RSBO v10 by Univille

v. 10 – n. 2 – April/June 2013

Indexed on the following databases: • BBO

(Brazilian Dental Bibliography)

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• LATINDEX

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• PORTAL DE PERIÓDICOS DA CAPES

(Coordination for the Improvement of Higher Education Personnel)

• ICI

(Index Copernicus International)

• DOAJ

(Directory of Open Access Journals)

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ISSN: Electronic version: 1984-5685

RSBO

Joinville – SC

v. 10

n. 2

95 p.

2013

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 Cleiton Vaz Dean for Administration Raul Landmann 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

Editorial............................................................................................................................................107 Guest editorial................................................................................................................................. 108

Original Research Articles Bone healing in titanium and zirconia implants surface: a pilot study on the rabbit tibia................... 110 Gabriel Marques; Luis Eduardo Marques Padovan; Mariza Akemi Matsumoto; Paulo Domingos Ribeiro Júnior; Elisa Mattias Sartori; Marcela Claudino

Endodontic treatment for necrotic immature permanent teeth using MTA and calcium hydroxide. A retrospective study....................................................................................................... 116 Claudio Maniglia-Ferreira; Fabio de Almeida Gomes; Nadine Luísa Soares de Lima Guimarães; Marcelo de Moraes Vitoriano; Tatyana Albuquerque Ximenes; Bruno Carvalho de Sousa; Roberto Alves dos Santos

In vitro analysis of the pH alteration of the dentine after using different calcium hydroxide-based pastes...............................................................................................................................................122 Cláudio Maniglia-Ferreira; Fabio de Almeida Gomes; Nadine Luísa Soares de Lima Guimarães; Marcelo de Moraes Vitoriano; Tatyana Albuquerque Ximenes; Roberto Alves dos Santos

Comparative evaluation of the accuracy of pick up transfer impressions performed with two different types of trays.....................................................................................................................128 Aline Treml; Gustavo Holtz Galvão; Allan Fernando Giovanini; Eduardo Christiano Caregnatto de Morais; Carla Castiglia Gonzaga; Enio Marcos da Silva

Aesthetic improvements in free gingival graft due to its association with frenectomy.......................135

Tertuliano Ricardo Lopes; Cassiana Nathalie Machado; Mariana Camargo Rogacheski; Thalyta Verbicaro; Allan Fernando Giovanini; Tatiana Miranda Deliberador

Evaluation of active chlorine releasing of sodium hypochlorite during seven days, stored at different temperatures......................................................................................................................143 Gladyvam Rabêlo Braitt; Evaldo de Almeida Rodrigues; Carlos Eduardo da Silveira Bueno; Antonio Henrique Braitt

Comparative evaluation of radiopacity of MTA Fillapex® endodontic sealer through a digital radiograph system............................................................................................................................149 Silvya Aparecida Vanso Bicheri; Fausto Rodrigo Victorino

In vivo evaluation of tissue response to new endodontic sealers........................................................153 Annecy Geib da Silva; Nádia Carolina Teixeira Marques; Natalino Lourenço Neto; Tamara Zeponi Fernandes de Melo; Vivian Agostino Biella Passos; Cleide Felício Carvalho Carrara; Thais Marchini Oliveira

Staining susceptibility of methacrylate and silorane-based materials: influence of resin type and storage time.....................................................................................................................................161

Leonardo Fernandes da Cunha; Lino Oliveira Carvalho de Santana; Samantha Schaffer Pugsley Baratto; José Mondelli; Gisele Aihara Haragushiku; Carla Castiglia Gonzaga; Adilson Yoshio Furuse

Literature Review Articles Gubernacular cord and canal – does these anatomical structures play a role in dental eruption?.......167 Danielly Cunha Araújo Ferreira; Ana Caroline Fumes; Alberto Consolaro; Paulo Nelson-Filho; Alexandra Mussolino de Queiroz; Andiara De Rossi

Ludwig’s angina: diagnosis and treatment.........................................................................................172 Tiago André Fontoura de Melo; Tiago Rücker; Marcos Paulo Dias do Carmo; Luis Eduardo Duarte Irala; Alexandre Azevedo Salles

Microbiological aspects of endoperiodontal lesion............................................................................176 Cristiane Tokunaga; Bruno Monguilhott Crozeta; Mariangela Schmitt Bonato; Beatriz Serrato Coelho; Flares Baratto-Filho; Flávia Sens Fagundes Tomazinho

Case Report Articles Apicoectomy after conventional endodontic treatment failure: case report........................................182 Lorena Oliveira Pedroche; Neisiana Barbieri; Flávia Sens Fagundes Tomazinho; Luciene Miranda Ulbrich; Denise Piotto Leonardi; Stephanie Martins Sicuro

Hidden caries’ challenge diagnosis: case report............................................................................... 188 Fernanda Mara de Paiva Bertoli; Bruno Marques da Silva; Mariana Dalledone; Estela Maris Losso

Importance of the comparative anatomy in Forensic Anthropology – case report..............................193 Rhonan Ferreira da Silva; Solon Diego Santos Carvalho Mendes; Décio Ernesto de Azevedo Marinho; Ademir Franco do Rosário Júnior; Marco Aurélio Guimarães

Editorial

Dear readers I have a friend, who I did not see for a long time, he was a kind of prophet, visionary and we often thought he was a little crazy, as every human being who is ahead of his time. Since the 90s, he stated: “Those who do not know computer in a few years or more cannot ride the elevator”. He liked to talk and look at the infinite, as if the wall which was less than two meters was something missing. At the beginning of the 90s, he spoke of the wonders of a connected world, with everyone talking; the speed of information exchange, but at the same time a world of loneliness, isolation, in which the machine would replace people. If we study the evolution of technology in human history, we note that the large jumps, such as radio, television, telephone, took a generation to disseminate. What we see now with the computer and all its digitized evolution is of unprecedented speed. I still remember my first computer, latest generation – as the seller told me –, surpassed only six months after its purchase. Today, the technology has no limits, we talk, we posted anything at any corner of the world, and we just need to have a connection and a smartphone. The latest economic reports bring us a drop of over 20% in sales of notebooks (here including iPad and its similar devices) as a function of the large-scale use of smartphones. Literally the world is now in your pocket. Some readers may be wondering: Where does he want to go with all this talk? Well, let’s go back to the editorial, because this subject would fill in pages and pages. The RSBO comes in large steps following the evolution of this speed and globalization of information. Its indexing in several important databases makes our readership increases in an exponential pace and at every corner of the world. This brings a growing number of manuscript submissions, which increasingly results in a scientific depth of RSBO. Over the past three years, the receiving of submissions almost tripled; this honors us a lot and brings a lot of responsibility. It is a clear sign that the journal is being increasingly read and researched. Submissions come from various countries and continents in terms of our free access online. This finding made us again to take an important decision regarding the RSBO’s destiny. From this number on, RSBO will only be published in digital media and we will not have its paper publishing. We will be making easier the online access of RSBO’s articles with a better viewing at the website of our University (www.univille.edu.br). You will also have full access to RSBO’s articles of the portal rev@donto, where the search for the articles is more dynamic. We understand that part of our readers would be still attached to paper for reading, but the evolution of communications is here, knocking our doors, and we can no longer ignore it. Good reading. Flares Baratto-Filho, MS, PhD Fabricio Scaini, MS Luiz Carlos Machado Miguel, MS, PhD RSBO Editors

Guest editorial Probiotics: harbinger of a new era of biological strategies for prevention/ treatment of periodontal diseases Root scaling and planing (RSP) is the therapy of choice for most of the clinicians and largely considered as the “gold standard” for the treatment of periodontitis. However, its isolate use frequently does not result in the desired clinical outcomes in severe cases of periodontal disease (PD). In these situations, the recolonization of pathogens and the disease relapse are very common. Because of the largely knowledge on the microbiological basis of periodontitis, the use of the antimicrobial therapy is becoming conventional as an adjunctive for RSP, especially in the treatment of the severest cases of PD. Additionally, due to the fact that the host’s response play an important role in the disease progression, treatments which suppress the inflammation, the so-called host response modulators, have been employed [1]. In summary, the use of adjunctive therapies in periodontal treatment aims either to treat the sites that do not respond to the conventional treatment or potentiate the results normally obtained with the mechanical instrumentation, therefore avoiding the need of additional surgical procedures to decrease the periodontal pockets. Considering RSP limitations in PD treatment, as well as the participation of several mechanisms regarding the innate and adaptive immune system of the host in the disease pathogenesis, the use of probiotics as a new adjunctive therapy for periodontitis has brought the interest of the dental scientific community, since the probiotics may modulate the immune-inflammatory response of the host and modify the bacterial environment. This subject was recently discussed in of the work groups of the 7th European Workshop in Periodontics, held in Spain, in 2011. Probiotics have been defined as live microorganisms, mainly bacterias, safe for consumption and capable of producing beneficial effects for the host’s health when ingested in sufficient amounts [3]. A recent study of “proof of concept” demonstrated that the mixing of Streptococcus species applied onto teeth of dogs with periodontitis, as adjunctive therapy to RSP, delayed the recolonization of periodontal pathogens and reduced the inf lammation [7]. The probiotics are, generally, regulated as dietary supplements and commercialized to improve or keep health. The main microorganisms used as this purpose have been the bacterias of genus Lactobacillus and Bifidobacterium. The probiotic therapy may provide advantages that are not observed when antimicrobials and/or mouthrinses are associated with RSP, because they did not cause bacterial resistance and they can interfere naturally on the local immune-inflammatory response of the host. The oral cavity has been only recently suggested as a relevant target to the application of probiotics. Until now, the probiotics was evaluated, mainly for the control of caries, where they can reduce the levels of Streptococcus mutans in saliva. The mechanisms of action of the probiotics on oral cavity seemed to be similar to those described for the balance of intestinal microflora [2]. These mechanisms can be an alternative, not only for the control of dental caries, but also for PD treatment. The microorganisms used for probiotic purposes may cause direct effects on the periodontal pathogens, affecting their growth, adhesion and colonization [5]. Probiotic bacterias can produce several components acting as antimicrobial agents, such as lactic acid, hydrogen peroxide, and bacteriocins. Other mechanism suggested to explain the probiotic action on PD treatment is the modulation of the immune-inflammatory response of the host [5]. Some studies have demonstrated that some probiotic species can attenuate the expression of interleukin (IL)-8 induced by periodontal pathogens in the oral epithelial cells [10], reduce the levels of proinflammatory cytokines(IL-8, IL-1�� β�� and tumor necrosis factor alpha) in the gingival crevicular fluid [8], affect the levels of salivary lactoferrin in periodontal highly susceptible individuals [6] and interfere on the activity of the polymorphonuclear elastase, as well as on the levels of myeloperoxidase and metalloproteinase-3 of the matrix present in the gingival crevicular fluid of individuals with gingivitis [4]. Considering that the main two strategies for periodontal disease treatment are the elimination of specific pathogens and the suppression of the destructive response of the host, the use of probiotics, undoubtedly, opens a new promising field to reach these goals, which can be of great values for the

prevention of the disease relapse in follow-up programs of the periodontal-affected patient. There are little in vivo studies evaluating the effects of probiotics on the PD control. Generally, these clinical studies demonstrated that the use of probiotics can promote a significant reduction of periodontal pathogens, improve the clinical periodontal thresholds in individuals with periodontitis, inhibit the development of gingivitis and potentiate the RSP effects during the non-surgical therapy [6, 4, 9]. Notwithstanding, these studies still need to be carefully considered, because their methodologies hinder to reach definitive conclusions capable of supporting safely the efficacy of probiotics for PD treatment. The market of probiotic products is worldwide increasing at exponential rate. Their introduction in the care of periodontal health will provide a significant impact because PD is considered as a public health problem around the world due to its high incidence and prevalence. Also, it is important to emphasize the relevance of studies on this field in order to find new alternatives of PD preventions, assuming an urgent character facing the current increasing evidences of the bidirectional relationship between PD and general systemic conditions of the individual. Thus, further studies are necessary to clarify better the role of the beneficial bacterias within oral cavity, identify new species of probiotic bacterias, establish periods and new administration vehicles and determine the real clinical significance of this therapy. The results of these studies in the field of Periodontics will be surely very useful for the development of new products for the chemical control of the bacterial plaque, as well it can create solid bases for the replacement of the systemic use of antimicrobials, therefore avoiding the development of resistant bacterias.

References 1. Berezow AB, Darveau RP. Microbial �� shift and periodontitis. Periodontol 2000. 2011;55:36-47. 2. Haukioja A. Probiotics and oral health. Eur J Dent. 2010;4:348-55. 3. Reid G, Jass J, Sebulsky MT, McCormick JK. Potential uses of probiotics in clinical practice. Clin Microbiol Rev. 2003;16:658-72. 4. Staab B, Eick S, Knöfler G, Jentsch H. The influence of a probiotic milk drink on the development of gingivitis: a pilot study. J Clin Periodontol. 2009;36:850-6. 5. Stamatova I, Meurman JH. Probiotics and periodontal disease. Periodontol 2000. 2009;51:141-51. 6. Shimauchi H, Mayanagi G, Nakaya S, Minamibuchi M, Ito Y, Yamaki K et al. Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21: a randomized, double-blind, placebo-controlled study. J Clin Periodontol. 2008;35:897-905. 7. Teughels W, Newman MG, Coucke W, Haffajee AD, Van Der Mei HC, Haake SK et al. Guiding periodontal pocket recolonization: a proof of concept. J Dent Res. 2007;86:1078-82. 8. Twetman S, Derawi B, Keller M, Ekstrand K, Yucel-Lindberg T, Stecksen-Blicks C. Short-term effect of chewing gums containing probiotic Lactobacillus reuteri on the levels of inflammatory media tors in gingival crevicular fluid. Acta Odontol Scand. 2009;67:19-24. 9. Vivekananda MR, Vandana KL, Bhat KG. Effect of the probiotic Lactobacilli reuteri (Prodentis) in the management of periodontal disease: a preliminary randomized clinical trial. J �� Oral Microbiol. 2010;2:2. 10. Zhang G, Chen R, Rudney JD. Streptococcus cristatus attenuates Fusobacterium nucleatum-induced interleukin-8 expression in oral epithelial cells. J Periodontal Res. 2008;43:408-16.

Michel Reis Messora PhD, MSc Department of Buccomaxillofacial Traumatology and Surgery and Periodontics School of Dentistry of Ribeirão Preto (FORP) University São Paulo (USP)

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):110-5

Original Research Article

Bone healing in titanium and zirconia implants surface: a pilot study on the rabbit tibia Gabriel Marques1 Luis Eduardo Marques Padovan2 Mariza Akemi Matsumoto2 Paulo Domingos Ribeiro Júnior2 Elisa Mattias Sartori3 Marcela Claudino1 Corresponding author: Marcela Claudino Instituto Latino-Americano de Pesquisa e Ensino Odontológico Rua Jacarezinho, n. 656 CEP 80710-150 – Curitiba – PR – Brasil E-mail: marcelaclaudino@hotmail.com Department of Post-graduation, Latin American Institute of Dental Research and Education – Curitiba – PR – Brazil. Department of Dentistry, Sagrado Coração University – Bauru – SP – Brazil. 3 Department of Oral Surgery and Implantology Pathology, Camilo Castelo Branco University – Fernandópolis – SP – Brazil. 1 2

Received for publication: August 14, 2012. Accepted for publication: December 12, 2012.

Keywords: dental implants; titanium; zirconia.

Abstract Introduction: Zirconia has been considered an alternative material to titanium for implant manufacturing, however the mechanisms regarding to bone healing in presence of zirconia implants remains poorly known. Objective: The objective of the investigation was to evaluate the bone healing surrounding titanium and zirconia implants in rabbits after 7, 14, 30, 45 and 60 days of implant placement through histological evaluation. Material and methods: Fifteen rabbits were used in this study and randomly subdivided into 5 groups, according to experimental periods. Titanium and zirconia implants were inserted into the right and left tibia, respectively. After healing periods of 7, 14, 30, 45 and 60 days, animals were euthanatized, the implants were removed and the samples were submitted to histological procedures. Results: Our histological results demonstrated similar bone healing surrounding titanium and zirconia implants after 7, 14 and 30 days after implant placement. After 45 days, a trend towards to earlier bone maturation was detected, remaining after 60 days. Inflammatory infiltrate, bone resorption and foreign body reaction were not observed in any periods and groups evaluated. Conclusion: Our findings demonstrated that zirconia and titanium presented a similar pattern of bone healing.

RSBO. 2013 Apr-Jun;10(2):110-5 – 111

Introduction Denta l impla nts have been considered a well-accepted and predictable alternative to the rehabilitation of edentulous patients [1, 6]. The clinical success of this treatment modality is strictly related to osseointegration, defined as a direct apposition of bone to the implant surface [8]. Titanium and its alloys have been largely used in implant manufacturing due to their excellent biocompatibilit y a nd mecha nica l properties. Moreover, favorable results concerning to bone apposition on titanium surface are well described, demonstrating the osseointegration of titanium dental implants. Therefore, titanium has been described as a reliable implant material for longterm use in the oral cavity [25]. Despite these data, several techniques and materials have been developed in order to achieve even better results, particularly as regards the aesthetic parameters. Indeed, titanium has been related to certain disadvantages such grayish color and the possible accumulation of titanium ions surrounding dental implants [7] and in local lymph nodes [28]. Based on these requirements, ceramic implants made of zirconia were developed, being considered a viable alternative to titanium [3, 16]. In fact, ceramic materials such zirconia are radiopaque, extremely hard, wear resistant and its ivory color is similar to the color of natural teeth compared to the gray color of titanium, which render it an important material for use in the esthetic zone [2, 5, 13]. Generally, zirconia ceramics present a high degree of biocompatibility and exhibit minimal ion release [4, 13, 14, 23]. In addition, higher fracture resilience and higher flexure strength were detected in zirconia dental implants [24]. Moreover, ma ny efforts have been made to eva luate t he putative differences bet ween zirconia and titanium surfaces concerning to osseointegration using different experimental models [10-12, 15, 16, 18]. Although several studies have been compared the osseointegration process of between titanium and zirconia implants, the putative differences concerning to biological mechanisms remain poorly known. Moreover, it would be interesting to evaluate the bone healing in presence of zirconia and titanium implants at different time points. Therefore, in the present study, we investigated the bone healing around titanium and zirconia implants in rabbits throughout a descriptive histological evaluation after 7, 14, 30, 45 and 60 days.

Material and methods Experimental animals and implants Fifteen New Zealand white mature male rabbits weighing 4 to 5 kg were used in this study. The animals were kept in individual cages, fed with a standard laboratory diet and given tap water ad libitum. In the sequence, rabbits were randomly subdivided into 5 groups, according to experimental periods (7, 14, 30, 45 and 60 days after implant placement). These procedures were performed under sterile conditions and the study protocol was approved by the Research Ethics Committee of the University of Sagrado Coração (116/09). Morse taper connection implants made of commercially pure titanium (Neodent, Curitiba, Brazil) with a length of 7 mm and diameter of 3.5 mm and machined surface were placed in the right tibia of each rabbit (group 1). Implants made of zirconia ceramic with machined surface were also inserted in the left tibia of each rabbit (group 2). These implants were manufactured with a modification in the platform, characterized by an insertion of a hexagon of 1.5 mm in order to provide the implant placement. Therefore, each rabbit received 2 implants, 1 in the left tibia and 1 in the right tibia. A total of 30 implants (15 titanium implants and 15 zirconia implants) were placed. Anesthesia, surgery and histological procedures Prior to surgery, the shaved skin in the tibial metaphysis area was cleaned with iodine solution at the surgical and surrounding area. The animals were anaesthetized through intramuscular injection of a combination of ketamine (Ketamina Agener®; Agener União Ltda., São Paulo, SP, Brazil) (0.35 mg/kg of body weight) and xylazine (Rompum® Bayer S.A. São Paulo, SP, Brazil) (0.5 mg/kg of body weight). Incisions of approximately 3 cm in length were performed in the left and right tibiae. After dissection, the bone surface of the tibial metaphysis was exposed and one implant was placed in each tibia. Implants were placed using a progressive sequence of drills under saline cooling, according to the manufacturer’s instructions. The soft tissues were sutured in separate layers and the animals received postoperatively a single intramuscular

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Marques et al. Bone healing in titanium and zirconia implants surface: a pilot study on the rabbit tibia

dose of antibiotic (Pentabiótico Pequeno Porte – Fort Dodge®, Campinas, SP, Brazil) (0.1 ml/kg of body weight). After 7, 14, 30, 45 and 60 days, animals were sacrificed by intramuscular injection of high dose of the anesthetic solution and the tibiae containing the implants were removed in terms of histological techniques. A fter the fi xation procedures, the implants were removed and the samples were decalcified for 8 weeks in 18% EDTA, washed, dehydrated and embedded in paraffin. Serial sections with 5 µm thickness were cut and stained with goldner’s trichrome. In the sequence, all histological sections were identified with a random numerical sequence to codify experimental periods and groups during the analysis procedures performed by a single calibrated investigator with a binocular microscope (Olympus Optical Co., Tokyo, Japan). Histological evaluation Descr ipt ive h istolog ic a l eva luat ion wa s performed in the section correspondent to central region of sockets. This evaluation includes analysis of presence of inflammatory infiltrate, formation of fibrous connective tissue, bone resorption and foreign body reaction.

