in the United Kingdom and Ireland. Eur J Dent Educ. 2012 Feb;16(1):e53-8. 2. Braga RR, Meira JB, Boaro LC, Xavier TA. Adhesion to tooth structure: a critical review of “macro” test methods. Dent Mater. 2010 Feb;26(2): e38-49.

4. Cunha LF, Dziedzic DSM, Nascimento BM, Baratto SSP, Gonzaga CC, Furuse AY et al. Influence of different surface treatments on the shear bond strength of a methacrilate composite repaired with silorane. RSBO. 2013. In press. 5. Cunha LF, Santana LOC, Baratto SSP, Mondelli J, Haragushiku GA, Gonzaga CC et al. Staining susceptibility of methacrylate and silorane-based materials: influence of resin type and storage time. RSBO. 2013;10(2):161-6. 6. Demarco FF, Correa MB, Cenci MS, Moraes RR, Opdam NJ. Longevity of posterior composite restorations: not only a matter of materials. Dent Mater. 2012 Jan;28(1):87-101.

8. El-Askary FS, Fawzy AS, Abd Elmohsen HM. Tensile bond strength of immediately repaired anterior microfine hybrid restorative composite using nontrimmed hourglass specimens. J Adhes Dent. 2009 Feb;11(1):41-7. 9. Furuse AY, Cunha LF, Benetti AR, Mondelli J. Bond strength of resin-resin interfaces contaminated with saliva and submitted to different surface treatments. J Appl Oral Sci. 2007 Dec;15(6):501-5. 10. Furuse AY, Gordon K, Rodrigues FP, Silikas N, Watts DC. Colour-stability and gloss-retention of silorane and dimethacrylate composites with accelerated aging. J Dent. 2008 Nov;36(11):945-52. 11. Furuse AY, Pirolo R, Rodrigues LK, Pizzatto E, Losso EM, Mondelli J. The efficacy of acid etching for removing contamination in layered dental restorations. Gen Dent. 2012 Sep-Oct;60(5): e312-4. 12. Giachetti L, Scaminaci Russo D, Baldini M, Goracci C, Ferrari M. Reparability of aged silorane with methacrylate-based resin composite: micro shear bond strength and scanning electron microscopy evaluation. Oper Dent. 2012 JanFeb;37(1):28-36.

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13. Gordan VV. Clinical evaluation of replacement of class V resin based composite restorations. J Dent. 2001 Sep;29(7):485-8. 14. Guggenberger R, Weinmann W. Exploring beyond methacrylates. Am J Dent. 2000 Nov;13(Spec No):82D-4D. 15. Hamano N, Chiang YC, Nyamaa I, Yamaguchi H, Ino S, Hickel R et al. Effect of different surface treatments on the repair strength of a nanofilled resin-based composite. Dent Mater. J 2011;30(4):537-45. 16. Krejci I, Lieber CM, Lutz F. Time required to remove totally bonded tooth-colored posterior restorations and related tooth substance loss. Dent Mater. 1995 Jan;11(1):34-40. 17. Luhrs AK, Gormann B, Jacker-Guhr S, Geurtsen W. Repairability of dental siloranes in vitro. Dent Mater. 2011 Feb;27(2):144-9. 18. Maneenut C, Sakoolnamarka R, Tyas MJ. The repair potential of resin composite materials. Dent Mater. 2011 Feb;27(2):e20-7. 19. Manhart J, Garcia-Godoy F, Hickel R. Direct posterior restorations: clinical results and new developments. Dent Clin North Am. 2002 Apr;46(2):303-39.

20. Mobarak EH. Effect of surface roughness and adhesive system on repair potential of silorane-based resin composite. J Advance Res. 2012;3:279-86. 21. Moncada G, Martin J, Fernandez E, Hempel MC, Mjor IA, Gordan VV. Sealing, refurbishment and repair of Class I and Class II defective restorations: a three-year clinical trial. J Am Dent Assoc. 2009 Apr;140(4):425-32. 22. Soderholm KJ. Flexure strength of repaired dental composites. Scand J Dent Res. 1986 Aug;94(4):364-9. 23. Tezvergil-Mutluay A, Lassila LV, Vallittu PK. Incremental layers bonding of silorane composite: the initial bonding properties. J Dent. 2008 Jul;36(7):560-3. 24. Weinmann W, Thalacker C, Guggenberger R. Siloranes in dental composites. Dent Mater. 2005 Jan;21(1):68-74. 25. Wiegand A, Stawarczyk B, Buchalla W, Taubock TT, Ozcan M, Attin T. Repair of silorane composite – using the same substrate or a methacrylate-based composite? Dent Mater. 2012 Mar;28(3):e19-25.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):28-40

Original Research Article

Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive – analysis by SEM and confocal laser scanning microscopy (CLSM) Aline de Oliveira Gonçalves1 Cinthia Sawamura Kubo1 Osmir Batista Oliveira Júnior1 Edson Alves de Campos1 Marcelo Ferrarezi Andrade1 Corresponding author: Aline de Oliveira Gonçalves Universidade Estadual Paulista (Unesp), Faculdade de Odontologia de Araraquara Rua Humaitá, n. 1.680, sala 310, 3.º andar CEP 14801-385 – Araraquara – SP – Brasil E-mail: alineogoncalves@hotmail.com 1

Department of Restorative Dentistry, São Paulo State University, School of Dentistry of Araraquara – Araraquara – SP – Brazil.

Received for publication: June 26, 2013. Accepted for publication: November 11, 2013.

Keywords: dental cavity preparation; confocal microscopy; lasers; scanning electron microscopy; smear layer; dentinal adhesives.

Abstract Introduction and Objective: This study aimed to evaluate the effect of Er:YAG (L) and diamond drills (DD) on: 1) the microshear bond strength (MPa); 2) the adhesive interface of two-step (TS) – Adper Scotchbond Multipurpose and one-step (OS) adhesives – Adper EasyOne, both from 3M ESPE. Material and methods: According to the preparation condition and adhesives, the samples were divided into four groups: DD_TS (control); DD_OS; L_TS and L_OS. 60 bovine incisors were randomly divided into experimental and groups: 40 for microshear bond strength (n = 10) and 20 for the adhesive interface morphology [6 to measure the thickness of the hybrid layer (HL) and length of tags (t) by CLSM (n = 3); 12 to the adhesive interface morphology by SEM (n = 3) and 2 to illustrate the effect of the instruments on dentine by SEM (n = 1)]. To conduct the microshear bond strength test, four cylinders (0.7 mm in diameter and 1 mm in height with area of adhesion of 0.38 mm) were constructed with resin composite (Filtek Z350 XT

29 – RSBO. 2014 Jan-Mar;11(1):28-40 Gonçalves et al. – Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive – analysis by SEM and confocal laser scanning microscopy (CLSM)

– 3M ESPE) on each dentin surface treated by either L or DD and after adhesives application. Microshear bond strength was performed in universal testing machine (EMIC 2000) with load cell of 500 kgf and a crosshead speed of 0.5 mm / min. Adhesive interface was characterized by thickness of hybrid layer (HL) and length of tags (t) in nm, with the aid of UTHSCSA ImageTool software. Results: Microshear bond strength values were: L_TS 34.10 ± 19.07, DD_TS 24.26 ± 9.35, L_OS 33.18 ± 12.46, DD_OS 21.24 ± 13.96. Two-way ANOVA resulted in statistically significant differences only for instruments (p = 0.047). Mann-Whitney identified the instruments which determined significant differences for HL thickness and tag length (t). Concerning to the adhesive types, these differences were only observed for (t). Conclusion: It can be concluded that 1) laser Er:YAG results in higher microshear bond strength values regardless of the adhesive system (TS and OS); 2) the tags did not significant affect the microshear bond strength; 3) the adhesive interface was affected by both the instruments for cavity preparation and the type of adhesive system used.

Introduction The technological advancement of the adhesive restorative materials has allowed the execution of more conservative cavity preparations in which the dentist only removes the carious tissue and obtains t he convenience form to access a nd instrument the cavity [13]. The use of diamond drills at high speed to perform cavity preparations has the following main advantages: fast and precise cutting of the tooth structure; obtainment of defined angles and walls; the presence of micro-grooves on the cavity walls, which can contribute to increase the retention of direct restorations [3]. However, its disadvantages comprise to cause: pain and physical/emotional discomfort to the patient; pulp damage because of heating the tooth structure due to friction; cross contamination and diseases because of the spray generated during preparation [4]. Also, it jeopardizes the visualization of the cavity during instrumentation. Because Er:YAG laser is silent and does not need the direct contact with the dental structure, there is not pain and discomfort to patient and it enables the execution of minimally invasive cavity preparations, which mostly are impossible to be performed with diamond drills [13, 18]. Er:YAG laser action is based on the ejection of fragments of toot h structure (ablation), because of t he

kinetic mechanical action of intense releasing of energy due to micro-explosions of water molecules irradiated by the laser beam [13]. This can affect the performance of adhesive systems since these have been developed to either remove or incorporate the smear layer resulting from the cavity preparations with drills on their surfaces. Considering that the hybrid layer seems to develop a critical role in adhesive Dentistry [7] and that the tooth substrate type can influence on its formation and on the adhesion mechanism [32], it can be stated that the tooth substrate obtained by several instruments may influence on the final result of the adhesive restoration, leading to the formation of different patterns of hybrid layer. This study aimed to evaluate the effect of Er:YAG laser and drills on the microshear bonding strength and the bonding interface of one- and t wo -step bondi ng a gent s t h roug h SEM a nd confocal laser scanning microscopy (CLSM).

Material and methods This study was submitted and approved by the Ethical Committee in Research of the São Paulo State University. Sixty bovine incisors without caries were stored into 0.2% thymol solution and kept under refrigeration at temperature of about 7ºC for seven days. The teeth were randomly distributed according to figure 1.

30 – RSBO. 2014 Jan-Mar;11(1):28-40 Gonçalves et al. – Effect of Er:YAG laser and diamond drill on hybrid layer morphology obtained with self-etch adhesive – analysis by SEM and confocal laser scanning microscopy (CLSM)

Figure 1 – Distribution and amount of teeth of each experiment and group

The teeth were prepared with the aid of a diamond drill (DD) no. 3131 (KG Sorensen) coupled to high speed handpiece (Kavo). Er:YAG laser (L) (Twinlight: Fotona, Slovenia) was used. Two-step (TS) bonding system used was Adper Scotchbond SE Multipurpose (3M ESPE, St. Paul, MN, USA), while onestep (OS) bonding system was Adper Easy One (3M ESPE, St. Paul, MN, USA) and both were applied onto the dentin surface of the samples.

Experiment 1 – Microshear bond strength Sample preparation The teeth had their roots and distal and mesial surfaces of their crowns removed with the aid of a diamond disc (11-4254, 4”x 0.012” / series 15LC, Diamond Blade, Buehler Ltd., Lake Bluf, IL, USA) and metallographic cutting machine (ISOMET 1000, Buehler, Lake Buff, IL, USA). The labial surface was worn and flattened with the aid of a polishing machine (DP-10 Panambra, Struers, Ballerup, Denmark), to obtain a smooth and flat tooth surface. The fragments were embedded into acrylic resin inside PVC

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tubes measuring 1.2 cm in height and 2.0 cm in diameter. After the resin setting, the surface was cleaned and again flattened in the polishing machine. Then, the samples were washed with water jet and stored into distilled water for 24 h. After this period, each fragment was submitted to a superficial weariness executed with either (L) or (DD). Adhesive procedures The bonding area was delimited onto a double-sided adhesive tape which was guided by four perforation of a tripartite matrix measuring 0.7 mm in diameter. The sequence of application of the bonding systems were performed according to the manufacturers’ instructions (table I). All light-curing procedures were carried out with the aid of a LED unit (LED Bluephase, Ivoclar Vivadent, Schann – Liechtenstein), at low intensity (± 800 mW/cm2). Polyethylene tubes were place onto the tripartite matrix with area of 0.38 mm2 (Tygon tubing, R-3603, Saint-Gobain Performance Plastics, Maime Lakes, FL, USA). This set was filled with resin composite (Filtek Z350 XT) and the four cylinders were light-cured for 40 s. The samples were stored into distilled water at 37°C for 24 h. Elapsed that time, the tubes were removed with the aid of a scalpel blade to obtain a resin composite cylinder. Table I – Composition and application of the bonding systems used

Adper™ Scotchbond™ Multipurpose (3M ESPE, St. Paul, MN, USA) Primer: 2-hydroxyethyl methacrylate and polyalkenoic acid. Composition Bonding agent: bisphenol a-glycidyl methacrylate (Bis-GMA), 2-hydroxyethyl methacrylate (HEMA), polymerization initiator system. 1. Acid etching on dentin for 15 seconds. Wash for 30 seconds; gently dry for 2 seconds; Application 2. Application of primer agent and dry for 5 seconds; 3. Light-cure for 10 seconds. Adper™ EasyOne™ (3M ESPE, St. Paul, MN, USA) 2 hydroxyethyl methacrylate, Bis-GMA, methacrylated phosphoric esters, 1,6 hexamediol dimethacrylate, Methacrylate functionalized Polyalkenoic acid Composition Vitrebond copolymer, finely dispersed bonded silica filler with 7 nm primary particle size, water, ethanol, initiators based on camphorquinone, stabilizers. 1. Apply the bonding agent for 20 seconds; Application 2. Gently air dry for 5 seconds; 3. Light-cure for 10 seconds. Microshear bond strength testing Elapsed the storage time period, the samples were individually coupled to a metallic device and fixed to an universal testing machine (EMIC DL 2000, São José dos Pinhais, PR, Brazil). Prior to testing, the device was carefully aligned to allow that the loading was applied as closest as possible to the bonding interface in the base of each cylinder. This loading was applied through a ring made of steel wire (0.2 mm in diameter). The test was performed with a load cell of 500 Kgf and crosshead speed of 0.5 mm/min until the fracture of the specimens. Microshear bond strength was calculated by dividing the maximum force registered during the testing (N) by the bonding area (0.38 mm2) and expressed into MPa. The results obtained were tabulated in an Excel sheet (Excel Microsoft) and analyzed statistically with the aid of SPSS Statistic V 19 software (IBM Corporation, Armonk, NY, USA).

Experiment 2 – Morphology of the bonding interface Morphology of the bonding interface Cavity preparations: two bovine incisors had the medium third of the crown prepared measuring 3.0 mm in width, 2.0 mm in height and 2.0 mm in deepness. One tooth was prepared through laser

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while the other through diamond drills. Next, the teeth were washed and dried with air jet for 1 minute. Aiming to avoid technique artifacts, the surfaces analyzed in SEM were replicated with epoxy resin (Epoxide; Buhler). Impression of the surfaces were taken with the aid of an addition silicone (Express XT Penta – 3MESPM) mixed according to the manufacturer’s instructions. After the material setting, the impressions were filled with Epoxide, mixed according to the manufacturer’s instructions. The set was stored at environment temperature and controlled humidity for 24 hours. The samples were longitudinally cut with the aid of metallographic cutting machine (ISOMET 1000, Buehler, Lake Buff, IL, USA) and diamond disc (11-4254, 4”x 0.012”/ series 15LC, Diamond Blade, Buehler Ltd., Lake Bluf, IL, USA), under water cooling. SEM: t he re pl ic a s w e re re move d f rom the impressions, identified and submitted to metallization, in which a layer of 24-carat gold powder with thickness varying from 50 to 100 Angstron was deposited by vaporization onto the samples. The bonding interface of each group was analyzed and characterized with the aid of a scanning electronic microscope (Jeol JSM – 6610 LV, Tokyo, Japan). The dentin morphology was described regarding to its structural characteristics. CLSM: Six incisors were divided into two groups (n = 3), according to the instrument type employed in cavity preparation (laser or diamond drill). At the medium third, two cavity preparations (3.0 x 2.0 x 2.0 mm) were executed. The incisal cavities (IC) were restored with resin composite (Filtek Z350 – 3M ESPE, St. Paul, MN, USA) and one-step (OS) bonding agent (AdperTM Easy One – 3M ESPE, St. Paul, MN, USA). The cervical cavities (CC) were restored with two-step (TS) bonding agent (AdperTM ScotchbondTM Multipurpose – 3M ESPE, St. Paul, MN, USA) and resin composite (AdperTM Z350 XT – 3M ESPE, St. Paul, MN, USA). Elapsed the 24 h storage in distilled water at 36±1oC, the samples were longitudinally cut to expose the bonding adhesive and characterized through diamond disc (11-4254, 4”x 0.012” / series 15LC, Diamond Blade, Buehler Ltd, Lake Bluf, IL, USA) with the aid of a cutting machine (ISOMET 1000 – Buehler, Lake Buff, IL, USA). The bonding agents for analysis in CLSM were pigmented with rhodamine B – C28H31CIN2O3 – (LABSYNTH Produtos para Laboratórios Ltda., Diadema, SP, Brazil) at 0.001 g/ml ratio. The right surface of the adhesive interface was polished with 1200-grit silicon carbide sandpaper. The samples were ultrasonically cleaned in water for 3 minutes to remove the debris coming from polishing.

Analysis of confocal laser scanning fluorescence microscopy (CLSFM) The hybrid layer and the configuration and the medium leng th of tags were assessed in confocal laser microscopy (Leica TCS SMD – Leica Microsystems – Integrated Research Center of the School of Dentistry of the University of São Paulo, Brazil). The thickness of the hybrid layer was quantitatively measured at different sites of the bonding interface. The obtained results were related to the perpendicular distances between the dentin-composite junction. To perform these measurements, UTHSCSA ImageTool software (http://en.bio-soft.net/draw/ImageTool.html) was used. The thickness of hybrid layer was measured at the incisal, cervical and a xial walls of the cavities. Five measurements were executed on each wall, totalizing ten measurements for each sample. The morphology of the bonding interface by SEM: the labial surfaces of 12 bovine incisors were worn in a polishing machine up to obtain their flattening and dentin exposure. Following, either L or DD weariness were executed according to which is established in figure 1. Next, the bonding sequence was applied and a thin layer of resin composite (Filtek Z350) was inserted and light-cured. To exposure the bonding interface, the samples were identified and longitudinally cut with the aid of a diamond disc (11-4254, 4”x 0.012” series 15LC, Diamond Blade, Buehler Ltd., Lake Bluf, IL, USA) and cutting machine (ISOMET 1000 – Buehler, Lake Buff, IL, USA). Additional cuts left the samples measuring 10.0 mm in width and 2.0 mm in height. The interface was polished with the aid of 1200-grit silicon carbide sandpaper. The samples were ultrasonically cleaned for 3 minutes. Next, the surface was submitted to the action of HCl 4M for 0.5 min, washed and exposed to 5% NaOCl for 10 min. Elapsed this time, the samples were washed and dried with the aid of air jet. Impressions were taken from the specimens with the same technique described above for the obtainment of the images of dentin surface. The morphology of the bonding interface (HL a nd t) was a na lyzed t h roug h sca n ni ng electronic microscope (Jeol JSM – 6610 LV, Tokyo, Japan, of the School of Dentistry of Araraquara – Department of Dental Materials and Prosthesis) at x450 magnification. The bonding interface morphology was comparatively described regarding to its structural features.

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Results Experiment 1 – Effect of Er:YAG laser and diamond drill on microshear bond strength of one- and two-step bonding agents The descriptive statistics of microshear bond strength testing (MPa) is seen in table II. Table II – Mean and standard-deviation (SD) of microshear bond strength (MPa) regarding to the instruments (diamond drill – DD; and Er:YAG laser – L) and adhesive systems: one- (OS) and two-step (TS)

Bonding agent Instrument

Mean

SD (±)

Mean

SD (±)

Laser

34.10

19.07

33.18

12.46

Drill

24.26

9.35

21.24

13.96

Kolmogorov-Smirnov (p(DD) = 0.20 and p(L) = 0.07) and Levene (p(DD, L) = 0.27 and p(OS and TS) = 0.68) tests confirmed the application of two-way ANOVA. Two-way Anova results are displayed in table III. Table III – Two-way ANOVA for microshear bond strength mean values (MPa) using two different bonding agents (one and two steps) applied after different dentin treatments (laser and drill) (p < 0.05) DF

h 2p

Instrument

795.575

4.399

.043

.109

.532

Bonding agent

44.542

.246

.623

.007

.077

Instrument* bonding agent

14.268

.079

.780

.002

.059

Error

6510.812

180.856

Total

41424.677

Variation source

* Based on the test results, the influence of the effect “bonding agent” and the interaction of the effects were considered as nonsignificant (p > 0.05). The influence of the effect “instrument” was considered as significant (p < 0.05) DF degrees of freedom; SS = sum of squares; MS = mean square; h2p = dimension of the effect and p = test power

These results are presented in graph 1, in which different letters mean statistically significant differences at p < 0.05.

Graph 1 – Means and standard-deviation (MPa) of microshear bond strength in relation to the instruments (L and DD) and bonding agents (OS and TS) * Different letters indicate statistic differences for p < 0.05

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Experiment 2 – Effect of Er:YAG laser and diamond drill on the morphology of the bonding interface of one- and two-step bonding agent – study by CLSFM and SEM The descriptive statistics for the effect of instruments and bonding agents on the bonding interface (HL) and (t) is seen in table IV.

Table IV – Mean, standard-deviation (SD) and confidence interval (CI95%) of the hybrid layer (HL) thickness and length of tags (t) measured in µm, according to the instruments diamond drill (DD) and Er:YAG laser (L) and one(OS) and two-step (TS) bonding agents

Bonding Interface Tags HL Tags UL

Variable

Mean

SD (±)

18.89

CI95% Lb

8.42

16.72

21.06

20.89

24.52

14.56

27.23

13.55

9.98

10.97

16.13

28.46

15.72

24.40

32.52

24.58

24.53

18.25

30.92

15.20

5.28

13.84

16.56

21.09

16.83

16.74

25.44

20.92

13.29

17.49

24.36

These data were analyzed through Mann-Whitney non-parametric test (U) because the parametric tests failed. Thus, the distribution of the different measurement of (HL) and (t) was compared for instruments and bonding agents. Instruments (DD and L) exhibited statistically significant differences in the thickness of the hybrid layer (HL) (U = 804.00; W = 2.63 and p = 0.00) and for the length of tags (t) (U = 2.26; W = 4.09 and p = 0.015). Bonding agents (OS and TS) did not formed hybrid layers with statistically significant thickness (U = 1.94; W = 3.77 and p = 0.46), but produced tags (t) with statistically different lengths (U = 1.35; W = 3.18 and p = 0.019).

SEM analysis Figure 2 illustrates the effect of (DD) and (L) on the bonding interface (HL) and (t) in the samples. Different morphologic bonding interfaces were observed. The hybrid layers, although present in all groups/samples, were thin and visually with similar thickness. The use of one-step bonding agent exhibited a more regular bonding interface, while two-step bonding agent showed thickness variations, deficiencies in the diffusion of the monomers and in the encapsulation of the collagen fibers. The specimens that received the drill action showed the occurrence of many “droplets”, which was not observed in the specimens receiving the laser action. Two-step bonding agent presented a clear formation of tags (t), which could not be observed in the specimens receiving one-step bonding agent. The group L_TS exhibited longer and irregular tags than those of the other groups.

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Figure 2 – Morphology of the bonding interface in relation to the instruments (DD and L) and bonding agents (OS and TS) RC = resin composite; D = dentin; HL = hybrid layer; t = tags; arrow = droplets

Analysis of the dentin surface The analysis of the dentin surface by SEM enabled the visualization of an amorphous and uniform smear layer covering the surface of the specimen receiving the action of the diamond drill (figure 3). The morphology of the cavity preparation executed with Er:YAG laser was very different. It was observed lack of smear layer, dentin with aspect of desquamation, with very irregular margins and superficial waves, and open dentinal tubules in great number. Also, neither the exposure of collagen fibers nor presence of peri- and/or intertubular dentin was observed.

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Figure 3 – SEM images of the dentin surface after the action of the diamond drill (DD) and Er:YAG laser (L) and prior to the application of the bonding agents (OS and TS). x500 and 3,000 magnification

Discussion and Conclusion It could be observed, similarly to the studies of Antunes et al. [3], Oliveira et al. [30] (2005), Oliveira et al. [29] and Semeraro et al. [34], that the surface treatment with diamond drill (DD) resulted in a regular substrate with some waves and grooves, presence of smear layer over all dentin and the closure of the dentinal tubules (figure 3). The use of Er:YAG laser (L) resulted in a very regular substrate forming craters, elevations and pores, with rugosities and granulations and with lack of smear layer (figure 3). This same morphological aspect was also described by Delmé

et al. [15], Esteves-Oliveira et al. [17], Shirai et al. [36], Nishimoto et al. [27], Shigetani et al. [35], Navarro et al. [26], and Ramos et al. [33]. These differences can be attributed to the mechanism of action of laser, which is characterized by the ablation of the tooth tissues. During its operation, the laser source emits photons inside a water-air jet towards the target tissue. The abrupt heating of the water closer to the surface results in a series of micro-explosions and ejection of hard tissues of the tooth [17, 18, 20, 26, 35]. The formation of a morphologically irregular dentin may favor the bonding procedure, since it increases the number of micromechanical retentions.