Figure 1 – Histological findings of bone healing in titanium (A) and zirconia (B) implants surface after 7 days of implant placement, presenting cortical bone (arrows) in contact with the implants (*)

It was also detected a more dense and organized bone in the region correspondent to implant surface after 14 days. Moreover, reversion lines were detected, characterizing the maturation phase. No differences in histological findings were observed between both groups (figure 2A and 2B).

Results Our histological results demonstrated similar bone healing surrounding titanium and zirconia implants after 7, 14 and 30 days after implant placement. After 45 days, a trend towards to earlier bone maturation was detected, remaining after 60 days. Inflammatory infiltrate, bone resorption and foreign body reaction were not observed at any periods and in any groups evaluated. After 7 days of implant placement, cortical bone was detected in the superficial regions, referred to the cortical bone of the tibia. In the deeper portions, medullary bone was found, consisting of thin and slender newly formed bone trabeculae in the impressions of the implant threads. Furthermore, a highly vascularized fibrous tissue permeating these regions was observed, while the cortical region showed a discreet layer of connective tissue. Inflammatory cells, bone resorption and foreign body reaction were not detected in both groups. No differences between titanium and zirconia implants were found, as illustrated in figure 1A and 1B.

Figure 2 – Histological findings of bone healing in titanium (A) and zirconia (B) implants surface after 14 days of implant placement presenting lamellar bone (Lb)

After 30 days, Haversian system was observed in the cortical bone of the region correspondent to implant surface, demonstrating the remodeling process. In addition, an intense vascularization was detected, accompanied by a strong presence of bone cells lineage. There no significant difference between titanium and zirconia implants (figure 3A and 3B).

RSBO. 2013 Apr-Jun;10(2):110-5 – 113

Figure 3 – Histological findings of bone healing in titanium (A) and zirconia (B) implants surface after 30 days of implant placement showing evident Haversian systems (Hv)

Figure 5 – Histological findings of bone healing in titanium (A) and zirconia (B) implants surface after 60 days of implant placement maintaining a cortical bone (#)

Discussion Moreover, the cortical bone detected in the superficial regions remains in the bone remodeling process at 45 days after implant placement. In the deeper portions, there was a predominantly medullar bone with thin trabeculae. No differences were observed between groups, as illustrated in figure 4A and 4B.

Figure 4 – Histological findings of bone healing in titanium (A) and zirconia (B) implants surface after 45 days of implant placement presenting mature cortical bone (#)

Finally, it was observed a mature bone tissue in remodeling process, characterized by reversion lines after 60 days. The corticalized aspect was more evidente in this period. As observed in other time points, no differences were detected in titanium and zirconia implants (figure 5A and 5B).

Ceramic materials have also been proposed for the use in dental implants manufacturing [3, 16]. The applicability of these materials is associated to a wide range of advantages as mechanical characteristics and tooth-like color, allowing their indication to aesthetics regions [2, 5, 13]. Among ceramic materials available, zirconium oxide partially stabilized with yttrium (yttrium-stabilized tetragonal polycrystals [Y-TZP]) has been considered an interesting material for dental implants [24]. Indeed, evaluations regarding to stability of YTZP oral implants have shown that this material may be able to withstand oral forces over an extended period [5, 17]. Several studies have been investigated the osseointegration of zirconia implants using different experimental models [10-12, 15, 16, 18]. However, it would be reasonable to eva luate t he bone apposition in ceramic surfaces at different time points after implant placement. Therefore, our study demonstrated histological findings of bone healing in presence of zirconia and titanium implants placed in rabbit tibia after 7, 14, 30, 45 and 60 days. Our histological examination revealed similar results regarding to bone healing in zirconia and titanium implants after 7, 14 e 30 days. Nevertheless, a trend of earlier bone maturation was found surrounding zirconia implants after 45 and 60 days. In fact, previous studies did not found differences in osseointegration process of zirconia implants when compared to titanium implants [11, 12, 16, 18]. Accordingly, no statistical differences were found between bone-implant contact rates in zirconia and

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titanium implants placed in monkeys. The mean mineralized bone-to-implant contact after 9 months of healing and 5 months of loading amounted to 72.9% for the titanium implants and 67.4% for zirconia implants [16]. A similar rate of bone apposition was reported after 2 and 4 weeks of zirconia and titanium implant placement using a rabbit model [11]. Further evaluations performed using the same experimental model in rabbits revealed comparable rates of bone apposition in the zirconia and titanium surfaces at 6 and 12 weeks of healing [12]. In the same way, other authors demonstrated similar bone-implant contact rates between zirconia and titanium implants placed in dogs [15]. Moreover, no significant differences were found regarding to histological and biomechanical results in the osseointegration of zirconia and titanium implants after 2 and 4 weeks of implant placement in rats [18]. In pigs, zirconia implants showed a slight delay in osseointegration in terms of bone-implant contact rates. Zirconia implants presented rates of 59.3% and 67.1% after 4 and 12 weeks, respectively. Moreover, titanium implants revealed 64.1% and 73.6% after 4 and 12 weeks, respectively. However, no significant differences were observed [20]. Our histological findings also demonstrated the absence of inflammatory infiltrate, bone resorption and foreign body reaction were not detected in any groups and periods evaluated, indicating the biocompatibility of both materials. In accordance with our data, some studies have been showed the biocompatibility of ceramic materials such as zirconia [3, 13, 23]. In monkey and rabbit models, a high degree of biocompatibility of zirconia implants was observed, characterized by high bone-implant contact rates [3, 23]. In fact, the biocompatibility of zirconia was prev iously described a fter implantation into muscles and bone, suggesting the absence of local and systemic effects in mice [21]. In vitro and in vivo experiments also revealed that bioceramics exhibited superior osteoconductive ability and biocompatibility [22]. Indeed, some authors related that the inflammatory response and bone resorption induced by ceramic particles were more discreet when compared to those induced by titanium alloys such Ti6A14V [27]. Our data demonstrate that both implant materials can be considered highly biocompatible and osteoconductive, with a similar pattern of bone healing in both surfaces. Therefore, zirconia implants seems to have good biological properties, characterizing high biocompatible as observed in

commercially pure titanium implants [20, 26]. Accordingly, substantial evidence demonstrated similar biocompatibility and osseointegration for zirconia and titanium implants [9, 11, 12, 15-18, 20, 29]. However, it is reasonable to consider that our sample is small, representing a limitation of our study. Other studies must be carried out to improve knowledge on this field, which may serve as a basis for development of more effective strategies for rehabilitation of edentulous patients.

Conclusion Our findings demonstrated that zirconia and titanium presented a similar pattern of bone healing.

References 1. Adell R, Eriksson B, Lekholm U, BrĂĽnemark PI, Jemt T. Long-term follow-up study of osseointegrated implants in the treatment of totally edentulous jaws. Int J Oral Maxillofac Implants. 1990 Winter;5(4):347-59. 2. Ahmad I. Yttrium-partially stabilized zirconium dioxide posts: an approach to restoring coronally compromised nonvital teeth. Int J Periodontics Restorative Dent. 1998 Oct;18(5):454-65. 3. Akagawa Y, Hosokawa R, Sato Y, Kamayama K. Comparison between freestanding and toothconnected partially stabilized zirconia implants after two yearsâ&#x20AC;&#x2122; function in monkeys: a clinical and histologic study. J Prosthet Dent. 1998 Nov;80(5):551-8. 4. Albrektsson T, Hansson HA, Ivarsson B. Interface analysis of titanium and zirconium bone implants. Biomaterials. 1985 Mar;6(2):97-101. 5. Andreiotelli M, Wenz HJ, Kohal RJ. Are ceramic implants a viable alternative to titanium implants? A systematic literature review. Clin Oral Implants Res. 2009 Sep;20 Suppl 4:32-47. 6. Buser D, Mericske-Stern R, Bernard JP, Behneke A, Behneke N, Hirt HP et al. Long-term evaluation of non-submerged ITI implants. Part 1: 8-year life table analysis of a prospective multi-center study with 2359 implants. Clin Oral Implants Res. 1997 Jun;8(3):161-72. 7. Bianco PD, Ducheyne P, Cuckler JM. Local accumulation of titanium released from a titanium implant in the absence of wear. J Biomed Mater Res. 1996 Jun;31(2):227-34.

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8. Brånemark PI, Adell R, Breine U, Hansson BO, Lindström J, Ohlsson A. Intra-osseous anchorage of dental prostheses. I. Experimental studies. Scand J Plast Reconstr Surg. 1969;3(2):81-100.

19. �� Landreneau RJ, Johnson JA, Marshall JB, Hazelrigg SR, Boley TM, Curtis JJ. Clinical �� spectrum of paraesophageal herniation. Dig Dis Sci. 1992 Apr;37(4):537-44.

9. Depprich R, Zipprich H, Ommerborn M, Naujoks C, Wiesmann HP, Kiattavorncharoen S et al. Osseointegration of zirconia implants compared with titanium: an in vivo study. Head Face Med. 2008 Dec;11(4):30.

20. Möller �� B, Terheyden H, Açil Y, Purcz NM, Hertrampf K, Tabakov A et al. A �� comparison of biocompatibility and osseointegration of ceramic and titanium implants: an in vivo and in vitro study. Int J Oral Maxillofac Surg. 2012 May;41(5):638-45.

10. Dubruille JH, Viguier E, Le Naour G, Dubruille MT, Auriol M, Le Charpentier Y. Evaluation of combinations of titanium, zirconia, and alumina implants with 2 bone fillers in the dog. Int J Oral Maxillofac Implants. 1999 Mar-Apr;14(2):271-7. 11. Hoffmann O, Angelov N, Gallez F, Jung RE, Weber FE. The zirconia implant-bone interface: a preliminary histologic evaluation in rabbits. Int J Oral Maxillofac Implants. 2008 Jul-Aug;23(4):691-5. 12. Hoffmann O, Angelov N, Zafiropoulos GG, Andreana S. Osseointegration of zirconia implants with different surface characteristics: an evaluation in rabbits. Int J Oral Maxillofac Implants. 2012 Mar-Apr;27(2):352-8. 13. Ichikawa Y, Akagawa Y, Nikai H, Tsuru H. Tissue compatibility and stability of a new zirconia ceramic in vivo. J Prosthet Dent. 1992 Aug;68(2):322-6. 14. Josset Y, Oum’Hamed Z, Zarrinpour A, Lorenzato M, Adnet JJ, Laurent-Maquin D. In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics. J Biomed Mater Res. 1999 Dec 15;47(4):481-93. 15. Koch FP, Weng D, Krämer S, Biesterfeld S, JahnEimermacher A, Wagner W. Osseointegration of onepiece zirconia implants compared with a titanium implant of identical design: a histomorphometric study in the dog. Clin Oral Implants Res. 2010 Mar;21(3):350-6. 16. Kohal RJ, Weng D, Bächle M, Strub JR. Loaded custom-made zirconia and titanium implants show similar osseointegration: an animal experiment. J Periodontol. 2004 Sep;75(9):1262-8.

21. Piconi C, Maccauro G. Zirconia as a ceramic biomaterial. Biomaterials. 1999 Jan;20(1):1-25. 22. Quan R, Yang D, Wu X, Wang H, Miao X, Li W. In vitro and in vivo biocompatibility of graded hydroxyapatite-zirconia composite bioceramic. J Mater Sci Mater Med. 2008 Jan;19(1):183-7. 23. Scarano A, Di Carlo F, Quaranta M, Piattelli A. Bone response to zirconia ceramic implants: an experimental study in rabbits. J Oral Implantol. 2003;29(1):8-12. 24. Sennerby L, Dasmah A, Larsson B, Iverhed M. Bone tissue responses to surface-modified zirconia implants: A histomorphometric and removal torque study in the rabbit. Clin Implant Dent Relat Res. 2005;7 Suppl 1:S13-20. 25. Sykaras N, Iacopino AM, Marker VA, Triplett RG, Woody RD. Implant materials, designs, and surface topographies: their effect on osseointegration. A literature review. Int J Oral Maxillofac Implants. 2000 Sep-Oct;15(5):675-90. 26. Wang RR, Li Y. In vitro evaluation of biocompatibility of experimental titanium alloys for dental restorations. J Prosthet Dent. 1998 Oct;80(4):495-500. 27. Warashina H, Sakano S, Kitamura S, Yamauchi KI, Yamaguchi J, Ishiguro N et al. Biological �� reaction to alumina, zirconia, titanium and polyethylene particles implanted onto murine calvaria. Biomaterials. 2003 Sep;24(21):3655-61.

17. Kohal RJ, Klaus G, Strub JR. Zirconia-implantsupported all-ceramic crowns withstand long-term load: a pilot investigation. Clin Oral Implants Res. 2006 Oct;17(5):565-71.

28. Weingart D, Steinemann S, Schilli W, Strub JR, Hellerich U, Assenmacher J et al. Titanium deposition in regional lymph nodes after insertion of titanium screw implants in maxillofacial region. Int J Oral Maxillofac Surg. 1994 Dec;23(6 Pt 2):450-2.

18. Kohal RJ, Wolkewitz M, Hinze M, Han JS, Bächle M, Butz F. Biomechanical and histological behavior of zirconia implants: an experiment in the rat. Clin Oral Implants Res. 2009 Apr;20(4):333-9.

29. Wenz HJ, Bartsch J, Wolfart S, Kern M. Osseointegration and clinical success of zirconia dental implants: a systematic review. Int J Prosthodont. 2008 Jan-Feb;21(1):27-36.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):116-21

Original Research Article

Endodontic treatment for necrotic immature permanent teeth using MTA and calcium hydroxide. A retrospective study Claudio Maniglia-Ferreira1 Fabio de Almeida Gomes1 Nadine Luísa Soares de Lima Guimarães² Marcelo de Moraes Vitoriano² Tatyana Albuquerque Ximenes² Bruno Carvalho de Sousa³ Roberto Alves dos Santos4 Corresponding author: Cláudio Maniglia-Ferreira Rua Bento Albuquerque, n. 685, apto. 1.102 CEP 60192-060 – Fortaleza – CE – Brasil E-mail: maniglia@unifor.br Department of Endodontics, University of Fortaleza – Fortaleza – CE – Brazil. ² School of Dentistry, University of Fortaleza – Fortaleza – CE – Brazil. ³ Department of Endodontics, Federal University of Ceará – Fortaleza – CE – Brazil. 4 Department of Endodontics, University of Pernambuco – Recife – PE – Brazil. 1

Received for publication: September 28, 2012. Accepted for publication: November 12, 2012.

Keywords: incomplete root formation; apical plug; MTA; calcium hydroxide.

Abstract Introduction: Endodontic treatment of young permanent teeth with incomplete root formation and necrotic pulp has been a challenge for the dentist. It is necessary to induce the formation of apical barrier (apical plug), enabling the complete root canal filling. Objective: This retrospective clinical study compared the protocols for treatment of teeth with incomplete root formation, using calcium hydroxide (CH) or mineral trioxide aggregate (MTA). Material and methods: 28 patients with incomplete root formation have undergone root canal treatments in the period from 2000 to 2009 were selected. The procedures for cleaning, shaping and intracanal medication (CH paste) were performed in a standardized manner. In 13 patients, after using the CH paste (14 days), apical plugs with MTA were made. In the remaining 15 teeth monthly exchanges with CH paste were executed until it was observed radiographically the formation of the apical barrier. In all cases the canals were filled conventionally with gutta-percha and sealer. Initially, follow-ups were made on a

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quarterly and semiannually at the end of first year. Results: All apical lesions showed apical healing between 4 and 13 months after starting treatment. There was not noted the continuing process of root formation. Conclusion: It was concluded that the treatment of teeth with incomplete root formation and necrotic pulp with the use of MTA as apical plug has the same clinical results compared with the use of CH, with the advantage of less clinical time.

Introduction Tooth trauma is the most frequent cause of pulpal necrosis in teeth with incomplete root formation and it requires an endodontic treatment [1, 10]. The strategies of treatment for young adults with immature dentition are important for the long-term prognosis of these teeth and they must aim to the integrity of the periapical tissues, and if possible, the vitality of the pulpal remnant, to assure the maturation and development of the roots of the teeth [22]. However, the rupture of the vascular-nervous bundle may occur definitively, resulting in pulpal necrosis and the paralyzation of the stimulus responsible for the induction of the root formation [13, 15]. Apexification refers to a process of induction of the calcified barrier in teeth with pulpal necrosis in which there was not the completion of the root apex [1]. Currently, there are studies showing another treatment approach which would be based in revascularization. When the apexification was executed and compared with calcium hydroxide (CH) and mineral trioxide aggregate (MTA), it exhibited a significantly longer increasing in the root lenght in addition to the survival rate of the teeth closer to 100% [12]. The use of CH as intracanal pulpal medication to induce the formation of a barrier of hard apical tissue has been reported and successfully applied since the 1960s [4, 9]. This induction occurs due to its high pH, which after causing a superficial contact necrosis with the periapical tissue guides the deposition of hard tissue on the area [20], by forming a barrier of an osteoid or cementoid material firmly adhered to the cementum and dentine [11]. Several clinical studies demonstrated that this treatment approach is efficient for the treatment of immature permanent teeth with pulpal necrosis and apical lesion [3, 4, 9, 15, 19]; however, this requires a longer treatment time [15]. The MTA is a powder composed of t hin hydrophilic particles that ag glutinates in the presence of humidity, forming a colloidal gel and setting [11], with a pH of 12.5, low compressive

strength, low solubility, and greater radiopacity than dentine [7, 18]. It has been successfully employed as both apical plug in teeth presenting incomplete root formation and direct pulpal dressing in pulpotomies because similarly to CH, it induces the apical closing without promoting an inflammatory reaction [16, 22], in addition to enable the immediate procedure of root canal [2, 11]. The aim of this study was to compare the treatment protocols (CH or MTA) of teeth with incomplete root formation and apical lesion visible radiographically. The following factors were taken into consideration: etiology of the pulpal necrosis, age, gender, size of the periapical lesion, symptomatology between appointments, and total time required for the complete closing of the apexes.

Material and methods This study was approved by t he Et hica l Committee in Research of the University of Fortaleza, under protocol number 10-290.

Selection of the patients Thirty-two patients, both genders, were identified and selected in the Dentistry course of the University of Fortaleza with need of endodontic treatment of the anterior teeth with incomplete root formation, pulpal necrosis and apical lesion, in the period between 2000 and 2010. Of 32 patients, it was possible to contact and follow-up 28 patients. The data regarding to the endodontic treatments were extracted from the individual files of the patients. In 13 patients, after the use of the CH paste for 14 days, MTA plugs were executed. In the other 15 teeth, the apexification was carried out every month with CH paste. The diagnosis of pulpal necrosis was determined through the dental history, clinical examination, pulpal sensitivity, in addition to the periapical radiographs from which it was seen the presence of apical lesion, an essential fact for the precise diagnosis severa l t imes associated w it h t he darkening of the crown [13].

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Maniglia-Ferreira et al. Endodontic treatment for necrotic immature permanent teeth using MTA and calcium hydroxide. A retrospective study

Treatment methods As apexification techniques, the pulpal chamber was accessed and a conservative biomechanical preparation was executed by respecting the thin walls of the root canals and 1 mm short of the radiographic apex, with the aid of standardized hand instruments. The irrigation was carried out with biocompatible solutions (saline and chlorhexidine), followed by the intracanal medication with CH paste associated with a viscous vehicle filling all pulp cavity. After the initial appointment, the treatment protocols were different, as follows: • Protocol 1 – Execution of monthly changes of intracanal medication aiming to renew the stimulus for the formation of the hard tissue. After the radiographic proof of the complete repair of the apical lesion and formation of the apical barrier of hard tissue, the filling procedures were executed; • Protocol 2 – It comprises the construction of an apical plug with MTA after intracanal medication with CH for 14 days, and next, the filling procedures were executed and ended normally at the second appointment or at the moment in which the clinical normality was reached (lack of signs and symptoms contraindicating it). Success was considered when the clinical normality and radiographic (partial or total) repair of the apical lesion. Based on the history and previous radiographic images, data were gathered and analyzed regarding to the clinical variables: (i) etiology of the pulpal necrosis, (ii) age, (iii) gender, (iv) initial size of the periapical lesion (longer axis in mm), (v) symptomatology between the appointments and (vi) total time for the complete closing of the apex.

Statistical analysis of the results A ll results obta ined were tabulated a nd statistically analyzed through BioEstat version 5.0 software (Instituto de Desenvolvimento Sustentável Mamirauá, Tefé, AM, Brazil) for Windows, by using ANOVA and t tests, with level of significance of p ≤ 0.05.

Results Twenty-eight patients were analyzed, resulting in 11 central incisors and 17 lateral incisors. The group of patients was composed by 24 male subjects and four female subjects. It is highlighted that the gender did not influence on the clinical variables

studied. Tooth trauma accounts for pulpal necrosis in all the cases studied. All teeth showed periapical lesions visible radiographically; seven patients reported clinical symptomatology (pain and swelling) between the appointments and for that reason, there was an extra urgency appointment. There was no need of retreatment or surgical interventional in none of the cases studied. Table I showed the mean of the following variables: age (years), size of the periapical lesion l (mm) and time (months) of the apical closing of all the cases analyzed, according with the different treatment protocols. Table II compares the values of three variables studied between the cases with or without clinical sy mptomatolog y bet ween t he appoi nt ments. Differences were not noted between the protocols studied regarding to the time required for the apexification process and the sizes of the lesions (p = 0.1582). Regardless of the protocol studied, the presence of the symptomatology between the appointments indicated a longer time for the formation of the apical barrier (p = 0.0003). Table I – Means and standard deviations of the following variables: age (years), size of the periapical lesion (mm) and time (months) for the apical closing of the cases analyzed in this study

Protocols

Age

Lesion

Time

9.3±1.3

9.0±1.6

7.7±2.4

MTA

9.5±1.4

8.9±2.5

7.9±3.1

Table II – Comparisons of the means and standard deviations of the sizes of the lesions (mm) and time (months) for the apical closing between the patients with and without clinical symptomatology between the appointments

Protocols

With symptomatology

Without symptomatology

Lesion

Time

11,3±0,4 10,3±1,9

8,3±1,0

8,1±2,4

MTA

12,7±1,2 12,3±0,5

9,0±1,2

6,6±2,0

Table II demonstrated that although there were not statistically significant differences between the protocols (p = 0.1582), the apical barrier was obtained faster with MTA in the cases of teeth without symptomatology and with CH in the cases with symptomatology. The symptomatology between appointments was noted only in teeth showing apical lesions with diameters greater than 11 mm. The age and

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the gender of the patients did not influence on the results. It is worth noting that the difficulty of scheduling and the failures in the radiographic documentation during the execution of the treatments were problems seen during the analysis of the files. There was only clinical failure in one case because of the relapse of trauma and occurrence of root fracture.

Discussion The evaluation performed in this present study is in agreement with other studies [1, 13, 15], in which the maxillary anterior teeth of male young adults have been most common involved in traumas, with more than 85%. Apexification was defined as the development of an apical barrier of hard tissue which enables the ending of the endodontic treatment without requiring the surgical intervention [1, 14]. In teeth with incomplete or open apexes, the biological sealing occurs through the deposition of a mineralized tissue similar to cementum varying in structure and thickness [10, 11, 20]. The occurrence or not of this process of induced formation of the root apex depends sensibly on the operative technique, the degree of root formation already present and the intensity of the infectious process installed [5, 14, 17, 19]. The treatment protocols of teeth with incomplete root formation is based on t he execution of maneuvers aiming to the induction of the biological sealing [22] through the use of substances of high pH, such as CH [19] and currently, MTA [7, 11]. The impossibility of obturation of the teeth with incomplete root formation guides the necessity of stimulating the continuation of the root development, filling the root canal temporal with medicaments [9, 10, 15, 19-21] or Portland cement [7, 18], to obtain the anatomical conditions that allows to perform the adequate filling, definitively [8]. The results of this present study demonstrated a high rate of success with the treatment of teeth with incomplete root formation, regardless of the protocol used, which has been demonstrated by the literature [10, 19]. However, caution should be taken in affirming that the clinical success of these cases when the adequate protocol is employed is of 100%, because the greatest etiologic agent of pulpal necrosis is tooth trauma, which also causes vertical and horizontal root fractures. This can hinder the clinical success and in several times they are not detectable. Thus, some cases can result in tooth extraction. This fact was verified in one

of the cases of this study treated with CH. In this study, clinical success was considered as the total or partial radiographic repair of the apical lesion had been achieved. Notwithstanding, the tooth must be restored, in function and with no painful symptomatology or presence of fistula. Otherwise, the tooth was considered as failure [16]. The option for the apexification technique with changing of CH paste is a treatment that lasts for months, until the verification of the apical formation of hard tissue closing the root apex [19], consequently the tooth is more vulnerable to coronal infiltration and fractures. The use of MTA as apical plug enabled the earlier obturation and restoration of the tooth [8] and with high success rate [6, 7, 13]. Because of the anatomic features of the teeth with incomplete root formation, the removal of both the necrotic tissues and local infection is more difficult with the use of the instrumentation, which should be executed the most conservative as possible because of the fragility of the root walls, making difficult the removal of the stimulus causing the inflammatory reaction [4]. This fact justifies the use of the intracanal medication for a period from seven to 15 days, aiming to stabilize the apical tissues (inflammatory process) and help the fight for the remnant infection [3, 8]. Some studies found a correlation between the size of the apical lesion with the presence of clinical symptomatology between the appointments [9, 15] and that the occurrence of flare-up between the appointments causes an increase of five months in the time required for the apical closing [15, 23]. The results of the present study demonstrated the occurrence of symptomatology in 25% of the patients studied, corroborating the findings of the literature, in addition to take a longer time for the repair. CH was used as intracanal medication based on the features of the substances used in Endodontics [14]. Several formulations containing CH have been already tested and there is no evidence of advantages of one formulation over another [1, 21]. It is known that the association of CH with antiseptic chemical agents, such as chlorhexidine, increases the antibacterial action of the medication even more [23]. The amount of time for the CH medication changing is variable: after six months [14], when there is the presence of clinical symptoms 1], when the decreasing of the paste is radiographically noted [5] or when there is only the formation of the apical barrier [19]. However, all reach the clinical success and agree with the fact that the CH must

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Maniglia-Ferreira et al. Endodontic treatment for necrotic immature permanent teeth using MTA and calcium hydroxide. A retrospective study

be filling temporal the root canal for a period not shorter than 14 days. In the teeth in which CH was used, the protocol of changing was at every month aiming to renew the stimulus in the periapical tissues [22]. In this group, after a mean period of eight months, the apical barrier of hard tissue started to be radiographically visualized and/or clinically verified with the aid of paper points during the root canal drying. The difference between the size of the lesion and the time required for the apical closing seems to be logical because the cases exhibiting the largest lesions showed the greatest amount of tissue to be reorganized until the deposition of mineralized tissue on the root apex. Caution was taken not to misdiagnosis pathological radiolucent areas with radiolucent areas related to the incomplete root formation Evaluation of the surrounding and contralateral teeth were carried out to avoid this type of error [23]. It was not necessary the complete regeneration of the periapical lesion to result in apexification, regardless of the type of protocol used. In many cases, the apexification occurred prior to the bone regeneration. Some very extensive radiolucent areas can take longer periods to regenerate completely. Differences in the success rates were not verified between the protocols applied, indicating that the advantages in the treatment with the apical plug should be taken into consideration because they enable the earlier definitive restoration of the tooth, preventing the occurrence of fractures and/or the clinical failure. Therefore, MTA is a safer alternative for apical closing in traumatized teeth [22]. Notwithstanding, it is worth noting that the dentist must be capable of executing such procedure so that the apical plug be restricted to the apical third and reach the proper sealing.

Conclusion According to the results, it can be concluded: • The treatment of teeht with incomplete root formation and necrotic pulp with MTA as apical plug exhibits the same clinical results of CH, with the advantage of shorter clinical time; • Tooth trauma is the main etiology for the pulpal necrosis of the ma xillary anterior teeth with incomplete root formation; The symptomatology between appointments has positive correlation with the size of the apical lesion; • All cases presented clinical and radiographic success w it hi n a period shorter t ha n eig ht months.

References 1. Andreasen JO, Andreasen FM, Andersson L. Textbook and color atlas for traumatic injuries to the teeth. 4. �� ed. São Paulo: Blackwell Publishing; 2006. 2. Castro A, Oliveira D, Diniz L, Eulália A, Paulillo L, Pereira G. Avaliação da utilização de MTA como plug apical em dentes com ápice aberto. Rev Bras Odontol. 2011 Jan/Jun;68(1):59-63. 3. Chueh L, Ho Y, Kuo T, Lai W, Chen Y, Chiang C. Regenerative endodontic treatment for necrotic immature permanent teeth. J Endod. 2009 Feb;35(2):160-4. 4. Cooke C, Rowbotham TC. Root canal therapy in nonvital teeth with open apices. British Dent J. 1960;108:147-50. 5. Cvek M. Prognosis of luxated non-vital maxillary incisors treated with calcium hydroxide and filled with gutta-percha. A retrospective clinical study. Endod Dent Traumatol. 1992 Apr;8(2):45-55. 6. Damle SG, Bhattal H, Loomba A. Apexification of anterior teeth: a comparative evaluation of mineral trioxide aggregate and calcium hydroxide paste. J Clin Pediatr. 2012 Sep;36(3):263-8. 7. De Deus G, Coutinho Filho T. The use of white Portland cement as an apical plug in a tooth with a necrotic pulp and wide-open apex: a case report. Int Endod J. 2007 Aug;40(8):653-60. 8. Desai S, Chandler N. The restoration of permanent immature anterior teeth, root filled using MTA: a review. J Dent. 2010;37:652-7. 9. Frank AL. Therapy for the divergent pulpless tooth by continued apical formation. J Amer Dent Assoc. 1966;72:87-93. 10. Goldstein S, Sedaghat-Zandi A, Greenberg M, Friedman S. Apexification & apexogenesis. N Y State Dent J. 1999;65(5):23-5�. 11. Gomes-Filho JE, Watanabe S, Bernabé PFE, Costa MTM. �� A mineral trioxide aggregate sealer stimulated mineralization. J Endod. 2009 Feb;35(2):256-60. 12. Jeeruphan T, Jantarat J, Yanpiset K, Suwannapan L, Khewsawai P, Hargreaves KM. Mahidol study 1: comparison of radiographic and survival outcomes of immature teeth treated with either regenerative endodontic or apexification methods: a retrospective study. J Endod. 2012 Oct;38(10):1330-6.

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13. Kvinnsland SR, Bardsen A, Fristad I. Apexogenesis after initial root canal treatment of an immature maxillary incisor – a case report. Int Endod J. 2010 Jan;43(1):76-83.

in vitro tests of mineral trioxide aggregate and regular and white Portland cements. J Endod. 2005 Aug;31(8):605-7.

14. Leonardo MR. Endodontia: tratamento de canais radiculares – princípios técnicos e biológicos. 1. ed. São Paulo: Artes Médicas; 2005.

19. Sheehy EC, Roberts GJ. Use of calcium hydroxide for apical barrier formation and healing in non-vital immature permanent teeth: a review. British Dent J. 1997 Oct;183:241-6.

15. Maniglia-Ferreira C, Gurgel-Filho ED, Fröner IC, Moraes IG, De Deus G, Coutinho-Filho T. Avaliação clínica e radiográfica da apicificação em dentes traumatizados. �� Stoma. 2004;73:51-6.

20. Tziafas D, Molyvdas I. The tissue reactions after capping of dog teeth with calcium hydroxide experimentally crammed into the pulp space. Oral Surg Oral Med Oral Pathol. 1988 May;65(5):604-8.

16. Mente J, Hage N, Pfefferle T, Koch MJ, Dreyhaupt J, Staehle HJ et al. Mineral trioxide aggregate apical plugs in teeth with open apical foramina: a retrospective analysis of treatment outcome. J Endod. 2009;35(10):1354-8.

21. Tronstad L, Andreasen JO, Hasselgreen G, Kristerson L, Riis L. pH changes in dental tissue after root canal filling with calcium hydroxide. J Endod. 1981;7(1):17-21.

17. Morse DR, O’larnic J, Yesilsoy C. Apexification: review of the literature. Quint Int. 1990;21(7):58998. 18. Ribeiro AD, D�� uarte MAH, Matsumoto MA, Marques MEA, Salvadori DMF. Biocompatibility ��

22. Trope M. Treatment of the immature toth with a non-vital pulp and apical periodontitis. Dent Clin N Am. 2010;54:313-24. 23. Walton RE, Torabinejad M. Principles and practice of endodontics. 2. ed. Philadelphia: WB Saunders Company; 1996.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):122-7

Original Research Article

In vitro analysis of the pH alteration of the dentine after using different calcium hydroxidebased pastes Cláudio Maniglia-Ferreira1 Fabio de Almeida Gomes1 Nadine Luísa Soares de Lima Guimarães2 Marcelo de Moraes Vitoriano2 Tatyana Albuquerque Ximenes2 Roberto Alves dos Santos3 Corresponding author: Cláudio Maniglia-Ferreira Rua Bento Albuquerque, n. 685, apto. 1.102 CEP 60192-060 – Fortaleza – CE – Brasil E-mail: maniglia@unifor.br Department of Endodontics, University of Fortaleza – Fortaleza – CE – Brazil. ² School of Dentistry, University of Fortaleza – Fortaleza – CE – Brazil. ³ Department of Endodontics, University of Pernambuco – Recife – PE – Brazil. 1

Received for publication: November 28, 2012. Accepted for publication: December 17, 2012.

Keywords: pH alteration; intracanal medications; calcium hydroxide; chlorhexidine; Endodontics.

Abstract Introduction and Objective: To analyze the pH increase at the external root surface after the use of different calcium hydroxide pastes (Calen, calcium hydroxide associated with 2% chlorhexidine gel, calcium hydroxide associated with saline) with and without EDTA as chelating agent before the topical application of the intracanal medication. Material and methods: One-hundred single-rooted extracted teeth were cleaned and shaped. They were randomly divided into six experimental groups (n = 15) and one control group (n = 10), according to the medication to be used. The teeth were kept immersed in saline solution and the pH measurements were weekly verified with the aid of a pH meter. Results: It was verified the pH increasing at the first week in almost all groups. Only the groups in which Ca(OH)2 was associated with 2% chlorhexidine gel exhibited a significant evolution in the pH increasing over time (p = 0.0116). The use of EDTA did not result in higher pH values (p = 0.2278). Conclusion: i) the pH increased in all associations used; ii) 2% chlorhexidine gel allowed the gradual pH increasing over time; iii) the smear layer removal did not influence on the pH increasing.

RSBO. 2013 Apr-Jun;10(2):122-7 – 123

Introduction The aim of the endodontic treatment is the complete removal of the pulpal tissues and/or microorganisms within this root canal system, enabling a good quality filling and consequently the regeneration/repairing of the periapical tissues [9, 20]. For this purpose, combinations of endodontic techniques of instrumentation, irrigation/aspiration, use of intracanal medicaments and pulp cavity filling which eliminates the microorganisms within the infected root canals [5, 31]. The success rate of the conventional endodontic treatment reported by the literature ranges from 80% to 85% [26]. Therefore, the failure quite frequently occurs and demands the execution of the endodontic retreatment, because there is an increasing need of the maintenance of the teeth on the arches [5]. The main causes of endodontic failure are coronal infiltration, incomplete cleaning and filling, anatomic alterations and occlusal trauma [9, 28]. Teeth exhibiting endodontic failure have a considerable diversity of microorganisms [20, 27]. Compositions of calcium hydroxide-based pastes have been proposed over time and they have shown very positive antiseptic activity against these bacterial strains [1, 5, 15, 31]. Studies utilizing calcium hydroxide pastes as intracanal medications have demonstrated that at minimum intervals of seven days there is a significant reduction in the endodontic microbiota [25], consume of CO 2 [11], alkalization of the dentinal tissue [2, 4, 29] and hydrolysis of the lipopolysaccharide [22]. Although the antibacterial activity of calcium hydroxide is dependent on the direct contact with the bacteria [23], Ørstavik and Haapasalo [18] demonstrated that calcium hydroxide is not effective in eliminating the bacterias deeply colonizing the dentinal tubules. Thus, calcium hydroxide depends on other characteristics to act against the present infection, such as its capacity of increasing the pH through the dissipation of calcium ions [16, 29]. Because of this failure in the antibacterial activity and its low solubility, many studies have searched to find other efficient substances that could

be associated with calcium hydroxide [24, 25, 32]. Chlorhexidine has been proposed as antiseptic agent in Dentistry for more than two decades and for some years as endodontic irrigating agent and intracanal medication [8, 24]. Chlorhexidine is biocompatible and it has a large antibacterial spectrum and action against lipoteichoic acid [12] associated with calcium hydroxide, demonstrating excellent results in clinical and laboratorial studies [7, 13-15, 30]. Several studies have demonstrated the good efficacy of the association of calcium hydroxide with several vehicles [19, 32]. Its association with chlorhexidine gel has been little studied and reported in literature regarding the releasing of hydroxyl ions. Thus, this study aimed to analyze in vitro the capacity of pH increasing of the external root surface of different associations of calcium hydroxide used as intracanal medications in extracted teeth.

Material and �� methods This was a n ex perimenta l, qua nt itat ive, transversal study of prospective character which aimed to analyze in vitro the pH increasing in the external surface of the root after the use of different calcium hydroxide pastes (Calen® paste, calcium hydroxide associated with 2% chlorhexidine gel and calcium hydroxide associated with saline solution), with and without the use of EDTA prior to the topical application of the intracanal medication.

Obtainment and selection of the specimens This study was executed on 100 single-rooted natural teeht that were extracted and kept in 10% formalin solution until their use. The origin data of the specimens are seen in a donation consent form attached to the research project referred to the Ethical Committee in Research of the University of Fortaleza. The project was approved under protocol number 364/2006.

Preparation of the specimens The cleaning and shaping procedures of the specimens were based on the crown-down technique described Maniglia-Ferreira et al. �� [13]. The �� irrigation

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Maniglia-Ferreira et al. In vitro analysis of the pH alteration of the dentine after using different calcium hydroxide-based pastes

procedures were standardized with the use of 5 ml of 1% sodium hypochlorite (Biodinâmica, São Paulo, Brazil) at every change of instrument. The irrigation in all groups of teeht was executed with the aid of a disposable syringe (5 ml) associated with a BD needle (20X0.55 mm). All canals were dried with absorbent paper points previously to the topical application of the calcium hydroxide pastes.

Preparation and composition of the calcium hydroxide – Ca(OH)2 pastes

ch lorhe x id i ne g el (Endo g el, Endo s upp or t , Itapetininga, SP, Brazil), at the proportion of 1 ml vehicle/1 gram of powder, up to a paste consistency. Association of calcium hydroxide with saline solution The paste was obtained by mixing Ca(OH)2 PA (Biodinâmica, São Paulo, Brazil) with saline solution (0.9% sodium chloride) (Gaspar Viana S.A., Fortaleza, CE, Brazil), at the proportion of 1 ml vehicle/1 gram of powder, up to a paste consistency.