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On the other hand, not even the increase of the micromechanical retention results in the increase of the bond strength [5, 9, 25]. The analysis of the figure 2 enables to observe a clear morphological difference in the hybrid layer formed by the different bonding systems on the surfaces prepared with either the drill or laser. The two-step bonding agent, in both substrates, formed a hybrid layer with similar thickness with the presence of many tags. It was also noted the presence of small lateral rami in these tags, namely micro-tags, indicating that the monomers from the bonding agent could propagate and penetrate deeply in the dentin etched [1]. This difference in the performance of one- and two-step bonding agents is explained by the etching of dentin with 37% phosphoric acid, which promoted a greater dentin demineralization, increasing the infiltration of the resin monomers. The group TS exhibited a hybrid layer less thick and practically without tags. The tendency is this bonding agent form uniform hybrid layers without failures, which is mandatory to prevent nanoleakage. Also, it could be noted in the bonding interface of OS bonding agent the presence of little droplets (figure 2), that is, the separation of a localized zone close to the bonding interface, which resulted from the water absorption coming from the dentin by osmosis due to low molecular weight of HEMA that makes the area fragile and compromises the effectiveness and longevity of the bonding agent [1]. According to Van Meerbeek et al. [39], mild one-step bonding agents dissolve the smear layer without demineralizing the surface in deepness, thus removing the hydroxyapatite from the interface. By preserving the hydroxyapatite, the collagen fibers are protected from external chemical aggression and calcium are available for the chemical bonding to the functional monomer of the bonding agent. The literature demonstrated that two-step agents similar to that used in this present study can produce high bond strength values to dentin [11, 36, 37]. The control group (DD_TS) showed a microshear bond strength mean value of 24.26 MPa, while the group exhibiting the highest mean was L_TS, with mean of 34.10 MPa. The higher bond strength of group L_TS than that of DD_TS can be explained by the different patterns of smear layer obtained by the instruments and by the greater dentin demineralization promoted by the total etching. However, despite of the favorable mechanical results, the removal of smear layer in the bonding technique of TS bonding agents

allowed the exudation of the dentinal fluid [32], which can interfere in the polymerization of the resin monomer and cause dentin hypersensitivity by affecting the hydrodynamic balance of dentin [10]. Thus, although group L_TS showed the highest bond strength value (16.10 MPa), its bonding interface presented excessive longer tags that during clinical situation may lead to dentinal hypersensitivity and pulp damage. Many studies obtained results similar to those of this present study, indicating that generally the bond strength value of one-step bonding agent is statistically similar to that of two-step bonding agents [10, 37]. Notwithstanding, because of the hydrophilic characteristic of one-step bonding agents, they have a tendency towards the deterioration of the bonding interface since the hydrophilicity of these systems leads to humidity absorption both from the oral environment and the pulp, causing hydrolytic degradation. This present study suggests the preparation of class V cavity by laser because the results demonstrated that laser produces a substrate with many micro-retentions and lack of smear layer, favoring the penetration of both types of bonding agents and the highest microshear bond strength values were obtained with the use of this instrument. Due to adhesion difficulty, cervical lesions are very employed to evaluate the clinical performance of adhesive systems. Barceleiro et al. [6] and Lizarelli et al. [22] obtained a greater success in laser performance than that of diamond drills through a microleakage study. In this present study, when two-step bonding agent is used, the preparation with laser favored the bonding process in comparison with those prepared with drills. In the substrate irradiated, the mean hybrid layer was 1.32 µm, while that obtained by drill preparation was 1.03 µm. The same behavior was observed when one-step bonding agent was used, with a greater effect for laser (1.42 µm) than for drill preparation (1.03 µm). It is worth observing that the mean thickness of the hybrid layer obtained through drills was the same for both bonding agents. These data are contrary to those of the studies conducted by Barceleiro et al. [6]. These authors by analyzing the surface either irradiated by laser or prepared by drills, obtained a deficient hybrid layer for the former. Barceleiro et al. [7] carried out a study with the same instruments and many adhesive systems and found a hybrid layer less homogenous when the dentin was prepared by drills, which was a result similar to that of this present study.

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The literature states that the formation of the hybrid layer on the surface irradiated by Er:YAG laser is more susceptible to the loss of tensile bond strength [17, 38], smaller thickness [6, 8, 16, 29, 36] and to the increasing of marginal leakage [19, 20]. The smaller bond strength of the surfaces irradiated by laser comes from the dentin abrasion which promotes the fusion of the collagen fibers, restricting resin diffusion within peritubular dentin [24]. Other authors, however, affirmed that laser is a good alternative to conventional drills because it promotes a good marginal sealing [19, 26, 35] and does not affect the bond strength of adhesive systems [7, 10, 14]. In the evaluation of the bonding interface by CLSFM, one- and two-step bonding agents showed non-expected behaviors regarding to the hybrid layer formation when submitted to laser, since they displayed similar thickness: 1.32 µm (TS) and 1.42 µm (OS). These results are related to the lack of smear layer and to the dentin morphology after the laser action. However, this behavior of the hybrid layer and tags of the samples irradiated by laser does not necessarily mean that the bonding to this substrate is more effective than that of the groups treated by drills. The micro-explosions, common features of the mechanism of action of laser could remove and damage the collagen fibers jeopardizing the adhesion. The control group (DD_TS) and group DD_OS showed the same hybrid layer thickness, which was not expected because the mechanism of action of the different bonding agents. Albaladejo et al. [1], Korkmaz et al. [20], Moura et al. [24] and Van Meerbeek et al. [39] already proved that the fact most affecting bonding strength is HL and not the length or number of tags. Additionally, the obtainment of longer tags may indicate the possibility of damaging to both the odontoblastic processes and pulp tissue by the monomers still present within the tags partially or totally unpolymerized. Because in this present study, groups L_TS and DD_TS exhibited numerous and long tags (means of 28.39 µm and 20.77 µm, respectively) and the laser determined the highest microshear bond strength values, it can be inferred that both mechanically and biologically the use of OS bonding agents is the most recommended [7, 29, 30]. Although the results of this present study showed the higher microshear bond strength of dentin treated by laser than that treated by diamond drills, it was observed a moderate significance (h2p = 0.10) for clinical practice with a low test power (π = 0.53). These results suggested that the number of specimens for

each group (n = 10) could have not be enough to evidence safely the effect of the instruments and bonding systems, despite this sampling number is greater than that of many studies already published: Ramos et al. [33] (n = 4), Mendez et al. [23] (n = 5), Lenzi et al. [21] (n = 5), Carvalho et al. [12] (n = 10), and Ali et al. [2] (n = 10).

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9. Brudevold F, Buonocore M, Wileman W. A report on a resin composition capable of bonding to human dentin surfaces. J Dent Res. 1956 Dec;35(6):846-51. 10. Cal-Neto JP, de Miranda MS, Dias KR. Comparative SEM evaluation of penetration of adhesive systems in human dentin with a non-rinse conditioner and a self-etching primer. Braz Dent J. 2004;15(1):19-25. 11. Cardoso MV, Coutinho E, Ermis RB, Poitevin A, Van Landuyt K, De Munck et al. Influence of dentin cavity surface finishing on micro-tensile bond strength of adhesives. Dent Mater. 2008 Apr;24(4):492-501. 12. Carvalho RC, de Freitas PM, Otsuki M, de Eduardo CP, Tagami J. Micro-shear bond strength of Er:YAG-laser-treated dentin. Lasers Med Sci. 2008 Apr;23(2):117-24. 13. Cozean C, Arcoria CJ, Pelagalli J, Powell GL. Dentistry for the 21st century? Erbium:YAG laser for teeth. J Am Dent Assoc. 1997 Aug;128(8):1080-7. 14. Dalia MP, Gomes PF, Menezes-Filho RP, Guimarães ALA, Mariz CHV. Ultraconservative dentistry – alternative methods of cavities preparations. RFO. 2009;14(2):168-73. 15. Delmé KI, De Moor RJ. Scanning electron microscopic evaluation of enamel and dentin surfaces after Er:YAG laser preparation and laser conditioning. Photomed Laser Surg. 2007 Oct;25(5):393-401. 16. Ergücü Z, Celik EU, Unlü N, Türkün M, Ozer F. Effect of Er, Cr:YSGG laser on the microtensile bond strength of two different adhesives to the sound and caries-affected dentin. Oper Dent. 2009 Jul-Aug;34(4):460-6.

19. Karaarslan ES, Usumez A, Ozturk B, Cebe MA. Effect of cavity preparation techniques and different preheating procedures on microleakage of class V resin restorations. Eur J Dent. 2012 Jan;6(1):87-94. 20. Korkmaz Y, Ozel E, Attar N, Bicer CO, Firatli E. Microleakage and scanning electron microscopy evaluation of all-in-one self-etch adhesives and their respective nanocomposites prepared by erbium:yttrium-aluminum-garnet laser and bur. Lasers Med Sci. 2010 Jul;25(4):493-502. 21. Lenzi TL, Tedesco TK, Soares FZ, Loguercio AD, Rocha RO. Chlorhexidine does not increase immediate bond strength of etch-and-rinse adhesive to caries-affected dentin of primary and permanent teeth. Braz Dent J. 2012;23(4):438-42. 22. Lizarelli RFZ, Silva PCG, Kurachi C, Porto Neto ST, Bagnato VS. Estudo-piloto comparativo da microinfiltração in vitro entre os preparos cavitários classe V, através da ponta diamantada em alta rotação ou laser de Er:Yag seguido ou não de ataque ácido. JBD. 2002;1(1):33-6. 23. Mendez JC, Pabon GE, Hilgenberg SP, Garcia EJ, Arana-Correa B. Effect of water storage on microtensile bond strength of a two-step self-etch adhesive and a two-step etch-and-rinse adhesive Acta Odontol Latinoam. 2012;25(2):176-80. 24. Moura SK, Santos JF, Ballester RY. Morphological characterization of the tooth/adhesive interface. Braz Dent J. 2006;17(3):179-85. 25. Nakabayashi N, Pashley DH. Hibridização dos tecidos dentais duros. São Paulo: Quintessence; 2000.

17. Esteves-Oliveira M, Zezell DM, Apel C, Turbino ML, Aranha AC, Eduardo CP et al. Bond strength of self-etching primer to bur cut, Er, Cr: YSGG and Er:YAG laser dental surfaces. Photomed Laser Surg. 2007 Oct;25(5):373-80.

26. Navarro RS, Gouw-Soares S, Cassoni A, Haypek P, Zezell DM, de Paula Eduardo C. The influence lf erbium:yttrim-aluminum-garnet laser ablation with variable pulse width on morphology and microleakage of composite restorations. Lasers Med Sci. 2010 Nov;25(6):881-9.

18. Hibst R, Keller U. Experimental studies of the application of the Er:YAG laser on dental hard substances: I. Measurement of the ablation rate. Lasers Surg Med. 1989;9(4):338-44.

27. Nishimoto Y, Otsuki M, Yamauti M, Eguchi T, Sato Y, Foxton RM et al. Effect of pulse duration of Er:YAG laser on dentin ablation. Dent Mater J. 2008 May;27(3):433-9.

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28. Oliveira WJ, Pagani C, Rodrigues JR. Comparação da adesividade de dois sistemas adesivos autocondicionantes em esmalte de dentes bovinos. Rev Fac Odontol S J dos Campos. 2001;4(1):43-6.

34. Semeraro S, Mezzanzanica D, Spreafico D, Gagliani M, Re D, Tanaka T et al. Effect of different bur grinding on the bond strength of self-etching adhesives. Oper Dent. 2006 MayJun;31(3):317-23.

29. Oliveira SS, Pugach MK, Hilton JF, Watanabe LG, Marshall SJ, Marshall Jr. GW. The influence of the dentin smear layer on adhesion: a self-etching primer vs. a total-etch system. Dent Mater. 2003 Dec;19(8):758-67.

35. Shigetani Y, Tate Y, Okamoto A, Iwaku M, Abu-Bakr N. A study of cavity preparation by Er: YAG laser. Effects on the marginal leakage of composite resin restoration. Dent Mater J. 2002 Sep;21(3):238-49.

30. Oliveira DC, Manhães LA, Marques MM, Matos AB. Microtensile bond strength analysis of different adhesive systems and dentin prepared with highspeed and Er:YAG laser: a comparative study. Photomed Laser Surg. 2005 Apr;23(2):219-24. 31. Paris S, Bitter K, Renz H, Hopfenmuller W, Meyer-Lueckel H. Validation of two dual fluorescence techniques for confocal microscopic visualization of resin penetration into enamel caries lesions. Microsc Res Tech. 2009 Jul;72(7):489-94. 32. Perdigao J, Swift Jr. EJ, Denehy GE, Wefel JS, Donly KJ. In vitro bond strengths and SEM evaluation of dentin bonding systems to different dentin substrates. J Dental Res. 1994 Jan;73(1):44-55. 33. Ramos TM, Ramos-Oliveira TM, Moretto SG, Freitas PM, Esteves-Oliveira M, de Paula Eduardo C. Microtensile bond strength analysis of adhesive systems to Er:YAG and Er,Cr:YSGG laser-treated dentin. Lasers Med Sci. 2013 Jan 26. [Epub ahead of print].

36. Shirai K, De Munck J, Yoshida Y, Inoue S, Lambrechts P, Suzuki K et al. Effect of cavity configuration and aging on the bonding effectiveness of six adhesives to dentin. Dent Mater. 2005 Feb;21(2):110-24. 37. Susin AH, Vasconcellos WA, Saad JR, Oliveira Junior OB. Tensile bond strength of self-etching versus total etching adhesive systems under different dentinal substrate conditions. Braz Oral Res. 2007 Jan-Mar;21(1):81-6. 38. Tonami K, Takahashi H, Kato J, Nakano F, Nishimura F, Takagi Y et al. Effects of laser irradiation on tensile strength of bovine dentin, Photomed Laser Surg. 2005 Jun;23(3):278-83. 39. Van Meerbeek B, Inokoshi S, Braem M, Lambrechts P, Vanherle G. Morphological aspects of the resin-dentin interdiffusion zone with different dentin adhesive systems. J Dent Res. 1992 Aug;71(8):1530-40.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):41-6

Original Research Article

Comparative evaluation of pH and solubility of MTA Fillapex® endodontic sealer Meiryelen Silva Finger1 Gislaine Faraoni1 Michel do Carmo Masson1 Rogério Aparecido Minini dos Santos1 Ana Claudia Baladelli Silva Cimardi1 Fausto Rodrigo Victorino1 Corresponding author: Fausto Rodrigo Victorino Rua Formosa, n. 489 – Centro CEP 86990-000 – Marialva – PR – Brasil E-mail: frvictorino@ig.com.br. 1

Dentistry Course, University Center of Maringá – Maringá – PR – Brazil.

Received for publication: June 20, 2013. Accepted for publication: November 14, 2013.

Keywords: pH; solubility; root canal obturation.

Abstract Introduction and Objective: To evaluate pH and solubility of MTA Fillapex ® cement. Material and methods: Patients were divided into four groups: GI (MTA Fillapex ®), GII (Sealer 26®), GIII (Sealapex®) and GIV (AH Plus®). Samples of each group with 10 mm in diameter and 2 mm in height were immersed into water at neutral pH and kept at 37ºC. After 3 hours, the first pH measurement was carried out and repeated at 24 hour intervals for seven days. The data were submitted to Anova (p < 0.05). To analyze the solubility, specimens with 20 mm in diameter and 1.5 mm in thickness were weighed after the setting time, and maintained in distilled water at 37ºC for seven days. After this period, the specimens were again weighed. The difference between their weighs represents the mass loss. The data were subjected to Student’s t test for paired samples (p < 0.05). Results: GI and GIII showed pH increase at the first 24 hours, with a significant reduction compared with the other cements. GIV was the smallest mass loss, followed by GII, GIII and GI. Conclusion: MTA Fillapex® has higher solubility than that of resin cements, but its pH remained above 10 for seven days.

42 – RSBO. 2014 Jan-Mar;11(1):41-6 Finger et al. – Comparative evaluation of pH and solubility of MTA Fillapex® endodontic sealer

Introduction The main goal of endodontic filling is to obtain the sealing of root canal system which favors the process of periapical process after endodontic therapy and prevents either coronal or apical marginal leakage. The improper sealing may result in the movement of fluids towards the cement gaps which may lead to periapical inflammatory reaction, compromising endodontic success [12]. Antimicrobial activity is also an important requirement of endodontic sealers and it is directly related to their releasing of hydroxyl ions leading to pH increasing and to the creation of an unfavorable environment for bacterial survivor [10]. Currently, it has been available in dental market many sealers with different formulations, and consequently different physical and chemical propert ies [16]. Endodont ic cements ca n be classified into: resin-, zinc oxide and eugenol-, calcium hydroxide-, and glass ionomer cementbased sealers. Mineral trioxide aggregate (MTA) was initially introduced in Endodontics for the sealing of root and retrofilling perforations because of its favorable physical, chemical, and biological properties [2, 13]. Notwithstanding, to be used as endodontic sealer, its formulation has to be upgraded to improve its flowing, setting time and bond strength [5]. MTA Fillapex® (Angelus, Londrina, PR, Brazil) is a MTA-based endodontic sealer currently launched in Brazilian dental market and little studies on its physical-chemical properties have been conducted. Therefore, the aim of this study was to evaluate pH and solubility of MTA Fillapex® and to compare its results with those of other endodontic sealers that have been used in clinical practice (Sealer 26®, Sealapex® and AH Plus®).

Material and methods The sealers were divided into four groups: group I – MTA Fillapex® (Angelus, Londrina, PR, Brazil), group II – Sealer 26® (Dentsply, Petrópolis, RJ, Brazil), group III – Sealapex ® (SybronKerr, Washington, USA) and group IV – AH Plus® (Dentsply, DeTrey, Konstanz, Germany).

pH test To construct the samples, poly(vinyl chloride) (PVC) rings with 10 mm in diameter and 2 mm in height were employed. The rings were placed onto a thin cellophane sheet supported by a glass

plate and filled with the sealers mixed according to each manufacturer’s instructions. Just after that, a nylon thread with about 0.5 mm in diameter was inserted into the material mass and another cellophane sheet and glass plate were placed onto the rings filled with the sealers. A mass of 100 g was placed over this set. The samples were kept in an environment with temperature of 37°C for up to three times the setting time of each sealer. Six samples were constructed for each group. Elapsed that time, the samples were immersed into flasks with 40 mm in diameter filled with 50 ml of distilled and deionized water (milli-Q type) whose pH was previously measured to prove its neutrality. The flasks were carefully closed to prevent that the samples were in contact with the flasks’ walls. All sets were maintained in an incubator at 37°C, and after 3 hours, the first pH measurement was performed. The following measurements were executed at 24 hour intervals for one week. At each evaluation period, the specimens were carefully removed, evaluated and again maintained into the same flasks, but with new water. The pH measurement was performed with the aid of a pH meter (W3b – BEL Engineering, Piracicaba, SP, Brazil) previously calibrated with solutions of known pH: 4 and 7. Data found were submitted to ANOVA and Tukey test with level of significance of 5%.

Solubility test PVC rings with 20 mm in internal diameter and 5 mm in thickness were employed. The rings were placed onto a thin cellophane sheet supported by a glass plate and filled with the sealers mixed according to each manufacturer’s instructions. Just after that, a nylon thread with about 0.5 mm in diameter was inserted into the material mass and another cellophane sheet and glass plate were placed onto the samples filled with the sealer. A mass of 100 g was placed over this set. The samples were kept in an environment with temperature of 37°C for up to three times the setting time of each sealer. After that, the samples were removed from the rings. Each sample was weighed in a precision scale (Ohaus Adventurer® – Toledo do Brasil, São Bernardo do Campo, SP, Brazil) and suspended through the nylon thread inside a large opening flask containing 50 ml of milli-Q ultrapure water. The ring was then placed into it without contacting the flasks’ walls. These samples were maintained into an incubator at 37°C for seven days.

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Elapsed that period, the samples were removed from the flask and washed in deionized water to remove possible residues. Then, the samples were placed into a desiccator for 24 hours for new weighing. The difference between the first and the second weighing represents the mass loss for each one of the specimens. Data were analyzed by Student’s t test for paired samples because it enables the analysis of results at two different periods, with level of significance of 5% (p < 0.05).

Results pH test Table I shows the pH mean values according to the experimental period for each sealer tested. ANOVA exhibited significant differences in pH among the periods tested for the same sealer; also, there were differences in pH among sealers at the same period (p < 0.05). Table I – pH mean values of endodontic sealers at different time periods

Time

Group

3 h

24 h

48 h

72 h

96 h

120 h

168 h

GI (MTA Fillapex )

9.5

10.8

10.3

10.4

GII (AH Plus®)

7.4a

7.2a

6.4

6.3

5.8

6.3

GIII (Sealer 26 )

11.0

10.5

9.2

GIV (Sealapex )

10.2

a b

11.5

8.6

10.8

a,b

8.6

10.8

9.0

10.8

8.7

10.9

11.1

Statistically significant difference from other time periods (p < 0.05) Statistically significant difference from other sealers (p < 0.05)

MTA Fillapex® and Sealapex® sealers exhibited a similar behavior with pH increasing of the solution at the first 24 hours, but with statistically significant reduction when compared with the other sealers. AH Plus® and Sealer26® also had a similar behavior because, unlikely the other sealers, they showed a pH reduction at 24 hours.

Solubility test Table II displays the mean values of the initial and final weighs. Table III exhibits the weight loss in milligrams and in percentage. Table II – Mean of the initial and final mass, in milligrams of the experimental groups

GI MTA Fillapex®

GII Seapalex®

GIII Sealer 26®

GIV AH Plus®

Initial

Final

Initial

Final

Initial

Final

Initial

Final

1.137

1.007*

1.089

0.952*

1.100

1.081

1.724

1.720

P value

0.0110

0.0070

0.0003

0.2703

* Significant difference, p < 0.05

Table III – Weight loss in milligrams and in percentage of each experimental group

GI MTA Fillapex®

GII Seapalex®

GIII Sealer 26®

GIV AH Plus®

0.130

0.137

0.019

0.004

11.4%

12.5%

1.7%

0.2%

44 – RSBO. 2014 Jan-Mar;11(1):41-6 Finger et al. – Comparative evaluation of pH and solubility of MTA Fillapex® endodontic sealer

Discussion Calcium silicate-based MTA sealer was firstly introduced in Endodontics by Lee et al. [15] in 1993 as retrofilling material and for the repairing of perforations [19]. Posteriorly, it has been indicated for pulp capping, apexification, and repairing of root resorption because of its favorable physical, chemical and biological properties [2, 13]. Its composition was based on hydrophilic particles that set in the presence of humidity. The hydration of the powder results in a colloidal gel that solidifies becoming a hard structure. MTA leads to an ideal physical sealing, being insoluble even in the presence of blood [3]. Most of the filling materials tend to contract moving away from the canal’s wall, creating a gap for which the contaminants may penetrate. MTA setting results in the hydration of the compounds of anhydrous mineral oxide to produce calcium silicate hydrate and phases of calcium hydroxide, which expands against the canal’s walls, improving the sealing and decreasing the leakage [12]. In order to use MTA as endodontic sealer, it was necessary to adjust its formulation to improving its flowing, setting time and bond strength [5]. Currently, a new MTA-based endodontic sealer was launched in the Brazilian dental market – namely MTA Fillapex® (Angelus Soluções Odontológicas, Londrina, PR, Brazil) –, whose composition, according to the manufacturer, is basically MTA, silicate resin, natural resin and bismuth oxide. The use of materials providing high alkalinity favors the mineralization of hard tissues and stimulates the body reaction, which quickens the process of repairing, clinically evidenced by biological sealing [4], as well as providing a good antimicrobial activity [11]. Antimicrobial activity is promoted by pH increasing resulting from the releasing of hydroxyl ions because the alkaline pH induces the loss of the integrity of the cytoplasmic membrane of the cells, promotes the inactivation of the enzymes involved in the cellular metabolism, and damages bacterial DNA [11]. The releasing of hydroxyl ions occurs by the interaction of the sealer with water [6]. Therefore, in this present study, the samples were placed into distilled water (milli-Q type) so that hydroxyl ions were formed consequently leading to pH alteration; also the water was renewed at every analysis period as previously reported by other authors [79], aiming to evaluate whether the ions releasing would occur at the intervals between the periods assessed and to reproduce which happens in vivo:

the constant changes of fluids and renovation of ionic concentrations [6]. In this present study, MTA Fillapex®, AH Plus®, Sealer 26® and Sealapex® sealers were evaluated at 3, 24, 48, 72, 96, 120 and 168 hours after their immersion into milli-Q water. There was an increasing in the pH value for MTA Fillapex® over time, beginning with a pH of 9.5 and reaching to 10.4 in t he last measurement. The sa me behavior was observed for Sealapex® sealer, which exhibited an initial pH of 10.2, finishing with a pH of 11.1. Such behavior can be explained by the solubilization of the surface of the samples during the successive water changes through dilution into water so that the highest the dilution the highest the degree of ionization is, that is, the highest the pH of the solution [6]. The pH peak of MTA Fillapex® and Sealapex® sealers was observed at 24 hours: 10.8 and 11.5 respectively for MTA Fillapex® and Sealapex®. This behavior was also observed by Kuga et al. [14] for MTA Fillapex® sealer, with pH value of 9.39 at the first 24 hours. These authors compared the pH value of MTA Fillapex® with that of grey and white MTA. The tested sealer evidenced a satisfactory alkalinity, but smaller than those of other sealers at the evaluation period. Morgental [18] reported a pH value for MTA Fillapex® around 10, which also corroborates the results evidenced by this present study. The highest pH values found in this present study were for MTA Fillapex® and Sealapex®, which were closer to the values established by McHugh [17] either to inhibit or eliminate E. faecalis, which is capable of surviving in an alkaline pH that normally would inhibit other bacteria [17]. As a physical property of a material, the insolubility can greatly impact on endodontic treatment success rate. Moreover, endodontic sealers must have low solubility because the leach i n g of t hei r component s ca n generate undesirable biological effects on the surrounding tissues [21]. The endodontic filling materials are designed to be kept inside root canals to promote an impermeable sealing at long term and to eliminate any communication route between oral cavity and periapical tissues. Consequently, the low solubility level for these materials is of extreme importance [3]. Scelza et al. [20] studied the physical-chemical properties of endodontic sealers and demonstrated that Sealer 26® was the one that solubilized the least, followed by AH Plus®; unlikely, Sealapex® showed the highest solubilization. Contrary to this

45 – RSBO. 2014 Jan-Mar;11(1):41-6 Finger et al. – Comparative evaluation of pH and solubility of MTA Fillapex® endodontic sealer

result, this present study demonstrated that AH Plus® sealer showed the smallest mass loss during the solubility test without statistically significant difference between the initial and final weighing, followed by Sealer 26®, which exhibited a mean mass loss of 1.7%. Borges et al. [3] described that AH Plus® and MTA® Angelus sealer demonstrated to be soluble within the recommended range, while MTA Fillapex® and Sealapex® sealers exhibited values higher than those recommended by the American National Standards Institute / American Dental Association [1], results that corroborate with the findings of this present study, in which Sealapex sealer presented a higher level of mass loss, reaching 12.5%, while MTA Fillapex® reached 11.4%. It is clear then that the most soluble sealers are composed by calcium hydroxide and because of this they show the highest pH values. Further studies on whether the releasing of calcium ions and pH increasing of MTA Fillapex® sealer would compensate its sealing capacity are necessary because these latter has been previously well presented by MTA used in retrofilling obturations.

Conclusion It seems fair to say that the results found in this study corroborates those of the literature, in which MTA Fillapex® sealer showed high solubility when compared with that of other resin sealers, above 3%, within the range recommended by the American National Standards Institute / American Dental Association. However, its pH was kept above 10 for up to seven days. Also, further studies on the evaluation of its sealing capacity and antimicrobial activity are necessary.

Acknowledges The authors thank to the University Center of Maringá by the granting of the Project of Scientific Initiation and to Dentsply of Brazil and Angelus Industry of Dental Products by providing the endodontic sealers.

References 1. American National Standards Institute / American Dental Association. Specification n. 57 for endodontic filling materials. Chicago: ADA; 2000.

2. Bogen G, Kuttler S. Mineral trioxide aggregate obturation: a review and case series. J Endod. 2009;35:777-90. 3. Borges RP, Sousa-Neto MD, Versiani MA, Rached-Júnior FA, De-Deus G, Miranda CES et al. Changes in the surface of four calcium silicatecontaining endodontic materials and epoxy resinbased sealer after a solubility test. Int Endod J. 2012;45:419-28. 4. Broon NJ, Bramante CM, Assis GF, Bortoluzzi EA, Bernardineli N, Moraes IG et al. Healing of root perforations treated with mineral trioxide aggregate (MTA) and Portland cement. J Appl Oral Sci. 2006;14:305-11. 5. Camilleri J. Evaluation of selected properties of mineral trioxide aggregate sealer cement. J Endod. 2009;35:1412-7. 6. Carneiro D, Barbosa SV. Avaliação do pH dos cimentos endodônticos e considerações clínicas. Robrac. 1998;7:6-10. 7. Duarte MAH, Demarchi ACCO, Yamashita JC, Kuga MC, Fraga SC. pH and calcium ion release of 2 root-end filling materials. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2003;95:345-7. 8. Duarte MAH, Martins CS, Demarchi ACOC, de Godoy LF, Kuga MC, Yamashita JC. Calcium and hydroxide release from different pulp-capping materials. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;104:66-9. 9. Eldeniz AU, Erdemir A, Kurtoglu F, Esener T. Evaluation of pH and calcium ion release of Acroseal sealer in comparison with Apexit and Sealapex sealers. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;103:86-91. 10. Estrela C. Antimicrobial and chemical study of MTA, Portland cement, calcium hydroxide paste, Sealepex and Dycal. Braz Dent J. 2000;11:3-9. 11. Estrela C, Sydney GB, Bammann LL, Felippe Júnior O. Mechanism of action of calcium and hidroxyl ions of calcium hydroxide on tissue and bacteria. Braz Dent J. 1995;6:85-90. 12. Gomes-Filho JE, Moreira JV, Watanabe S, Lodi CS, Cintra LT, Dezan Jr. E et al. Sealability of MTA and calcium hydroxide-containing sealers. J Appl Oral Sci. 2012;20:347-51.

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13. Gomes-Filho JE, Watanabe S, Estrada Bernabe PF, Costa MTM. A mineral trioxide aggregate sealer simulated mineralization. J Endod. 2009;35:256-60. 14. Kuga MC, Campos EA, Viscardi PH, Carrilho PZ, Xaviér FC, Silvestre NP. Hydrogen ion and calcium releasing of MTA Fillapex® and MTA-based formulations. RSBO. 2011;8:271-6. 15. Lee SJ, Monsef M, Torabinejad M. Sealing ability of mineral trioxide aggregate for repair of lateral root perforations. J Endod. 1993;19:541-4. 16. Leonardi DP, Battisti JC, Klimiont DT, Tomazinho PH, Baratto-Filho F, Haragushiku GA et al. Avaliação in vitro da ação antimicrobiana de alguns cimentos endodônticos. RSBO. 2009;6:367-73. 17. Mchugh CP. pH required to kill Enterococcus faecalis in vitro. J Endod. 2004;30:218-9.

18. Morgental RD. Antibacterial activity of two MTA-based root canal sealers. Int Endod J. 2011;44:1128-33. 19. Santos AD, Moraes JC, Araújo EB, Yukimitu K, Valério Filho WV. Physico-chemical properties of MTA and a novel experimental cement. Int Endod J. 2005;38:443-7. 20. Scelza MFZ, Scelza P, Costa RF, Câmara A. Estudo comparativo das propriedades de escoamento, solubilização e desintegração de alguns cimentos endodônticos. Pesq Bras Odontoped Clín Integr. 2006:6:243-7. 21. Schäfer E, Zandbiglari T. Solubility of rootcanal sealer in water and artificial saliva. Int Endod J. 2003;36:660-9. 22. Torabinejad M, Higa RK, McKendry DJ, Pitt Ford TR. Dye leakage of four root-end canal filling materials: effects of blood contamination. J Endod. 1994;20:159-63.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):47-51

Original Research Article

Analysis of blood pressure during tooth extraction Alessandro Hyczy Lisboa¹ Chigueyuki Jitumori¹ Evaldo Artur Hasselmann Júnior¹ Rafael Pes¹ Gibson Luis Pilatti² Corresponding author: Rafael Pes Avenida Vedolino Neves, n. 400 – Centro CEP 84530-000 – Teixeira Soares – PR – Brasil E-mail: rafaeps2@hotmail.com ¹ Department of Dentistry, Center of Higher Education of Campos Gerais – Ponta Grossa – PR – Brazil. ² Department of Dentistry, State University of Ponta Grossa – Ponta Grossa – PR – Brazil.

Received for publication: July 4, 2013. Accepted for publication: November 18, 2013.

Keywords: blood pressure; anesthesiology; tooth extraction.

Abstract Introduction: Surgical procedures have a history of dental pain, apprehension and fear reported by patients. Because of these reasons, they trigger a series of modifications that lead to changes in blood pressure and stress occurrences. Objective: This study aimed to monitor changes in blood pressure pre-, trans-, and postoperatively and assess whether the differences would be significant. Material and methods: One hundred and ninety-four patients, both genders, aged between 14 and 65 who were undergoing surgery for tooth extraction were selected. They had their systolic and diastolic blood pressures measured at four different times: preoperative (M1), post-anesthesia (M2), post-extraction (M3) and at the end, after the suture (M4), with the aid of a digital tensiometer. Results: There were statistically significant differences between systolic blood pressure changes among all times, except between M1 and M4. Concerning the diastolic blood pressure measurement, there were statistical differences among all times except between M1xM4 and M2xM3. Conclusion: There were changes in blood pressure between different moments of extractions.

48 – RSBO. 2014 Jan-Mar;11(1):47-51 Lisboa et al. – Analysis of blood pressure during tooth extraction

Introduction Blood pressure (BP) is understood as that within arteries with communication to the walls. It can be calculated by multiplying the total peripheral resistance by the cardiac output and it can vary from a maximum value during systoles and a minimum value during diastoles [23]. These measurements can undergo alterations because of systemic, behavioral, and physiological modifications when one faces stimuli caused by oral surgery, for example [8]. The current dental surgeries have a close relationship with blood pressure changes, since patients potentially correlate dental treatment with pain [4], resulting in fear [24], anxiety and stress, which is a body response to negative [27] and/or stress [19, 22, 26] stimuli many times imperceptible to the dentist. Consequently, one of the most evident effects in modifying the physiologic balance is the blood pressure change, resulting from the stress due to dental procedure [9]. In Brazil, there has been found a prevalence of 15% of patients with dental anxiety [10], which can reach to 95% when tooth extraction is mentioned [11]. Heart diseases are a great risk for world population health [15] reaching from 22% to 41% of Brazilian population [2, 6], and it has been an extremely prevalent condition particularly in patients who will be submitted to surgeries [25]. The disease is currently explained by several genes interacting with many factors, among them, inheritance, obesity, and food [12, 13]. The dentists must pay attention to any symptom and they play an important role in diagnosing the disease [16]. There exists also a relationship between bacterial endocarditis and oral surgery, which can results in heart alterations [30]. It is also possible to cite “white coat hypertension”, a condition of blood pressure change only noted in clinical practice, where the patient is anxious and stressed waiting to be seen by either the dentist or the doctor, but it is within normal range during other daily situations [16]. Another relationship extremely important in dental clinics and mainly during oral surgeries is the use of anesthetic solutions associated with vasoconstrictor drugs because the following are the most used ones in dental practice [14]: epinephrine, felypressin, phenylephrine and noradrenaline. These vasoconstrictor drugs have been associated with anesthetic salts and have directly acted on the system of blood vessels of the area anesthetized, resulting in the vasoconstriction as main effect, but exerting a side effect on blood pressure and/or

heart rate [3], in addition to the pain and anxiety during anesthetic application [18]. According to Trento et al. [28] and Tucci et al. [29], these drugs inside the circulatory system can cause alterations. Adrenaline raises the systolic pressure and the heart rate, causing excitatory movements in the myocardium, palpitations and tachycardia, but these symptoms are transitory and not serious [29]. On the other hand, phenylephrine and noradrenaline can significantly raise both systolic (SP) and diastolic (DP) pressure. Felypressin does not affect directly the myocardium, but it is a powerful vasoconstrictor of the heart, which can lead to angina attacks with myocardial ischemia in patients with some heart circulation deficiency [17]. With the advent of vasoconstrictor drugs, it has been noted that most of the heart complications occur during or after the surgical procedure [21]. Generally, these acts and symptoms cause blood pressure alterations. The dentist should control and pay special attention mainly in hypertensive and elderly patients and those with any heart alteration [1]. This study aimed to measure and analyze the result of systolic and diastolic blood pressure values at four moments of possible significant alterations during tooth extractions.

Material and methods This study was submitted and approved by the Ethical Committee in Research of the Center of Higher Education of Campos Gerais (Cescage), under protocol no. 976/CEP. The study randomly selected 194 volunteers, both genders (91 male and 103 female), aged between 14 and 65 years-old, who were referred to tooth extraction in the Discipline of Oral Surgery of the School of Dentistry of the Center of Higher Education of Campos Gerais, Ponta Grossa, Paraná, Brazil. All patients participating in the study were carefully evaluated by anamnesis, physical and radiographic examination, and when necessary by complementary laboratorial tests. Inclusion criteria comprised the referral for at least one tooth extraction and good general health to be submitted to the procedure. Patients who were not within the parameters to be submitted to tooth extraction; continuously using anticoagulant and/or antiplatelet drugs for the last six months; pregnant women; presenting pericoronitis, hepatic dysfunction, uncontrolled diabetes mellitus, and hypertension; and using substances capable of altering blood pressure were excluded from the

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study. All measurements were executed by a single examiner previously trained after the signing of a Free and Clarified Consent Form by all patients who were willing to participate in the study. The measurements were carried out with the aid of a digital tensiometer (G-Tech Model GS100® – Genexel Medical Instruments, South Korea), previously calibrated, verified and approved by Inmetro. The systolic and diastolic blood pressure were measured (in mmHg), at four different moments: waiting room, ten minutes prior to the patient was sat on dental chair (M1), five minutes after the application of the first anesthetic tube (1.8 ml of 2% mepivacaine with epinephrine 1:100.000) (M2), after tooth extraction (M3) and after suture (M4). After the analysis of the data, the following statistical tests were applied: Analysis of Variance for Repeated Measures Tukey-Kramer Multiple Comparisons, with level of significance of 5%.

Results By evaluating the data of systolic measurements, the highest mean was at M1 followed by M4, M3 and M2. Comparing the systolic measurements between two different moments, only at M1xM4 there were no statistically significant differences (p > 0.05). At the other measurements, there were statistically significant differences between M1xM2 (p < 0.001), M1xM3 (p < 0.001), M2xM3 (p < 0.01), M2xM4 (p < 0.001) and M3xM4 (p < 0.001) (table I). The value of p < 0.0001 is considered as extremely significant. Table I – Mean and standard deviation of systolic blood pressure for different moments

Group

N. of patients

Mean

Standard deviation

Systolic M1

194

119.2

15.982

Systolic M2

194

112.2

14.315

Systolic M3

194

115.1

15.141

Systolic M4

194

118.5

14.693

* Equal letters mean that there were no statistically significant differences; different letters mean significant differences

Concerning to the diastolic measurement, the highest mean was seen at M4, followed by M1, M3 and M2. Comparing these moments, at two comparisons there were no statistically significant differences: M1xM4 (p > 0.05) and M2xM3 (p > 0.05). At the other comparisons, there were statistically significant differences between M1xM2 (p < 0.001), M1xM3 (p < 0.001), M2xM4 (p < 0.001) and M3xM4 (p < 0.001) (table II). Table II – Mean and standard deviation of diastolic blood pressure for different moments

Group

N. of patients

Mean

Standard deviation

Diastolic M1

194

77.3

14.532

Diastolic M2

194

71.3

12.099

Diastolic M3

194

73.6

12.189

Diastolic M4

194

77.8

11.848

* Equal letters mean that there were no statistically significant differences; different letters mean significant differences

Discussion The knowledge of the patient’s general health is of extreme importance for dentists, since systemic alterations can influence on dental treatment. Heart diseases play an important role in this context, mainly hypertension, which is the most common diseases in adults [8, 15] and a growing condition in Brazil, affecting 30% of the population [2, 6, 10].

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In this study, it could be observed that during a dental surgical procedure existed a significant alteration of blood pressure, because the peak of SP occurred at the first moment, that is, at the pre-operative time and before the anesthetic solution injection, characterizing the “white coat hypertension” [16], in addition to the anxiety generated by the effects of oral surgery can cause to patients, for example, pain, fear, and stress [4, 11, 19, 22, 24, 26]. Salles et al. [20], by studying the influence of vasoconstrictor drugs on blood pressure, affirmed that there were not significant alterations after the application of 2% lidocaine with noradrenaline, 3% prilocaine with felypressin and 2% mepivacaine with epinephrine. According to Dantas et al. [7], who evaluated the effect of mepivacaine with epinephrine on blood pressure, only the diastolic pressure slightly increased about 1.5 mmHg, which is within the patterns of normal measurements: 120 for SP and 80 for DP. In this present study, the mean and standard deviation of both systolic and diastolic blood pressure measurements were within the patterns of physiologic normality. Santello et al. [21] affirmed that most of the cardiac risks for patients submitted to dental surgeries occur after the ending of the procedure. In this present study, the DP peak was just after the ending of the procedure.

Conclusion Within the results of this study, it can be concluded: • There were alterations in blood pressure between different moments of tooth extraction; • T he h i g he st me a n of s y stol ic pre s su re measurements was during the pre-operative moment; • The h i g hest mea n of dia stol ic pressu re measurements was after the ending of the procedure; • There were significant differences in systolic pressure measurements between M1xM2, M1xM3, M2xM3, M2xM4 and M3xM4, when compared between each other; • There were significant differences in systolic pressure measurements between M1xM2, M1xM3, M2xM4 and M3xM4, when compared between each other.

References 1. Alencar CRB, Andrade FJP, Catão MHCV. Cirurgia oral em pacientes idosos: considerações clínicas, cirúrgicas e avaliações de riscos. RSBO. 2011 Apr-Jun;8(2):200-10. 2. Bronzo ALA. Procedimentos odontológicos em pacientes hipertensos com ou sem o uso de anestésico local prilocaína associada ou não ao vasoconstritor felipressina. Dissertação [Mestrado]–Faculdade de Medicina da Universidade de São Paulo, São Paulo; 2005. 3. Cáceres MTF, Ludovice ACPP, Brito FS, Darrieux FC, Neves RS, Scanavacca MI et al. Efeito de anestésicos locais com e sem vasoconstritor em pacientes com arritmias ventriculares. Arq Bras Cardiol 2008 Fev;91(3):142-7. 4. Cardoso CL, Loureiro SR. Estresse e comportamento de colaboração em face do tratamento odontopediátrico. Psicologia em Estudo. 2008 Jan-Mar;13(1):133-41. 5. Cardoso CL, Loureiro SR, Nelson-Filho P. Pediatric dental treatment: manifestations of stress in patients, mothers and dental school students. Braz Oral Res. 2004 Mar;18(2):150-5. 6. Cavagioni LC, Pierin AMG. Hipertensão arterial em profissionais que atuam em serviços de atendimento pré-hospitalar. Texto Contexto Enferm. 2011 Jul-Sep;20(3):235-44. 7. Dantas MVM, Gabrielli MAC, Hochuli-Vieira E. Efeito da mepivacaína 2% com adrenalina 1-100.000 sobre a pressão sanguínea. Rev Odontol Unesp. 2008 Aug;37(3):223-7. 8. Ferraz EG, Carvalho CM, Jesuíno AA, Provedel L, Sarmento VA. Avaliação da variação da pressão arterial durante procedimento cirúrgico. Rev Odontol Unesp. 2007;36(3):223-9. 9. Ganhoto APA, Cabral AM, Vasquez EC, Ganhoto MRA, Lima EG. Monitorização ambulatorial da pressão arterial em indivíduos submetidos à cirurgia periodontal. Rev Bras Hipertens. 2006 Mar;13(2):97-103. 10. Kanegane K, Penha SS, Borsatti MA, Rocha RG. Ansiedade ao tratamento odontológico em atendimento de urgência. Rev Saúde Pública. 2003 Jul;37(6):786-92.

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11. Lisboa AH, Kindl C, Pilatti GL. Nível de ansiedade em pacientes submetidos a procedimentos cirúrgicos odontológicos. Full Dent Sci. 2012;3(12):400-7.

21. Santello JL. Atualização em hipertensão arterial: risco cirúrgico – hipertensão, fatores associados e prognósticos. J Bras Nefrol. 2001 Feb;23(1):60-3.

12. Loffredo LCM, Telarolli Jr R, Basso MFM. Prevalência de hipertensão arterial sistêmica em estudantes da faculdade de Odontologia de Araraquara – Unesp. Rev Odontol Unesp. 2003 Jul-Dec;32(2):99-104.

22. Santos FA, Santos LA, Melo DO, Alves Jr A. Estresse e estratégias de enfrentamento em pacientes que serão submetidos à cirurgia de colecistectomia. Interação em Psicologia. 2006 Jan-Jun;10(1):63-73.

13. Lolio CA. Epidemiologia da hipertensão arterial. Revista Saúde Pública. 1990 Apr;24(5):425-32.

23. Santos TS, Acevedo CR, Melo MCR, Dourado E. Abordagem atual sobre hipertensão arterial sistêmica no atendimento odontológico. Odontologia Clín-Científ. 2009 Apr-Jun;8(2):105-9.

14. Montan MF, Cogo K, Bergamaschi CC, Volpato MC, Andrade ED. Mortalidade relacionada ao uso de anestésicos locais em Odontologia. RGO. 2007 Apr-Jun;55(2):197-202. 15. Neves CAF, Couto GBL, Botelho KVG, Vasconcelos MMVB, Soares RPF, Cavalcanti JB et al. Avaliação da pressão arterial de crianças e adolescentes atendidos em clínica odontopediátrica. Odontologia Clín-Científ. 2007 Apr-Jun;6(2):163-7. 16. Oliveira AEM, Simone JL, Ribeiro RA. Pacientes hipertensos e a anestesia na Odontologia: devemos utilizar anestésicos locais associados ou não a vasoconstritores? HU Revista. 2010 JanMar;36(1):69-75. 17. Paiva LCA, Cavalcanti AL. Anestésicos locais em Odontologia: revisão de literatura. Publ UEPG Ci Biol Saúde. 2005 Jun;11(2):35-42. 18. Possobon RF, Carrascoza KC, Moraes ABA, Costa Jr AL. O tratamento odontológico como gerador de ansiedade. Psicologia em Estudo. 2007 Sep-Dec;12(3):609-16. 19. Rocha R, Porto M, Morelli MYG, Maestá N, Waib PH, Burini RC. Efeito de estresse ambiental sobre a pressão arterial de trabalhadores. Rev Saúde Pública. 2002 May;36(5):568-75. 20. Salles CLF, Martinez AC, Pavan AJ, Daniel NA, Cuman RKN. Influência de vasoconstritores associados a anestésicos locais sobre a pressão arterial de ratos hipertensos e normotensos. Acta Scientiarum. 1999 Apr;21(2):395-401.

24. Singh KA, Moraes ABA, Bovi Ambrosano GM. Medo, ansiedade e controle relacionados ao tratamento odontológico. Pesq Odont Bras. 2000 Apr-Jun;14(2):131-6. 25. Slullitel A. Hipertensão arterial e anestesia. RGO. 2007 Apr-Jun;55(2):197-202. 26. Tanno AP, Marcondes FK. Estresse, ciclo reprodutivo e sensibilidade cardíaca às catecolaminas. Rev Bras Ciên Farm. 2002 Jul-Sep;38(3):273-89. 27. Tomita LM, Costa Jr AL, Moraes ABA. Ansiedade materna manifestada durante o tratamento odontológico de seus filhos. Psico-USF. 2007 Jul-Dec;12(2):249-56. 28. Trento CL, Gaujac C, Santos F, Lima GDN. Complicações no uso de anestésicos locais – uma revisão. POS – Perspect Oral Sci. 2010 Aug;2(2):43-50. 29. Tucci CJ, Sposto MR, Mendes AJD. Influência da n ora dre n a l i n a con t i da n os a n es t é si c os odontológicos sobre a pressão arterial de pacientes normotensos: estudo clínico. Rev Odontol Unesp. 1986 Mar;15(16):123-9. 30. Veronese EL, Silva FBR, Silva-Netto CR. Profilaxia e ocorrência de endocardite bacteriana por procedimentos odontológicos: uma revista da literatura. FOL – Faculdade de Odontologia de Lins / Unimep – Universidade Metodista de Piracicaba. 1999 Jan-Jun;11(2):45-51.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):52-7

Original Research Article

Immunoexpression of BMP-2 protein on bone repair of critical size defects treated with autogenous macerated adipose tissue Clayton Luiz Gorny Junior1 Allan Fernando Giovanini1 Juliana Vieira1 João Cézar Zielak1 Felipe Rychuv Santos1 Carmen Lucia Mueller Storrer1 Tatiana Miranda Deliberador1 Corresponding author: Tatiana Miranda Deliberador Mestrado em Odontologia, Universidade Positivo Rua Professor Pedro Viriato Parigot de Sousa, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: tdeliberador@gmail.com 1

Program of Professional Master Course in Clinical Dentistry, Positivo University – Curitiba – PR – Brazil.

Received for publication: October 12, 2013. Accepted for publication: November 20, 2013.

Keywords: adipose tissue; calvaria; bone regeneration.

Abstract Introduction: The adipose tissue is an important reservoir of adult stem cells which have capacity of differentiating in osteoblasts with potential implication in reaching bone regeneration. The evaluation of the osteoblastic differentiation can be verified through immunohistochemical markers such as bone morphogenetic protein2 (BMP-2). Objective: To evaluate the immunoexpression of BMP-2 protein on the bone repairing of critical size defects (CSD) surgically created in rat calvaria and treated by autogenous macerated adipose tissue. Material and methods: Forty male rats had a CSD measuring 5 mm created on their calvaria. The animals were randomly divided into two groups: group C (control) and group AT (macerated adipose tissue grafting). In group C, the defect was filled with only blood clot. In group AT, the defect was filled with autogenous macerated adipose tissue. The groups were subdivided into two subgroups (n = 10) for euthanasia at 7 and 90 post-operative days. Histological and immunohistochemical analyses were carried out. Data were submitted to descriptive statistics (mode). Results: In group AT, both at 7 and 90

53 â&#x20AC;&#x201C; RSBO. 2014 Jan-Mar;11(1):52-8 Gorny Junior et al. â&#x20AC;&#x201C; Immunoexpression of BMP-2 protein on bone repair of critical size defects treated with autogenous macerated adipose tissue

post-operative days, the main healing type was the presence of dense conjunctive tissue exhibiting bundles of collagen fibers disposed in beams permeating the remaining adipose tissue with rare heterotopic bone formation associated to fibrosis and different types of tissue necrosis. In group C, the repair was achieved by the formation of bundles of collagen fibers oriented parallelly to the surface of the wound at the two post-surgical periods. The immune-staining of BMP-2 was present only in group C (7 and 90 post-operative days). Conclusion: Within the limits of this present study, it can be concluded that the adipose tissue grafting did not favor bone neoformation in critical size defects and BMP-2 signaling was not observed.

Introduction The repair of extensive bone defects, caused by either traumas or pathologies, constituted a great problem for both Medicine and Dentistry. Despite of the great potential of repair by bone tissue, in some situations, according to the proportion of defects, the regeneration cannot be complete because the defect can be invaded by the surrounding conjunctive tissue which has a proliferation and cellular migration speed faster than those of bone tissue. Thus, the use of bone grafting can collaborate with the repair process. To achieve better results, the graft should preferentially osteogenic, osteoinductive and osteoconductive [6]. Tissue bioengineering is a new branch of Science aiming to investigate and integrate techniques of tissue regeneration thorugh new biomaterials including natural markers (growth factors) and tissue and cellular components (mesenchymal stem cells). Thus, the search for new materials helping the bone healing process have found different ways from those of the use of autogenous and xenogenous bones; bioceramics and bioglasses, raising great interest in the research with adipose tissue as a reservoir of adult stem cells, generating good results. Mesenchymal stem cells have the ability to differentiate in osteoblasts with potential implications for the engineering of human bone tissue [11]. The stem cells coming from the adipose tissue can be an important source of progenitor mesenchymal stem cells which can be employed in tissue engineering for bone repairing [8]. Currently, Zou et al. [13] verified that stromal cells (CD 34+) derived from the adipose tissue has the potential of osteogenic differentiation both in vitro and in vivo, being considered as a promising cellular therapy for the healing of bone defects. The adult unilocular subcutaneous adipose cells have the potential in vitro to redifferentiate in osteoblast

(transdifferentiation). This transdifferentiation capacity can hypothetically favor bone repairing procedures [3]. The presence of osteoblasts is an important requisite in the effectiveness and success of the bone repairing. This evaluation of osteoblastic differentiation is commonly evaluated through different protein markers, among them bone morphogenetic protein-2 (BMP-2). Currently, there are little studies evaluating whether the adipose tissue is really a safe option to reach bone repairing. For this reason, studies on animals can clarify the quality and type of bone healing when adipose tissue grafting is used. The aim of this study was to evaluate the immunoexpression of BMP-2 protein on bone repairing of critical size bone defects surgically created in rat calvaria, treated with macerated adipose tissue grafting.

Material and methods This present study was submitted and approved by the Ethical Committee in Research of Positivo University, Curitiba (PR), under protocol no. 019/2009. Forty male rats (Rattus norvegicus, albinus, Wistar), aged from about 5 to 6 months, weighing from 400 to 600 grams were selected. The animals were randomly divided into two groups: group C (control) and group AT (macerated adipose tissue). The animals had good health conditions. They were kept at every 4 inside a box in the vivarium of Positive University and received solid food and mineral water ad libitum.

Anesthetic protocol To conduct the surgical and experimental procedures, the animals were individually placed inside a campane and submitted to inhaling

54 – RSBO. 2014 Jan-Mar;11(1):52-8 Gorny Junior et al. – Immunoexpression of BMP-2 protein on bone repair of critical size defects treated with autogenous macerated adipose tissue

anesthesia with oxygen and isoflurane (Cristália, Itapira, SP, Brazil) and then anesthetized by intramuscular injection at the posterior part of the thigh with 2.3 g of xylazine (0.52 mg/kg) (Vetbrands, Paulínia, SP, Brazil) and 1.16 g ketamine (1.04 mg/kg) (Vetbrands, Paulínia, SP, Brazil).