Calen® paste (S.S.White, Rio de Janeiro, Brazil) Product kept in tubes with the following composition: Ca(OH)2, polyethylene glycol 800 and colophony. Association of calcium hydroxide with 2%chlorhexidine gel (CX) The paste was obtained by mixing Ca(OH)2 PA (Biodinâmica, São Paulo, Brazil) with 2%

Study groups After the instrumentation procedures, all teeht were randomly divided into six experimental groups of 15 specimens. The teeth of each group were filled with a different calcium hydroxide-based (Ca(OH)2) paste, according to table I. Ten specimens were used to compose the control group, in which there is no application of the intracanal medication.

Table I – Study groups according to the Ca(OH)2 paste used Groups

Pastes used

Calen (S.S. White, Brazil)

Calen after EDTA use

III

Ca(OH)2 + CX

Ca(OH)2 + CX after EDTA use

Ca(OH)2 + saline

Ca(OH)2 + saline after EDTA use

Control

Canals instrumented and kept empty

For groups II, IV and VI, EDTA was used as auxiliary chemical agent, for 4 minutes, to remove the smear layer, prior to the final irrigation. The filling of the root canals with the different calcium hydroxide pastes was carried out with the aid of irrigation syringes for the specimens of the groups III to VI, while the specimens of groups I and II (Calen pate) a special syringe suitable for the application of this medicament was employed according to the manufacturer’s instructions.

Verification of the pH alteration All teeth received provisional restorations with Super Bonder glue (3M do Brazil, Campinas, SP, Brazil), suspended onto individual holders in Becker flasks containing saline solution for the analysis of the pH alteration. The pH meter (Micronal® – model B474) was connected to the liquid involving the specimens of each group through its terminal portion, so-called electrode. The readings were performed at every week for 4 weeks. All values were recorded in sheets for statistical analysis.

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Results The results were statistically analyzed with the aid of Bioestat 5.0 software and did not follow the normal curve distribution. Data was submitted to Kruskal-Wallis test, with level of significance of 5%. The pH value means for the different groups and time periods studied are seen in table II. Table II â&#x20AC;&#x201C;pH value means of the different groups and time periods analyzed

Groups

pH 1st week

pH 2nd week

pH 3rd week

pH 4th week

Calen

8.03

8.20

8.23

7.45

Calen + EDTA

8.12

8.40

8.18

7.94

CX Ca(OH)2

7.69

8.76

8.90

8.37

CX Ca(OH)2 + EDTA

8.07

8.81

8.98

8.68

Saline Ca(OH)2

8.84

8.32

8.01

7.34

Saline Ca(OH)2 + EDTA

8.98

8.67

8.27

8.01

Negative control

7.39

7.57

7.56

7.71

Except for group III, all materials used promoted significant increasing in pH values at the first week (p = 0.0095) in comparison with the control group which did not exhibited pH alteration at the different time periods analyzed (p = 0.3789). Groups III and IV showed significant evolution in the pH increasing over time (p = 0.0116), differing from the other groups which demonstrated a decrease in the pH values over time. The use of ED TA d id not p rov ide d h i g he r pH v a lue s (p = 0.2278).

Discussion The hypothesis that the diffusion of hydroxyl ions from calcium hydroxide through the dentinal tubules would increase the pH of the dental tissues was evaluated by Tronstad et al. [29], by using an experimental animal model. The authors verified that the insertion of calcium hydroxide within root canal has a direct influence on the pH increase of the external root surface, mainly in areas of root resorption or incomplete root formation, making impossible the osteoclastic activity and stimulating the repairing of the local tissues. In this present study, a previous analysis of the provisional restorative materials was executed so that they did not influence on the results. Because Super Bonder glue did not show any influence on pH, it was the option of choice for the coronal and apical sealing. In a pilot study with different provisional restorative materials, we found many influences on pH, both separately and when they were applied

as restoration of teeth with intracanal medication of calcium hydroxide. It is known, however, that the provisional restoration with ideal properties of neutral pH is almost impossible in Dentistry, but a substance closer to that desired conditions must be searched and if possible, found. The time intervals selected for the measurement of the pH were based on technical reasons related to the routine of the dental office, determined by studies in the literature [8, 24, 25]. The methodology used is valid once the hydroxyl ions on the external root surface are immediately noted by the electrode of the pH meter. Some studies [21, 29] were carried with similar methodology. The results of this present study differ from those of the literature [6, 21], which demonstrated that there was not pH alteration at periods shorter than 14 days with use of viscous vehicles, such as Calen paste and chlorhexidine gel. The dissociation of calcium hydroxide is directly proportional to the vehicles used, because the aqueous vehicles enable to reach a pH close to 12.6, due to a faster dissociation and diffusion velocity of the hydroxyl ions. Because the viscous vehicles exhibit smaller dissociation and diffusion velocity of hydroxyl ions they require longer times to reach high pH thresholds [3]. Concerning to the seven groups of the study, each one with a different intracanal medication, they were tested as aforementioned described. The sealing of the apical foramen was carried out to avoid to the direct contact of the medication with the solution (saline) in which the specimens were immersed, therefore avoiding influences on the results. This occurs because which causes the pH

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Maniglia-Ferreira et al. In vitro analysis of the pH alteration of the dentine after using different calcium hydroxide-based pastes

alteration of the samples would be, according to the literature, the diffusion of the intracanal medication through the dentinal tubules of the dentinal tissue, reaching the periodontal tissues consequently with pH increase of the external root surface. It is necessary to emphasize that for the medication diffusion through dentinal tubules to occur and consequently alter the pH of the external environment, the tubules should be the most preserved as possible and this was obtained with the maximum conservation of the teeth through their washing with saline just after the extraction and storage in 10% formalin solution. The results of the pH measurements revealed that the most beneficial association to increase the pH progressively over time was calcium hydroxide with 2% chlorhexidine gel, regardless of the use of EDTA after the ending of the instrumentation procedures of the root canals. The use of EDTA prior to the application of the intracanal medication has a chelating function of the removal of the smear layer, opening of the dentinal tubules and facilitation of the diffusion of the intracanal medication inside the canals [5, 6, 8, 18]. However, the results of this present study demonstrated there was no influence of EDTA on the process of the hydroxyl ion diffusion through the dentinal tissue. Although the Calen® paste is for many dentists more practical for usage and demonstrated adequate initial results it did not show the maintenance of the pH increasing, as expected with pastes with viscous vehicles. Probably, the fact of the paste is ready for use, i. e., it had been mixed long time ago, the calcium and hydroxyl ions react internally and promote the formation of calcium carbonate, which is stable. There is no ideal intracanal medication, that is, one that is able to influence directly on the clinical signs and symptoms of the patients. Notwithstanding, the researches must be conducted to guide the best choices of the existing materials as well as their associations. The association of biocompatible substances with high antimicrobial capacity directs the ideal intracanal medication because the endodontic treatment aims to eliminate of the aggressive agent within the root canals and to promote its tridimensional sealing. Further studies analyzing the profile of the medicaments over time and which verify their antimicrobial activities and formation of compounds should be executed with the goal of searching the ideal substances and associations for dental and endodontic purposes.

Conclusion According to the results obtained, it can be concluded that: • All calcium hydroxide pastes used demonstrated the capacity of releasing hydroxyl ions at the initial period of use, with pH increasing; • The use of 2% chlorhexidine gel provided the gradual pH increasing over time; • The removal of the smear layer did not influence on the pH of the root surface of teeth with canals filled with calcium hydroxide paste.

References 1. Atila-Pektas B, Yurdakul P, Gülmez D, Görduysus Ö. Antimicrobial effects os root canal medicaments against Enterococcus faecalis and Streptococcus mutans. Int Endod J. 2012;45(12):1-6. 2. Barekatain B, Hasheminia SM, Shadmehr E, Attary Z. The effect of calcium hydroxide placement on ph and calcium concentration in periapical environment: an in vitro study. Indian J Dent Res. 2012;23(2):226-9. 3. Barreto SS, Luisi SB, Fachin EVF. Importância �� da dissociação dos ions cálcio e hidroxila de pastas de hidróxido de cálcio. Rev Clín Pesq Odontol. 2005;1(4):37-46. 4. Duarte MAH, Balan NV, Zeferino MA, Vivan RR, Morais CAH, Tanomaru-Filho M et al. �� Effect of ultrasonic activaction on pH and calcium released by calcium hydroxide pastes in simulated external root resorption. JOE. 2012;38(6):834-7. 5. Ercan E, Dalli M, Dülgergil ÇT, Yaman F. Effect of intracanal medication with calcium hydroxide and 1% chlorhexidine in endodontic retreatment cases with periapical lesions: an in vivo study. �� J Formos Med Assoc. 2007;106(3):217-24. 6. Estrela C, Pécora JD, Souza Neto MD, Estrela CRA, Bammann LL. �� Effect of vehicle on antimicrobial properties of calcium hydroxide pastes. Braz Dent J. 1999;10:63-72. 7. Farhad AR, Barekatain B, Allameh M, Narimani T. Evaluation of the antibacterial effect of calcium hydroxide in combination with three different vehicles: an in vitro study. Dent Res J. 2012;9(2):167-72. 8. Ferraz CCR, Gomes BPFA, Zaia AA, Souza-Filho FJ. In vitro assessment of the antimicrobial action and the mechanical ability of chlorhexidine gel as an endodontic irrigant. J Endod. 2001;27:452-5.

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9. Gomes BPFA, Lilley JD, Drucker DB. Associations of endodontic symptoms and signs with particular combinations of specific bacteria. Int Endod J. 1996;29(2):69-75. 10. Kazemiroor M, Tabrizizadeh M, Dastani M, Hakimian R. The effect of retreatment procedure on the changes at the surface of root dentin using two different calcium hydroxide pastes. J Conserv Dent. 2012;15(4):346-50. 11. Kontakiotis E, Nakou M, Georgopoulou M. In vitro study of the indirect action of calcium hydroxide on the anaerobic flora of the root canal. Int Endod J. 1995;28(6):285-9. 12. Lee J-K, Baik JE, Yun C-H, Lee K, Han SH, Lee W et al. Chlorhexidine gluconate attenuates the ability of lipoteichoic acid from Enterococcus faecalis to stimulate toll-like receptor 2. J Endod. 2009;35(2):212-5. 13. Maniglia-Ferreira C, Bonifácio KC, Fröner IC, Ito IY. Evaluation of the antimicrobial activity of three irrigating solutions in teeth with pulpal necrosis. Braz Dent J. 1999;10:15-27. 14. Maniglia-Ferreira C, Rosa OPS, Torres SA, Ferreira FBA, Bernardineli N. Activity of endodontic antibacterial agents against selected anaerobic bacteria. Braz Dent J. 2002;13(2):118-22.

do pH de três diferentes pastas de hidróxido de cálcio. Rev �� Clín Pesq Odontol. 2008;4(3):169-73. 22. Safavi KE, Nichols FC. Alteration of biological properties of bacterial lipopolysaccharide by calcium hydroxide treatment. J Endod. 1994;20(3):127-9. 23. Siqueira Jr JF, Lopes HP. Mechanisms of antimicrobial activity of calcium hydroxide: a critical review. Int Endod J. 1999;23:361-9. 24. Siqueira Jr JF, Uzeda M. Intracanal medicaments: evaluation of the antimicrobial effects of chlorhexidine, metronidazole, and calcium hydroxide associated with three vehicles. J Endod. 1997;23:167-9. 25. Sirén EK, Haapasalo MPP, Waltimo TMT. In vitro antibacterial effect of calcium hydroxide combined with chlorhexidine or iodine potassium iodine on Enterococcus faecalis. Eur J Oral Sci. 2004;112:326-31. 26. Sjögren U, Figdor D, Persson S. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int Endod J. 1997;30:297-306.

15. Mohammadi Z, Shalavi S. Is clorhexidine an ideal vehicle for calcium hydroxide? A microbiologic review. Iran Endod J. 2012;7(3):115-22.

27. Sundqvist G, Figdor D, Persson S. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998;85:86-93.

16. Montero JC, Mori GG. Assessment of ion diffusion from a calcium hydroxide-propolis paste through dentin. �� Braz Oral Res. 2012;26(4):318-22.

28. Torabinejad M, Ung B, Kettering JD. In vitro bacterial penetration of coronally unsealed endodontic treated teeth. J Endod. 1990;16:566-9.

17. Mori GG, Ferreira FC, Batista FRS, Godoy AMS, Nunes DC. Evaluation �� of the diffusion capacity of calcium hydroxide pastes through the dentinal tubules. Braz Oral Res. 2009;23(2):113-8.

29. Tronstad L, Andreasen JO, Hasselgren G, Kristerson L, Riis I. pH changes in dental tissue after root canal filling with calcium hydroxide. J Endod. �� 1981;7(1):17-21.

18. Ørstavik D, Haapasalo M. Disinfection by endodontic irrigants and dressing of experimentally infected dentinal tubules. Endod Dent Traumatol. 1990;6:142-9.

30. Vianna ME, Gomes BP, Sena NT, Zaia AA, Ferraz CC, Souza Filho FJ. �� In vitro evaluation of the susceptibility of endodontic pathogens to calcium hydroxide combined with different vehicles. Braz Dent J. 2005;16:175-80.

19. Pacios MG, de La Casa ML, Bulacio M de los A, Lopez ME. �� Influence of different vehicles on the pH of calcium hydroxide pastes. �� J Oral Sci. 2004;46(2):107-11. 20. Pinheiro ET, Gomes BPFA, Ferraz CCR, Sousa ELR, Teixeira FB, Souza-Filho FJ. �� Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J. 2003;36(1)1-11. 21. Repeke HP, Westphalen VPD, Silva Neto UX, Carneiro E, Fariniuk LF, Sousa MH et al. Estudo

31. Vianna ME, Horz HP, Gomes BPFA, Conrads G. In vivo evaluation of microbial reduction after chemo-mechanical preparation of human root canals containing necrotic pulp tissue. Int Endod J. 2006;39:484-92. 32. Yücel AC, Aksoy A, Ertas E, Güvenc D. The pH changes of calcium hydroxide mixed with six different vehicles. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;103(5):712-7.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):128-34

Original Research Article

Comparative evaluation of the accuracy of pick up transfer impressions performed with two different types of trays Aline Trem�l1 Gustavo Holtz Galvão1 Allan Fernando Giovanini1 Eduardo Christiano Caregnatto de Morais1 Carla Castiglia Gonzaga1 Enio Marcos da Silva1 Corresponding author: Carla Castiglia Gonzaga Universidade Positivo Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: carlacgonzaga@gmail.com 1

Master of the Science Program in Clinical Dentistry, Positivo University – Curitiba – PR – Brazil.

Received for publication: September 12, 2012. Accepted for publication: December 12, 2012.

Keywords: dental implants; dental impression technique; dental casts.

Abstract Introduction: The success of implant-supported prostheses is related to the passive union between the prostheses’ connections and the implants, so it is necessary to obtain an accurate working cast. Objective: To evaluate the accuracy of the transfer impressions of implants performed with individual acrylic resin trays and conventional trays using polyvinyl siloxane. Material and methods: To carry out the transfer impressions, a master cast with four external hexagon implants parallel to each other was used. Five impressions were made with individual acrylic resin trays and five other impressions with modified conventional trays, opened in the region of the implants. The linear measurements between the implants were made with a digital caliper with an accuracy of 0.01 mm and the results were statistically evaluated (alpha �� = 5%)��. Results: For the points AB and BC, the measurements obtained with the individual trays were statistically similar to the master cast. For the points CD and DA no statistical differences among the three groups were observed. Conclusion: Given the obtained results and the methodology used, it can be concluded that the impressions performed with individual trays presented higher accuracy compared to the ones obtained with conventional trays.

RSBO. 2013 Apr-Jun;10(2):128-34 – 129

Introduction The osseointegrated implants have been used for the rehabilitation of edentulous patients aiming to replace conventional complete dentures [3, 13, 22]. The success of implant-supported prostheses is directly related to the passive union between the prostheses’ connections and the implants themselves. Thus, it is necessary to obtain a cast reliable to the positioning of the implants in oral cavity and on which the most adequate transfer technique of the prosthetic abutments is performed [5, 20]. The transfer impression for osseointegrated implant aims to record, transfer and reproduce with certain accuracy the relationship between the implants and the surrounding structures through the connection of transfer copings to the abutments installed [24]. Next, to obtain the working cast, the implant analogues or replica are connected to the mucosal transfer copings [10]. To achieve the properly adaptation of the prosthesis, an accurate impression must be executed to prevent either mechanical or biological failures. Mechanical failures would comprise the maladjustment of the prosthesis, loosening or fracture of the screw, fracture of the implant and incorrect occlusion [11]. On the other hand, the biological failures may be related to soft tissue irritation, pain, marginal bone loss and osseointegration loss [6]. Concerning to the conventional impression techniques of fixed dentures, the transfer impression of the implants can present a relative easy execution, once devices for gingival tissue retraction as impression caps and retraction threads are not required. For the impression of the abutments of implantsupported prostheses, either for multiple or single cases, two main techniques are employed: impression technique with the aid of conventional trays (closed trays), in which transfer copings without retention are used; and the impression technique with the aid of customized trays (open trays) in which a customized acrylic resin tray is perforated and trimmed at the implant area, with the aid of transfer copings with retention [9, 18]. When employing the closed trays, the dentist should use cone-shaped transfer copings, which are kept in the mouth after the impression is removed and then replaced onto the impression to construct the artificial gingiva and pour the stone [18]. This type enables that the replica could be screwed outside the impression to obtain a better visualization of the adaptation between the two components [21]. Notwithstanding it may cause distortions in the impression material at the moment of its removal because the greater the discordance [2, 16] and the contact among the abutments [16], the greater the inaccuracy of the impression.

For the transfers with opened trays, square transfer copings are used, which need the union with the aid of dental floss and acrylic resin inside the mouth. These transfers correspond to the perforations of the trays and they are directly removed together with the impression, therefore receiving the application of the artificial gingiva to construct the working cast [18]. As advantages of the use of the opened tray, it can be cited the minimum error rate and the fact of working from the beginning to the ending on the same cast; as disadvantages: the high cost of the customized tray and one more appointment for the procedures [20]. The conventional plastic tray is very used in daily clinical practice, therefore avoiding the execution of a previous impression with irreversible hydrocolloid and the construction of a customized acrylic resin tray. However, in literature, there are not studies demonstrating the application of these trays in the technique with square transfers in comparison with the customized trays. The immobilization of transfer copings (square), together with an intraoral pre-fabricated self-cured acrylic resin bar has shown better results when one desires a certain accuracy in the impression [5, 9, 12, 17]. Theoretically, this splinting technique is performed to prevent the displacement of the copings during the impression [12]. Among the several impression materials to be utilized in the transfer of implants, polyether and addition silicone have been the most indicated [2, 8, 15, 24], because they show a greater dimensional stability, greater hardness and elastic recovery [2, 8, 15]. Addition silicone, because of its excellent physical properties, has a privileged post among the impression materials. Its introduction in the market caused a restriction in the use of condensation silicone which because of the elimination of a sub-product – ethanol – results in unreliable impressions with the need of the immediate pouring of the plaster [15, 23]. During the obtainment of the working cast, this material enables accuracy in the reproduction of small details, mainly at the cervical area, in addition to its dimensional stability and easier visualization of the cervical margins [15, 19]. Thus, the aim of this study was to evaluate the accuracy of the transfer impressions of implants performed with individual acrylic resin trays and conventional trays using polyvinyl siloxane.

Material and methods Four implants (4.1 x 13 mm) with hexagon platform (Osteofit, Dental Special Produtos Industriais Ltda., Campo Largo, PR, Brazil) were parallely fixed with the aid of a parallelometer and the transfer copings were

130 –

linked through dental floss and red acrylic resin (figure 1a). The implants were then placed onto a pre-fabricated rubber mold with the shape of a dental arch and fixed with addition silicone (Stern Tek, Sterngold, Germany). After the setting of this material, to construct the master cast, a colorless acrylic resin (Jet Clássico, Artigos Odontológicos Clássico Ltda., São Paulo, SP, Brazil) was used to simulate the installation of the implants to construct the protocol prosthesis (figure 1b).