Surgical procedures After anesthesia, all animals were submitted to trichotomy on the calvaria; the animals of group AT also received trichotomy on the abdominal area (to collect the adipose tissue). Next, antisepsis was performed and a U-shaped incision was executed with the aid of a size 15c scalpel blade to access the calvarium area to raise a total flap towards the posterior direction. A critical size defect (CSD) measuring 5 mm in diameter, trans-osseous [10], was created with the aid a trephine bur coupled to a counter-angle handpiece for implant (20:1, Kavo, Joinville, SC, Brazil), under copious irrigation with sterile saline solution. The defect reached a portion of the sagittal suture. The removal of the bone block was carefully executed with the aid of spatulas to avoid the meninges rupture. With the aid of a millimetric probe (PCPUNC 15, Hu-Friedy, Chicago, IL, USA) and a number 701 conical trunk carbide drill coupled to a straight handpiece was used to create two L-shape marks: 2 mm towards anterior and 2 mm towards posterior of the margins of the surgical defect. The long axis of each L was located onto an imaginary longitudinal line that divided the surgical defect by half. The other marks were filled with dental amalgam (Permite – SDI, Victoria, Australia) both to identify the middle of the original surgical defect during laboratorial processing and to locate its original bone margins during histological analysis (figure 1). In group C, the surgical defect was filled with blood clot. In group AT, the defect was filled with macerated adipose tissue (figure 1). To obtain autogenous adipose tissue, an incision was performed with the aid of a size 15 scalpel blade, parallelly to the right posterior limbs onto the abdominal area with size of about 1 cm. The adipose tissue of the exposed area was carefully removed with the aid of scissors and tweezers and then macerated with size 15c scalpel blade through random cuts into different directions and immediately grafted into the surgical defect.

Figure 1 – Group AT. Critical size defect surgically created on the calvaria on animals with the amalgam marks and the defect filled with the autogenous macerated adipose tissue

The soft tissues were then repositioned and sutured with silk thread 4-0, (Ethicon, Johnson & Johnson, São José dos Campos, SP, Brazil) to obtain a primary closure of the wound both in the donor (abdominal area) and receptor (calvaria) sites. To control post-operative pain, the animals received morphine sulphate (3 mg /kg) (União Q u í m ic a , Ja baqu a ra , SP, Bra z i l), t h rou g h intramuscular injection, at the ending of the surgery. Analgesia was kept with 20 drops of paracetamol (200 mg/kg) diluted into 400 ml of water placed into the drinking fountain for three days. The groups were divided into subgroups (n = 10), for euthanasia at 7 and 90 post-operative days. The animals were positioned in a gas chamber (CO2) and maintained for 10 minutes which was the time amount required to the complete euthanasia.

Tissue processing The area of the original surgical defect and the surrounding tissues were removed in block. The pieces were fixed into 10% neutral formaldehyde, washed in running water and decalcified in 20% formic acid. Then, each piece was hemi-sectioned at the center parallelly to the sagittal suture by using the long axis of each L-shaped amalgam mark. Thus, it was possible to determine precisely the limits of the original surgical defect for histological and immunohistochemical analyses. The pieces were processed and included into paraffin. Next, serial longitudinal cuts measuring 3 µm in thickness were executed, initiating at the center of the original surgical defect. The cuts were stained by hematoxylin and eosin (HE) for light microscopy analyses.

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Histological analysis Two histological cuts representing the center of the original surgical defect of each animal were selected for histological analysis. All analyses were conducted by a single operator previously trained. Qualitative histological analysis was carried out by optical microscope (021/3 Quimis, Diadema, SP, Brazil), and the following parameters were assessed: the closure of the bone defect; the type of the neoformed bone; the characteristics of conjunctive tissue; the presence of osteoid matrix; the presence of chronic and acute inflammatory infiltrate; the progression of the repairing type present in the defect created surgically; and the thickness of the neoformed tissues compared with the original bone of calvaria. The closure of the defect was considered as complete when all its extension was filled by neoformed bone tissue. Also, the presence of remnants of macerated adipose tissue graft was assessed.

Immunohistochemical analysis Two histological cuts representing the center of the original surgical defect of each animal were selected for immunohistochemical analysis. All analyses were conducted by a single operator previously trained. Immunohistochemical antiperoxidase analysis through BMP-2 antibody was carried out. The immunoexpression of the protein was obtained through streptavidin-biotin-immunoperoxidase. The images for each field were obtained by light microscopy at x400 magnification, under fixed light focus. The images visualized on a TV screen coupled to a computed system were digitized by camera system (SDC-310 Samsung, Corrêa) coupled to the light microscope (021/3 Quimis, Diadema, SP, Brazil). The manual counting of the nucleus expressing immune-staining was executed for the protein studied. To evaluate the data of the immunohistochemical analysis (quantitative analysis), it was employed a semi-quantitative score, ranging from: “-” for the lack of staining to 10% of immunopositivity; “+” for 10-25%; “++” for 25-50%; “+++” for 50-75% and “++++” for more than 75%. The semi-quantitative immunohistochemical analysis was chosen because BMP-2 protein was expressed both in the cells and extracellular matrix.

Statistical analysis Data was submitted to descriptive statistics (mode). Mode is the value representing the greatest number of observations, that is, the most frequent

value or values. The mode is not necessarily one, rather than mean and median. It is especially useful when the values or observation are not numerical, since the mean and median cannot be well defined.

Results Histological analysis Group AT: 7 post-surgical days At this period, there was not the completely closure of the wound. Mostly, the extension of the defect was occupied by remnants of adipose tissue graft permeated by the presence of beams and capsule of dense fibrous conjunctive tissue (figure 2), which was also permeated by intense presence of chronic inflammatory infiltrate predominantly with lymphocytes and plasmocytes. In the body of the defect, it was also observed the sound architecture of the anuclear adipose tissue, suggesting ischemic/coagulative necrosis karyolysis. It was not verified any bone neoformation in none of the specimens analyzed.

Figure 2 – Group AT (7 days): surgical defect filled by remnant of adipose tissue permeated by the presence of beams and capsule of fibrous conjunctive tissue (hematoxylin and eosin, original x40 magnification)

Group AT: 90 postoperative days It was not verified the complete closure of the bone defect. The reparative histology showed the presence of the dense conjunctive tissue exhibiting bundles of collagen fibers disposed in beams permeated in the remnant adipose tissue and presence of heterotopic bone formation associated with fibrosis (figure 3).

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coincides with the lack of bone neoformation in this group. In group C, the immune-staining occurred at the margins of the defect, in areas containing the bone remaining (figure 5). Group AT – 7 days

Figure 3 – Group AT (90 days): defect filled by conjunctive tissue disposed in collagen fibers permeated in the remnant of adipose tissue with heterotopic bone formation (hematoxylin and eosin, original x40 magnification)

Group AT – 90 days

Group C: 7 and 90 postoperative days The microscopic features at these periods did not show the closure of the bone defect. All healing area was composed by dense conjunctive tissue with bundles of collagen fibers parallelly to the surface of the wound (figure 4), permeated by intense chronic lymphoplasmocytic inflammatory process at 7 postoperative days. A small amount of neoformed bone tissue was seen at the defect margins at 90 postoperative days.

Group C – 7 days

Group C – 90 days

Figure 4 – Group C (90 days): presence of collagen fibers disposed parallelly to the wound surface (hematoxylin and eosin, original x40 magnification)

Qualitative immunohistochemical analysis At both experimental periods, all specimens treated with adipose tissue (groups AT), did not exhibited the presence of BMP-2 protein. This result

Figure 5 – Immune-staining of BMP-2 protein between groups at 7 and 90 postoperative days (original x40 magnification). C = control and AT = adipose tissue

57 – RSBO. 2014 Jan-Mar;11(1):52-8 Gorny Junior et al. – Immunoexpression of BMP-2 protein on bone repair of critical size defects treated with autogenous macerated adipose tissue

Quantitative immunohistochemical analysis The results of quantitative immunohistochemical analysis are seen in table I. Table I – Score (mode) regarding to the immune-staining of BMP-2 proteins at 7 and 90 postoperative days for all groups analyzed

Protein BMP-2

Period

Group AT

Group C

7 days

90 days

Discussion The aim of this present study was to evaluate the immunoexpression of BMP-2 protein on the bone repairing of critical size defects surgically created in rat calvaria, treated with macerated adipose tissue. In this study, it could be observe that the macerated adipose tissue hindered the bone repair. This result is similar to those found recently by Zanicotti et al. [12], Matsubara et al. [5] and Oliveira et al. [7]. Zanicotti et al. [12] and Matsubara et al. [5] evaluated the use of non-processed adipose tissue in the treatment of peri-implantar bone defects. According to these authors, the adipose tissue seems to prevent the bone formation around the implant. Oliveira et al. [7] evaluated the influence of macerated adipose tissue on the bone repairing of bone defects created surgically on rabbit calvaria. The results demonstrated that macerated adipose tissue grafting did not favor positively the amount of bone neoformation. The negative result of the adipose tissue in the bone repairing was evident in this present study because there was no immune-staining of BMP-2 protein, which evaluates the osteoblastic di f ferent iat ion a nd has been considered as osteogenesis modulators. BMP-2 was seen in group C at small amount at 7 and 90 postoperative days, showing that there exists the presence of osteoblastic cells. In this sense, it can be said that the adipose tissue formed a physical necrotic barrier impeding the bone neoformation. Moreover, in group AT, there was either ischemic or microcystic necrosis. These different necrosis types favored the negative response of bone neoformation. The transdifferentiation of mature adipocytes into osteoblasts is already documented in literature, both in in vivo and in vitro studies, confirming that adipocytes can be good candidates for tissue engineering protocols aiming to bone tissue

development [3]. Notwithstanding, in this present study, this transdifferentiation process could not be proved. The transplant of adipose tissue to ectopic site has the self-capacity of heterotopic mineralization [4]. Areas of heterotopic mineralization were observed in some specimens of groups AT. It is important to emphasize that these areas cannot be considered as osteoid tissue. The rationale behind this presence is that all necrotic fat is caused by the saponification which attracts calcium salts [9]. Studies have shown that the adipose tissue can secrete proteins so-called adipokine, and the stem cells derived from this tissue are “tripotent”, with potential capacity of adipogenic, chondrogenic and osteogenic differentiation [1, 2, 14]. Notwithstanding, in this present study, the use of autogenous macerated adipose tissue favored the necrotic rather than osteogenic process.

Conclusion Within the limits of this present study, the macerated adipose tissue grafting did not favor bone neoformation in critical size defects by the suppression of BMP-2 protein signaling.

References 1. Fruhbeck G, Gomez-Ambrosi J, Muruzabal FJ, Burrell MA. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am J Physiol Endocrinol Metab. 2001 Jun;280(6):E827-47. 2. Gimble JM, Katz AJ, Bunnell BA. Adiposederived stem cells for regenerative medicine. Circ Res. 2007 May 11;100(9):1249-60. 3. Justesen J, Pedersen SB, Stenderup K, Kassem M. Subcutaneous adipocytes can differentiate into bone-forming cells in vitro and in vivo. Tissue Eng. 2004 Mar-Apr;10(3-4):381-91. 4. Kaplan FS, Glaser DL, Hebela N, Shore EM. Heterotopic ossification. J Am Acad Orthop Surg. 2004 Mar-Apr;12(2):116-25. 5. Matsubara FB, Zanicotti DG, Zielak JC, Giovanini AF, Gonzaga CC, de Andrade Urban C. et al. Nonprocessed adipose tissue graft in the treatment of dehiscence bone defects in rabbit tibiae: a pilot study. Implant Dent. 2012 Jun;21(3):236-41.

58 â&#x20AC;&#x201C; RSBO. 2014 Jan-Mar;11(1):52-8 Gorny Junior et al. â&#x20AC;&#x201C; Immunoexpression of BMP-2 protein on bone repair of critical size defects treated with autogenous macerated adipose tissue

6. Misch CE, Dietsh F. Bone-grafting materials in implant dentistry. Implant Dent. 1993 Fall;2(3):158-67.

7. Oliveira LD, Giovanini AF, Abuabara A, Klug LG, Gonzaga CC, Zielak JC, et al. Fragmented adipose tissue graft for bone healing: histological and histometric study in rabbits calvaria. Med Oral Patol Oral Cir Bucal. 2013 May 1;18(3):510-5.

8. Qu CQ, Zhang GH, Zhang LJ, Yang GS. Osteogenic and adipogenic potential of porcine adipose mesenchymal stem cells. In Vitro Cell Dev Biol Anim. 2007 Feb;43(2):95-100.

9. Requena L. Normal subcutaneous fat, necrosis of adipocytes and classification of the panniculitides. Semin Cutan Med Surg. 2007 Jun;26(2):66-70.

10. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop Relat Res. 1986 Apr;(205):299-308. 11. Weinzierl K, Hemprich A, Frerich B. Bone engineering with adipose tissue derived stromal cells. J Craniomaxillofac Surg. 2006 Dec;34(8):466-71. 12. Zanicotti D, Brugin Matsubara F, Zielak JC, Giovanini AF, de Andrade Urban C, Miranda Deliberador T. J Oral Implantol. 2011 Nov 21. 13. Zou J, Wang G, Geng D, Zhu X, Gan M, Yang H. A novel cell-based therapy in segmental bone defect: using adipose derived stromal cells. J Surg Res. 2011 Jun 1;168(1):76-81. 14. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001 Apr;7(2):211-28.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):59-65

Original Research Article

Analysis of tensile strength of poly(lactic-coglycolic acid) (PLGA) membranes used for guided tissue regeneration Bruno Gasparini Betiatto de Sousa¹ Gabrielle Pedrotti¹ Ana Paula Sponchiado1 Rafael Schlögel Cunali1 Águedo Aragones2 João Rodrigo Sarot3 João Cézar Zielak¹ Bárbara Pick Ornaghi¹ Moira Pedroso Leão¹ Corresponding author: Bárbara Pick Ornaghi Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: bpo@up.com.br 1 2 3

School of Dentistry, Positivo University – Curitiba – PR – Brazil. School of Dentistry, Federal University of Santa Catarina – Florianópolis – SC – Brazil. School of Dentistry, Federal University of Paraná – Curitiba – PR – Brazil.

Received for publication: September 12, 2013. Accepted for publication: November 20, 2013.

Keywords: PLGA membranes; tensile strength test; guided tissue regeneration.

Abstract Introduction: The challenge of restoring patient’s function that presented some loss of an organ or tissue encourages the Tissue Engineering and Biotechnology to develop materials that promote bone regeneration. Poly(lactic-co-glycolic acid) (PLGA) copolymer is among of the most biomaterials used. Objective: To evaluate the tensile strength of PLGA membranes at different conditions of humidity and temperature. Material and methods: PLGA membranes were hourglass-shape cut and prepared at three different conditions of temperature and humidity (n = 10): (I) dry membrane at environment temperature of about 20ºC (control group), (II) moist membrane plasticized at 55ºC, (III) moist membrane plasticized at 55ºC, which subsequently underwent cooling. Subsequently, the membranes were subjected to tensile tests in a universal testing machine (DL-2000, EMIC) at 1.0 mm/min. Data was submitted to ANOVA and Tukey’s test (p < 0.05). Results: Group I showed the highest tensile strength mean (16.7 ± 1.9a MPa, p = 0.0022). There was no statistically significant difference between the means of groups II (14.6 ± 1.4 MPab) and III (13.9 ± 1.7 MPab). Conclusion: The dried PLGA membranes showed higher tensile strength than the membranes that were only either plasticized or cooled.

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Introduction The loss of bone tissue resulting from lesions or other damages impacts the patient’s life. The reconstruction of such structures through synthetic materials many times does not return the function and aesthetics required, making this a clinical challenge. The use of autogenous bone grafting collected from the patient is efficient; however, there is the need of a second surgical site because of the donor area. The most common donor areas used in Dentistry for bone grafting are: the skull bone, chin, iliac crest, retromolar area, and the maxillary tuberosity [2, 6]. Consequently, this cause greater morbidity to patient, contraindicating the surgical procedure [8]. Allogeneic (from individuals of the same species) and xenogeneic (from one species and transplanted to other species) grafting has the advantages of not necessitating another second surgical site; however, they have disadvantages such as incompatibility of the host, risk of disease transmission and greater chance of resorption and consequently loss of the bone gain [2, 17]. Other procedures can be executed aiming to increase the bone volume, such as osteogenic distraction (surgical induction of the bone fracture and splitting into two fragments so that a new bone is formed between them), osteoinduction with growth factors and/or stem cells, osteoconduction by the use of substrates for cellular development (scaffolds) and guided tissue regeneration (GTR) with the aid of membranes [18]. GTR is an alternative basically based on the installation of mechanical barriers to protect the area of neoformed tissue avoiding that other tissues, e.g., connective and clots, invade and jeopardize bone formation [10] (figure 1).

Figure 1 – Schematic drawing of the membrane action as a physical barrier to impede that other tissues and blood clots invade the site of bone

The membranes should be hard enough to maintain the space and support the tissues of the surgical area. Thus, it is needed that the constituting material of the membranes is malleable to provide the specific geometry for the functional reconstruction and hard to support external forces, such as those from mastication [7, 18]. Moreover, it is of great importance that they are totally biocompatible to not damage the surrounding tissues. Also, they should be porous, because it is through the pores that the fluids, nutrients, oxygen, and bioactive substances for cellular growth are changed. On the other hand, the diameter of the pores should be controlled. If they are very large, they can provide the leakage of fibroblasts, thus inhibiting the proliferation of stem cells and acting as route to bacteria [28]. The membranes can be constituted by either a single material or a combination of materials, such as the association of polymers with either collagen or hydroxyapatite. According to Pereira Neto et al. [15], still there is no consensus on which biomaterial would display the best performance in the tissue engineering. Commercially, resorbable and non-resorbable membranes have been found. Among the resorbable membranes, those constituted by polymers such as glycolic acid (PGA), polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) are the most used. Their advantage is to not require a second surgical procedure decreasing the morbidity of the patient. One of the disadvantages is the possibility of collapse during degradation, resulting in the loss of the barrier function and consequently the invasion by other tissues on the regeneration area, leading to the procedure failure. Non-resorbable membranes are

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composed by titanium and polytetrafluoroethylene net. Although they require a second surgical procedure, they are stable and do not undergo collapse and act as a barrier until their removal, reducing the risk of complications [18]. Therefore, PLGA membra nes a re a good alternative for this purpose because they are biomaterials serving as physical support to guide tissue neoformation. Based on this information, the aim of this study was to evaluate the tensile strength of a resorbable PLGA membrane at different conditions of humidity and temperature.

Material and methods Construction of the specimens The membranes employed in this study were produced with PLGA copolymer and obtained by solvent evaporation technique [16]. PLGA copolymer (Resomer, Evonik Ind., Essen, Germa ny), at 82:18 (m:m) ratio, was diluted in organic solvent dichloromethane formaldehyde (ChCl2, Synth – LabSynth, Diadema, Brazil). This solution was poured into rectangular moulds measuring 2.0 cm in width and 3.0 cm in length. After the solvent evaporation, the pieces were cut in rectangles (1.5 cm in width and 3.0 cm in length) to obtain samples with thickness ranging from 16 to 30 micrometers (figure 2). Next, the membranes were sterilized by gamma radiation (CBE, Cotia, Brazil).

Figure 3 – Hourglass-shape PLGA copolymer membrane in front of the resin composite guide

Prior to the tensile strength tests, the samples were submitted to three different humidity and temperature conditions: (I) membranes dried at environmental temperature of about 20ºC (control group); (II) moist membranes and plasticized; (III) moist membranes and plasticized which were cooled subsequently. The membranes of groups II and III were plasticized for two minutes in 0.9% saline solution (Segmenta, Ribeirão Preto, Brazil), heated at constant temperature of 55ºC (model CRC-5AC2W, PolyScience, Niles, USA) (figure 4). The membranes of group III were cooled in 0.9% saline solution (Segmenta, Ribeirão Preto, Brazil) at 10ºC for 30 seconds.

Figure 2 – PLGA copolymer membrane

To perform the tensile strength test, the membranes were cut in hourglass shape (5.0 mm in width at the central portion and 25.0 mm in length) with the aid of a guide of resin composite (figure 3).

Figure 4 – Thermal cycler (model CRC-5AC2W, PolyScience, Niles, EUA) for the membranes plasticization to keep constant the temperature of saline solution (55ºC)

62 – RSBO. 2014 Jan-Mar;11(1):59-65 Sousa et al. – Analysis of tensile strength of poly(lactic-co-glycolic acid) (PLGA) membranes used for guided tissue regeneration

Tensile strength test The tensile strength tests were conducted in a universal testing machine (model DL 2000, EMIC, São José dos Pinhais, Brazil), in which two self-lock claws distant 15.0 mm between each other were placed with constant cross-head speed of 1.0 mm/s [1,5] (figure 5A). To calculate the tensile strength (in MPa), the maximum load tension (in N) was divided by the value of the area of the central portion of the sample (in mm2). The thickness of each sample used for the area calculation was the mean of the measuring at three points on the central section of the sample, performed with the aid of a digital caliper (model 799, Starret, Itu, Brazil) (figure 5B).

Figure 5 – A: Universal testing machine used for tensile strength tests (model DL 2000, EMIC, São José dos Pinhais, Brazil); B: Digital caliper employed for measuring the thickness of the samples (model 799, Starret, Itu, Brazil)

Statistical analysis The data of the tensile strength test were submitted to one-way ANOVA and Tukey’s test to compare the mean values. The level of significance was set at 5% (p < 0.05).

Results There were statistically significant differences among groups (p = 0.0022, figure 6). Group I (dry membranes) showed the highest tensile strength mean values (16.7 ± 1.9 a MPa). There were no statistically significant differences between groups II (plasticized membranes: 14.6 ± 1.4b MPa) and III (plasticized and cooled membranes: 13.9 ± 1.7 b MPa).

Figure 6 – Tensile strength means (MPa): (I) dry membranes at environmental temperature (control group); (II) moist membranes plasticized at 55ºC; (III) moist membranes plasticized at 55ºC and then cooled

63 â&#x20AC;&#x201C; RSBO. 2014 Jan-Mar;11(1):59-65 Sousa et al. â&#x20AC;&#x201C; Analysis of tensile strength of poly(lactic-co-glycolic acid) (PLGA) membranes used for guided tissue regeneration

Discussion The first use of polyglycolic acid (PGA) was in the construction of totally resorbable suture threads [9, 12]. Poly-lactic acid (PLA) is presented as distinct stereoisomers, dextro-gyrate (D) and levogyrous (L): -PLA, D-PLA and DL-PLA [26]. In this study, L-PLA L was the polymer used to obtain PLGA because this is preferentially employed in materials requiring mechanical resistance and toughness [12]. PLGA copolymer membranes have been largely researched and studied because their degradation time can be controlled by the alteration of the concentrations of PLA and PGA copolymers and its molecular weight [19, 22]. Moreover, they are excellent mechanical barriers because they avoid the invasion of soft tissues and can be used as delivery system of drugs, skin replacements, vascular stents, and cell scaffolds [14]. Also, they have the approval of Food and Drug Administration (FDA). Sefat et al. [21] reported that dehydrated PLGA copolymer membranes present a hydrophobic feature, which makes difficult the cellular adhesion, therefore demanding a prior hydration before its use. The time recommended for this hydration is from 10 to 30 minutes in buffer phosphate-saline solution so that the process of cellular adhesion is more efficient [21]. However, according to the manufacturer, these membranes can be employed: (1) moist; (2) plasticized in heated solution; (3) cooled after plasticization. If the case does not require the molding of the membrane to the surgical site, the manufacturer advises only to place it over the receptor site for tissue regeneration and perform suture. If the case exhibits a surgical site of irregular morphology, it is advised to plasticize the membrane in solution heated at 55ÂşC, that is make it malleable, and adapt it over the site. Alternatively, the membrane can be cooled with saline solution after plasticization to memorize the desirable position. The results of this present study showed t hat t here were no st at ist ica l ly si g n i f ica nt differences among tensile strength means after the plasticization regardless whether they had been cooled. Notwithstanding, the best results were achieved with the dried membrane. However, this is not advisable because it jeopardizes cellular aggregation. Thus, the literature has reported the association of hydroxyapatite with PLGA copolymer to improve the mechanical properties of the membranes,

achieving a force and hardness similar to that of t he t issue su rrou ndi ng t he su rg ica l site [1, 21]. Moreover, this association could neutralize the acids produced by the degradation of PLGA copolymer and promote better bone neoformation than that of pure polymers due to its cellular adhesion capacity [1, 22, 27, 29]. With regard to cellular aggregation, most of the cells do not grow satisfactorily on the surface of PLGA membranes when compared with collagen membranes [4, 11]. Therefore, PLGA can be classified as a poor substrate for in vitro cellular growth [4, 16]. Another important factor to be reported is that the byproducts of PLGA copolymer, resulting from its degradation, are relatively strong acids (lactic acid and glycolic acid), which can accumulate on the surgical site and cause a late inflammatory response, thus negatively interfering in bone neoformation process [3, 12, 13, 19, 24]. Based on the aforementioned discussion, further studies are suggested aiming to analyze the degradation time and the residues coming from PLGA copolymer membrane after undergoing different humidity and temperature conditions similar to those of this study, since many studies have pointed out a late inflammatory response [3, 12, 13, 19]. Moreover, future studies are needed to verify the behavior of PLGA membranes as stem cell scaffolds by assessing the capacity of cellular adhesion to the substrate and cellular proliferation.

Conclusion Based on the results obtained, it can be concluded that dried PLGA membranes show the greatest tensile strength compared with membranes only plasticized or cooled after plasticization.

Acknowledgment The authors would like to thank Genius Biomateriais of Baumer S.A. for the PLGA copolymer membranes.

References 1. Asti A, Gastaldi G, Dorati R, Saindo E, Conti B, Visai L et al. Stem cells grown in osteogenic mediumon PLGA, PLGA/HA and titanium scaffolds for surgical applications. Bioinorg Chem Appl. 2010.

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2. Bayat M, Momen-Heravi F, Marjani M, Motahhary P. A comparison of bone reconstruction following application of bone matrix gelatin and autogenous bone grafts to alveolar defects: an animal study. Journal of Cranio-Maxillo-Facial Surgery. 2010;38:288-92.

11. Kim SY, Kanamori T, Noumi Y, Wang PC, Shinbo T. Preparation of porous poly(D,L-lactide) and poly(D,L-lactide-coglycolide) membranes by a phase inversion process and investigation of their morphological changes as cell culture scaffolds. J Appl Polym Sci. 2004;92:2082-92.

3. Bergsma EJ, Rozema FR, Bos RRM, Debruijn WC. Foreign body reaction to resorbable poly(Llactic) bone plates and screws used for the fixation of unstable zygomatic fractures. J Oral Maxillofac Surg. 1993;51:666-70.

12. Kohn J, Abramson S, Langer R. Biomaterials science an introduction to materials in medicine. 2. ed. San Diego, CA: Elsevier; 2004. p. 115-20.

4. Chen G, Liu D, Maruyama N, Ohgushi H, Tanaka J, Tateishi T. Cell adhesion of bone marrow cells, chondrocytes, ligament cells and synovial cells on a PLGA collagen hybrid mesh. Mater Sci Eng. 2004 Dec;24(6-8):867-73. 5. Chen G, Xia Y, Lu X, Zhou X, Zhang F, Gu N. Effects of surface functionalization of PLGA membranes for guided bone regeneration on proliferation and behavior of osteoblasts. J Biomed Mater Res. Part A. 2013;101A:4139-47. 6. Del Valle RA, Carvalho ML, Gonzales MR. Estudo do comportamento de enxerto ósseo com material doador obtido dos bancos dos tecidos músculo-esqueléticos. Revista de Odontologia da Universidade de São Paulo. 2006 MayAug;18(2):189-94. 7. Fujihara K, Kotaki M, Ramakrishna S. Guided bone regeneration membrane made of polycaprolactone/calcium carbonate composite nano-fibers. Biomaterials. 2005;4139-47. 8. Griffin TJ, Cheung WS, Zavras AI, Damoulis PD. Postoperative complications following gingival augmentation procedures. J Periodontol. 2006;77:2070-9.

13. Martin C, Winet H, Bao JY. Acidity near eroding polylactidepolyglycolide in vitro and in vivo in rabbit tibial bone chambers. Biomaterials. 1996;17(24):2373-80. 14. Pan D, Liu LF, Wang BY. Factors affecting the guided tissue regeneration in periodontal tissue. Chin J Aesthet Med. 2005;14:509-14. 15. Pereira Neto ARL, Cruz ACC, Aragones A, Simões AMO, Souza JGO, Sella GC et al. Proliferation and viability of gingival human fibroblast cultured on membranes. In: IADR General Session, 2011, San Diego, California. Journal of Dental Research. 2011. v. 25. 16. Pezzin APT, Zavaglia CAC, Duek EAR. Estudo da degradação in vitro de blendas de poli(pdioxanona)/poli(I-ácido láctico) (PPD/PLLA) preparadas por evaporação do solvente. Polímeros: Ciência e Tecnologia. 2002;12(4):285-94. 17. Pinto JGS, Ciprandi MTO, Aguiar RC, Lima PVP, Hernandez PAG, Silva Júnior AN. Enxerto autógeno x biomateriais no tratamento de fraturas e deformidades faciais – uma revisão de conceitos atuais. RFO. 2007 Sep-Dec;12(3):79-84.

9. Hollander AP, Hatton PV. Methods in molecular biology: biopolymer methods in tissue engineering. Totowa: The Humana Press Inc; 2003. p. 1-10.

18. Rakhmatia YD, Ayukawa Y, Furuhashi A, Koyano K. Current barrier membranes: titanium mesh and other membranes for guided bone regeneration in dental applications. Journal of Prosthodontic Research. 2013;57:3-14.

10. Kikuchi M, Koyama Y, Yamada T, Imamura Y, Okada T, Shirahma N et al. Development of guided bone regeneration membrane composed of b-tricalcium phosphate and poly(l-lactideco-glycolide-coe-caprolactone) composites. Biomaterials. 2004;5979-86.

19. Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials. 2006 Jun;27(18):3413-31.

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20. Seal BL, Otero TC, Panitch A. Polymeric biomaterials for tissue and organ regeneration. Mater Sci Eng: R: Rep. 2001;34:147-230. 21. Sefat F, Mckean R, Deshpande P, Ramachandran C, Hill CJ, Sangwan VS et al. Production, sterilization and storage of biodegradable electrospun PLGA membranes for delivery of limbal stem cells to the cornea. In: 3rd International Conference on Tissue Engineering; 2013; Leiria, Portugal. Elsevier; 2013. p. 101-16. 22. Song X, Ling F, Ma L, Yang C, Chen X. Electrospun hydroxyapatite grafted poly(L-lactide)/ poly(lactic-co-glycolic acid) nanofibers for guided bone regeneration membrane. Composites Science and Technology. 2013;79:8-14. 23. Stevens B, Yang Y, Mohandas A, Stucker B, Nguten KT. A review of materials, fabrication methods, and strategies used to enhance bone regeneration in engineered bone tissues. J Biomed Mater Res. Part B: Appl Biomater. 2008;85B:573-82. 24. Sung HJ, Meredith C, Johnson C, Galis ZS. The effect of scaffold degradation rate on three-dimensional cell growth and angiogenesis. Biomaterials. 2004 Nov;25(26):5735-42.

25. Takechi M, Ohta K, Ninomiya Y, Tada M, Minami M, Takamoto M et al. 3-dimensional composite scaffolds consisting of apatite-PLGAatelocollagen for bone tissue engineering. Dent Mater J. 2012;31(3):465-71. 26. Tokiwa Y, Calabia BP, Ugwu CU, Aiba S. Biodegradability of plastics. Int J Mol Sci. 2009 Sep;10(9):3722-42. 27. Wu C, Zhang Y, Fan W, Ke X, Hu X, Zhou Y et al. CaSiO3 microstructure modulating the in vitro and in vivo bioactivity of poly(lactide-coglycolide) microspheres. J Biomed Mater Res A. 2011;98(1):122-31. 28. Zhang M. Biocompatible of materials. In: Shi D, Wang M, Zhang M, Clare A, Kasuga T, Liu Q, editors. Biomaterials and tissue engineering. Berlin/Heidelberg: Springer-Verlag; 2004. 29. Zhang P, Hong Z, Yu T, Chen X, Jing X. In vivo mineralization and osteogenesis of nanocomposite scaffold of poly(lactide-co-glycolide) and hydroxyapatite surface-grafted with poly(Llactide). Biomaterials. 2009 Jan;30(1):58-70.

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Original Research Article

Analysis of salivary pH, flow rate, buffering capacity, concentrations of calcium, urea and total proteins in 2-8 years-old children with Down’s syndrome Gizele Franco1 Rafaella Saab1 Luciani Variani Pizzatto1 Maria Fernanda Torres2 Andréa Paula Fregoneze3 João Armando Brancher1 Corresponding author: João Armando Brancher Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: brancher@up.com.br 1 2 3

Positivo University – Curitiba – PR – Brazil. Federal University of Paraná – Curitiba – PR – Brazil. Pontifical Catholic University – Curitiba – PR – Brazil.

Received for publication: October 14, 2013. Accepted for publication: November 18, 2013.

Keywords: Down syndrome; saliva; salivary flow.

Abstract Introduction: Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. It is the most common chromosomal abnormality found in humans. Despite the motor difficulties and biofilm accumulation, individuals with DS have low caries prevalence. In this context it is assumed that saliva plays an important role in maintaining oral health. Objective: To evaluate the following salivary components: pH, buffering capacity and salivary flow volume in children with DS aging 2-8 years-old in the city of Curitiba (PR). Material and methods: Saliva samples were collected from 20 children with DS. The following parameters were evaluated: buffering capacity, flow rate, pH, and concentrations of calcium, urea and total proteins. Results: There was a normal distribution among the variables and the values observed were not statistically significant (p > 0.05). Conclusion: The results of this study revealed that there were no statistically significant differences in salivary flow, pH, buffering capacity, urea, calcium and total proteins in the subjects studied.

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Introduction Down syndrome (DS) is a genetic disturb caused by the trisomy of chromosome 21 [12]. It is the most common chromosomal anomaly found in humans. In Brazil, at every 700 births, one child has born with DS, regardless of the race, gender or social class [6]. The term “syndrome” indicates the set of characteristics which identifies the carrier. In DS, the obvious signs are related to the motor and cognitive impairment with physical features characterized by rounded or flattened face, oblique eyes, strabismus, small nose with flattening of the tip, large tongue and short neck [16]. Congenital heart diseases, recurrent respiratory infections, gast rointest ina l disorders, neurologica l a nd endocrine alterations can also be seen [5]. Within oral cavity, narrow maxilla, high palate, delay and alteration in tooth eruption, and tooth agenesis are frequent. Clinically, it is evident the biofilm accumulation onto the teeth, a determinant factor for t he beginning a nd prog ression of caries disease [24] and a high rate of periodontal disease, probably due to a low immunological response [20]. Despite of the motor impairment and biofilm accumulation, DS individuals have low caries prevalence [7]. In this context, it is believed that saliva plays and important role in maintaining oral health because of its organic and inorganic components influencing on the fragile balance of oral microbiota [13, 25]. Although the scientific literature has published many studies on saliva, this has received little attention by dentists. Therefore, the aim of this study was to verify the saliva of DS individuals from the city of Curitiba, Brazil.

Material and methods This study was submitted and approved by the Ethical Committee in Research of Positivo University, under protocol number 480602/2012, according the Guideline number 196/96 of the Brazilian Health Council. Only the children whose parents or legal responsible person signed the free and clarified consent forms participated in the study. Thus, 20 DS children, both genders, aged from 2 to 8 years-old, studying at the School of

Special Education, Development and Stimulation (Cedae/Apae), in the city of Curitiba (PR, Brazil) p a r t ic ip at e d i n t he st udy. Two e x a m i ners performed all clinical examinations, during the school period of the children, according to the international guidelines established by World Hea lth Orga nization. A ll children ex hibiting oral health problems were referred to the dental clinics of the Schools of Dentistry closer to the school. The sa liva sa mples of 20 chi ldren were col lected i n a si lent env i ron ment, w it hout interferences or external stimulations, with the aid of micropipette with disposable tips, without stimulus, for 10 minutes. Salivary f low was determined by dividing the volume collected by the time of aspiration. All saliva produced was stored into a sterile universa l collector vial. The measurement of pH was performed with the aid of a portable pH meter (Digimed Analytical Instrumentation, DM 23). Salivary buffering capacit y was determined w it h t he aid of Caritest ® –SL kit (Technew Comércio e Indústria Ltda.). For this purpose, 1 ml of the collected saliva was added to a f lask containing 3 ml of 0.005N HCl solution, and the reading of the samples strictly followed the manufacturer’s instructions. The sa liva r y concent rat ions of ca lcium, urea a nd proteins were determined t hrough colorimetric tests (Labtest Diagnóstica, Vista Alegre, MG, Brazil). The salivary biochemical tests were conducted always in triplicate. The obtained data were obtained by applying KolmogorovSmi rnov test for norma lit y, Levene test for homogeneity of variances and Student t test. The level of significance was set at 0.05.

Results The a na ly zed va r iables showed nor ma l d i s t r i but i o n b e c au s e t he v a lue s w e re not statistically significant (p > 0.05) (table I). Levene test exhibited that the variables were homogenous between genders without statistically significant differences (p > 0.05). The mean values of both genders were compared through Student t test for independent samples, without statistically significant differences (table II).

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Table I – Descriptive statistics for total sample

Age (years)

SFR (ml/min)

Calcium (mg/ml)

Urea mg/ml

TP (g/dL)

Total sample (n = 20)

4.72 (±1.8)

6.7 (±1.1)

0.41 (±0.18)

2.45 (±0.85)

23.55 (±7.01)

0.231 (±0.065)

Boys (n = 12)

4.58 (±1.8)

6.7 (±1.1)

0.39 (±0.18)

2.19 (±0.78)

23.61 (±6.68)

0.226 (±0.056)

Girls (n = 8)

4.87 (±1.8)

6.6 (±1.1)

0.45 (±0.18)

2.85 (±0.84)

23.16 (±5.63)

0.238 (±0.040)

SFR: salivary flow rate; TP: total proteins There were no statistically significant differences (p > 0.05)

Table II – Descriptive statistics for pH, salivary flow rate (SFR), calcium, urea and total proteins according to the distribution between boys and girls

Variable pH SFR (ml/min) Calcium (mg/dL) Urea (mg/dL) TP (g/dL)

Group

Mean

Boys Girls Boys Girls Boys Girls Boys Girls Boys Girls

12 8 12 8 12 8 12 8 12 8

6.750 6.688 0.394 0.450 2.191 2.850 23.610 23.163 0.226 0.238

Standard deviation 1.105 1.105 0.186 0.186 0.780 0.846 6.686 6.789 0.065 0.065

Standard error 0.030 0.045 0.018 0.019 0.099 0.108 0.289 0.319 0.088 0.099

CI (95%) LB 6.535 6.473 0.369 0.374 1.998 2.678 21.956 21.844 0.156 0.198

CI (95%) UB 6.975 6.903 0.419 0.462 2.245 2.915 25.399 25.248 0.235 0.257

CI: confidence interval; LB: lower bound; UB: upper bound There were no statistically significant differences (p > 0.05)

Discussion The sa liva plays i mport a nt roles i n t he maintenance of the oral health because it can prevent bacterial invasion, growth and metabolism through different mechanisms [25]. Also, it can modulate the bacterial adhesion to teeth and attenuate the deleterious effects of the production of metabolites by oral microbiota [23], due to its organic and inorganic components, contributing for oral health maintenance [11]. In this present study, 20 saliva samples of DS children aged from 2 to 8 years-old (12 boys and 8 girls) were verified. Salivary pH, flow, buffering capacit y as well as ca lcium, urea a nd tota l proteins were assessed. There were no statistically significant differences for all variables between genders (p > 0.05). Salivary flow is a very individualized measurement and varies according to circadian rhythm. It is known that the constant salivary flow can efficiently dilute and eliminate the products of the bacterial metabolism within oral cavity. Low salivary flow has been associated with high caries prevalence [28]. The data found in this study confirmed the results from

prior researches in which the mean salivary flow values were significantly smaller for DS individuals [21, 26]. In this context, special care should be given to DS individual, since low salivary flow, sugar consumption and the natural motor impairment can contribute for caries development. pH did not show significant alteration (p > 0.05) and ranged from 6.7 (±1.1) for boys and 6.6 (±1.1) for girls. There is a consensus in the literature that oral pH varies from 6.8 to 7.2, in average, in the different world population, with little alterations, regardless of the age [11]. When the analyzed sample is from nonstimulated saliva, pH tends to be low, about 5.6; however, pH increases when salivary flow is high [4]. Prior studies have already demonstrated that salivary pH values from DS individuals were not statistically significant when compared with those without the syndrome [2, 26]. Salivary buffering capacity is the ability of saliva in keeping stable oral pH, within the limits of normality, that is, it is the capacity of saliva in neutralizing the acids and/or bases within oral cavity, contributing to oral health [15]. All studied individuals exhibited good buffering capacity.

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Salivary proteins have many functions, among them, the bacterial aggregation [1, 8]; oxidation of hydrogen peroxide; antiviral, antimicrobial and antifungal activity. They also inhibit mineral precipitation and aids in remineralization [11]. On the other hand, salivary collagenases can also account for the increase of periodontal disease in DS individuals [9]. The measurement of proteins individually requires elaborated and laborious techniques and methodologies. Thus, in this present study, salivary total proteins were quantified. It was observed no statistically significant differences between boys and girls, with values found within the thresholds described in the literature for individuals without the syndrome. Urea is an organic component and plays an important role in salivary biochemical because when metabolized by bacteria within the biofilm, there is the releasing of ammonia, which is capable of neutralizing the acids produced by the biofilm and assures certain immunity against caries [27]. Previous studies have demonstrated the relationship between caries and salivary urea and found a smaller caries prevalence in individuals with chronic kidney disease than in normal people, probably because the higher concentration of salivary urea [18, 19]. The urea concentration in total saliva of healthy individuals is of about 30 mg/dL [14]. The urea elevation can indicate systemic alteration, mainly in the elderly [17] and nephropathic individuals [3]. It is possible that the reduction in the salivary urea concentration is associated with morphofunctional changes in salivary glands. Therefore, further studies are necessary to prove scientifically the congenital anomalies involving these glands. The results obtained in this study on DS individuals revealed that the concentration of salivary urea in these children was 23.55 mg/dL (±7.01). Although there are not salivary urea values from DS children within this age range, the results were close to those of people without the syndrome. There were no statistically significant differences between boys and girls. Although DS individuals have some motor impairment and consequently biofilm accumulation onto the teeth, caries prevalence is low. A possible rationale behind this is the high concentration of salivary calcium even with low salivary flow. High levels of salivary calcium determine a protector factor on the teeth [22]. Calcium concentration is inf luenced according to t he sa liva ry f low value. According to Jenkins and Hargreaves [10], the higher the salivary flow, the higher the

concentration of calcium ions within saliva. This present study revealed that despite of the reduced salivary flow, the levels of salivary calcium are high in DS individuals; however, this association was not statistically significant.

Conclusion The salivary variables studied have been largely used to determine the risk of oral diseases, specially caries and periodontal disease. None of these variables showed statistically significant differences when compared with data of the literature from individuals without DS. The results revealed that there were no statistically significant differences in salivary flow, pH, buffering capacity, urea, calcium, and total proteins between boys and girls.

References 1. Antti SA, Tenovuo J. Association between mother-infant salivary contacts and caries resistance in children: a cohort study. Pediatric Dentistry. 1994;16(2):9-14. 2. Cogulu D, Sabah E, Kutukculer N, Ozkinay F. Evaluation of the relationship between caries indices and salivary secretory IgA, salivary pH, buffering capacity and flow rate in children with Down’s syndrome. Arch Oral Biol. 2006;51(1):23-8. 3. Courts FJ, Tapley PM. Relationship of salivary urea to caries incidence in CRF patients. J Dent Res. 1984;63(2):184-98. 4. Dawes C, Jenkins GN. The effects of different stimuli on the composition of saliva in man. J Physiol. 1964;170:86-100. 5. Desai SS. Down syndrome: a review of the literature. Oral Surgery Oral Medicine Oral Pathology Oral Radiology. 1997;84:279-85. 6. Ministério da Saúde. Ministério da Saúde, Secretaria de Atenção à Saúde, Departamento de Ações Programáticas Estratégicas. Diretrizes de atenção à pessoa com síndrome de Down. Brasília; 2012. 7. Fiorati SM, Spósito RA, Borsatto MC. Prevalência de cárie dentária e doença periodontal em pacientes com síndrome de Down. Odontol 2000. 1999;3(2):58-62.

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8. Gahnberg L, Krasse B. Salivary immunoglobulin A antibodies and recovery from challenge of Streptococcus mutans after oral administration of Streptococcus mutans vaccine in humans. Infect Immun. 1983(39):514-9. 9. Halinen S, Sorsa T, Ding Y, Ingman T, Salo T, Konttinen YT et al. Characterization of matrix metalloproteinase (MMP-8 and -9) activities in the saliva and in gingival crevicular fluid of children with Down’s syndrome. Journal of Periodontology. 1986(67):748-54. 10. Jenkins GN, Hargreaves JA. Effect of eating cheese on Ca and P concentrations of whole mouth saliva and plaque. Caries Res. 1989;23(3):159-64. 11. Kidd E, Fejerskov O. What constitutes dental caries? Histopathology of carious enamel and dentin related to the action of cariogenic biofilms. Journal of Dental Research. 2004(83):35-8. 12. Lejeune J, Turpin R, Gautier M. Mongolism: a chromosomal disease (trisomy). Bulletin de l’Acadèmie Nationale de Mèdecine. 1959;143:256-65. 13. Lenander-Lumikari M, Loimaranta V. Saliva and dental caries. Adv Dent Res. 2000;14:40-7. 14. Macpherson LM, Dawes C. Urea concentration in minor mucous gland secretions and the effect of salivary film velocity on urea metabolism by Streptococcus vestibularis in an artificial plaque. J Periodontal Res. 1991;26(5):395-401. 15. Mandel ID. Impact of saliva on dental caries. Compendium of Continuing Education in Dentistry. 1989(1):476-81. 16. Moraes ME, Moraes LC, Dotto GN, Dotto PP, Santos LR. Dental anomalies in patients with Down syndrome. Braz Dent J. 2007;18(4):346-50. 17. Pajukoski H, Meurman JH, Snellman-Gröhn S, Keinänen S, Sulkava R. Salivary flow and composition in elderly patients referred to an acute care geriatric ward. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1997;84(3):265-71.

18. Peterson S, Woodhead J, Crall J. Caries resistance in children with chronic renal failure: plaque pH, salivary pH, and salivary composition. Pediatr Res. 1985;19(8):796-9. 19. Ruchi A, Bhumi S. Estimation of salivary urea levels and its relation with dental caries in children with chronic renal failure. Journal of Oral Health Research. 2010;1(2):70-5. 20. Santos P. Pacientes com síndrome de Down apresentam doença periodontal precoce. Rev Sobrape. 2003;81:53-7. 21. Siqueira WL, Bermejo PR, Mustacchi Z, Nicolau J. Buffer capacity, pH, and flow rate in saliva of children aged 2-60 months with Down syndrome. Clin Oral Investig. 2004;9(1):26-9. 22. Sewon L, Makela M. A study of the possible correlation of high salivary calcium levels with periodontal and dental conditions in young adults. Arch Oral Biol. 1990;35:211-2. 23. Tenovuo J. Clinical applications of antimicrobial host proteins lactoperoxidase, lysozyme and lactotransferrin in xerostomia: efficacy and safety. Oral Diseases. 2002;8:23-9. 24. Van Houte J. Role of micro-organisms in caries etiology. J Dent Res. 1994;73(3):672-81. 25. Van Nieuw Amerongen A, Bolscher J, Veerman EC. Salivary proteins: protective and diagnostic value in cariology? Caries Research. 2004;38:247-53. 26. Yarat A, Akyüz S, Koç L, Erdem H, Emekli N. Salivary sialic acid, protein, salivary flow rate, pH, buffering capacity and caries indices in subjects with Down’s syndrome. J Dent. 1999;27(2):115-8. 27. Zabokova E, Sotirovska IA, Ambarkova V. Correlation between salivary urea level and dental caries. Prilozi. 2012;33(1):289-302. 28. Zijinge V, Van Leewen B, Degener J, Abbas F, Thurnheer T. Oral biofilm architecture on natural teeth. 2010;5:1-9.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):71-6

Original Research Article

Adhesion and formation of tags from MTA Fillapex compared with AH Plus® cement Marina Samara Baechtold1 Ana Flávia Mazaro1 Bruno Monguilott Crozeta1 Denise Piotto Leonardi1 Flávia Sens Fagundes Tomazinho1 Flares Baratto-Filho1 Gisele Aihara Haragushiku1 Corresponding author: Gisele Aihara Haragushiku Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: gisele.haragushiku@gmail.com 1

Department of Dentistry, Positivo University – Curitiba – PR – Brazil.

Received for publication: October 12, 2013. Accepted for publication: November 21, 2013.

Keywords: endodontic cement; adhesion; scanning electronic microscopy.

Abstract Introduction and Objective: The aim of this study was two-fold: 1) to evaluate, in vitro, the shear bond strength of two sealers by push-out test and 2) to assess the failures after displacement. Additionally, the formation of tags was observed by SEM. Material and methods: Forty mandibular premolars were selected and the canals were subjected to biomechanical preparation with rotary instruments. These specimens were divided into two groups according to the sealer (n = 20): GI – MTA Fillapex and GII – AH Plus. All roots were filled with sealer only, without gutta-percha. After a period corresponding to three times the setting time of the sealer, the roots were sectioned transversely into slices of 1 mm thickness, to obtain one slice from the cervical third, to be used in the push-out test. Following, two slices of each group were randomly chosen for ultrastructural analysis by scanning electron microscopy (SEM). The data obtained in shear bond strength test were subjected to statistical analysis. Results: AH Plus cement exhibited higher shear bond strength values (1.332±0.75 MPa) than MTA Fillapex (0.071±0.07 MPa), with statistically significant differences. Conclusion: MTA Fillapex has a low bond strength with less formation of tags than AH Plus.

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Introduction

Material and methods

One of the desirable physical-chemical properties of the endodontic cements is adhesivity to the root canal walls [6]. Thus, when meeting this feature, a hermetic filling can be obtained through the sealing of root canal, promoting the apical repair, and avoiding the percolation of fluids to the periapical tissues and consequently preventing endodontic reinfections [3, 12]. Currently, the association of the endodontic cement with gutta-percha points is the gold standard in endodontic obturation, mainly because of lack of adhesion of the gutta-percha to the dentinal walls. The flowing property of the endodontic cement should be also taken into consideration, in order to fill the spaces between the gutta-percha and the canal wall, therefore providing a sealing with better quality [4], and enabling the filling of lateral canals and isthmuses [15]. The cements most commonly used today are based on epoxy resin, calcium hydroxide, zinc oxide and eugenol, and glass ionomer. Recently, MTA cement has been also employed and studies have aimed to evaluate the sealing capacity of resin-based cements and the biological repairing of mineral trioxide aggregate which is the new filling material. The following clinical characteristics of MTA-based cement have been reported: higher radiopacity; easy removal in cases of retreatment; excellent f lowing providing the easy filling of depressions and lateral canals; low solubility; releasing of calcium ions, which induces the bone regeneration; high alkalinity, which results in an antibacterial material inducing neoformation of peri-radicular cementum. Because of its composition, MTA-based cements exhibit an excellent biocompatibility to human tissues, making it an attractive material to both the professionals and researchers. Notwithstanding, little has been known on its adhesivity, which is fundamental for endodontic treatment success. M o s t o f t h e e n d o d o nt i c c e m e nt s h a v e demonstrated inadequate biological activity and adhesive capacity [2, 7]. Consequently, many studies have been constantly conducted to assess their physical, chemical and biological properties, which vary according to the composition of each material. Therefore, the aim of this study was to evaluate the capacity of adhesion to dentinal walls and the formation of tags of MTA Fillapex compared with AH Plus cement.