(a)

(b)

Figure 1 – (a) Transfer copings linked with the aid of a parallelometer; (b) Master cast constructed in acrylic resin with the implants placed

The impressions were performed with plastic conventional trays (Tray Aways, Harry J. Bosworth Company, Skokie, IL, USA) and customized acrylic resin trays (figure 2). The conventional trays were relieved at the implant areas with the use of a tungsten carbide bur. To construct the customized trays a copy of the master cast was obtained. The master cast was relieved with two dental wax laminas number 7 (Artigos Odontológicos Clássico Ltda., São Paulo, SP, Brazil) and retentions were made onto its lateral sides to assure a space for the impression material. This model was then impressed with an irreversible hydrocolloid (Cavex, Color Change, Cavex, Haarlem, Holand), and the cast was poured with type IV stone (Durone IV, Dentsply, Rio de Janeiro, RJ, Brazil). This model was used to construct customized colorless self-cured acrylic resin trays.

(a)

(b)

Figure 2 – (a) Plastic conventional tray; (b) Customized tray constructed in colorless acrylic resin

RSBO. 2013 Apr-Jun;10(2):128-34 – 131

Impression copings for open tray (square copings) for external hexagon, of 4.1 mm diameter, were used and linked with dental floss and self-cured acrylic resin (Pattern, Harry J. Bosworth Company, Skokie, IL, USA) (figure 3).

(a)

(b)

Figure 3 – (a) Transfer copings linked with dental floss; (b) Stabilized with red self-cured acrylic resin

The heavy and light addition silicone (Elite HD+, Zhermack SpA, Rovigo, Italy) was distributed and handled according to the manufacturer’s recommendations, by executing a single-step impression. The tray was placed by applying a digital pressure. After the impression material setting, the impression was separated from the master cast. The analogues were placed onto the transfer copings to obtain working casts by pouring type IV stone (Durone IV, Dentsply, Rio de Janeiro, RJ, Brazil), mixed and handling according to the manufacturer’s recommendations, under constant vibration. After two hours, the impressions were separated from the casts. Then, the linear measurements of all distances among the implants were established (figure 4) through a digital caliper (Starrett, Itu, SP, Brazil) with accuracy of 0.01 mm. For each condition, five casts were obtained and for each one three measurements were executed.

(a)

(b)

Figure 4 – (a) Distances among the implants; (b) Determination of the measurements with a digital caliper

132 –

The values obtained in the casts were compared with the measurement of the master cast and the results were statistically evaluated through the analysis of variance and Tukey’s test, with level of significance of 5%.

Results The results for the distances measured among the four implants are seen in table I. For the points AB and BC, the measurements obtained with the customized trays were statistically similar to the master cast. For the points CD and DA there was not statistically significant difference among the three groups. Table I – Means, standard-deviations and coefficient of variation for the measurements performed (mm) among the four points for the groups studied. Values followed by the same letters are statistically similar (p > 0.05)

Measurements among the points (mm) AB

CoT

17.55 ± 0.06 (0.32%) b

18.45 ± 0.01 (0.05%) d

18.95 ± 0.02 (0.11%) e

42.14 ± 0.02 (0.04%) f

CuT

17.65 ± 0.01 (0.08%) a

18.46 ± 0.01 (0.04%) cd

18.96 ± 0.01 (0.07%) e

42.15 ± 0.01 (0.02%) f

17.66 ± 0.02 (0.09%) a

18.47 ± 0.01 (0.07%) c

18.96 ± 0.01 (0.07%) e

42.16 ± 0.02 (0.05%) f

CoT – conventional tray, CuT – customized tray, MM – master- model

Discussion The hypothesis to be test in this study was that there would not be difference in the accuracy of the casts obtained from impressions through conventional or customized trays. However, this hypothesis was rejected, once, for two measurements analyzed, the customized tray was more accurate than the conventional tray. The most used impression techniques are the close tray/cone-shaped transfer copings and open tray/square transfer copings, which may or may not be linked [11]. Notwithstanding, the literature has reported that when acrylic resin is used to link transfer copings, the cast is more accurate [1]. The impressions utilized in this study were through open acrylic resin customized trays and conventional trays with square transfer coping linked through dental floss and self-cured acrylic resin. The master cast was constructed in the shape of a dental arch to the best approximation with the clinical reality and poured with acrylic resin to avoid any deformation in the casts obtained from them. A digital caliper was used to measure the distances among the implants, because this device allowed a precise reading between the extension of the points of choice and also makes the reading easier for the examiner. To perform the measurements, the points of choice must be fixed and stable in

all casts and the force applied onto them must be minimum. Aiming to obtain a precise mean of these distances, three measurements for each cast was executed. In the analysis of the results, it could be observed a statistically significant difference at the points AB and BC for the groups of conventional trays (CoT). However, the literature reports studies with similar methodologies in which the comparison of the accuracy of three different impression techniques that there was not difference among groups [11, 21]. It should be highlighted that it was not found studies that had conducted the same study design, using the same impression technique and only varying the tray. In this present study, it could be said that the dimensional alteration in points AB and BC could have occurred by some motives, such as: the use of a modified conventional tray, relatively small number of samples (n = 5), lack of control during the force application at the moment of the mensuration or even because the measurements were not executed exactly at the same point of choice. Concerning to the impression technique, the literature does not have a consensus. Some authors indicate that the utilization of square transfer copings in the direct technique tend to exhibit a greater dimensional accuracy regarding to the coneshaped transfer copings [3, 7, 12, 17]. However,

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Humphires et al. [14] reported that the impression techniques with square transfer copings linked to acrylic resin showed greater accuracy regarding to the impression techniques with cone-shaped transfer copings. On the other hand, Carr [7] and Pinto et al. [21] did not observe differences regarding the accuracy of the pick-up impression technique in relation to the transfer impression technique. The use of cone-shaped transfer coping provides that the analogue could be screwed outside the impression, therefore allowing a better visualization of the adaptation between the two components [21]. According to Michalakis et al. [16], the impression material distortion could occur in this impression type during the removal, which results in its permanent deformation because the greater the divergence among the analogues and the proximity of the abutments, the greater the impression inaccuracy. Because these reasons, in this present study, the use of impressions with open trays was opted because the possibility of a correct positioning of the implant is greater due to the fact that the transfer coping be removed with the impression, by avoiding the positioning stage and its insertion within the impression, which may result in displacement [20]. The impressions executed with customized and modified conventional trays showed a statistically significant difference, since when the customized tray is used the study cast was considered more accurate when compared to the master cast. According to Burns et al. [4], this difference was also observed because in their study with three different types of open trays (conventional polycarbonate tray, customized rigid tray with relief, and customized rigid tray without relief) the customized rigid tray with relief exhibited the most accurate casts and it was not seen statistically significant differences between the other two types of customized trays. The literature reports that conventional trays did not provide an accurate adjustment since the material thickness is not homogenous; also some trays are not flexible. Notwithstanding, the customized trays are rigid and more stable and allowed a homogenous thickness of the material and consequently more accurate casts [8]. In addition to the tray type to be used in the impression of the implants, the material is also of fundamental importance to enable a passive seating for the prosthesis. In a study conducted to evaluate the morpho-dimensional behavior of the materials, six addition silicones, one polyether, one polysulphate, one condensation silicone, and one irreversible hydrocolloid were related and employed in an impression technique of the transfer of the dental implants. In the analysis

of the data obtained, the study demonstrated that all materials tested showed statistically significant dimensional alterations. The addition silicones exhibited a smaller dimensional alteration while the irreversible hydrocolloid showed the greatest alteration. All addition silicones produced similar casts, followed by polyether, polysulphate, condensation silicone and irreversible hydrocolloid [25]. Finally, it should be remembered that during the conduction of the clinical procedures, such as the transfer impression for implant-supported prostheses, the dentist should not only chose the technique, the impression and cast material, but also have the knowledge on the “pros” and “cons” of the materials and techniques attempting to minimize the undesirable errors and enable a more satisfactory final outcome.

Conclusion Within the limits of this present study, it can be concluded that the pick up impression technique for the transfer of implants with customized trays was more accurate than that executed with conventional trays at determined measurement points.

Acknowledgements The aut hors t ha n k t he compa n ies DSP Biomedical, Labordental and Dental News (Bosworth Company) for donating the materials for the execution of the study.

References 1. Assif D, Marshak B, Nissan J. A modified impression technique for implant-supported restoration. J Prosthet Dent. 1994 Jun;71(6):58991. 2. Assunção WG, Gennari Filho H, Zaniqueli O. Evaluation of transfer impressions for osseointegrated implants at various angulations. Implant Dent. 2004 Dec;13(4):358-66. 3. Assunção WG, Cardoso A, Gomes EA, Tabata LF, Santos PH. �� Accuracy of impression techniques for implants. Part 1 – Influence of transfer copings surface abrasion. Journal of Prosthodontics. 2008 Dec;17(8):641-7. 4. Burns J, Palmer R, Howe L, Wilson R. Accuracy of open tray implant impression: an in vitro comparison of stock versus custom

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trays. The Journal of Prosthetic Dentistry. 2003 March;3(89):250-5.

material in combination with transfer caps for the Frialit-2 system. J Oral Rehabil. 2000;27:629-38.

5. Cabral LM, Guedes CG. Comparative analysis of 4 impression techniques for implants. �� Implant Dentistry. 2007 Jun;16(2):187-94.

16. Michalakis KX, Kalpidis CD, Kang K, Hirayama H. A simple impression technique for dental implants placed in close proximity or adverse angulations. J Prosthet Dent St Louis. 2005 Oct;94(3):293-5.

6. Campi Junior L, Nagem Filho H, Fares NH, Missaka R, Fiuza CT, D’Azevedo MTFS. Passividade da prótese sobre implante. Innov Implant J. 2010 Sep/Dec;5(3):53-9. 7. Carr AB. A comparison of impression techniques for a five-implant mandibular model. Int J Oral Maxillofac Implants. 1991;6:448-55. 8. Cristensen GJ. Implant prosthodontics contribute to restorative dentistry. J �� Am Dent Assoc. 1990 Nov;121(5):582. 9. Del’Acqua MA, Chávez AM, Compagnoni MA, Mollo Jr FA. �� Accuracy of impression techniques for an implant-supported prothesis. The Internacional Journal of Oral and Maxillofacial Implants. 2010 Oct;4(25):715-21. 1 0 . D i n a t o J C , P o l i d o W D . I�� mplantes osseointegrados: cirurgia e prótese. São Paulo: Artes Médicas; 2001. 11. Faria JCB, Concília LRS, Neves ACC, Miranda ME, Teixeira ML. Evaluation �� of the accuracy of different transfer impression techniques for multiple implants. �� Braz Oral Res. 2011 Mar/ Apr;25(2):163-7. 12. Gennari Filho H, Mazaro JVQ, Vedovatto E, Assunção WG, Santos PH. Accuracy �� of impression techniques for implants. Part 2 – Comparison of splinting technique. Journal of Prosthodontics. 2009 Feb;18(2):172-6. 13. Goiato MC, Gennari Filho H, Fajardo RS, Assunção WG, Dekon SFC. Comparação �� entre três materiais de moldagem e três técnicas de moldagem de transferência para implantes. �� Revista Cir Implantodont. 2002 Apr/Jun;9(34):164-8. 14. Humphries RM, Yaman P, Bloem TJ. The accuracy of implant master casts constructed from transfer impressions. Int J Oral Maxillofac Implants. 1990;5:331-6. 15. Lorenzoni M, Pertl C, Penkner K, Polansky R, Sedaj B, Wegscheider WA. Comparison of the transfer precision of three different impression

17. Naconecy MM, Teixeira ER, Shinkai RS, Frasca LC, Cervieri A. Evaluation of the accuracy of 3 transfer techniques for implant-supported prostheses with multiple abutments. �� Int J Oral Maxillofac Implants. 2004 Mar/Apr;19(2):192-8. 18. Neves FD, Fernandes Neto AJ, Oliveira MRS, Lima JHF, Galbiatti MAD. Seleção de intermediários para implantes Branemark compatíveis parte II: casos de implantes individuais. Rev Bras Implantodont e Prótese Implant. 2000 Apr/ Jun;7(26):76-87. 19. Nishioka RS, Almeida EES, Andreatta Filho OD, Balducci I. Avaliação da alteração dimencional entre um silicone de polimerização por adição e outros por condensação. Rev Odontol. 2000 Jan/ Dec;29(1/2)93-104. 20. Pieralini ARF, Lazarin AA, Segalla JCM, Silva RHBT, Pinelli LAP. Técnica de moldagem para implantes. Salusvita. 2007 Jan;27(2):169-78. 21. Pinto JHN, Valle AL, Scolaro JM, Bonfante G, Pegoraro LF. Estudo comparativo entre técnicas de moldagem para implantes odontológicos. Rev Fac Odontol. 2001 Jul/Dec;9(3/4)167-72. 22. Prithviraj DR, Pujari ML, Garg P, Shruthi DP. Accuracy of the implant impression obtained from different impression materials and techniques: review. �� Journal section: Clinical and Experimental Dentistry. 2011 Dec;3(2)106-11. 23. Rode SM, Dutra CR, Matson E. Controle clínico da alteração dimensional dos elastômeros. Rev Assoc Paul Cir Dent. 1987 Sep/Oct;41(5):266-9. 24. Silva MM, Mima EGO, Del’Acqua MA, Segalla JCM, Silva RHBT, Pinelli LAP. Técnicas de moldagem em prótese sobre implantes. Revista de Odontologia da UNESP. 2008 May;37(4):301-8. 25. Valle AL, Coelho AB, Scolaro JM. Avaliação do comportamento morfodimensional de materiais de moldagem utilizados em implantes dentais. Rev Fac Odontol Bauru. 2001 Jan/Jun;9(1/2):41-8.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):135-42

Original Research Article

Aesthetic improvements in free gingival graft due to its association with frenectomy Tertuliano Ricardo Lopes1 Cassiana Nathalie Machado1 Mariana Camargo Rogacheski1 Thalyta Verbicaro1 Allan Fernando Giovanini1 Tatiana Miranda Deliberador1 Corresponding author: Tatiana Miranda Deliberador Mestrado Profissional em Odontologia Clínica, Universidade Positivo Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: tdeliberador@gmail.com 1

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

Received for publication: November 11, 2012. Accepted for publication: December 1, 2012.

Keywords: Periodontics; labial frenulum; gingiva; histology; clinical stage.

Abstract Introduction: The insufficient amount of attached gingiva and the abnormal insertion of the labial frenulum may be related. When this occurs, it is common to associate frenectomy techniques with free gingival graft (FGG). Objective: To evaluate the clinical and histological staining difference and blood flow when the FGG is or is not associated with frenectomy. Material and methods: Ten patients were selected in the Dentistry Clinics of the Positivo University and divided into two groups (n = 5): group GF (graft/frenectomy), with association of FGG and frenectomy in the V sextant, and group G (graft), in which only the FGG was performed in the V sextant. Clinical examination, initial standardized photographs and post-surgery following-up for seven, 14 and 21 days were executed. For histological analysis the excesses of the graft tissues of a patient from each group were removed. Results: Clinically, it was observed in both groups after seven and 14 postoperative days, superficial necrosis of the tissue and color similar to the surrounding soft tissue. At 21 days, there is the total reduction of the necrosed tissue, and it was not seen the color difference between groups. Histologically, the difference between groups is related to the epithelial tissue, which was thicker in group G. Conclusion: Clinical (color) and histological (vascularization) significant differences were not observed between groups GF and G.

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Introduction In the past, the “ideal” amount of attached gingiva was considered by many experts, an important factor for periodontal health [11, 17, 18, 24, 27, 30]. According to Lindhe et al. [22], after conducting many researches, it was proven that the periodontal health is not related to the amount of attached gingiva, but the plaque is the main etiological factor of periodontal disease and the insertion loss is not dependent on the extent of attached gingiva. The keratinized gingiva has the function of support between the alveolar mucosa and free gingival margin as well as being resistant to traumatic procedures such as toothbrushing [34] and orthodontic forces [13]. There may be a relationship between the highest insertion of the labial frenulum and insufficient attached gingiva [7, 25]. If such a relationship occurs, it may result in gingival recession, because it makes the process of cleaning difficult, leading to the retention of biofilm and culminating in a chronic inflammatory process [36]. In cases of abnormal insertion of the labial frenulum that cause deleterious effects such as gingival recession, the frenectomy is indicated [29]. This technique involves surgical excision of the labial frenulum. The frenectomy technique may be associated with the surgical procedure of free gingival graft (FGG), with the purpose of alleviating the relapse of labial frenulum, in addition to increase the width of attached gingiva [3, 9, 16] and to deepen the vestibule [6].

However there are disadvantages to associate the two techniques. One is the necessity of a surgical field to obtain the graft (donor site) [3]. Moreover, the use of FGG generates an unfavorable aesthetics. This is because the donor site of the graft, in most cases, is the palate (keratinized gingiva) and at the time of transplantation, the receptor site receives the genetic features of the palate [22], leaving the grafted area with a whitish shade. According to Carranza et al. [12], at the moment of the removal and transplantation of the graft, its color changes to a grayish white ischemic color, because the blood vessels of the graft are empty, i.e., without adequate vascularization. The aim of this study was to evaluate clinically and histologically, the difference in color and blood supply of FGG with or without frenectomy.

Material and methods This study was approved by t he Et hica l Committee in Research of the Positivo University (Curitiba, Brazil), under protocol number 26/12. For this present study, ten patients were selected from the Dentistry Clinics of the Positivo University, divided into two groups: group GF (five patients requiring FGG and frenectomy in the V sextant) and group G (five patients requiring only FGG in the V sextant). The pat ients were submitted to cli nica l examination and initial standardized photographs (figure 1a and figure 1b).

Figure 1a – Initial photograph of the GF group: patient A, patient B, patient C, patient D and patient E

Figure 1b – Initial photograph of the G group: patient A, patient B, patient C, patient D and patient E

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In the group GF, the association of the two techniques were performed comprising frenectomy procedures, preparation of the receptor site, obtainment of the donor site (palate) and FGG technique (figure 2). In group G only the receptor site preparation, the obtainment of the donor site (palate) e and FGG technique were executed. During the surgical procedure, the patient was submitted to bilateral mental nerve anesthesia and at the donor site an infiltrative anesthesia was performed. The labial frenulum was removed through the frenectomy technique by using the single clamping. With a curve hemostatic clamp and a n. 15c scalpel blade, an incision was executed along the upper surface of the hemostatic clamp surpassing the tip of the clamp a (figure 2-B). Following, a similar incision along the lower surface of the hemostatic clamp consequently result in the labial frenulum removal. Next, a transversal incision in the periosteum (periosteum fenestration), at the height of the alveolar mucosa was executed to avoid a future reinsertion of the frenulum. Then, the preparation of the receptor site was performed and with the aid of a n. 15c scalpel blade, the deepening of the vestibule was carried out and it was limited to the area to be grafted (figure 2-C). A sterile metallic surgical template (figure 2-D) was used to delimit the area to be graft

(map). Previously to the removal of the palate graft, a compressive suture at the posterior area of the palate was executed to compress the greater palatine artery to avoid possible bleeding. Next, the map was placed onto the area between the premolars and an incision perpendicular to the palate was executed, approximately of 1 mm thick, delimiting the graft size. Posteriorly, an incision parallel to the palate was performed to remove the FGG. The graft was adapted to the receptor site through two simple sutures: one at the mesial and the other at the distal side, followed by a compressive “V” suture (figure 2-E). The area was compressed with moist gauze for 5 minutes to eliminate possible clots between the receptor site and the graft. The sutures were removed after seven days. In g roup �� G, �� t he surg ica l sequence was t he sa me of t he g roup GF, but w it hout t he frenectomy. To cont rol t he post- operat ive pa i n a nd infection, the following drugs were prescribed: anti-inflammatory (Nimesulide – 100 mg, every 12 hours, for 5 days), antibiotics (Amoxicillin – 875 mg, every 12 hours, for 7 days or Clindamycin – 300mg, every 8 hours, for 7 days) and oral antiseptics solution (0.12% chlorhexidine digluconate , every 12 hours, for 7 days) [28].

Figure 2 – Surgical sequence. A: initial photograph; B: frenulum removal; C: delimitation of the area to be grafted; D: metallic surgical template; E: suture

Some post-operative recommendations were instructed to the patients: ice bandages on the receptor site at the first 2 post-surgical hours; not to expose to sun; not execute physical efforts; not vigorously rising; cold liquid and pasty food at the first 48 post-surgical hours. The patients submitted to the procedures were clinically followed up with periodical appointments at seven, 14 a nd 21 days. At t hese periods, standardized photographs were performed aiming to compare the clinical healing between groups. At 21 post-operative days, in two patients (one of the group GF and the other of the group G) it was observed that the FGG was with a thickness

wider than that desired. Thus, a second surgical procedure was carried out 84 days after the first surgery, in which a gingivoplasty was executed aiming to reduce the graft thickness. A s a g ra ft piece was removed i n t he gingivectomy procedure, this tissue was used to evaluate the histological differences in the healing (mainly the vascularization) between both groups. To conduct the histological analysis, the tissue removed was fixed in 10% formaldehyde, washed in water and included in paraffin. Serial cuts of 3 µm of thickness was performed which were stained in hematoxylin and eosin for analysis in light microscopy.

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Results Clinical findings The results were obtained through clinical and photographic analysis, by observing the color acquired by the graft after the surgery. After seven post-operative days, in most patients of both groups, it was seen a superficial necrosis of the tissues. The graft color was similar to that of the surrounding soft tissue (figure 3a and figure 3b).

Figure 3a – Seven day following up appointment of group GF: patient A, patient B, patient C, patient D and patient E

Figure 3b – Seven day following up appointment of group G: patient A, patient B, patient C, patient D and patient E

At 14 post-operative day, in some patients of both groups, it was observed yet, small areas of tissue necrosis (figure 4a and 4b). The colors of most of the grafts were similar among each other and with the surrounding soft tissue. However, in some patients of both groups, it was seen a more whitish shade than the surrounding tissue (figure 4a – patient A and E and figure 4b – patient A and D).

Figure 4a –14 day following up appointment of group GF: patient A, patient B, patient C, patient D and patient E

Figure 4b – 14 day following up appointment of group G: patient A, patient B, patient C, patient D and patient E

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At 21 post-operative days, it was verified the total regression of the necrosis in both groups and it was not observed clinical differences of the color between groups (figure 5a and 5b). In group GF, two patients exhibited the graft color very closer to that of the surrounding tissue (figure 5a – patients B and C). In group G, three patients showed the graft color very closer to that of the surrounding tissue (figure 5b – patients B, C and E).

Figure 5a –21 day following up appointment of group GF: patient A, patient B, patient C, patient D and patient E

Figure 5b –21 day following up appointment of group G: patient A, patient B, patient C, patient D and patient E

Histological findings The aim of the histological analysis was to compare the difference in vascularization (amount of the blood vessels) between the groups GF and G. In the histological cuts of the group G, it was seen a dense and fibrous connective tissue, covered by an epithelium, verifying intense pseudoepitheliomatous hyperplasia and high keratinization. Area of a discrete chronic inflammatory infiltrate in the connective tissue of the focal area was observed (figure 6b). In the histological cuts of the group GF, it was noted a dense and fibrous connective tissue, in addition to a thinner epithelial tissue (figure 6a).

Figure 6a – Histological cut of a patient of group GF

Figure 6b – Histological cut of a patient of group G

Source: Allan F. Giovanini (2012)

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It was not observed a vascular difference between the groups. The main distinction between the groups was the amount of the epithelial tissue, which was greater in group G.

Discussion T he l a bi a l f renu lu m, when a bnor m a l ly inserted may cause damage to the periodontium [4, 5, 10]. Such damaging effects comprising from area hygiene difficulty because of the bad positioning of the toothbrush to gingival recession caused by the traction of the mucosa [2, 8, 14, 15, 20, 21, 23, 32]. One of t he t reat ments recom mended by these cases is the frenectomy (total removal of the labial frenulum) [1]. However, the healing of this surgical procedure results in an area of little keratinized gingiva. Thus, it is common to associate the frenectomy procedure with the FGG, which maintain the frenulum insertion far from the gingival margin, in addition to lead to an increasing of the attached gingiva [19]. FGG surg ica l procedure consists i n t he removal of the gingival tissue from the palate (donator site) and its transference for the area with lack of keratinized tissue (receptor site) [19]. The advantages of this type of procedure are related to the increasing of the area and tissue thickness, graft predictability, technique simplicity and post-operative no painful symptomatology of the receptor site. There are some disadvantages, such as unfavorable aesthetics, second intention healing of the palate, in addition to more painful post-operative period and risks for complications [33]. Because FGG has the disadvantage of lack of aesthetics at the post-operative period, this present study hypothesized if the possibility of its association with the frenectomy procedure would show a healing improvement, due to a g reater vascula ri zat ion of t he a rea (coming from the frenectomy) and consequently better aesthetics in relation to the color of the graft. However, this hypothesis was not confirmed after the observation of the clinical and histological results. For a longer longevity of the graft, it is necessa ry t hat it has proper dimensions, that is, it must be sufficient thin to enable its

nutrition (through propagation of the f luids) through the receptor site. A very thinner graft has great chances of necrosis and exposure of the receptor area [26, 31]. However, if the graft is very thicker, its superficial layer may be at risk, once the excess of tissue will hinder an adequate nutrition [35]. In this present study, a gingivoplasty was necessary in two patients because the gingival grafts were thicker than the desired thickness However, the histological result did not demonstrate an inadequate nutrition, regardless of the thickness of the grafts. The healing of the FGG comprises three phases: 1) initial phase, from 0 to three days. At this stage, the epithelium of the grafted tissue undergoes a superficial necrosis and receives its avascular plasmatic nutrition from the receptor site [22], because just after its removal from the donator site, the blood vessels of the graft are empty [12]; 2) revascularization phase, from two to 11 days. There is the union (anastomosis) of the pre-existing vessels and the appearance of new vessels, in addition to the beginning of the new re-epithelization; 3) maturation phase, from 11 to 42 days. The â&#x20AC;&#x153;maturationâ&#x20AC;? of both the vessels and epithelium occurs with keratin deposition [22]. In this present study, the gingival tissue of both groups was gathered for histological analysis 84 days after the initial surgical procedure. Therefore, it cou ld be obser ved t he t issue healing and the maturation phase. The groups exhibited a similar maturation and amount of blood vessels. Additionally, in the histological aspect, it was verified that the connective tissue repair in both groups was similar. Either fibrosis or disturbances of the vascular new formation was not detected. In group G, it was noted a greater focal epithelial hyperplasia (frenulum area), which was not a primordial factor for the color changing in the representative part of the surgical site. In the tissue remnant, the amount of epithelium is normal. One hypothesis for this focal hyperplasia can be related to the frenulum which was not removed in this group, whose epithelium is superimposed to that of the grafted tissue. Notwithstanding, clinically, this variation is not visible.

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Conclusion Within the limits of this study, it can be concluded that in both groups, there was not clinical (color) and histological (vascularization) significant differences between the association or not of the frenectomy and FGG techniques.

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33. Segundo TK, Alves R. Emprego do enxerto gengival epitélio-conjuntivo no recobrimento radicular. Rev �� Gaúcha Odontol. 2005 JanMar;54(1):81-3.

29. Nogueira GRF, Benatti BB, Bittencourt S, Peruzzo DC, Casati MZ. Frenectomia �� associada ao enxerto gengival livre. �� Rev Gaúcha de Odontol. 2005 Apr-Jun;53(2):85-164. 30. Ochsenbein C. Newer concept of mucogingival surgery. J Periodontol. 1960;31:175-85. 31. Pennel BM, Tabor JC, King KO, Towner JD, Fritz BD, Higgason JD. Free �� masticatory mucosa graft. J Periodontol. 1969 Mar;40(3):162-6. 32. Raveli DB, Chiavini PCR, Pinto AS, Sakima MT, Martins LP, Melo ACM. Diastema interincisal.

34. Stoner JE, Mazdyasna S. Gingival recession in the lower incisor region of 15 year old subjects. J Periodontol. 1980 Feb;51(2):74-6. 35. Ward VJ. A clinical assessment of the use of the free gingival graft for correcting localized recession associated with frenal pull. �� J Periodontol. 1974 Feb;45(2):78-83. 36. Yared KFG, Zenobio EG, Pacheco W. A etiologia multifatorial da recessão periodontal. R Dental Press Ortodon Ortop Facial. 2006 NovDec;11(6):45-51.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):143-8

Original Research Article

Evaluation of active chlorine releasing of sodium hypochlorite during seven days, stored at different temperatures Gladyvam Rabêlo Braitt1 Evaldo de Almeida Rodrigues1 Carlos Eduardo da Silveira Bueno2 Antonio Henrique Braitt1 Corresponding author: Gladyvam Rabêlo Braitt Av. Aziz Maron, n. 1.117, sala 703 – Jardim União CEP 45605-415 – Itabuna – BA – Brasil E-mail: gladybraitt@hotmail.com Program of Specialization in Endodontics of the Institute of Health Sciences, Funorte/Soebras, Núcleo Ilhéus – Ilhéus – BA – Brazil. 2 São Leopoldo Mandic Center of Post-Graduation – Campinas – SP – Brazil. 1

Received for publication: November 5, 2012. Accepted for publication: December 14, 2012.

Keywords: Endodontics; sodium hypochlorite; irrigating substances.

Abstract Introduction: The sanitation of the canal system through irrigation/ aspiration, at the changing of the endodontic instruments aims to the excised material, removal of microorganisms and the cleaning of the walls of the canals. One of the substances used in endodontic treatment of root canals, sodium hypochlorite, used at different concentrations of active chlorine and pH, has gained popularity due to its physical chemical properties. Objective: To analyze the active chlorine content of sodium hypochlorite solution at 6.0%. Material and methods: 80 samples were obtained from two litres of sodium hypochlorite at 6%, obtained at every hour: one liter was stored at room temperature and one liter at refrigerated environment, between the morning and afternoon shifts, except on Saturday and Sunday. The free residual chlorine was determined, using as variables: the temperature, length of time in storage and the handling of the substance in the Endodontic clinic for seven days. The data obtained were submitted to analysis of variance according to the linear regression model to test the effect of time of storage condition and interaction between the main effects on the chlorine content of the

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Braitt et al. Evaluation of active chlorine releasing of sodium hypochlorite during seven days, stored at different temperatures

stocked solution (p < 0.01). Results: The results showed that the sodium hypochlorite solution is quite unstable, with considerable loss of active chlorine (58.33%), depending on the storage conditions and storage time and temperature. The temperature interferes in the free residual chlorine concentration contained in sodium hypochlorite solution, that is, the higher the temperature, the lower the lifetime of free residual chlorine in sodium hypochlorite solutions. Conclusion: Storage in refrigerated environment proved to be the best option to avoid the marked loss of active chlorine in sodium hypochlorite of concentration at 6%.

Introduction The irrigating substance should make ease the removal of the organic and inorganic remnants (including microorganisms), promoting the root canal and dentinal tubules sanitation simultaneously to the absence of damages to periapical tissue [4]. Hypochlorite is bleaching agents commonly used to bleach textiles or papers and to disinfect the solutions. The solution normally contain 10 to 15% of available chlorine (equivalent to 100 to 150 g/L), but rapidly loses its force during the storage. Because the solution is very affected by the heat, light, pH and heavy metals, the solution needs to be regularly controlled. Although variations exist regarding to the concentration recommended, the active principle of hypochlorite is the same. It is a very unstable substance and this instability have been already highlighted by Dakin [6] and confirmed by several studies conducted [8, 11, 12]. Introduced in Dentistry by Barret [1], in 1917, sodium hypochlorite (NaOCl) is a very efficient irrigating solution of the root canals because of its properties such as deodorant, bactericidal and solvent of the tissues [2, 10]. Because sodium hypochlorite is the irrigating substance with the best physical-chemical property among the armamentarium of substances used in the cleaning and shaping of root canals, it is the irrigating agent most utilized in Endodontics. Gomes et al. �� [8], by analyzing the concentration of active chlorine in sodium hypochlorite solutions at dental offices, concluded that most of the solutions were the Dakin’s liquid which however showed the greatest variation of loss of active chlorine. The storage most found was in white plastic flasks stored at the counter or cabinet. The Milton’s solution and chlorinated soda was the most stable. The pH of the samples was inside the ideal values for stability; however, most of the professionals did not use the real concentrate they believe to be using. Fabro et al. [6] observed that the studies conducted in Brazil have demonstrated

that the dentists have preferred sodium hypochlorite solutions in concentrations at around 0.5 to 1.0%; however, because of the constant chemical reaction, there is the loss of active chlorine and at the moment of its use they are very weaker and do not exhibit the desired action. Bra itt et al. �� [3], referri ng to t he rot a r y instrumentation of the root canal treatment, they emphasized the necessity of a copious irrigation concurrent with the use of the instruments otherwise organic and inorganic material (smear layer) and putrefaction material and microorganisms will stay adhered on root canal wall, which prevents the sanitation of the root canal system and impedes a complete obliteration of the canal during the endodontic obturation. Most of the studies regarding the temperature and storage time of the sodium hypochlorite employed in the endodontic clinics was performed with the liquid stored in different temperatures; notwithstanding, the samples for evaluation of the sodium hypochlorite stability were carried out at relatively longer intervals once it was not considered that in the endodontic clinic this substance is handled, in mean, eight times per day with interval of two hours between the morning and afternoon shifts and pause of generally two days per week (Saturday and Sunday). Aiming to contribute for the study of the temperature influence on the releasing of chlorine which can lead to the decrease of the free chlorine and consequently to the loss of the bactericidal power, this present study was conducted to establish which would be the storage option more suitable for the maintenance of the active chlorine from 6% sodium hypochlorite used in the endodontic clinics, by analyzing the content of this solution.

Material and methods The concentration of active chlorine of sodium hypochlorite solutions at 6.0% was determined

RSBO. 2013 Apr-Jun;10(2):143-8 – 145

by titration, iodometry method, through the determination of the free residual chlorine. The following variables were utilized: storage time and the substance handling in the endodontic clinics for seven days, by observing the number of the daily collection, pause between the morning and afternoon shifts as well as the lack of handling during the Saturday and Sunday. The reagents and solutions employed in the methodolog y execution for samples A and B comprised a set for 100 tests for lixivium (HI 3843, Hanna Instruments, São Paulo, Brazil), with gamma specification from 50 to 150 g/L as chlorine (C12), smaller increment of 5 g/L (0.5%) as chlorine (C12), with size of the sample of 1 ml (figure 1). It is also used a universal indicator set of pH 1-14 with 200 bands (J Prolab Ind. e Com. de Produtos para Laboratório Ltda., São José dos Pinhais, Brazil).

The presence of the sodium hypochlorite makes the solution to be dark orange. Slowly, drops of titration reagent HI 3843C-0 were added, and after each drop the solution was agitated. Each drop was counted until the solution changing from dark orange to colorless solution, as seen in figure 2.

Figure 1 – Materials used in the samples A and B

Stages of the procedure Sample A – environment temperature In about 70-75 ml of distilled water within an Erlenmeyer flask, six drops of the reagent HI 3843A-0 were added, carefully agitated for mixing. Following, to obtain a pH lower than 3, a package of the reagent HI 3843B-0 was dissolved within this mixture and the pH was measured in the Erlenmeyer flask with the aid of litmus paper. In a plastic flask, 25 ml of sodium hypochlorite from sample A (environment temperature) was added, with the measurement of the temperature at the moment of the collection. From this sample, with the aid of a syringe, 1 ml of sodium hypochlorite was collected and added to the Erlenmeyer flask.

Figure 2 – Changing of the solution color from orange to colorless

Sample B – refrigerated environment The same procedure steps executed in sample A were adopted, and 25 ml of hypochlorite from sample B (refrigerated environment) were added within the plastic flask, with temperature measurement at the refrigerated environment at the moment of the collection. To obtain the chlorine concentration at % of the sample, the number of drops of the reagent C of the titration was multiplied by 0.5 to make a

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Braitt et al. Evaluation of active chlorine releasing of sodium hypochlorite during seven days, stored at different temperatures

colorless solution, according to the manufacturer’s instructions, in which # of drops x 0.5= % of chlorine. The calculations were performed with the use of the procedures of SAS system– INSTITUTE Inc (The SAS system – release 9.2.SAS Insitut82ute Inc. Cary:NC. 2011.2008). The �� data were statistically analyzed through analysis of variance with level of significance of 5%.

Results This present study was conducted to evaluate the alterations observed over time of the solutions stored in two conditions: environment and refrigerator. In table I are seen the main data regarding to the evaluations of the sodium hypochlorite in relation to the factors of temperature variation and storage time, whose graphic representation is shown in graph 1.

Table I – Main results of the free residual chlorine concentration in 6% sodium hypochlorite in relation to temperature, storage time and number of handlings

SAMPLE A Vol. ml Collection Hour

SAMPLE B

Temp. (oC)

# drops x 0.5 =

% Cl

Temp. (oC)

# drops x 0.5 =

% Cl

1000

8:00

27.7

12 x 0.5 =

6.0

12 x 0.5 =

6.0

900

14:00

30.8

11 x 0.5 =

5.5

12 x 0.5 =

6.0

800

8:00

28.9

9 x 0.5 =

4.5

12 x 0.5 =

6.0

325

11:00

28.8

8 x 0.5

4.0

12 x 0.5%

6.0

250

16:00

27.6

8 X 0.5

4.0

11 X 0.5%

5.5

125

11:00

28.2

7 X 0.5

3.5

11 x 0.5

5.5

17:00

27.2

7 X 0.5

3.5

11 x 0.5

5.5

The analysis of the variance of the effects planed as determinants of the variations of the chlorine content of the solutions studied is seen in table II. Table II – Analysis of the variance according with the linear regression model to test the linear effect of time and storage condition as dummy variable and of interaction among the main effects on the chorine content (Cl%) of the stocked solution

Sum of the squares

Mean Squares

F Statistics

p-Value

51.517217

17.172406

192.41

0.0001

Environment

36.450000

408.42

0.0001

Time

10.923793

122.40

0.0001

4.143424

46.43

0.0001

Residue

6.782783

0.089247

Corrected total

58.300000

Effect Model

Environment*Time

Coefficient of determination (R2): 88.37%

Coefficient of variation (CV): 5.74%

The tests from each effect separately strongly indicates (p < 0.01) that all are significant, i.e., both the time and the storage condition and the interaction among the main effects are not casual. The regression model is shown as graph.

RSBO. 2013 Apr-Jun;10(2):143-8 – 147

Graph 1 – Equations obtained through the process of linear regression with the dummy variable

For the execution of the analysis of the data and obtainment of the subsides to the decisions inherent to the research, it was adopted to the technique of the analysis of the linear regression with the dummy variable (storage condition), with a continue variable (time) and the interaction between them. Such technique enables the estimation of the parameters of a model that allows the prediction of the variation of the amount of chlorine and the testing of the significance of the effects listed in the model.

Discussion Since its introduction in Endodontics, sodium hypochlorite always has a detached position during the preparation phase of the root canal, once in addition to combine with several other substances in the search for the greatest bactericidal effect, it has qualities such as the tissue compatibility, tissues solvency, increase of the dentinal permeability and root canal cleaning. Most of the studies regarding to the temperature and storage time of the sodium hypochlorite employed in the endodontic clinics were conducted with the liquid stored at different temperatures; however, the collections for the evaluation of the sodium hypochlorite stability were performed in relatively longer intervals, once it was not considered that in endodontic clinics this substance is handled, in mean, eight times per day with interval of 2 hours between the morning and afternoon shifts, with pause of generally two days per week (Saturday and Sunday). Also Clarkson et al. �� [4] pointed out the necessity that sodium hypochlorite be stored in opaque and closed flasks because the constant opening

of the f lasks causes a greater loss of chlorine. Considering the necessity of the constant opening of the hypochlorite solution stored in the endodontic clinics and that the hypochlorite solution stored in and refrigerated environment (between 9 and 13oC) only started to lose active chlorine at the collection number 31 (750 ml), it is recommended that, for safe, the product be stored in flasks of 500 ml. Accord i n g to t he Br it ish Pha r macopeia Commission, the titration process, also called iodometry, it is the most efficient and reliable method to determine the concentration of the active chlorine. Through titration, iodometry method, it is possible to determine the amount of active chlorine in a sodium hypochlorite solution. The iodine moves the active chlorine present in the solution at the proportion of 1 mole to 1 mole. When sodium thiosulfate is added to the solution, an oxi-reduction reaction of iodine occurs so that it is possible to determine the amount of this substance present. Thus, which is being titrated is the iodine, but as it is present at the same proportion than chlorine, its concentration is easily determined. The chlorine available is related to the chlorine released by the action of the acid diluted in the hypochlorite. According to the manufacturer’s instruction, the hypochlorite solution is treated with potassium iodide and highly acidified. The amount of iodine generated is equivalent to the chloride from the sample. The iodine concentration is then calculated by the titration of the thiosulfate ions which reduces the iodine back to the iodide ions. Although the aim of this present study had been to analyze the content of the sodium hypochlorite, through the determination of the free residual chloride, using variables as temperature, storage time and the substance handling in the endodontic clinics, it is worth highlighting that, by analyzing the calculus to obtain the concentration with the aid of the kit, it was verified that the minimum value that can be measured is 0.5%, since there is not possible to use less than a drop. This fact demonstrates that with the calculation, in none of the hypotheses would be possible to achieve values lower than 0.5. Such fact displays the possibility of an experimental error of the method at the rate of ±0.25%; for example, in a result of 0.5%, the measurement can be 0.25% or 0.75%, values which can be very large in the variation of the concentration used in Endodontics.