This study was submitted and approved by the Ethical Committee in research of the Positivo University under protocol number 088/11. Forty mandibular human premolars were selected with minimum root length of 11 mm, determined through digital caliper (Starret 799, Athol, USA) and radiographed at buccal-lingual direction. Inclusion criteria comprised: lack of endodontic treat ment, bone resorptions a nd calcifications; and complete formation of root apex. After selection, the teeth were extracted, cleaned with the aid of a periodontal curette and kept into 0.1% thymol solution at temperature of 9ºC. Before the study, the teeth were washed into running water for 24 hours, aiming to eliminate the thymol remnants. Following, the teeth were cut with the aid of carborundum discs mounted into a straight handpiece at low speed (Kavo do Brasil, Chapecó, Brazil) close to the enamel-cementum junction so that all roots measured 11 mm in length. Then, the specimens were kept into 0.9% saline solution in an incubator at temperature of 9ºC to avoid dehydration. The work i n g len g t h of a l l sa mples wa s determined at 10 mm. Crown-down technique was used with apical stop of 0.60 mm for all specimens. During all preparation, 2.5% sodium hypochlorite (Asfer Indústria Química Ltda., São Caetano do Sul, Brazil) was used as irrigant solution. Final irrigation was executed with 10 ml of 17% ethylenediaminetetraacetic acid (EDTA) (FarmáciaEscola Universidade Positivo, Curitiba, Brazil), followed by irrigation with 10 ml of distilled water and drying with absorbent paper points (DentsplyMaillefer, Petrópolis, Brazil). The specimens were randomly divided into two groups s (n = 20) according with the endodontic cement used: GI – AH Plus (DeTrey Dentsply, Konstanz, Germany), GII – MTA Fillapex (Angelus, Londrina, Brasil). The canals were filled only with endodontic cement, without using gutta-percha points so that gutta-percha/cement interface did not interfere in the shear bond strength test. The roots were cut at 1 mm slices, with the aid of diamond discs mounted into cutting machine (Isomet 1000 – Buehler, Lake Bluff, USA). A cervical third slice of each specimen was selected to be tested in the universal testing machine (Emic

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DL2000 – EMIC, São José dos Pinhais, Brazil), at crosshead speed of 0.5 mm/min. A stainless steel device was used to place the samples so that the surface of smaller diameter of the root canal was turned up, aligned with the rod employed to push the cement until the sample displacement. The rods had tips with 1 mm in diameter. The force (F) required to displace the filling material, in kilonewtons (kN), was transformed into Newton (N), and expressed in megapascal (MPa) by dividing the force value (N) by the adhesion area of the filling material (SL), in mm2. Thus, the formula employed to relate these measures was: s = F / SL. The calculation of the area (SL) was obtained according to the following formula:

SL = lateral area of the cone trunk; π = 3.14; R = mean radius of the coronal canal, in mm; r = mean radius of the apical canal, in mm; h = height related to the side of the cone trunk, in mm. After push-out test, the cuts were assessed with the aid of a stereoscopic lens (ZEISS; Stemi 2000-C, Germany), at x40 magnification, to verify the failure type, which was classified as: 1) adhesive – when the root canals were free of filling material; 2) cohesive – when the filling material completely covered the canal walls; 3) mixed – when there were areas covered by and free from filling material. Data were submitted to statistical analysis to verify the sample normality and determine the proper statistical test. Next, two specimens of each group were randomly selected for ultra-structural analysis in scanning electronic microscopy (SEM): one sectioned at the longitudinal direction and other at the cross-sectional direction in order to analyze the tags of cements within the tubules. The specimens for SEM analysis were kept into 2.5% glutaraldehyde solution, buffered with 0.1 mol/l sodium cacodylate (pH = 7.4) for 12 hours in an incubator at 4ºC. Following, the specimens were submitted to three baths in 0.1 mol/l sodium cacodylate (pH = 7.4) (for 20 min each) and dehydrated in increasing ethanol (Farmácia-Escola Universidade Positivo, Brazil): 25%, 50%, 75%, 95% (for 20 min of immersion into each solution) and 100% for 1 hour. The specimens were dried in an incubator at 37ºC for 24 hours, placed into a vacuum chamber and covered by gold of about 300 Aº (Bal-Tec SCD 030; Leica Microsystems, Germany). The analysis was performed in scanning electronic microscopy (Jeol JSM-6360LV, JEOL, Milestones, USA). In the qualitative analysis of the photomicrographies, the formation of cement tags and their aspect were analyzed.

Results Push-out test The values obtained by push-out test, in kN were transformed into MPa and submitted to statistical analysis with SPSS software (IBM, Armonk, USA). Table I – Mean, standard deviation, minimum and maximum values, confidence interval for push-out test

Standard Standard deviation error

95% Confidence interval Lower Upper bound bound

Mean

MTA Fillapex

0.0719

0.0708

0.0224

0.0212

AH Plus

1.3321

0.7557

0.2389

20 0.7020

0.8311

0.1858

Total

Minimum

Maximum

0.1226

0.01

0.19

0.7915

1.8728

0.69

2.66

0.3130

1.0910

0.01

2.66

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Based on the normality of the samples, one-way ANOVA was chosen (table II). Table II – One-way ANOVA

Sum of squares

Degree of freedom

Mean of squares

Groups

7.941

27.566

Within groups

5.185

0.288

Total

13.125

According to one-way ANOVA, there were statistically significant differences (p < 0.05) between AH Plus and MTA Fillapex cements. AH Plus cement exhibited the highest bond strength values (1.332±0.75 MPa) than those of MTA Fillapex cement (0.071±0.07 MPa). The analysis of the failures observed in stereomicroscopy is seen in graph 1.

Graph 1 – Failure types after shear bond strength test. Data per group

There was the predominance of the cohesive failure for AH Plus and mixed failure for MTA Fillapex. Adhesive failure occurred in both groups, with greater prevalence for MTA Fillapex.

Figure 1 – Images obtained by stereoscopic lens, at x40 magnification for failure type analysis. A) cohesive failure, when the filling material completely covered by the canal walls; B) adhesive failure, when the root canal walls were completely free from the filling material; C) mixed failure, when there were areas covered by and free from filling material

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Scanning electronic microscopy (SEM) SEM analysis showed a greater formation of tags in the teeth filled with AH Plus, while the teeth filled with MTA Fillapex exhibited little or none formation of tags (figure 2). At higher magnification, it was possible to observe the aspects of each cement: AH Plus was smoother and compact and MTA Fillapex was rougher and sparse.

Figure 2 – Photomicrographies at x500 and x5000 magnification. A) Cross-sectional cut of AH Plus specimen: tags within tubules; B) Longitudinal cut of AH Plus specimen: smooth and compact aspect of the tags; C) Cross-sectional cut of MTA Fillapex specimen: lack of tags within the tubules and cement detached from the dentinal wall; D) Longitudinal cut of MTA Fillapex cement: rough and sparse aspect of the cement

Discussion The association of the endodontic cement with gutta-percha points is the gold standard in the filling of root canals. The bonding capacity of the filling material to the dentinal wall is desirable for maintaining the integrity of the cement/dentine interface during displacement forces, as those occurring in the preparation of intraradicular posts, aiming to prevent marginal leakage [8]. In this present study, AH Plus cement exhibited better statistically significant results than those of MTA-based cement. The best adhesion force of epoxy resin-based cements have been studied through the comparison with other endodontic cements [10, 11, 14]. Prior studies have explained that the highest bond strength values obtained by the epoxy resinbased cements are because the capacity of creating a covalent bonding with an opened epoxy ring to any amine group exposed in collagen, giving long-

term dimensional stability and low polymerization tension [5, 9, 14]. The chemical composition of MTA-based cement could also influence on its bonding capacity [13]. A recent study discovered that the rationale behind the low bonding strength of MTA Fillapex is its low bonding capacity to dentinal tubules because of the formation of apatite by MTA, over its own surface, thus creating a similar structure that is different from that of the tag which prevents its leakage [11]. When exposed to scanning electronic microscopy, AH Plus exhibited longer and uniform tags, showing its higher mechanical imbrication and resulting in greater bonding capacity [10], while MTA Fillapex cement displayed little or none formation of tags, confirming the studies of Sagsen et al. [11]. Based on the results of this present studies, it could be observed that the material composition direct ly inf luences on its physica l-chemica l behavior.

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Conclusion This present study concluded that MTA Fillapex cement has low bond strength and little formation of tags compared with AH Plus cement.

References 1. Assmann E, Scarparo RK, Böttcher DE, Grecca FS. Dentin bond strength of two mineral trioxide aggregate-based and one epoxy resin-based sealers. J Endod. 2012 Feb;38(2):219-21. 2. Bouillaguet S, Shaw L, Barthelemy J, Krejci I, Wataha JC. Long term sealing ability of pulp canal Sealer, AH Plus, GuttaFlow and Epiphany. Int Endod J. 2008 Mar;41(3):219-26. 3. Brosco VH, Bernardineli N, Torres SA, Consolaro A, Bramante CM, Morais IG et al. Bacterial leakage in obturated root canals. Part 2: a comparative histologic and microbiologic analyses. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010 May;109(5):788-94. 4. Cobankara FK, Orucoglu H, Sengun A, Belli S. The quantitative evaluation of apical sealing of four endodontic sealers. J Endod. 2006 Jan;32(1):66-8. 5. Fischer MA, Berzins DW, Bahcall JK. An in vitro comparison of bond strength of various obturation materials to root canal dentine using a push-out test design. J Endod. 2007 Jul;33(7):856-8. 6. Haragushiku GA, Sousa-Neto MD, Silva-Sousa YT, Alfredo E, Silva SC, Silva RG. Adhesion of endodontic sealers to human root dentine submitted to different surface treatments. Photomed Laser Surg. 2010;28(3):405-10.

7. Huang TH, Yang JJ, Li H, Kao CT. The biocompatibility evaluation of epoxy resin-based root canal sealers in vitro. Biomaterials. 2002 Jan;23(1):77-83. 8. Huffman BP, Mai S, Pinna L, Weller RN, Primus CM, Gutmann JL et al. Dislocation resistance of ProRoot Endo Sealer, a calcium silicate-based root canal sealer, from radicular dentine. Int Endod J. 2009 Jan;42(1):34-46. 9. Koh ET, McDonald F, Pitt Ford TR, Torabinejad M. Celular response to mineral trioxide aggregate. J Endod. 1998 Aug;24(8):543-7. 10. Lee KW, Williams MC, Camps JJ, Pashley DH. Adhesion of endodontic sealers to dentin and gutta percha. J Endod. 2002 Oct;28(10):684-8. 11. Sagsen B, Ustün Y, Demirbuga S, Pala K. Pushout bond strength of two new calcium silicate-based endodontic sealers to root canal dentine. Int Endod J. 2011 Dec;44(12):1088-91. 12. Schilder H. Filling root canals in three dimensions. J Endod. 1967 Apr;32(4):281-90. 13. Torabinejad M, Hong CU, McDonald F, Pitt Ford TR. Physical and chemical properties of a new root-end filling material. J Endod. 1995 Jul;21(7):349-53. 14. Vilanova WV, Carvalho-Junior JR, Alfredo E, Sousa-Neto MD, Silva-Sousa YT. Effect of intracanal irrigants on the bond strength of epoxy resin-based and methacrylate resin-based sealers to root canal walls. Int Endod J. 2012 Jan;45(1):42-8. 15. Venturi M, Di Lenarda R, Prati C, Breschi L. An in vitro model to investigate filling of lateral canals. J Endod. 2005 Dec;31(12):877-81.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):77-82

Literature Review Article

Cell adhesion in bone grafts associated to nanotechnology: a systematic review Haroldo Gurgel Mota-Filho1 Amanda Alencar Cabral2 Diego Moura Soares3 Fernanda Ginani4 Carlos Augusto Galvão Barboza4 Corresponding author: Carlos Augusto Galvão Barboza Universidade Federal do Rio Grande do Norte Centro de Biociências – Departamento de Morfologia Av. Salgado Filho, n. 3.000 – Campus Universitário CEP 59072-970 – Natal – RN – Brasil E-mail: cbarboza@cb.ufrn.br Graduation Graduation 3 Program of 4 Program of 1 2

Course in Dentistry, Federal University of Rio Grande do Norte – Natal – RN – Brazil. Course in Biological Sciences, Federal University of Rio Grande do Norte – Natal – RN – Brazil. Post-Graduation in Dentistry, Federal University of Pernambuco – Natal – RN – Brazil. Post-Graduation in Oral Pathology, Federal University of Rio Grande do Norte – Natal – RN – Brazil.

Received for publication: July 18, 2013. Accepted for publication: November 11, 2013.

Keywords: cell adhesion; bone and bones; nanotechnology.

Abstract Introduction: Tissue engineering aims at the development of biological substitutes that can restore, maintain, or improve the functionality of damaged tissue or organs. To this end, molecular and cellular interactions may influence the tissue reactions to biomaterials. In order to be effective and integrated to the receiving area, the bone graft is required to allow a strong cell adhesion, interacting with several molecules to induce migration, differentiation, and thus the mineralization of the new bone on the graft. These cell adhesion molecules (CAM) will mediate the contact between two cells or between cells and the extracellular matrix, an essential process to the success of the implant. Objective: This paper is a systematic review of the literature on the mechanisms of cell adhesion to bone grafts associated to nanotechnology, describing the importance and the role of those molecules in the adhesion and thus in tissue regeneration. Literature review: After the use of search strategies, 18 articles that describe processes of cell adhesion to bone grafts were selected. Results: The main reported mechanisms involve cell

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adhesion molecules (CAMs) and extracellular matrix components. Conclusion: Several molecules are involved in the process of cell adhesion to bone grafts, highlighting the role of integrins, the focal adhesion mechanism, the influence of the collagen matrix, and the activity of alkaline phosphatase in bone matrix formation. Accurate identification of these mechanisms of cell adhesion is essential for further advancement in tissue engineering, such as the production of biological bone substitutes that achieve a better clinical outcome.

Introduction Bone regeneration is a complex and continuous process aiming at the anatomic and functional restoration and it can demand the use of biomaterials to promote a fast bone formation. Likely other tissues, many events occur when a given biomaterial is in contact with the biological bone environment, with molecular and cellular interactions influencing on the tissue features surrounding the biomaterial. In its presence, growth factors either adsorb or moisten the surface of the bone substitutes, promoting an adequate integration with the host bone [13]. Tissue engineering is a strategy very used to obtain functional repairing through the development of biological substitutes that can restore, keep or substitute damaged tissues or organs [31], through the combination of scaffolds biocompatible with live cells and/or bioactive molecules [19]. In this procedure it is possible to use stem cells obtained from different sources. The use of nanotechnology plays an important role in tissue engineering because the properties that this technology adds to the material, such as the greater surface area and greater roughness, improve the physical-chemical properties which mimic those of the natural tissues and organs [31]. These properties will act mediating the action of cell adhesion proteins, regulating cell behavior and causing tissue regeneration. Within bone tissue, the use of nano-modified scaffolds is a promising alternative aiming to accelerate the repair process and to reestablish the height and volume of the bone lost. Notwithstanding, to achieve the effectiveness and integration of the grafted tissue to the receptor site, it is necessary a strong cell adhesion, so that it demands many molecules interaction to induce cell differentiation and the bone matrix mineralization formed onto the graft. Some of these molecules are glycoproteins expressed on the cell surface, socalled cell adhesion molecules (CAM), which mediate either the contact between two cells or the contact

among cells and the extracellular matrix, being therefore of fundamental importance for adhesion [3]. CAMs have been classified into: cadherins, the immunoglobulin superfamily, integrins and selectins. To occur the adhesion of CAMs to the biomaterial, it is necessary their interaction with some components of extracellular matrix, such as proteoglycans, collagen and proteins [23]. Many of the properties giving biocompatibility to materials are closer related to the reaction of cells during contact, mainly in adhesion to their surface. The first interaction phase between the cell and biomaterial is characterized by events, such as approximation, adhesion and “spreading”. The quality of this first phase will influence on the cell capacity to proliferate and differentiate in contact with the grafted material. This is essential for graft effectiveness, in order to establish a mechanically solid surface, by the complete fusion between the material and bone tissue and without the presence of a fibrous interface [3]. The aim of this study was to analyze the most updated studies on the mechanisms of cell adhesion to bone grafts using nanotechnology by modifying the surface that potentiates these processes. Also, it is described the importance and role of CAMs in cell adhesion, and consequently in tissue regeneration.

Literature review The electronic search of the studies was executed until the ending of March of 2012, within journals available on PubMed/Medline database and published from 2007 to 2011. To perform the search, the search tool «advanced», available at NCBI site, was used by combining the descriptors All fields «nanotechnology and tissue engineering» and Date publication «2007/01/01 to 2011/12/31». Inclusion criteria comprised the use of studies which the complete text could be accessed, in English language, classified as experimental studies with

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main objective of the use of nanotechnology applied to biomaterials for bone grafts, which described the process of cell adhesion. The selection of the studies began by the evaluation of their titles identified by the application of the search strategy. The studies whose titles did not meet the aim of this present systematic review were excluded. All other studies were pre-selected and had their abstracts analyzed. Both the studies whose abstracts met the inclusion criteria and those which the abstracts did not provide enough data had their complete text analyzed. After the reading of the complete text, the studies describing experiments on other tissues or those which adhesion mechanism were not clear were excluded. After the application of the search strategy, 781 studies displaying the used terms were found. By applying a careful analysis and exclusion, 18 studies were selected, according to figure 1.

781 studies Exclusion of the review studies whose titles did not meet the aim of this present study

103 studies Exclusion of the studies which provided insufficient data or had been conducted on other tissues rather than bone tissue

18 studies selected Figure 1 – Systematic review for the selection of studies according to inclusion and exclusion criteria

Many molecules and mechanisms related to the processes of cell adhesion in bone grafts were reported by the articles selected. Among the processes described, it could be observed an emphasis on the role of integrins [9, 24, 26, 27, 30], alkaline phosphatase (ALP) [11, 12, 15, 22, 24, 26, 28] and on the mechanism of focal adhesion [4-6, 9]. These three mechanisms were the most cited because they have fundamental roles in the success and evaluation of adhesion, since integrins have significant influence on osteoblastic cell adhesion, ALP provides the differentiation degree of these cells, and focal adhesion acts as anchoring structure. The other molecules and mechanisms described are summarized in table I. Table I – Summary of the literature review, reporting the main molecules and cell adhesion mechanisms

Authors

Year published

Results

Comisar et al. [9]

2007

Integrin focal adhesion

Oliveira et al. [24]

2007

Integrin, fibronectin and ALP

Marletta et al. [22]

2007

ALP and collagen

Dalby et al. [10]

2007

Genes

Biggs et al. [4]

2008

Focal adhesion

Biggs et al. [5]

2008

Focal adhesion

Heo et al. [15]

2008

ALP

Prabhakaran et al. [27]

2009

ECM protein and collagen

Liuyun et al. [21]

2009

Filopodium and lamellipodium

Gupta et al. [14]

2009

ALP

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Table I (continued)

Authors

Year published

Results

Zhou et al. [32]

2009

Vitronectin, fibronectin and laminin

Biggs et al. [6]

2009

Focal adhesion

Francis et al. [12]

2010

ALP

Duan et al. [11]

2010

ALP and filopodium/lamellipodium

Ravichandran et al. [28]

2011

ALP and integrin

Polini et al. [26]

2011

ALP, proteins in general and integrin

Wang et al. [30]

2011

Integrin

Abd El-Fattah et al. [1]

2011

Collagen

Generally, the processes of adhesion described by the authors involved some cell-to-cell and cell-tomatrix adhesion molecules (table II). Among these processes, filopodium is emphasized because it is vital for the establishment of cell-to-cell contact in epithelial cells and it is also involved in the metastasis of neoplastic cells [16], and lamellipodium, which has an important function in cell migration, including embryonic development, inflammation and metastasis of malign cells [25]. Table II â&#x20AC;&#x201C; Molecules involved in the process of cell-to-cell and cell-to-matrix adhesion cited in the literature

Cell-to-cell

Cell-to-matrix

Integrin Filopodium/lamellipodium

Focal adhesion Collagen Fibronectin Alkaline phosphatase Laminin Vitronectin

Discussion O ne of t he ch a racter i st ic s of ad he sion molecules is their capacity of interacting with specific ligands, which can be present in the cell membrane or extracellular matrix (ECM) [3]. The main molecule of cell adhesion described by many authors selected in this study is from integrin family [9, 24, 26, 28, 30]. These molecules account for either cell-to-cell or cell-to-matrix adhesion. Integrins are located in the interface between intra-and extracellular medium and consequently can translate the sings from external to internal medium, promoting the adhesion, spreading or migration of cells, therefore regulating the cell growth and differentiation [3, 18]. According to the data searched in the literature, it is inferred that integrins have a significant inf luence on the adhesion of osteoblastic cells [3, 9], because its amount can affect the degree of maturity of focal adhesion, the recruitment or

proteins from the cytoskeleton and still influence the signaling of the molecules [9]. It must have a primary cell-to-matrix interaction to occur cell differentiation. This process is known as focal adhesion, characterized by a strong contact through integrin receptors, between the cell and the material to which the adhesion will take place [23]. Thus, the site of focal adhesion acts as an anchoring structure [23] which will influence on the formation of a fibronectin matrix [17]. Concerning to ECM, the collagen is one of the main components cited by the authors [1, 22, 27], since it is an essential candidate to compose ECM due to its properties of providing mechanical resistance to the tissue and inf luence on cell adhesion and differentiation [23]. The studies selected also cited other essential components of ECM which also acts in cell adhesion, such as laminin and vitronectin [22] and fibronectin [24, 32]. According to Iilic et al. [17], fibronectin/ vitronectin are fundamental for focal adhesion

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because they have the capacity of communicating with integrins through a sequence of amino acids known as RGD (arginine, glycine and asparagine), allowing the cell “anchoring”. Cowles et al. [8] also emphasized such importance and related it with the initial stages of osteoblast differentiation. According to Carvalho et al. [7], cell adhesion capacity is related with the presence of collagen and fibronectin/vitronectin within ECM, while laminin acts as a limiting factor of cell adhesion within ECM. Therefore, it can be affirmed that these proteins are essential for culture cell adhesion and consequently for tissue development. Additionally, other proteins were reported in the studies [26, 27], but they were not specified by the authors. Some authors observed the lamellipodium and filopodium formation as fundamental mechanisms for cell anchoring and proliferation, because they have been used to aid in cell adhesion and elongation [11, 21]. Other important mechanism of cell adhesion is ALP [11, 12, 15, 22, 24, 26, 28], which participating in the cleavage of organic phosphate esters and it is a fundamental component of bone matrix because is important during the formation of hydroxyapatite and calcium crystals [2]. The expression of ALP activity in the cells shows the bone capacity of forming osteoblast regardless of scaffolds [12] and providing the degree of osteoblastic differentiation [15]. Concerning to gene expression described in the study of Dalby et al. [10], this has been a funda menta l factor to occur a ll adhesion, growth, and differentiation of the cells. A better understanding of these mechanisms of guidance and cell-to-cell or cell-to-matrix interaction is essential for the construction of biomaterials with nano-specific properties to provoke a better cell response and consequently a greater applicability in tissue engineering [29]. To compare the results better, the standardization of the studies is necessary so that the same adhesion analyses are employed and the results are observed in the same design (in vitro / in vivo). The limitation in this type of study is the comparison of the quantitative and qualitative data due to the variety of protocols which makes difficult the correlation among the studies.

Conclusion The process of cell adhesion in bone grafts occur through many molecules and cell mechanisms (integrins, focal adhesion, collagen matrix and ALP). To know these cell mechanisms is fundamental

to identify and quantify a good cell adhesion, consequently leading to great advancements in tissue engineering.

References 1. Abd El-Fattah HMDDS, Helmy YBDS, El-Kholy, Marie M. In vivo animal histomorphometric study for evaluating biocompatibility and osteointegration of nano-hydroxyapatite as biomaterials in tissue engineering. Journal of the Egyptian Nat Cancer Inst. 2011;22(4):241-50. 2. Anderson MC, Ochsner KN, Kuhl B, Cooper J, Robertson E, Gabrieli SW et al. Neural systems underlying the suppression of unwanted memories. Science. 2004;303:232-5. 3. Anselme K. Osteoblast adhesion on biomaterials. Biomaterials. 2000;21:667-81. 4. Biggs MJP, Richards RG, Gadegaard N, Mcmurray RJ, Affrissman S, Wilkinson CDW et al. Interactions with nanoscale topography: adhesion quantification and signal transduction in cells ofosteogenic and multipotent lineage. J Biomed Mater Res A. 2008;91(1):195-208. 5. Biggs MJP, Richards RG, McFarlane CDW, Wilkinson CDW, Oreffo ROC, Dalby MJ. Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330 nm deep microgrooves. J R Soc Interface. 2008;5:1231-42. 6. Biggs MJP, Richards RG, Gadegaard N, Wilkinson CDW, Oreffo ROC, Dalby MJ. The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signaling in STRO-1þ enriched skeletal stem cells. Biomaterials. 2009;30:5094-103. 7. Carvalho MV, Alves PM, Oliveira RS, Ramalho LS, Queiroz LMG. Estudo imuno-histoquímico da tenascina-C e fibronectina em lesões proliferativas não-neo-plásicas de mucosa oral. Odontol ClínCientíf. 2009;8(4):353-7. 8. Cowles EA, Derome ME, Pastizzo G, Brailey LL, Gronowicz GA. Mineralization and the expression of matrix proteins during in vivo bone development. Calcif Tissue Int. 1998;62(1):74-82. 9. Comisar WA, Kazmers NH, Mooney DJ, Linderman JJ. Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: a combined computational and experimental approach. Biomaterials. 2007;28:4409-17.

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10. Dalby MJ, Gadegaard NJ, Tare R, Andar A, Riehle MO, Herzyk P et al. The control of human mesenchymal cell differentiation using nanoscale symmetry and disorder. Nature Materials. 2007;6:997-1003.

22. Marletta G, Ciapetti G, Satriano C, Perut F, Salerno M, Baldini M. Improved osteogenic differentiation of human marrow stromal cells cultured on ion-induced chemically structured polye-caprolactone. Biomaterials. 2007;28(6):1132-40.

11. Duan B, Wang M, Zhou WY, Cheung WL, Li ZY, Lu WW. Three-dimensional nanocomposite scaffolds fabricated via selective laser sintering for bone tissue engineering. Acta Biomaterialia. 2010;6(12):4495-505.

23. Meyer U, Buchter A, Wiesmann HP, Joos U, Jones DB. Basic reactions of osteoblasts on structured material surfaces. Eur Cell Mater. 2005;9:39-49.

12. Francis L, Venugopal J, Prabhakaran MP, Thavasi V, Marsano E, Ramakrishna S. Simultaneous electrospin–electrosprayed biocomposite nanofibrous scaffolds for bone tissue regeneration. Acta Biomaterialia. 2010;6(10):4100-9.

24. Oliveira PT, Zalzal SF, Beloti MM, Rosa AL, Nanci A. Enhancement of in vitro osteogenesis on titanium by chemically produced nanotopography. Journal of Biomedical Materials Research Part. 2007;80(3):554-64.

13. Garofalo GS. Autogenous, allogenetic and xenogenetic grafts for maxillary sinus elevation: literature review, current status and prospects. Minerva Stomatol. 2007;56(7-8):373-92.

25. Pinco KA, He W, Yang JT. α4β1 integrin regulates lamellipodia protrusion via a focal complex/focal adhesion-independent mechanism. Molecular Biology of the Cell. 2002;13:3203-17.

14. Gupta D, Venugopal J, Mitra S, Dev VRG, Ramakrishna S. Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts. Biomaterials. 2009;30(11):2085-94.

26. Polini A, Pisignano D, Parodi M, Quarto R, Scaglione S. Osteoinduction of human mesenchymal stem cells by bioactive composite scaffolds without supplemental osteogenic growth factors. PLoS ONE. 2011;6(10):e26211.