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Braitt et al. Evaluation of active chlorine releasing of sodium hypochlorite during seven days, stored at different temperatures

Conclusion Based on the analysis of the results, it can be concluded that: • Sodium hypochlorite is a very unstable solution, with considerable loss of active chlorine, depending on the storage conditions and time; • The storage temperature interferes in the content of free residua l chlorine w it hin t he sodium hypochlorite. The higher the temperature, the shorter the life span of the free residual chlorine within the sodium hypochlorite; the storage in refrigerated environment was the best option to avoid the marked loss of active chlorine from 6% sodium hypochlorite.

References 1. Barret MT. The Dakin-Carrel antiseptic solution. Dental Cosmos. 1917;59(4):446-8. 2. Baumgartner JC, Cuenin PR. Efficacy of several concentrations of sodium hypochlorite for root canal irrigation. J Endod. 1992;18(12):605-12. 3. Braitt AH, Cunha RS, Martin AS, Bueno CES. Evaluation of cleaning efficacy of a nickel-titanium rotary system, with or without 17% EDTA passive ultrassonic activation: a scanning electron microscopic study. RSBO. 2012;9(1):38-43.

4. Clarkson RM, Moule AJ, Podlich HM. The shelflife of sodium hypochlorite irrigating solutions. Aust Dent J. 2001;46(4):269-76. 5. Dakin HD. On the use certain antisseptic substances in the treatment of infected wounds. Brit Med J. 1915;2(28):318-20. 6. Fabro RMN, Britto MLB, Nabeshima CK. Comparação de diferentes concentrações de hipoclorito de sódio e soro fisiológico utilizados como soluções irrigadoras. �� Odontol Clín Cient. 2010;9(4):365-8. 7. Gambarini G, De Luca M, Gerosa R. Chemical stability of heated sodium hypochlorite endodontic irrigants. J Endod. 1998;24(6):432-4. 8. Gomes MCP, Britto MLB, Nabeshima CK. Análise da concentração de cloro ativo em soluções de hipoclorito de sódio encontradas em consultórios odontológicos. �� Rev APCD. 2010;64(2):150-4. 9. Grossman LI, Maiman BW. Dissolution of pulp tissue by chemical agents. J Amer Dent Ass. 1941;28:223-5. 10. Johnson BR, Remeikis NA. Effective shelf-life of prepared sodium hypochlorite solution. J Endod. 1993;19(1):40-3. 11. Pécora JD, Guerisoli DMZ, Silva RG. Shelf-life of 5% sodium hypochlorite solutions. Braz Endod J. 1997;(2):43-5. 12. Piskin B, Tükün M. Stability of various sodium hypochlorite solutions. J Endod. 1995;21(5):253-5.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):149-52

Original Research Article

Comparative evaluation of radiopacity of MTA Fillapex® endodontic sealer through a digital radiograph system Silvya Aparecida Vanso Bicheri1 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

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

Received for publication: November 28, 2012. Accepted for publication: December 17, 2012.

Keywords: root canal obturation; digital dental radiograph; Endodontics.

Abstract Introduction: The radiograph represents one of the evaluation tools of the endodontic procedure. Therefore, radiopacity is an essential property of filling materials. Objective: To evaluate the MTA Fillapex® radiopacity, as compared to that of Sealer26®, Sealapex® and AH Plus®. Material and methods: Five specimens were prepared from each endodontic sealer, which were then positioned in the digital sensor (Kodak RVG 6100 – Digital Radiography System) and radiographed with a Kodak 2200 Intraoral Device Rx System, operating at 70 Kvp, 10 mV, with an exposition time of 0.071 tenth of second and a distance of 20 cm from the sensor. The radiopacity was assessed using the software Image Tool 3.0, which determines gray shades ranging from 0 to 255 pixels. For statistical analysis, ANOVA test was used, followed by Tukey’s test at a significance level of 5%. Results: The MTA Fillapex® presented the lowest radiopacity between all tested sealers, while AH Plus® was the most radiopaque one. Sealapex® and Sealer 26® showed intermediate radiopacity and statistically did not differ from each other. Conclusion: MTA Fillapex® presented intermediate radiopacity. When compared with AH Plus®, Sealer 26® and Sealapex® endodontic sealers, it exhibited the smallest result.

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Bicheri and Victorino Comparative evaluation of radiopacity of MTA Fillapex® endodontic sealer through a digital radiograph system

Introduction Root ca na l obturat ion is t he endodont ic treatment phase where the goal is the sealing of the root canal system to prevent a possible recontamination of the root canal [9]. The evaluation of the filling procedure is performed through radiographic image. Thus, the radiograph is an indispensable tool for the evaluation of the endodontic treatment executed. The radiopacity is a physical property required by the endodontic sealers which enables to assess the contrast of the tooth structure on a radiograph [15] as well as the filling of the root canal system. Gutta-percha points in association with endodontic sealers are the most accepted approach for the root canal filling [14]. The radiopacity of the endodontic sealer highly influences on the radiographic image of the filling [8]. To analyze the radiopacity, the specification n. 57 of the American Dental Association (ADA) [1] suggests an evaluation of the optical density in radiographic films through photodensitometer. Notwithstanding, the sealer radiopacity should be compared with that of a device with aluminum steps, so called penetrometer. The results are equivalent to millimeters of aluminum. The digital radiograph has been much utilized for the analysis of the radiopacity of the sealers [14, 16], because it is a technique of fast visualization dispensing the chemical processing, which avoids errors of this phase, reduces the time of exposure to radiation and still enables a better visualization of the radiographic contrast. Recently launched into the Brazilian market, MTA Fillapex® (Angelus, Londrina, PR, Brazil) is a Mineral Trioxide Aggregate (MTA)-based endodontic sealer, with little studies on it. For this reason, the aim of this present study was to assess the level of radiopacity of MTA Fillapex® sealer and compare it to that of AH Plus® (Dentsply, DeTrey, Konstanz, Germany), Sealer 26® (Dentsply, Petrópolis, RJ, Brazil) and Sealapex® (Sybron Kerr, Washington, USA) sealers through digital radiograph.

rings of 10 mm of internal diameter and 1.5 mm of thickness. The rings were placed onto a thin lamina of cellophane over a glass plate.�� The sealers were proportionated and mixed according to the manufacturer’s instructions and the rings were filled up to their upper border, with the aid of a dental vibrator. Additionally, a mass of 100 g was placed over the set. The samples were stored in an incubator at 37ºC. Elapsed three times the setting time of each material, the samples were removed from the incubator and the aluminum rings. Then, they were positioned on a sensor (Kodak RVG 6100 – Digital Radiography System – Kodak, Rochester, New York, USA) and radiographed with an x-ray device (Kodak 2200 Intraoral X – Ray System), operating at 70 Kvp, 10 mV, with exposure time of 0.071 tenth of second, at a distance of 20 cm from the sensor. On the radiographic images, the measurements of the optical density or gray shades of the sealers were executed on ImageTool 3.0 software (UTHSCSA, San Antonio, Texas) (figure 1). To measure the gray shades, which varied from 0 to 255 pixels, the function “histogram” was employed by using a standardized area of 20 x 19 pixels always equally positioned for all images. For the statistical analysis of the results, ANOVA and Tukey test was applied with level of significance 5%.

Figure 1 – Use of ImageTool 3.0 software in the determination of the pixel values through the histogram tool

Material and methods

Results

The sealers were divided into four groups: group I – MTA Fillapex® (Ângelus, Londrina, PR, Brazil), group II – AH Plus® (Dentsply, DeTrey, Konstanz, Germa ny), group III – Sea ler 26 ® (Dentsply, Petrópolis, RJ, Brazil) and group IV – Sealapex® (Sybron Kerr, Washington, USA). Five samples were constructed for each sealer with aluminum

Table I shows the mean radiopacity values in pixels and percentage for the endodontic sealers, using as reference the minimum and maximum pixel values of the histogram. The results demonst rated t hat A H Plus ® was the most radiopaque sealer (169.7 pixels), equivalent to 66.5% of the total of 255 pixels,

RSBO. 2013 Apr-Jun;10(2):149-52 – 151

while Sealapex® and Sealer 26® sealers exhibited intermediate radiopacity and did not statistically differ between each other, 60% (153.7 pixels) and 58.1% (148.3 pixels), respectively. MTA Fillapex® sealer significantly differed from the other sealers, presenting a radiopacity of 49.8% (127 pixels). Table I – Mean of the radiopacity, in pixels and in percentage of MTA Fillapex®, Sealer 26®, Sealapex®, AH Plus® sealers

Sealer

Radiopacity Pixels*

Black

MTA Fillapex®

127.0b

49.8%

Sealer 26®

148.3a

58.1%

Sealapex

153.7

60.2%

AH Plus

169.7

66.5%

White

255.0

100%

* Different letters represent statistically significant differences, p < 0.05

Discussion A great technological advancement of Radiology is the use of the digitized image, a technique of fast visualization which has made easy the work of endodontists because it enables the storage of the images in the computer and to perform adjustments in the brightness and contrast, therefore improving the evaluation of the procedures executed and careful interpretation, such as anatomic details, accessory root canals, precise measurements, periapical and bone alterations [4, 5, 10, 12]. Additionally, it does not employ the chemical processing it avoids the errors inherent to this phase; also, it reduces the time of exposure to radiation decreasing the risks of effects deleterious to patients [7]. Because the digital radiograph is a more sensible method than the conventional radiograph film, requires less radiation [11] and has been increasingly used, this present study opted to evaluate MTA Fillapex® radiopacity through this method. In this present study, the digital radiographic images of the endodontic sealers were evaluated through the histogram analysis of the gray shades, ranging from 0-255 pixels, in which 0 is black color and 255 is the white color. Therefore, the greater the pixel value, the more radiopaque is the

sealer. To obtain radiopacity values of the sealers in percentage, 255 pixels were considered as 100%. Considering the results, AH Plus® was the most radiopaque sealer (66.5%), which corroborates the results of Sydney et al. [16], �� Aznar et al. [2] and Vidotto et al. [18]. These �� authors demonstrated that the resin sealers are characterized by the highest radiopacity than that of the calcium hydroxideand MTA-based sealers. Sealapex ® and Sealer 26® sealers did not show statistically significant differences between each other, but a significantly smaller difference than AH Plus® and greater than MTA Fillapex®. Sealer 26® is a sealer containing calcium hydroxide and having good biological and physical-chemical characteristics. It shows a higher biocompatibility than that of the other sealers; however, it exhibits low radiopacity according the studies of Tanomaru-Filho et al. �� [17] and Rosa et al. [14]. The Sealapex® is a calcium hydroxide-based sealer and it is characterized by exhibiting good biocompatibility, control of the inflammatory root resorption and due its high pH shows excellent bacterial action [3]. However, studies demonstrated that Sealapex® radiopacity is also smaller than that of the other endodontic sealers[2, 8]. MTA Fillapex® exhibits the smallest radiopacity value (127 pixels), which represents 49.8% of the total of the histogram. A very similar result was found by Vidotto et al. [18], who obtained a value of 125.6 pixels. In the study of these authors, MTA Fillapex® exhibited the best performance because the sealers such as Acroseal®, Endomethasone® and Roekoseal® showed radiopacity smaller than 120 pixels. In this present study, the smallest radiopacity of o MTA Fillapex® than that of the other sealers is related to its composition, once it only contains bismuth oxide as radiopacifier. Materials with this component have obtained low radiopacity values, as demonstrated by Rasimick et al. �� [13]. On �� the other hand, AH Plus® sealer has zirconium oxide as radiopacifying agent and Sealer 26® and Sealapex® sealers has bismuth trioxide and titanium oxide.

Conclusion According to the results found, MTA Fillapex® has an intermediate radiopacity (49.8%) and, when it is compared with AH Plus®, Sealer 26® and Sealapex® sealers, it obtained the smallest result.

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Bicheri and Victorino Comparative evaluation of radiopacity of MTA Fillapex® endodontic sealer through a digital radiograph system

References 1. American National Standard / American Dental Association – ANSI/ADA. Specification n. 57: Endodontic sealing materials. Modified adoption of ISO 6876:2000. Dental root canal sealing materials. Chicago; 2000. 2. Aznar FDC, Bueno CES, Nishiyama CK, Martin AS. Radiopacidade de sete cimentos endodônticos avaliada através de radiografia digital. RGO. 2010;58(2):181-4. 3. Bezzera da Silva LA, Leonardo MR, Da Silva RS, Assed S, Guimarães LFL. Calcium �� hydroxide root canal sealers: evaluation of pH, calcium ion concentration and conductivity. Int Endod J. 1997;30:205-9. 4. Botelho TL, Mendonça EF, Cardoso LLM. Contribuição da radiografia digital na clínica odontológica. Robrac. 2003;12(33):55-9. 5. Coclete GA, Tavano O, Pavan AJ. Comparação das densidades ótica e radiográfica, analisadas pelo fotodensiômetro MRA e pelo sistema digital Digora. Rev Odontol UNESP. 2003;32(1):93-8. 6. Costa RF, Scelza MFZ, Costa AJO. Radiopacidade de cimentos endodônticos: avaliação pela intensidade de pixel. J Bras Clín Odontol Integr. 2002;6(32):137-9. 7. Cruz GA, Morais LC, Médici Filho E, Castilho JCM. Utilização de radiografia digital em Odontologia. Rev ABO Nac. 2004:12(5):283-6. 8. Ferreira FBA, Silva e Souza PAR, Vale MS, Tavano O. Radiopacidade de cimentos endodônticos avaliados pelo sistema de radiografia digital. Rev Fac Odontol Bauru. 1999;7(1/2):55-60. 9. Kopper PMP, Santos RB, Viegas APK, Reis Só MV, Grecca FS, Figueiredo JAP. Estudo do selamento dos canais radiculares obturados com

AH Plus® ou Endofill®, com e sem cimento nos cones acessórios. Rev Fac Odontol Univ Passo Fundo. 2007;12(1):52-5. 10. Kreich EM, Leal GA, Slusarz PAA, Santini RM. Imagem digital na Odontologia. Ciênc Biol Saúde. 2005;11(3/4):53-61. 11. Parks ET, Williamson GF. Digital Radiology: an overview. The Journal of Contemporary Dental Practice. 2002;3(4):1-13. 12. Pasquali AAG, Matson MR, Raitz R. Estudo comparativo da densidade radiográfica de cimentos resinosos. Rev Odontol Univ São Paulo. 2009;21(3):239-43. 13. Rasimick BJ, Shah RP, Musikant BL, Deutsch AS. Radiopacity of endodontic materials on film and a digital sensor. J �� Endod. 2007;33(9):1098-101. 14. Rosa RA, Bier CAS, Pereira CC, Só MVR, Wolle CFB. �� Simulation of soft and hard tissues and its effects on radiopacity of root canal sealers. Rev Odonto Ciênc. 2011;26(4):326-30. 15. Salzedas LMP, Louzada MJQ, Oliveira Filho AB. Radiopacity of restorative materials using digital imagens. J Appl Oral Sci. 2006;14(2):14752. 16. Sydney GB, Ferreira M, Leonardi DP, Deonizio MDA, Batista A. Análise da radiopacidade de cimentos endodônticos por meio de radiografia digital. Rev Odonto Ciênc. 2008;23(4):338-41. �� 17. Tanomaru-Filho M, Jorge EG, Tanomaru JMG, Gonçalves M. Radiopacity evaluation of new root canal filling materials by digitalization of images. J Endod. 2007;33(3):249-51. 18. Vidotto APM, Cunha RS, Zeferino EG, Rocha DGP, Martin AS, Bueno CES. Comparison of MTA Fillapex radiopacity with five root canal sealers. RSBO. 2011;8(4):404-9.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):153-60

Original Research Article

In vivo evaluation of tissue response to new endodontic sealers Annecy Geib da Silva1 Nádia Carolina Teixeira Marques2 Natalino Lourenço Neto2 Tamara Zeponi Fernandes de Melo2 Vivian Agostino Biella Passos1 Cleide Felício Carvalho Carrara1 Thais Marchini Oliveira2 Corresponding author: Thais Marchini Oliveira Faculdade de Odontologia de Bauru – Universidade de São Paulo Departamento de Odontopediatria, Ortodontia e Saúde Coletiva Alameda Dr. Octávio Pinheiro Brisolla, 9-75 CEP 17012-901 – Bauru – SP – Brasil E-mail: marchini@usp.br Sector of Pediatric Dentistry, Hospital of Rehabilitation of Craniofacial Anomalies of Bauru, University of São Paulo – Bauru – SP – Brazil. 2 Department of Pediatric Dentistry, Orthodontics and Collective Health, Discipline of Pediatric Dentistry, School of Dentistry of Bauru, University of São Paulo – Bauru – SP – Brazil. 1

Received for publication: October 22, 2012. Accepted for publication: December 18, 2012.

Keywords: biocompatibility; tissue response; root filling materials; Portland cement; MTA.

Abstract Introduction: The sealers can be in direct contact with the periapical tissues. Thus, these materials must have appropriate physical and biological properties, providing conditions for repair to occur. Objective: The aim of this study was to evaluate the response of rat subcutaneous tissue to endodontics sealers. Material and methods: Three materials comprised the groups: group I – Zinc Oxide, Eugenol and Iodoform paste, group II – Portland cement with propylene glycol, and group III – MTA Fillapex® (Angelus). These materials were placed in polyethylene tubes and implanted into dorsal connective tissue of Wistar rats for seven and 15 days. The specimens were stained with hematoxylin and eosin and evaluated regarding to inflammatory reaction parameters through a light microscope. The data were compared using Kruskal-Wallis test with significance level of 5%. The intensity of inflammatory response against the sealers

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was analyzed by two blinded and previously calibrated observers for all experimental periods. Results: The histological evaluation showed that all the materials caused a moderated inflammatory reaction at seven days which decreased with time. A greater inflammatory reaction was observed at seven days in group I. The other specimens had significantly less inflammatory cells when compared to this group. Tubes with MTA Fillapex® presented some giant cells, macrophages and lymphocytes after seven days. At 15 days, the presence of fibroblasts and collagen fibers was observed indicating normal tissue healing. The group II showed similar results to those observed in MTA Fillapex® already at seven days. At 15 days the inflammatory reaction presented was almost absent at the tissue, with many collagen fibers indicating normal tissue healing. Statistical analysis showed a significant statistical difference amongst the group I (seven days) and II (15 days) (p < 0.05). In the other groups no significant statistical differences were observed. Conclusion: MTA Fillapex® and Portland cement with propylene glycol were more biocompatible than the other tested cements.