15. Heo SJ, Kim SE, Wei J, Kim DH, Hyun YT, Yun HS et al. In vitro and animal study of novel nanohydroxyapatite=poly(e-Caprolactone) composite scaffolds fabricated by layer manufacturing process. Tissue Eng Part A. 2008;15(5):977-89. 16. Hoffmann B, Schafer C. Filopodial focal complexes direct adhesion and force generation towards filopodia outgrowth. Cell Adhesion & Migration. 2010;4(2):190-3. 17. Iilic D, Kovacic B, Johkura K, Schlaepfer DD, Tomase-Vic N, Han Q et al. FAK promotes organization of fibronectin matrix and fibrillar adhesions. J Cell Sci. 2004;117(Pt 2):177-87. 18. Krauser A, Cowles EA, Gronowicz G. Integrinmediated signaling in osteoblasts on titanium implant materials. J Biomed Mater. Res. 2000;52 (4):738-47. 19. Kubinová S, Syková E. Nanotechnologies in regenerative medicine. Minimally Invasive Therapy. 2010;19:144-56.

27. Prabhakaran MP, Venugopal J, Ramakrishna S. Electrospun nanostructured scaffolds for bone tissue engineering. Acta Biomaterialia. 2009;5(8):2884-93. 28. Ravichandran R, Venugopal JR, Sundarrajan S, Mukherjee S, Ramakrishna S. Precipitation of nanohydroxyapatite on PLLA/PBLG/Collagen nano fibrous structures for the differentiation of adipose derived stem cells to osteogenic lineage. Biomaterials. 2011;33(3):846-55. 29. Toh YC, Ng S, Khong YM, Zhang X, Zhum Y, Lin PC et al. Cellular responses to a nanofibrous environment. Nanotoday. 2006;1(3):34-43. 30. Wang G, Zheng L, Zhao H, Miao J, Sun C, Liu H et al. Construction of a fluorescent nanostructured chitosan-hydroxyapat ite scaffold by nanocryst allon induced biomimetic mineralization and its cell biocompatibility. ACS Appl Mater Interfaces. 2011;3:1692-701.

20. Lian JB, Stein GS. Development of the osteoblast phenotype: molecular mechanisms mediating osteoblast growth and differentiation. Lowa Orthop J. 1995;15:118-40.

31. Zhang L, Wbster T. Nanotechnology and nanomaterials: promises for improved tissue regeneration. Nano Today. 2009;4(1):66-80.

21. Liuyun J, Yubao L, Chengdong X. Preparation and biological properties of a novel composite scaffold of nano-hydroxyapatite/chitosan/ carboxymethyl cellulose for bone tissue engineering. Journal of Biomedical Science. 2009;16(6):1-10.

32. Zhou WY, Guo B, Liu M, Liao R, Rabie ABM, Jia D. Poly(vinyl alcohol)/halloysite nanotubes bionanocomposite films: properties and in vitro osteoblasts and fibroblasts response. J Biomed Mater Res A. 2009;93(4):1574-8.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):83-7

Case Report Article

Masseteric cysticercosis: an uncommon appearance diagnosed on ultrasound Vinod Vijay Chander1 Sridevi Koduri1 Atul Kaushik1 Manpreet Kalra2 Renu Tanwar1 Sukeerat Mann1 Corresponding author: Sridevi Koduri Department of Oral Medicine & Radiology S.G.T. Dental College, Hospital and Research Institute Mobile No. 09885248434 – Gurgaon – Haryana – India E-mail: drsrident@gmail.com 1 2

Department of Oral Medicine & Radiology, S.G.T. Dental College, Hospital and Research Institute – Gurgaon – Haryana – India. Department of Oral Pathology, S.G.T. Dental College, Hospital and Research Institute – Gurgaon – Haryana – India.

Received for publication: April 18, 2013. Accepted for publication: August 1, 2013.

Keywords: masseter cysicercosis; ultrasonography; nonsurgical management.

Abstract Introduction: Cysticercosis occurs when man is infested by the larvae of Taenia solium. Cysticercosis involving the maxillofacial region is not only an uncommon event, but also represents a difficulty in clinical diagnosis. Objective: The present manuscript highlights masseteric cysticercosis as a differential diagnosis of chronic maxillofacial swellings and the role of high resolution sonog raphy as a n importa nt noninvasive a nd non-ioni zing imaging modality. Case report: We present a case of masseteric cysticercosis in an eight year old Indian boy, its clinical features, the imaging modalities required and adequate management of the case. Conclusion: Cysticercosis should always be a part of the differential diagnosis of maxillofacial swellings. A thorough case history, clinical examination, proper investigations and conservative treatment forms the mainstay for management of affected patients.

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Introduction C yst icercosis is a n i n fect ion caused by cysticercus (Gr. kystis: bladder + kerkosi: tail), a larval form of Taenia species. Among various species, the larval form of Taenia Solium i.e., cysticercus cellulosae infest human beings [1]. The location of cysticercosis in the order of frequency is central nervous system, subcutaneous tissue, striated muscle, vitreous humor of the eye and rarely other tissues [2]. Cystcerci in muscles generally do not cause symptoms, however oral and maxillofacial region is not a frequent site of occurrence for cysticercosis despite the presence of abundance of muscular tissue. This article presents a case of cysticercosis involving the masseter as a facial swelling, diagnosed with high-resolution ultrasonography and managed conservatively.

Case report An 8-year-old boy reported to the Department of Oral Medicine & Radiology, SGT Dental College & Hospital, Gurgaon with a complaint of swelling in his left cheek since two years. There was a history of intermittent reduction in size of swelling followed by medication by a local practitioner. There was also associated occasional fever and irritability. Medical and dental history revealed that this was the first visit to a dental office and patients’ father reported history of weight loss and reduced appetite since last 2 years. Although the patient was not a vegetarian, there was no history of consumption of pork and the family history was non-contributory. On general examination, he was conscious and coherent with normal gait and was poorly built. Signs of pallor were appreciated in palpebral conjunctiva and nail bed. His body was febrile with temperature raised to 37ºC. His height was 1.21 m and he was underweight by 4 kg. E x t r a or a l e x a m i n at ion re v e a le d f a c i a l asymmetry due to the swelling in left lower third of face. On palpation the dome shaped swelling was tender, firm and non-fluctuant measuring 3.5 cm superoinferiorly 4.0 cm anteroposteriorly. On clenching, the swelling was slightly bulged and hence suggested masseter involvement (figure 1). Left submandibular and preauricular nodes were palpable, one on each side, oval, firm, tender and freely movable.

Figure 1 – Depicts the involvement of masseter by showing the prominence on clenching

Intraoral examination revealed a healthy mixed dentition phase without any carious lesion or periodontal pathology. A provisional diagnosis of juvenile fibroma was given and differential diagnoses of fibroma, cysticercosis, intramuscular hemangioma and intramuscular lipoma were considered. For subsequent i nvest i gat ions, informed consent from the patient’s parents and approval from ethical committee of the institute were taken. The panoramic radiograph was unremarkable (f i g u re 2) a nd t he pat ient wa s referred for ultrasonography (USG). USG was performed with a Voluson 730 scanner (GE Healthcare) using 12 Mhz linear transducer. Left masseter revealed a mixed lesion with characteristic 3-4 hyperechoic f lecks surrounded by an irregular hy poechoic pat tern (f i g u re 3) su g gest ive of cysticercosis. Enlarged lymph nodes measuring 2-6 mm were noted. Incisional biopsy of the lesion was performed which confirmed the diagnosis of left masseteric cysticercosis with subacute inflammation (figure 4). The patient was managed conservatively with 200 mg Albendazole for 28 days. A follow-up ultrasound was done after 15 days which revealed reduction in size of the lesion and a third follow-up ultrasound was done after 3 months which revealed complete resolution of the lesion (figure 5).

85 – RSBO. 2014 Jan-Mar;11(1):83-7 Chander et al. – Masseteric cysticercosis: an uncommon appearance diagnosed on ultrasound

Figure 2 – Panoramic radiograph revealing healthy mixed dentition phase

Figure 4 – Histopathology image suggestive of presence of cysticercii

Figure 3 – USG image revealing 3-4 hyperechoic flecks (calcified cysticercii) within the irregular hypoechoic cystic lesion

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Figure 5 – 3-month follow-up ultrasound after the administration of Albendazole 200 mg revealed complete resolution of the lesion

Discussion C yst icercosis i n hu ma ns is a pa ra sit ic infestation caused by the larval stage of the pork tape worm (Taenia Solium), cysticercus cellulosae. It results from the ingestion of the tape worm eggs through either fecally contaminated food and water as well as self-contamination by ref lux of the proglattid from the intestine or contaminated hands [3-5]. The oncospheres develop from the eggs, penetrate into the intestinal wall and via lymphatic or vascular circulation and reach various tissues where larvae develop and become the cysticerci or “bladder worm” a f luid filled cyst [3]. The mode of ent r y of nematode in the present case reported could be attributed to the ingestion of contaminated food /water. The la rvae ca n remain v iable at this stage for as long as ten years in humans. Living larvae evade immune recognition and do not elicit inf lammation. When the larva dies, it induces a vigorous granulomatous inf lammatory response that produces symptoms, depending on the anatomic location. Cysticercosis can affect various organs including brain, spinal cord, orbit, muscle and heart.

Oral and maxillofacial region is not a frequent site of occurrence for cysticercosis despite the presence of abundant muscular tissue. The age of incidence varies from young children to older adults with equal gender predilection. The common sites of involvement included tongue, lips and buccal mucosa [6]. The clinical features and differential diagnosis depend on the number and location of cysticerci as well as the extent of associated inflammatory response or scarring. Neurological manifestations, known as neurocysticercosis, are most common and may manifest as seizures, hydrocephalus and signs of raised intracranial tension [7]. Muscular cysticercosis may clinically present i n t hree dist i nct t y pes: mya lg ic, mass-like, pseudotumor or abscess-like as well as the rare pseudohypertrophic type. During the death of the larva, there is leakage of fluid from the cyst. The resultant acute inflammation may cause local pain and myalgia. Alternatively, degeneration of the cyst may result in intermittent leakage of fluid, eliciting a chronic inflammatory response, with collection of fluid around the cyst leading to a mass-like type as seen in our case. Alternatively, the cyst retracts, its capsule thickens and the scolex calcifies [8]. Ultrasonography can be effectively used to diagnose the cases of soft tissue cysticercosis besides various other methods such as: radiologic imaging, serology, computed tomography, magnetic resonance imaging [9], and fine needle aspiration cytology [4]. There are only few reports of the ultrasound features of muscular cysticercosis [1012]. Four different sonographic appearances in cysticercosis have been described by Vijayaraghvan [10]. On ultrasound, cysticercosis can appear as the cysticercus cyst with an inflammatory mass around it, as a result of the death of the larva. The second type can be seen an irregular cyst with very minimal fluid on one side, indicating the leakage of fluid. The eccentric echogenic protrusion from the wall due to the scolex is not seen within the cyst. It may be due to escape of the scolex outside the cyst or partial collapse of the cyst. The third appearance is a large irregular collection of exudative fluid within the muscle with the typical cysticercus cyst containing the scolex, situated eccentrically within the collection. The fourth sonographic appearance as seen in the present case reported here is that of calcified cysticercosis. It appears as multiple elliptical calcifications in soft tissue similar to the pathognomonic millet seed shaped elliptical calcifications in soft tissues described

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on plain radiography. These four appearances on high resolution ultrasonic are pathognomonic of cysticercosis and a definitive diagnosis can be made with great confidence [10]. Surgical removal is indicated for localized lesions that cause obvious symptoms, however medica l ma nagement w ith a ntihelminthetics such as either praziquantel or albendazole has been recommended for neurocysticercosis and subcutaneous cysticercosis [12, 13]. The case reported has been managed conservatively only with albendazole. Notwithstanding, preventive measures are also important including proper sanitation, good personal hygiene, thorough cooking of pork and all vegetables along with early detection and complete removal of the worm.

Conclusion Cysticercosis should always be a part of the differential diagnosis of maxillofacial swellings. High-resolution, non-invasive and non-ionizing ultrasound plays an important role in diagnosing soft tissue cysticercosis. However, histopathology is still the gold standard for confirmatory diagnosis. Although surgical removal is indicated for localized lesions, conservative medical management can also be considered along with the preventive measures.

References 1. Asrani A, Morani A. Primary sonographic diagnosis of disseminated muscular cysticercosis. J Ultrasound Med. 2004;23:1245-8. 2. Elias FM, Martins MT, Foronda R, Jorge WA, Araujo NC. Oral cysticercosis: case report & review of literature. Rev Inst Med Trop. 2005;47(2):95-8. 3. Jacobs RA. Infectious diseases: protozoal and helminthic. In: Tierney LM, Mephee SJ, Papadakes MAS (eds). Continuous medical diagnosis and treatment. 45th ed. New York: McGraw-Hill; 2006. p. 1463-536.

4. Richards Jr. F, Schantz PM. Laboratory diagnosis of cysticercosis. Clin Lab Med. 1991;11:1011-28. 5. Romero Deleon E, Aguirra A. Oral cysticercosis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1995;79:572-7. 6. Saran RK, Rattan V, Rajwanshi A, Nijkawan V, Gupta SK. Cysticercosis of the oral cavity: report of five cases and a review of literature. Int J Paediat Dent. 1998;8:273-8. 7. Schmidt DKT, Jordaan HF, Schneider JW. Cerebral and subcutaneous cysticercosis treated with albendazole. Int J Dermatol. 1995;34:574-9. 8. Scully RE, Mark EJ, McNeely WF. Case records of the Massachusetts General Hospital Weekly clinicopathological exercises, case 26, 1994. N Engl J Med. 1994;330:1887. 9. Sidhu R, Nada R, Palta A, Mohan H, Suri S. Maxillofacial cysticercosis: uncommon appearance of a common disease. J Ultrasound Med. 2002;21:199-202. 10. Sivapathasundharam B, Gururaj N. Mycotic infections of the oral cavity. In: Rajendran R, Sivapathasundharam B. Shafer, hine, levy. Shafers textbook of oral pathology. 5 th ed. India: Elsevier; 2006. 11. Timosca G, Gavrilita L. Cysticercosis of the maxillofacial region. A clinicopathological study of five cases. Oral Surg Oral Med Oral Pathol. 1974;37:390-400. 12. Vijayraghavan SB. Sonographic appearences in cysticercosis. J Ultrasound Med. 2004;23:423-7. 13. White AC, Wella PF. Cestodes. In: Kasper DL, Braunwald E, Fauci AS, Hansea SL, Lango DL, James JL. Harrisons principles of internal medicine. 16th ed. New York: McGraw Hill; 2004. p. 1272-6.

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Case Report Article

Unusual transmigration of canines – report of two cases in a family Sulabha A. Narsapur1 Sameer Choudhari2 Shrishal Totad3

Corresponding author: Sulabha A. Narsapur Department of Oral Medicine and Radiology, Al-Ameen Dental College and Hospital, Athani Road Bijapur-586108 – Karnataka – India E-mail: sulabha595@gmail.com Department of Oral Medicine and Radiology, Al-Ameen Dental College and Hospital, Rajiv Gandhi University – Bijapur-586108 – Karnataka – India. 2 Department of Oral and Maxillofacial Surgery, Al-Ameen Dental College and Hospital, Rajiv Gandhi University – Bijapur586108 – Karnataka – India. 3 Department of Conservative Dentistry and Endodontics, Al-Ameen Dental College and Hospital, Rajiv Gandhi University – Bijapur-586108 – Karnataka – India. 1

Received for publication: August 12, 2013. Accepted for publication: October 14, 2013.

Keywords: familial occurrence; observation; simultaneous transmigration.

Abstract Introduction: Transmigration is pre-eruptive migration of tooth across the midline. The etiology of this rare anomaly is unknown. Transmigration is largely related to mandibular canines. Although maxillary canine transmigrations are found in the literature, they are still a rare entity. Objective: The aim of the present paper is to report two cases of unusual transmigrations of canines in two immediate members of a family and to report the first case of simultaneous transmigration of maxillary and mandibular canines in an Indian adolescent. Case report: A rare case of simultaneous transmigration of ma xillary and mandibular canine in Indian adolescent along with bilateral transmigration of mandibular canines in her father is described here. Conclusion: Transmigration of canines in two immediate members of family needs to be further studied for familial occurrence of transmigration.

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Introduction Maxillary canine impaction is a well-known dental anomaly and the incidence is in the range of 0.8%-2.8%. Mandibular canine impaction is less frequent and the incidence was reported to be 20 times lower than that for maxillary canines. Migration of a tooth across the midline is an even rarer anomaly [3]. This rare phenomenon of intra osseous tooth crossing the midline is known as dental transmigration and it occurs almost exclusively with mandibular canines with an incidence of 0.1% [6, 10]. Ma xillary canine transmigration is still a rarer phenomenon. The exact mecha nism of transmigration is not clear [4, 6]. This anomaly is most often asymptomatic with no pain or over pathology, and usually cannot be detected during the routine clinical examination. Hence radiographs are essential for detection of such anomalies. Panoramic radiographs plays vital role in detection of transmigrations [9]. In human beings canines are very important for the facial esthetics a nd masticatory function a nd t heir absence may compromise both. Hence the unerupted or transmigrated canines must be diagnosed early with timely treatment plan which help to preserve these canines implicating better esthetics and functions [6]. Our search in the international literature did not revea l a ny reports on simulta neous transmigration of ma xillary and mandibular canines in a single patient. The present paper reports first case of simultaneous transmigration of right maxillary and left mandibular canines in adolescent and bilateral transmigration of mandibular canines in her father. To our best of knowledge simultaneous transmigration of maxillary and mandibular canines and familial occurrence of transmigration has not been reported in the literature yet.

Case report A 13 old young Indian female was referred to the department of oral medicine and radiology for oral prophylaxis. No extra oral abnormality was detected. Intraoral examination revealed clinical

absence of right permanent maxillary canine, left maxillary canine and left mandibular canine. Panoramic view revealed retention of right, left maxillary and left mandibular primary canines along with the impaction of permanent maxillary right canine, maxillary left canine and mandibular left canine (figure 1). The right maxillary canine had transmigrated with its crown just crossing the midline (figure 2). The left mandibular canine was transmigrated and horizontally impacted near inferior border of mandible with its crown positioned below the root apices of right canine. The crow ns of t ra nsm i g rated ca n i nes were surrounded by enlarged follicular space. All the third molars were missing. Left first premolar was rotated by 90º. With these findings a diagnosis of simultaneous transmigration of maxillary and mandibular canine was made. Patient was informed of the condition and possible treatment plans. Since patient was not interested in any type of treatment, no treatment was given except from oral prophylaxis. Patient was informed about the consequences and radiographic monitoring was strongly recommended. Examination of her immediate family members revealed that her father had missing lower canines bilaterally. Right lower lateral incisor was slightly extruded form its socket with slight mobility. Intra oral or extra oral swelling or tenderness was not noted in region of missing canines. Panoramic radiograph was taken which revealed bilateral transmigration of lower canines. There was irregular radiolucency around the left canine which was placed below the right lateral incisor and first premolar extending superiorly to the distal side of right lateral incisor (figure 3). Horizontal bone loss was seen till almost middle one third with lower anterior teeth with vertical bone defect along the distal aspect of the right lateral incisor and upper central incisor. With these findings a diagnosis of bilateral transmigration of mandibular canines with follicular cyst in association with left canine was made. Patient was informed about the consequences. Patient was not interested in any treatment type. None of other family members were affected. Unfortunately both the patients never returned for their follow up appointments and were lost to recall.

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Figure 1 – Panoramic view showing simultaneous canine transmigration in adolescent female

Figure 2 – Occlusal view showing the maxillary canine transmigration in adolescent female

Figure 3 – Panoramic view showing the bilateral transmigration with periodontitis and follicular cyst with left canine in her father

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Discussion Although migration of teeth is well documented, ectopia, pre-eruptive migration of tooth across the midline of jaw is termed as transmigration. The term transmigration was first used by Ando et al. in 1964 [7]. Tarsitano et al. defined transmigration as the phenomenon of an unerupted mandibular canine through midline. Joshi and Acluck suggested that the tendency of a canine to cross the midline suture is a more important consideration than the actual distance of migration after crossing the midline [4]. The etiology and exact mechanism are still not clear. Number of factors has been suggested. Abnormal displacement of the dental lamina in the embryonic life [6], abnormally strong eruptive force, conical shape of the canine which drives canine through dense symphysis, [4] agenesis of the adjacent teeth in particular the lateral incisor [11], presence of cyst around the crown of the canine facilitating the migration process [1], premature loss of deciduous teeth, retention of deciduous teeth, inadequate space, crowding, excessively large crowns, odontoma, fractures, cysts tumors, unfavorable alveolar length, genetics, and root stump obstacle would be sufficient to divert a tooth from its original path of eruption [4, 5, 8]. In these present cases the primary canines were retained in young female patient, and in her father, follicular cyst was noted together with the left canine in order that the occurrence of transmigration in two immediate family members needs to be further studied for familial occurrence of transmigration. Most of reports in the literature have described single cases [10]. Transmigration of unerupted tooth is generally a unilateral phenomenon, although bilateral transmigrations have been reported [10]. Transmigration is rarely found in maxillary arch. Aydin and Yilmaz reported the first case of maxillary canine transmigration in the literature in 2003 [2]. Kumar et al. reported maxillary canine transmigration of maxillary canine along with upper lateral incisor [6]. Till date only 31 cases of maxillary canine have been reported in the literature with only two cases in Indian population [6] before this; however, simultaneous mandibular and maxillary canine transmigration has not been reported in the literature. Bilateral transmigration of mandibular canines along with periodontitis is also not reported. The large cross-sectional area of the anterior mandible compared with the anterior maxilla may

be the reason for higher frequency of mandibular transmigration. The maxilla transmigration of canines may be prevented by a shorter distance between the roots of the maxillary incisors and the floor of the nasal fossa as well as by the restriction of path of tooth movement by the roots of adjacent teeth, the maxillary sinus and midpalatal suture which act as a barrier [6]. Mupparapu described the five patterns for transmigrated mandibular canine [6, 7]. Type 1 – Canine is impacted mesioangularly across the midline, labial or lingual to the anterior teeth with crown portion of tooth crossing the midline. Type 2 – Canine is horizontally impacted near the inferior border of the mandible below the apices of the incisors. Type 3 – Canine has erupted either mesial or distal to opposite canine. Type 4 – Canine is horizontally impacted near the inferior border of the mandible below the apices of the premolar or molar on the opposite side. Type 5 – Canine is positioned vertically in the middle with long axis of the tooth crossing the midline. In the child patient, the mandibular canine transmigration was type 2 and transmigrated ma x illa r y ca nine in t he child a nd bilatera l transmigration in her father could not be classified as there was no classification system proposed until now in the literature. There have been several treatment options proposed for this anomaly [4]. Surgical extraction appears to be the most favored, rather than heroic effort to bring the tooth back to its position. It is also indicated in the existence of pressure resorption towards the roots of adjacent teeth, periodontal problems, infections, cysts, prosthetic problems, neuralgic symptoms etc. Contralateral nerve should be anesthetized for extraction of transmigrated tooth as they maintain their nerve supply from original side. If the mandibular incisors are in normal position and space for the transmigrated canine is sufficient, transplantation may be undertaken. Orthodontic treatment can be done to bring back the labially impacted transmigrated canine to position. However, if the crown of such tooth migrates past the opposite incisor area or if the apex is seen to have migrated past the apex of adjacent lateral incisor it is impossible to bring back it into original place.

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Some authors believe that symptom less non erupted teeth can be left in place. In the present cases as both of our patients were not interested in any treatment, we opted to follow up the cases, but unfortunately the patients never came back for their appointments.

Conclusion Transmigration is a rare event and simultaneous transmigration of ma xillary and mandibular canines in a single patient is still rarer entity. Transmigration in two immediate members of family needs to be further studied for familial occurrence. Early radiographic examination of patient is important for treatment planning. Future studies and reports of more cases of canine transmigration would help to better understand t heir et iolog y, mecha nism of mig rat ion a nd improvement of classification criteria.

References

4. Camilleri S, Scerri E. Transmigration of mandibular canines – a review of the literature and report of five cases. Angle Orthod. 2003 Dec;73(6):753-62. 5. Joshi MR. Transmigrant mandibular canine: a record of 28 cases and retrospective review of the literature. Angle Orthod. 2001 Feb;71(1):12-22. 6. Kumar S, Urala AS, Kamath AT, Jayaswal P, Valiathan A. Unusual intraosseous transmigration of impacted tooth. Imaging Sci Dent. 2012 Mar;42(1):47-54. 7. Mupparapu M. Patterns of intraosseous transmigration and ectopic mandibular canine: review of literature and report of nine additional cases. Dentomaxillofac Radiol. 2002 Nov;31(6):335-60. 8. Peck S. On the phenomenon of intraosseous migration of nonerupting teeth. Am J Orthod Dentofacial Orthop. 1998 May;113(5):515-7.

1. Al-Waheidi EM. Transmigration of unerupted mandibular canine with a literature review and a report of five cases. Quintessence Int. 1996 Jan;27(1):27-31.

9. Shapira Y, Kuftinec M. Unusual intraosseous transmigration of a palatally impacted canine. Am J Orthod Dentofacial Orthop. 2005 Mar;127(3):360-3.

2. Aydin U, Yilmaz HH. Transmigration of impacted canines. Dentomaxillofac Radiol. 2003 May;32(3):198-200.

10. Sumer A, Sumer M, Ozden B, Otan F. Transmigration of mandibular canines: a report of six cases and a review of the literature. J Contemp Dent Prat. 2007 Mar;8(3):104-10.

3. Aydin U, Yilmaz HH, Yildirim D. Incidence of canine impaction and transmigration in patient population. Dentomaxillofac Radiol. 2004 May;33(3):164-9.

11. Vichi M, Franchi L. The transmigration of the permanent lower canine. Minerva Stomatol. 1991 Sep;40(9):579-89.

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Case Report Article

Maxillary squamous cell carcinoma: diagnosis and evolution of an inoperable case Luana Beber Yoshizumi¹ Mariah Scotti Alérico¹ José Miguel Amenábar Céspedes² Cleto Mariosvaldo Piazzetta² Cassius Carvalho Torres-Pereira² Corresponding author: Luana Beber Yoshizumi Rua José Rietmeyer, n. 471, bloco 1, apto. 204 – Guabirotuba CEP 81510-630 – Curitiba – PR – Brasil E-mail: luana.yoshizumi@gmail.com 1 2

Department of Dentistry, Federal University of Paraná – Curitiba – PR – Brazil. Department of Stomatology, Federal University of Paraná – Curitiba – PR – Brazil.

Received for publication: May 30, 2013. Accepted for publication: September 30, 2013.

Keywords: mouth neoplasms; diagnosis; squamous cell carcinoma.

Abstract Introduction: Squamous cell carcinoma (SCC) is a malignant neoplasia, originating from the oral lining epithelium, responsible for approximately 90-95% of malignancies in this location. Incidence and mortality rates for oral SCC may vary due to personal habits, socioeconomic characteristics, environmental factors and quality of health care. This study reports the aggressive evolution of SCC with a delayed diagnosis and the impossibility of immediate curative therapy. Case report: A 47-year-old brown male patient, smoker and drinker was referred by a primary health care center to the Stomatology Clinic at UFPR (Federal University of Paraná). The diagnosis of well-differentiated SCC was confirmed by histopathological examination. The patient was referred to a specialized service where an oncology team defined the case as inoperable due to lung function limitations and advanced tumor staging. The patient underwent lung physiotherapy sessions until reaching clinical conditions to undergo oncologic therapy which included radiotherapy and chemotherapy. Conclusion: The reported case highlights the importance of establishing an early diagnosis for SCC. There is a need of facilitating and speeding the access to both health care services and information that lead patients to seek professional assistance as soon as the first disease signs appear.

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Introduction Squamous cell carcinoma (SCC), sometimes referred to “spinocellular carcinoma” or “epidermoid carcinoma”, accounts for 90% of the malign neoplasias occurring in the oral mucosa [2, 6, 7, 9, 15]. It more frequently affects male individuals between the fourth and sixth decade of life, more commonly occurring at the tongue and mouth floor [2, 7, 12, 15]. Its etiology is multifactorial comprising environmental, social, and behavioral factors with smoking and drinking habits accounting for its main etiological agents [6, 7, 9, 12, 15]. The initial clinical aspect can vary among either leukoplakic, erythroplastic or leukoerythroplastic aspect. Its growth can be either exophytic or endophytic, leading to an aspect with stiffened base and areas of necrosis. In most advanced cases, in which the underlying bone was affected, the radiographic examination can show radiolucent areas without defined limits. The definitive diagnosis is obtained through histopathological examination which also determines the type and stage of the lesion [6, 7]. Early diagnosis is fundamental to choose the treatment management, its onset, and consequently the prognosis and the patient’s survival [2, 13, 15]. The treatment choice – surgery, chemotherapy and/or radiotherapy – depends on the clinical staging and the degree of histopathological differentiation of the tumor [6]. The best index of SCC prognosis is the moment of its diagnosis so that the advanced staging has been frequently associated with high mortality rates [13]. The survival rate of patients with head and neck malign neoplasia is of 76% in early diagnosis cases, 41% in cases of metastasis in cervical lymph nodes and 9% in cases of metastasis in the neck area [2]. Notwithstanding, the advancements in therapy and care patterns seem to play an important role in the moderate increasing of the survival rate [13]. Although early diagnosis can be performed by either the dentist during routine appointments or the patient through self-examination, most cases are late diagnosed [5, 9, 11, 13, 15]. The aim of this present study is two-fold: to report a clinical case of malign neoplasia affecting the maxilla, aggressive and inoperable, in a patient with prolonged history of exposition to the main risk factors; to discuss the treatment managements and its consequences in late diagnosing and advanced staging of the disease, considering the comorbidities identified in the comprehensive health state of the patient.

patient, the swelling had appeared after the removal of a maxillary tooth. During anamnesis, the patient reported he consumed alcoholic beverage daily and smoked three packages per day since his 18 years old. He also reported to not practicing exercises and not having any systemic alterations. At extraoral clinical examination, it was observed a significant volume increasing at the right zygomatic area with signs of inflammation (figure 1A and B). At intraoral clinical examination, an irregular nodular mass was seen measuring 3 cm in its higher diameter, localized at the maxillary right side on the alveolar ridge, vestibule bottom and palate with a color among pinkish, reddish and whitish, firm to palpation and asymptomatic. The oral hygiene was poor with advanced periodontal destruction (figure 2). Panoramic, lateral, profile and posterior-anterior radiographs were obtained and revealed an extensive radiolucent area with vaguely defined limits located in the area of teeth #13 to #16 closer to the lesion (figure 3). A

Figure 1 – Initial extraoral clinical aspect: A – frontal view; B – lateral view

Case report A 47-year-old male patient was referred to the Discipline of the Stomatology of the Federal University of Paraná by a primary care center with main complaint of a “swelling in the face”. According to the

Figure 2 – Initial intraoral clinical aspect

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Figure 3 – Initial radiographic image showing large bone destruction

The diagnosis hypothesis was suggested as malign neoplasia of conjunctive tissue. At the same appointment, an incisional biopsy was carried out whose histopathological examination described welldifferentiated, invasive and ulcerated squamous cell carcinoma (figure 4A and B).

Figure 4 – Histological image: A – x100 magnification; B – x400 magnification

The patient was referred to the Oncolog y Service of the Erasto Gaertner Hospital, in the city of Curitiba, Paraná, Brazil. The hospital team considered the lesion as inoperable because of its dimensions, therefore opting by radiotherapy treatment as the first choice. During stomatology revaluation, after three weeks, it was observed the increasing of the lesion (figure 5A and B), with poor oral hygiene, right nose suppuration, without painful symptomatology

(figure 6). It was prescribed Amoxicillin 500 mg and 0.12% Chlorhexidine Digluconate to prevent secondary infection. The patient returned 6 weeks later showing visible weight loss increasing of the swelling worst oral hygiene with foul odor, displacement of the teeth near to the lesion, nasal suppuration and pain (figure 7). Notwithstanding, the surgery was still impossible. The aforementioned prescription was maintained until the surgery would be possible.

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Figure 5 – Extraoral clinical aspect three weeks after the first appointment: A – frontal view; B – lateral view

Figure 6 – Intraoral clinical aspect three weeks after the first appointment

Figure 7 – Intraoral clinical aspect six weeks after the first appointment

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The patient was diagnosed with limited respiratory function and underwent 40 sessions of pulmonary physiotherapy. Only after that, the oncological treatment could be started. At first, this comprised chemotherapy for four appointments, which had to be ended because the comprehensive health of the patient had worsened. Then, radiotherapy was chosen. The radiotherapy appointments (n = 35) were daily executed together with chemotherapy (figure 8).

Figure 8 – Timeline comprising from the initial appointment to the case conclusion

Parenteral nutrition was introduced during radiotherapy, once the patient had difficulty in chewing and swallowing. Palliative treatment was continued for pain, swelli ng a nd i n fect ion cont rolli ng (codei ne, de x a met ha sone, ben zoi l met ron ida zole a nd omeprazol). Also, the physiotherapy exercises were maintained to preserve the muscle and lung condition. After 45 weeks since the definitive diagnosing at the Erasto Gaertner Hospital, the tumor mass reduced at the right zygomatic area but the patient still exhibited nasal suppuration with skin fenestrations which had been treated with metronidazole ointment and cleaning with saline solution (figure 9). At intraoral examination, there was advancement of the maxillary involvement including the left side, with degree 3 mobility in the teeth #24 and #25. The area directly affected by radiotherapy presented extensive areas of tissue necrosis. The patient still showed poor hygiene

with dental calculus, and hairy tongue (figure 10). The prescription of 0.12% chlorhexidine digluconate was maintained during all the stages of the treatment.

Figure 9 – Extraoral clinical aspect 45 weeks after the definitive diagnosis

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Figure 10 â&#x20AC;&#x201C; Intraoral clinical diagnosis 45 weeks after the definitive diagnosis

In February, 24, 2013 the patient passed away because of neoplastic cachexia.

Discussion Similarly to other malign neoplasias, the earlier the diagnosis of squamous cell carcinoma, the better it prognosis [7, 8]. Notwithstanding, in this case report, the patient only sought for help when the disease was at an advanced stage. Such fact has been frequently described in the literature [2, 7, 9, 12], and late diagnosis will result in more aggressive, expensive and disfiguring treatment, with poor quality of life and shorter survival rate [3, 7]. The lack of attention of both the patient and the dentist accounts for the delay in obtaining the diagnosis [11]. Because of the lack of symptomatology at the initial stages of the disease, the patients failed in seeking for treatment [9, 11]. The most frequent first symptoms are not shocking and can be easily attributed to simple causes by patients with little information [3]. In this case report, the patient sought for treatment only when he had difficulty in swallowing and because of the pressure of his relatives due to his halitosis. Tucci et al. [15] still added as causes in treatment delay the fear of receiving the diagnosis even noting alterations in the mucosa. It has been also emphasized the situations in which the patients only seek for treatment because there is a deformity caused by the tumor growth [15]. The social-economical level, difficulties of access to health care, and lack of information may play a role in treatment delay [10]. In this case report, the diagnosis delay is compatible with such situation because the patient had low education level and lived far from the primary health care center.

Santos et al. [11] pointed out that this situation is the result of the lack of public policies aiming to the prevention and information regarding to oral cancer. According to Torres-Pereira et al. [14], oral cancer problem would be better managed if the health managers assumed it as a responsibility of each Brazilian city. In this case report, the diagnosis delay occurred preponderantly because of the postponement in seeking for primary care and just after that because the patient failed in immediately attending to the referral for secondary attention, which took about two weeks. Other relevant factor was the delay in the beginning of the oncologic treatment. Most of the specialized health services are located in the capitals of the Brazilian states, which implies in difficulty of access of patients living in the country and even metropolitan regions. According to the literature review of Santos et al. [11], many authors already reported that the time from the perception of symptoms and diagnosis until the treatment interferes in the evolution and prognosis of the disease and also in the quality of life of the patients. This present case report strengthens this perception suggested in some studies on the positive relationship between the difficulty of access and failure in the early cancer diagnosis. Still considering the delay in oncological treatment, the referral to public health care may be an aggravating factor, since many times the referral time is long and bureaucratic, interfering in the treatment and consequently in the prognosis and survival of the patient [1]. In this case reported, the need of pulmonary physiotherapy was an additional factor for the delay in oncological treatment onset. Moreover, all the comorbidities resulting from the long exposition to tobacco and alcohol caused important loss of the pulmonary capacity and physical fragility, consequently a preparation phase was required until the patient exhibited minimum conditions to treat oral malign neoplasia. Because of the tumor dimensions, the oncological team considered it as inoperable. According to Brener et al. [4], surgical treatment is the first choice in the treatment of oral malign neoplasia, varying with the size and staging of the disease. Radiotherapy has been chosen if there are no clinical conditions to submit the patient to surgery or if the patient does not accept the sequelae left by the treatment [4]. The treatment timeline of this case report illustrates very well the difficulties faced during the decision making process of the treatment. The aesthetic and functional impact of an eventual surgical resection

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together with the physical fragility caused by the disease itself and the comorbidities hindered the adoption of a curative approach. At all stages, the disease management aimed to decrease the neoplasia volume and keep the patient in a condition of living together with his relatives. Concerning to the prognosis, Seoane-Romero et al. [13] affirmed that variables such as age, comorbidities, immunological or nutrition state, tumor size and location, expression of oncogenes, proliferation markers, and DNA content have been evaluated as independent prognostic markers for oral cancer. However, the diagnosis stage is the most important marker for oral and oropharynx squamous cell carcinoma prognosis so that advanced stages have been frequently associated with high mortality rates. The case here reported corroborates the situation described by other authors [13].

Conclusion This case reported highlights the importance of establishing an early SCC diagnosis. Therefore, an easier access to health services and more information on oral cancer prevention are required so that patients would seek for treatment at the first signs of the disease. Moreover, both the dentists and the health managers play an important role in decreasing the diagnosis delay by improving the referral system among the health-attention levels in Brazilian health services.

References 1. Abdo EM, Garrocho AA, Barbosa AA, Oliveira EL, Filho LF, Negri SLC et al. Time elapsed between the first symptoms, diagnosis and treatment of oral cancer patients in Belo Horizonte, Brazil. Med Oral Patol Oral Cir Bucal. 2007 Nov 1;12(7):469-73. 2. Akbulut N, Oztas B, Kursun S, Evirgen S. Delayed diagnosis of oral squamous cell carcinoma: a case series. J Med Case Rep. 2011 Jul;6(5):291. 3. Baykul T, Yilmaz HH, Aydin U, Aydin MA, Aksoy M, Yildirim D. Early diagnosis of oral cancer. J Int Med Res. 2010 Jun;38:737-49. 4. Brener S, Jeunon FA, Barbosa AA, Grandinetti HAM. Carcinoma de células escamosas bucal: uma revisão de literatura entre o perfil do paciente, estadiamento clínico e tratamento proposto. Rev Bras Cancerol. 2007;53(1):63-9.

5. Costa EGC, Migliorati CA. Câncer bucal: avaliação do tempo decorrente entre a detecção da lesão e o início do tratamento. Rev Bras Cancerol. 2001;47(3):283-9. 6. Daniel FI, Granato R, Grando LJ, Fabro SML. Carcinoma de células escamosas em rebordo alveolar inferior: diagnóstico e tratamento odontológico de suporte. J Bras Patol Med Lab. 2006 Aug;42(4):279-83. 7. Gaetti-Jardim EC, Pereira CCS, Guastaldi FPS, Shinohara EH, Garcia Jr IR, Gaetti-Jardim Jr E. Carcinoma de células escamosas de grandes dimensões. Rev Odont Araçatuba. 2010 JulDec;31(2):9-13. 8. Gao W, Guo CB. Factors related to delay in diagnosis of oral squamous cell carcinoma. J Oral Maxillofac Surg. 2009 May;67(5):1015-20. 9. Gervásio OLAS, Dutra RA, Tartaglia SMA, Vasconcellos WA, Barbosa AA, Aguiar MCF. Oral squamous cell carcinoma: a retrospective study of 740 cases in a Brazilian population. Braz Dent J. 2001;12(1):57-61. 10. Gómez I, Seoane J, Varela-Centelles P, Diz P, Takkouche B. Is diagnostic delay related to advanced-stage oral cancer? A meta-analysis. Eur J Oral Sci. 2009 Oct;117(5):541-6. 11. Santos LCO, Batista OM, Cangussu MCT. Caracterização do diagnóstico tardio do câncer de boca no estado de Alagoas. Braz J Otorhinolaryngol. 2010;76(4):416-22. 12. Sassi LM, Oliveira BV, Pedruzzi PAG, Ramo GHA, Stramandinoli RT, Gugelmin G et al. Carcinoma espinocelular de boca em paciente jovem: relato de caso e avaliação dos fatores de risco. Rev Sul-Bras Odontol. 2010 Mar;7(1):105-9. 13. Seoane-Romero JM, Vázquez-Mahía I, Seoane J, Varela-Centelles P, Tomás I, López-Cedrún JL. Factors related to late stage diagnosis of oral squamous cell carcinoma. Med Oral Patol Oral Cir Bucal. 2012 Jan 1;17(1):35-40. 14. Torres-Pereira CC, Angelim-Dias A, Melo NC, Lemos Jr. CA, Oliveira EMF. Abordagem do câncer da boca: uma estratégia para os níveis primário e secundário de atenção em saúde. Cad Saúde Pública [online]. 2012 Mar;28:s30-s9 [cited 2012]. Avaiable from: URL:http://www.scielo.br/scielo. php?script=sci_arttext&pid=S0102-311X20120 01300005&lng=en&nrm=iso>. 15. Tucci R, Borges FT, Castro PHS, Aburad A, Carvalhosa AA. Avaliação de 14 casos de carcinoma epidermoide de boca com diagnóstico tardio. RSBO. 2010 Jun;7(2):231-8.

ISSN: Electronic version: 1984-5685 RSBO. 2014 Jan-Mar;11(1):100-6

Case Report Article

Oral rehabilitation of patient with severe early childhood caries: a case report Luciana Pedroso1 Camila Zucuni1 Letícia Westphalen Bento1 Juliana Yassue Barbosa da Silva2 Bianca Zimmermann Santos1 Corresponding author: Bianca Zimmermann Santos Rua Guilherme Cassel Sobrinho, n. 275, apto. 902 – Nossa Senhora das Dores CEP 97050-270 – Santa Maria – RS – Brasil E-mail: biancazsantos@hotmail.com 1 2

Department of Dentistry, Franciscan University Center – Santa Maria – RS – Brazil. Department of Dentistry, Positivo University – Curitiba – PR – Brazil.

Received for publication: October 21, 2013. Accepted for publication: November 14, 2013.

Keywords: dental caries; childhood; oral health; quality of life.

Abstract Introduction: Severe early childhood caries (S-ECC) is very common in pre-school children and shows a pattern of development which is defined and symmetrical, beginning on the cervical third of labial surface of maxillary anterior teeth. Accordingly, it can damage speech, swallowing, feeding, development, esthetics and self-esteem of the child. Objective: To report a case of a 5-year-old female patient with S-ECC on teeth #51, #52, #61 and #62. Case report: The patient came to dental clinic of the university with her mother for dental care. During the interview, the mother reported that her daughter used to drink milk in baby bottle at day and night with sugar content. Moreover, oral hygiene was not performed after bottle feeding at night. On clinical examination, the teeth #51, #52, #61 and #62 presented coronal destruction, and the mucosa associated at these teeth was very inflamed. Radiographically, it was found that carious lesions were limited to inner dentin. The treatment plan included education on oral hygiene and diet guidance. Rehabilitation with acetate matrixes was the treatment chosen for teeth destroyed by caries. Conclusion: This study demonstrated that the use of acetate matrixes is an effective alternative to return aesthetics and functionality to teeth of patients with severe early childhood caries.

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Introduction

Case report

Currently, the aiming of Pediatric Dentistry is that the patient reaches maturity free of diseases affecting in oral cavity. It is important that its action initiates during pregnancy and continues up to the birth of the baby. Thus, preventive measures of oral diseases should begin at the pregnancy period through guidance for an adequate feeding of the mother, rich in vitaminic elements required for tooth formation. Additionally, information on oral health related to both the mother and infant is part of the prenatal examination, assuring that the infant has a childhood free of caries [9]. However, unfortunately this is not the reality faced by all children. Tooth caries is the most common chronic disease in childhood and a great problem for world public health [22]. Severe early childhood caries (S-ECC) is common and damages speech, swallowing, feeding, development (height and weight), esthetics and emotional (self-esteem) of the child. Habits, such the unrestricted use of baby bottle, mainly with sugar content during night, are associated with S-ECC [12]. The difficulty in performing ora l hygiene of t he child a nd maintenance of food remnants during longer periods, mainly during sleepiness, also contributes for the establishment of the disease [21]. The init ia l clinica l sig n of ca ries is t he presence of opaque a nd white spots. If not controlled, the process evolves to the appearance of cavities, and this can lead to the destruction of all tooth crown and initiate infectious root processes because of pulp involvement. ECC i nit iates on t he cer v ica l t hi rd of t he labia l surfaces of the ma xillary anterior teeth and concomitantly affects the occlusal surface of the maxillary and mandibular first molars, maxillary and mandibular canines and second molars [14]. At more advanced stages, it affects also the mandibular incisors [15]. Considering the impact on the quality of life and the possibility to prevent, arrest or even treat ECC, it is of great importance that the dentists, especially Pediatric Dentists, are capable of diagnosing and treating this pathology, returning the oral health and smile aesthetics to these children [3]. Therefore, the aim of this study was to report a clinical case of a female patient, aged 5 years old, with severe early childhood case on teeth #51, #52, #61 and #62.

Pat ient K.B.A, fema le, aged 5 yea rs old, came to the Dentistry Clinics of the Franciscan University Center (short Unifra), in the city of Santa Maria (RS/Brazil), taken by her mother. The chief complaint was “pain in the front teeth and the first appointment at the dentist”. Moreover, according to the mother, the patient suffered bullying at school because of the appearance of her teeth. During anamnesis, the mother of the child reported t hat her pregna ncy was unevent ful with neither medical nor dental treatments and without use of medications. The child was born by normal delivery at nine months of pregnancy. The mother also reported that the child had good general health. With regard to diet, the mother informed that the patient was fed with breast milk until 3 years and 6 months of age and then began to use baby bottle at day and night. Its content was usually yogurt. At three months of age, she began to eat fruits and other foods rich in saccharose and carbohydrates. Oral hygiene was performed by both the mot her a nd t he ch i ld, t h ree t i mes per day (morning, afternoon and evening), with fluoride toothpaste but without flossing. Du ri ng cli n ica l ex a m i nat ion of t he soft tissues, melanin pigmentation was observed. In the examination of tooth tissues, the following alterations were observed: active carious cavity on teeth #54 and #64; crown destruction of teeth #51, #52, #61 and #62; and the other teeth showing non-cavitated active lesions possible to be arrested by controlling the etiological factors and fluoride therapy (figure 1).

Figure 1 – Initial image of the patient’s smile

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Radiographically, tooth #54 showed a radiolucent lesion on the root furcation, evolving the tooth germ of the permanent successor; tooth #64 exhibited carious lesion on the inner half of dentine and the cervical third of the root; teeth #51, #52, #61 and #62 also had carious lesions at the inner half of the dentine (figure 2).

To rehabilitate teeth #52, #51, #61 and #62, acetate matrixes (Coroas decíduas – TDV, Brazil) were chosen.

Sequence of clinical procedures for teeth #52, #51, #61 and #62 Prior to the procedure, a study cast was obtained for the initial adjustment of the matrixes (figure 4).

Figure 2 – Radiographic examination evidencing carious lesions at the inner half of the dentine of teeth #51, #52, #61 and #62

After the analysis of anamnesis, clinical and radiographic examination, a careful treatment pla ning was elaborated, including educative measures of oral hygiene, diet counseling and f luoride therapy. The extraction of teeth #54 and #64 (figure 3) was chosen because of bone rarefaction at the furcation of tooth #54 and the radiolucent lesion on the cervical third of tooth #64. Consequently, the patient was referred to early preventive orthodontics.

Figure 3 – Occlusal view showing the clinical aspect of the maxillary arch after the extraction of teeth #54 and #64

Figure 4 – Study cast of the maxillary arch for adjusting the acetate matrixes on the incisors

With the aid of straight scissors, the acetate matrixes were adjusted for each tooth on the study cast (figures 5 and 6).

Figure 5 – Adjustments of the acetate matrix of tooth #52 on the study cast

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Figure 6 – Acetate matrix adapted on tooth #52 on the study cast

Next, shade B1 of the resin composite (Opallis, Brazil) was chosen. Following, carious tissue was removed with the aid of dentin excavators (figure 7).

Figure 8 – Checking of the marginal adaptation of the acetate matrix on tooth #52, after caries removal

Figure 9 – Orifice on the palatal surface of the acetate matrix for flowing of the resin composite excess

Figure 7 – Removal of carious tissue from tooth #52 with dentin excavators

At the mouth, the marginal adaptation of the acetate matrixes were checked (figure 8) and then, orifices on the palatal surface of the matrixes (figure 9) were executed to enable the flowing of the resin composite.

Under relative isolation, the tooth remnant was etched with 37% phosphoric acid (Condac, FGM, Brazil) for 7 seconds. Then, with the aid of a thin microbrush, the adhesive system (Single Bond 2, 3M ESPE, Sumaré, São Paulo, Brazil) was applied, according to the manufacturer’s instructions. The resin composite was then inserted into the acetate, atrix (figure 10) up to its entire filling and, then, placed onto the tooth.

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Figure 10 – Acetate matrix completely filled with resin composite

Figure 12 – Removal of the acetate matrix with the aid of scalpel blade

After the removal of excesses of resin, lightcuring was executed onto each surface for one minute with the acetate matrix in position (figure 11).

The patient reported to be very satisfied with the treatment because the restorative treatment gave back tooth function and aesthetics (figure 13).

Figure 13 – Final image of the patient’s smile

This case report was submitted and approved by the Ethical Committee in Research of the Franciscan University Center under protocol no. 235.049. Figure 11 – Light-curing of the labial surface of tooth #52

Discussion

After the polymerization of the resin, the acetate matrix was removed with the aid of a scalpel blade (figure 12). Finishing procedures were executed with polishing drills. Polishing procedures were executed at another appointment.

ECC is considered as any tooth smooth surface with caries but with or without cavities, in children aged lesser than 3 years of age. It is also considered as ECC if the child has more than four, five and six surfaces affected on anterior primary teeth at 3, 4, and 6 years of age, respectively [11]. Thus, ECC

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is a particular caries type affecting primary teeth. It usually begins on maxillary incisors, followed by maxillary and mandibular molars, and affecting all teeth sequentially as they erupt in oral cavity. Carious lesions can rapidly progress if not treated, resulting in pain and infection [17]. Despite of the world ECC decreasing, it is still prevalent. It is estimated that 8% of children at 2 years old have at least one tooth damaged. This prevalence increases for 48% in children at 7 years old [20]. Concerning to the etiologic factors, although breast feeding is related to t he g row t h a nd development of the infant at the first months of life and it is considered as the baby’s main source of nutrients [1], either longer breastfeeding or bottle feeding together with the frequent consumption of substrates rich in carbohydrates may cause ECC [18]. In the case reported herein, all these factors were present associated with a poor oral hygiene. Moreover, this condition was also associated with the use of sugar on pacifiers and the frequent use of medications with sugar. Other factors strongly related to ECC are: social-economic status, level of education of the parents, and predisposing conditions of the host [6, 18]. Therefore, it is necessary that the dentists know the real risk factors associated with caries in both infants and pre-school children [13], so that they can interfere in the disease process in the best possible way. Various alterations are observed in children with ECC, mainly at advanced stage, which can compromise their quality of life. Among these alterations, it can be cited: nutritional problems, reduction of height and weight, pain, aesthetic and mastication impairment, swallowing and speech problems. Either isolated or in conjunction, these factors can compromise the child’s self-esteem during an important period of the development [10]. ECC treatment implies in the compliance among the dentist, mother, family, and child so that a new food routine is introduced and a new perspective regarding to oral health is accepted [17, 23]. Concerning to the oral rehabilitation of the patient, it is important to identify the stage in which the caries lesions are to achieve a better planing [5]. Minimum tooth destruction enables the remineralization through f luoride therapy [15]. The American Academy of Pediatric Dentistry recommends the modified atraumatic restorative treatment (ARTm) if there is the slow progression of the caries on dentin, considering the little age of the patients and the easy execution of this type of procedure [2].

In cases that the tooth remnant is very destroyed by the disease, the choosing for the aesthetic restorative technique will depend on the age of the child, social-economic status, the compliance of both the parents and child, and the size of tooth remnant [24]. Stainless steel and polycarbonate crowns; acetate or celluloid matrixes; resin facets; autogenous and heterogeneous bonding; pivot crowns with metallic posts; resin or metallic posts; partial fixed dentures or total dentures, among other techniques have been the treatment options currently available [4]. The acetate matrixes filled with resin composite were chosen for the oral rehabilitation in this case report and have been a good option according to literature [7], especially in cases in which tooth crowns are much destructed. This technique enables that the resin covers all structure of the tooth remnant, which results in the improvement of the resistance and the protection of the tooth against the biofilm and further caries [4]. Moreover, this restoration type provides resistance and aesthetics [8].

Conclusion Based on the information above, it is important that the dentists are capable of early diagnosing and treating ECC. The treatment should provide a positive psycho-social impact on these patients, not only by oral health recovering but also by the reestablishment of a better quality of life [3]. In the case here reported, it was possible to achieve a satisfactory result, because both tooth function and aesthetics were reestablished, consequently with the improvement in the child’s self-esteem.

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