Introduction The endodontic cements can be in direct contact with the periapical tissues. Thus, these materials should present physical and biological properties, providing conditions for the occurrence of the repair [9, 10, 18, 19]. For this purpose, it is very important to know the biological compatibility of the sealers because if they were very irritating to the periapical tissues, all endodontic treatment will be damaged and, consequently, the capacity of repairing of the periapical region will undergo significant interferences [10]. Considering the importance of knowing the compatibility of these materials, several studies using implants on conjunctive tissues of rats have been carried out aiming to evaluate the biological characteristics of the endodontic sealers [6-8, 10, 12, 18, 23, 26, 28-30]. For deciduous teet h, i n add it ion to t he compatibility of the sealers employed, one should considers the use of resorbable sealers, i.e., which can be phagocytized by the cells of the tissues of the periapical area; also, the deciduous teeth undergo the physiologic root resorption and at this phase they can be in direct contact with the permanent tooth bud, therefore it is fundamental that they do not cause any alteration in this process [1, 24]. The mineral trioxide aggregate (MTA) sealer has been the subject of a series of experimental studies analyzing both their physical-chemical and biological properties with good clinical and laboratorial results [2, 4, 11, 14, 16, 20, 22, 25, 29]. Until recently, the MTA does not exhibited an

adequate formulation to be used as endodontic sealer; however, a new MTA formulation so-called MTA Fillapex® (Angelus®) has been indicated to be utilized as endodontic sealer in root canal filling. Because it has a chemical composition similar to that of the MTA cement, a similar biological response is also expected. Another material receiving attention in the scientific literature is the Portland cement, because it exhibits the composition and performance similar to MTA. It also enables a neoformation of the mineralized tissue and the maintenance of the vitality of the pulpal remnant [2, 3, 15, 17]. Aiming to assess other therapeutic options for endodontic filling of the root canals of deciduous teeth, the aim of this study was to evaluate the tissue response of endodontic sealers on the subcutaneous tissue of rats.

Material and methods This present study was approved by the Ethical Committee in Animal Research of the School of Dentistry of Bauru (University of São Paulo), under process number. 025/2010.

Selection of the animals and surgery Twelve Wistar male rats, weighing between 200 and 250 g (approximately 60 days), coming from the Central Vivarium of the School of Dentistry of Bauru (University of São Paulo). On the dorsum of each animal, three tubes containing each material

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was introduced and divided as follows: GI – zinc oxide (80%) + eugenol + iodoform (20%); GII – Portland cement + propylene glycol; GIII – MTA Fillapex (Angelus®). It was opted to use the lateral side of the polyethylene tube as negative control of the inflammation. The animals were anesthetized with ketamine hyd ro ch lor ide a nd x yl a z i ne hyd ro ch lor ide (Dopalen® – 0.4 ml/kg and Anasedan® – 0.02 ml/kg, intramuscular route). Following, the hand trichotomy on the dorsal region of the animal and the antisepsis with gauze embedded in 5% iodinated alcohol were executed. Next, two longitudinal incisions on the dorsum of the animal, one anterior e another posterior were carried out. After the divulsion of the tissues with the aid of blunt scissors, the polyethylene tubes were implanted on the dorsum of the animal with the aid of a surgical trochar. In the anterior region, one tube was implanted at each side of the animal, with distance of 5 cm between each other and one tube was inserted at the posterior area. The materials of groups I and II were mixed onto glass plates, comprising: the measuring spoon of the MTA kit (Angelus®), and two drops of eugenol for group I and two drops of propylene glycol for group II, obtaining a final consistence of paste. The MTA Fillapex® was used directly from its package as recommended by its manufacturer. All sealers were introduced into the polyethylene tubes with 10 mm of lenght, 1.5 mm of internal diameter and 2 mm of external diameter. The tubes were initially kept for 1 hour submersed in 1% sodium hypochlorite and then were washed with saline solution and dried with sterile gauze.�� The tubes were filled with the material following the aforementioned division of the groups. The samples were left in the dorsum of the animal for seven and 15 days. After these experimental periods, the animals were killed with the aid of an excessive dose of anesthetic drug to obtain the cuts for histological analysis.

Histological analysis The pieces containing the implants were removed and fixed in 10% formalin and kept for 48 hours in individual and identified flasks. Elapsed the period of 48 hours for fixation, the pieces were washed in 70% alcohol and the tubes were carefully removed. Next, the pieces were

dehydrated in consecutive baths with alcohol at increasing concentrations (70%, 80%, 90% and absolute), diaphanized with xylol and included in paraffin to obtain blocks. Then, the serial cuts were processed with 5 µm of thickness in a microtome (Leica RM 2165); next, the material cut was distended and put in laminas which were kept in an incubator at 60ºC for deparaffinization. The laminas were immersed in Harris’ hematoxylin and washed in running water to remove the dye excess. After the removal of the water excess, the cuts were immersed in eosin for five minutes, dehydrated, clarified and the laminas were mounted to the reading in optical microscope (Olympus CH-2). The histological evaluation was double blinded. It was verified the intensity of the inflammatory infiltrate and repair tissue. The materials were histologically analyzed and the tissue and cellular morpholog y was described rega rding to t he experimental period.

Results The evaluation of the histological events was performed by two previously calibrated examiners (Kappa = 0.96), through optical microscope, at x10 to x40 magnification, taking into consideration the following histopathological events inflammatory i n f i lt rate, cel lu l a r it y, v a s c u l a r i z at ion a nd macrophagic activity in the reactional capsule.

Group I – zinc oxide, eugenol and iodoform (ZOE + iodoform) At seven days, a n i ntense mononuclea r i n f l a m m at or y i n f i lt rat e (l y mpho c y t e s a nd plasmocytes), macrophages and giant cells were seen. Great amount of connective tissue and congested blood vessels.�� The �� presence of the remnant material was constant in all laminas observed. Conjunctive tissue in repairing process with considerable presence of fibroblasts on the region inflamed (figures 1.1 and 1.2). In one of the laminas observed, it was noted areas of tissue necrosis with presence of polymorphonuclear neutrophils. At 15 days, it could be still noted the constant presence of material, indicating its difficult resorption, associated with a mononuclear inflammatory infiltrate, congested blood vessels and disorganized conjunctive tissue (figures 2.1 and 2.2).

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Figure 1.1 – Group I – zinc oxide, eugenol, iodoform at seven days: intense mononuclear inflammatory infiltrate��, macrophages and giant cells �� (circle area) and presence of remnants of the material (*) – x10 magnification

Figure 2.2 – Group I – zinc oxide, eugenol, iodoform at 15 days: presence of material being encompassed by giant cells (arrows) – x40 magnification

Group II – Portland + propylene glycol

Figure 1.2 – Group I – zinc oxide, eugenol, iodoform at seven days: conjunctive tissue in process of repairing with presence of fibroblasts in the inflamed area (circle area) – x40 magnification

Figure 2.1 – Group I – zinc oxide, eugenol, iodoform at 15 days: inflamed area with persistence of the remnant material, mononuclear inflammatory infiltrate, congested blood vessels and disorganized conjunctive tissue (circle area) – x10 magnification

At seven days, there was a great amount of material, discreet mononuclear inf lammatory infiltrate, giant cells and congested blood vessels. Conjunctive tissue in formation, still very immature on the inflamed area of the opening of the tube (figure 3). At 15 days, there was the predominance of mature fibrous conjunctive tissue, exhibiting greater amount of collagen fibers and fewer cells in the areas of repairing (figure 4). Absent or discreet inflammatory infiltrate were observed, and when present they were involving the remnants of the material (foreign body-like multinucleated giant cells).

Figure 3 – Group II – Portland + propylene glycol at seven days: presence of the remnants of the material (*), congested vessels (arrows) and inflammatory infiltrate (circle area) – x10 magnification

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Figure 4 – Group II – Portland + propylene glycol at 15 days: presence of collagen fibers (arrows), absence of inflammatory infiltrate – x10 magnification

Figure 5.2 – Group III – MTA Fillapex�� ®�� at seven days: areas with inflammatory infiltrate (circle area), presence of remnant material involved by giant cells (*) – x40 magnification

Group III – MTA Fillapex® At seven days, a fibrous conjunctive tissue was observed with moderate, predominantly mononuclear inflammatory infiltrate and presence of multinucleated giant cells, especially close to the remnants of the material. Presence of congested blood cells. Formation of connective tissue close to the opening of the tube (figures 5.1 and 5.2). At 15 days, it was verified a connective tissue richly cellularized, exhibiting discreet mononuclear inflammatory infiltrate and blood vessels, some congested (figure 6). It was still noted the remnant material involved by multinucleated giant cells (figure 7).

Figure 5.1 – Group III – MTA Fillapex�� ®�� at seven days: presence of moderate inflammatory infiltrate (circle area), remnant material (*) and congested vessels (arrows) – x10 magnification

Figure 6 – Group III – MTA Fillapex�� ®�� at 15 days: discreet inflammatory infiltrate (circle area) and presence of collagen fibers (arrows) indicating repair of the tissue – x10 magnification

Figure 7 – Group III – MTA Fillapex�� ®�� at 15 days: tissue with satisfactory repairing process, over and area with the presence of the remnant of the material involved by multinucleated giant cells (circle area) – x40 magnification

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Discussion It is recognized that the endodontic sealers can be very irritating to the tissues of the periapical region, cause inflammation or promote the tissue necrosis in this area [8, 10]. The methodology employed with polyethylene tubes for the study of endodontic sealers is a tool demonstrably satisfactory for biocompatibility tests, once it mimics the root canal conditions because the ending of the tube containing the material is in contact with the conjunctive tissue, representing the apical foramen/apical tissue relationship [3, 9, 10]. Concerning to the evaluation time, there is not a consensus among the authors in the current literature. The study period was based on the studies of Gomes-Filho et al. �� [9], Gomes-Filho et al. [10] and Mori et al. [17]. According �� with the literature, the lateral side of the polyethylene tube was used as negative control, once it was not in contact with the materials tested, therefore resulting in the histological analysis of the direct contact of the tissue with the material of the tube [30]. In studies involving the conjunctive tissue, the biocompatibility is demonstrated by the formation of the fibrous capsule surrounding the tubes implanted, indicating the repairing activity and suggesting the good acceptance of the material by the tissues [3, 4, 12, 15, 27]. All sealers implanted provoked a tissue reaction characterized by a chronic inf lammatory response from mild to moderate, fibroangioblastic proliferation formation of collagen fibers, evidencing the biocompatibility of the materials over time [6, 23, 28, 30]. Although these tests are not even ideal, the data obtained is of great value for the evaluation of new materials that are launched into the market [4, 21]. The group of ZOE + iodoform was chosen because this material is largely employed in Endodontics of deciduous teeth and iodoform is a radiopacifying agent added to ZOE [1]. This material exhibited a biological behavior less favorable than that of the other groups and an inf lammatory infiltrate at the initial periods that persisted during all the period evaluated. In the analyses of 15 days, it was still observed great amounts of the remnants of the material in the tissue, in addition to a discreet formation of collagen fibers [8-10]. The morphological analysis of the laminas obtained from the materials tested demonstrated that the biological behavior of Portland cement and MTA Fillapex® sealer was similar at almost periods evaluated. Both sealers induced a discreet inflammatory infiltrate at the initial periods (seven days), which decreased over time, and significant fibroblastic and collagen fiber proliferation at the

final periods (15 days). Such findings corroborate with several studies which demonstrated the similarities in the basic chemical composition, mechanism of action and cytotoxicity of both materials tested, differing only in the addition of some resin and radiopacifying components in the MTA Fillapex® formula [3, 9, 15, 18, 19, 25, 31]. Aiming to reach a greater similarity between the sealers tested, the propylene glycol was added to the Portland cement, once such viscous vehicle is still little studied in association with this cement but it results in a better flowing and longer setting time, without compromising its physical-chemical properties and consequently giving in this present study characteristics very similar to those showed by MTA Fillapex® [5]. Despite the similarity between Portland cement and MTA, the latter can in some cases show more favorable results because it is fabricated under controlled conditions free of impurities. The Portland cement can contain some impurities coming from the mineral from which it is extracted [2, 11, 16, 20, 25, 30]. Other authors demonstrated in their studies that MTA can exhibit a less favorable result with greater occurrence of inflammation in the initial analyses because of its high pH and temperature at the initial moments of its setting, which does not occur with the Portland cement [2, 13]. Such fact is in agreement with the results of this study, justifying the data found.

Conclusion The Portla nd cement a nd MTA Fillapex ® exhibited the best biological behavior when the provoked inflammatory intensity and the tissue repairing evolution was evaluated; however, further studies are necessary for the most accurate indication of these materials.

Refer�� e�� nc�� e� s 1. Barcelos R, Santos MP, Luiz RR, Maia LC. ZOE paste pulpectomies outcome in primary teeth: a systematic review. �� J Clin Pediatr Dent. 2011;35(3):241-8. 2. Conti TR, Sakai VT, Fornetti AC, Moretti ABS, Oliveira TM, Lourenço Neto N et al. �� Pulpotomies with portland cement in human primary molars. J Appl Oral Sci. 2009;17(1):66-9.

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3. Coutinho-Filho T, De-Deus G, Klein L, Manera G, Peixoto C, Gurgel-Filho ED. Radiopacity �� and histological assessment of Portland cement plus bismuth oxide. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2008 Dec;106(6):69-77. 4. Danesh F, Tootian Z, Jahanbani J, Rabiee M, Fazelipour S, Taghva O et al. Biocompatibility and mineralization activity of fresh or set white mineral trioxide aggregate, biomimetic carbonated apatite, and synthetic hydroxyapatite. J Endod. 2010 Jun;36(6):1036-41. 5. Duarte MA, Alves de Aguiar K, Zeferino MA, Vivan RR, Ordinola-Zapata R, Tanomaru-Filho M et al. �� Evaluation of the propylene glycol association on some physical and chemical properties of mineral trioxide aggregate. Int Endod J. 2012 Jun;45(6):565-70. 6. Garcia LFR, Lia RCC, Lopes RA, Oliveira DA, Pires-De-Souza FCP, Santos HSL. Análise morfológica e morfométrica do tecido subcutâneo de ratos submetidos à ação de pasta de hidróxido de cálcio e óleo de Ricinus communis. �� Ciênc Odontol Bras. 2008 Jul/Sep;11(3):47-54. 7. Gencoglu N, Sener G, Omurtag GZ, Tozan A, Uslu B, Arbak S et al. Comparision of biocompatibility and cytotoxicity of two new root canal sealers. Acta Histochem. 2010 Nov;112(6):567-75. 8. Ghanaati S, Willershausen I, Barbeck M, Unger RE, Joergens M, Sader RA et al. Tissue reaction to sealing materials: different view at biocompatibility. Eur J Med Res. 2010 Nov;15(11):483-92. 9. Gomes-Filho JE, Watanabe S, Lodi CS, Cintra LT, Nery MJ, Filho JÁ et al. Rat �� tissue reaction to MTA Fillapex®. Dent Traumatol. 2012 Dec;28(6):452-6.

13. Koh ET, Torabinejad M, Pitt Ford TR, Brady K, McDonald F. Mineral trioxide aggregate stimulates a biological response in human odontoblasts. J Biomed Mater Res. 1997;37(3):432-9. 14. Lotfi M, Vosoughhosseini S, Saghiri MA, Mesgariabbasi M, Ranjkesh B. Effect of white mineral trioxide aggregate mixed with disodium hydrogen phosphate on inflammatory cells. J Endod. 2009 May;35(5):703-5. 15. Martínez Lalis R, Esaín ML, Kokubu GA, Willis J, Chaves C, Grana DR. Rat �� subcutaneous tissue response to modified Portland cement, a new mineral trioxide aggregate. Braz Dent J. 2009;20(2):112-7. 16. Moretti AB, Sakai VT, Oliveira TM, Fornetti AP, Santos CF, Machado MA et al. Effectiveness �� of mineral trioxide aggregate, calcium hydroxide and formocresol for pulpotomies in primary teeth. Int Endod J. 2008;40(8):653-60. 17. Mori GG, Moraes IG, Nunes DC, Castilho LR, Poi W, Capaldi ML. Biocompatibility evaluation of tendrovate paste in rat’s subcutaneous tissue. Dent. Traumatol. 2009;25(2):209-12. 18. Nassri MRG, Lia RCC, Bombana AC. Análise da resposta tecidual de dois cimentos endodônticos. J Appl Oral Sci. 2003;11(1):9-14. 19. Okino Neto K, Moura AAM, Davidowicz H. Estudo comparativo da reação tecidual conjuntiva de ratos, frente a três cimentos endodônticos resinosos. J Health Sci Inst. 2010;28(1):67-70. 20. Oliveira TM, Sakai VT, Silva TC, Santos CF, Machado MA, Abdo RC. �� Repair of furcal perforation treated with mineral trioxide aggregate in a primary molar tooth: 20-month follow-up. �� J Dent Child. 2008 May-Aug;75(2):188-91.

10. Gomes-Filho JE, Gomes BP, Zaia AA, Ferraz CR, Souza-Filho FJ. �� Evaluation of the biocompatibility of root canal sealers using subcutaneous implants. J Appl Oral Sci. 2007 Jun;15(3):186-94.

21. Parirokh M, Mirsoltani B, Raoof M, Trabrizchi H, Haghdoost AA. Comparative study of subcutaneous tissue response novel root-end filling material and white and gray mineral trioxide aggregate. Int Endod J. 2011;44(4):283-9.

11. Holland R, de Souza V, Nery MJ, Faraco Júnior IM, Bernabé PF, Otoboni Filho JA et al. Reaction �� of rat connective tissue to implanted dentin tube filled with mineral trioxide aggregate, Portland cement or calcium hydroxide. Braz Dent J. 2001;12(1):3-8.

22. Peng L, Ye L, Tan H, Zhou X. Evaluation of the formocresol versus mineral trioxide aggregate primary molar pulpotomy: a meta-analysis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2006;102(6):40-4.

12. Khashaba RM, Moussa MM, Chutkan NB, Borke JL. The response of subcutaneous connective tissue to newly developed calcium phosphate-based root canal sealers. Int Endod J. 2011 Apr;44(4):342-52.

23. Perassi FT, Pappen FG, Bonetti Filho I, Leonardo RT, Ykeda F, Ramalho LTO. Estudo morfológico da resposta tecidual a quatro cimentos endodônticos. Rev Odont UNESP. 2008;37(2):117-24.

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24. Queiroz AM, Assed S, Consolaro A, NelsonFilho P, Leonardo MR, Silva RA et al. �� Subcutaneous connective tissue response to primary root canal filling materials. �� Braz Dent J. 2011;22(3):203-11. 25. Sakai VT, Moretti AB, Oliveira TM, Fornetti AP, Santos CF, Machado MA et al. Pulpotomy �� of human primary molars with MTA and Portland cement: a randomised controlled trial. �� Br Dent J. 2009 Aug 8;207(3):E5; discussion 128-9. 26. Scarparo RK, Grecca FS, Fachin EV. Analysis of tissue reactions to methacrylate resin-based, epoxy resin-based, and zinc oxide-eugenol endodontic sealers. J �� Endod. 2009 Feb;35(2):229-32. 27. Scarparo RK, Haddad D, Acasigua GA, Fossati AC, Fachin EU, Grecca FS. �� Mineral trioxide aggregate-based sealer: analysis of tissue reactions to a new endodontic material. J �� Endod. 2010;36(7):1174-8. 28. Silva-Herzog D, Ramírez T, Mora J, Pozos AJ, Silva LA, Silva RA et al. Preliminary �� study

of the inflammatory response to subcutaneous implantation of three root canal sealers. Int Endod J. 2011 May;44(5):440-6. 29. Sumer M, Muglali M, Bodrumlu E, Guvenc T. Reactions of connective tissue to amalgam, intermediate restorative material, mineral trioxide aggregate, and mineral trioxide aggregate mixed with chlorhexidine. J Endod. 2006 Nov;32(11):1094-6. 30. Zafalon EJ, Versiani MA, de Souza CJ, Moura CC, Dechichi P. In vivo comparison of the biocompatibility of two root canal sealers implanted into the subcutaneous connective tissue of rats. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007 May;103(5):88-9. 31. Zmener O, Martinez Lalis R, Pameijer CH, Chaves C, Kokubu G, Grana D. �� Reaction of rat subcutaneous connective tissue to a mineral trioxide aggregate-based and a zinc oxide and eugenol sealer. J Endod. 2012 Sep;38(9):1233-8.

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Original Research Article

Staining susceptibility of methacrylate and silorane-based materials: influence of resin type and storage time Leonardo Fernandes da Cunha1 Lino Oliveira Carvalho de Santana2 Samantha Schaffer Pugsley Baratto2 José Mondelli1 Gisele Aihara Haragushiku2 Carla Castiglia Gonzaga2 Adilson Yoshio Furuse2 Corresponding author: Adilson Yoshio Furuse Universidade Positivo – Pós-graduação em Odontologia Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: ayf@up.com.br Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry – Bauru – São Paulo – Brazil. 2 Master Student of the Science Program in Clinical Dentistry, Positivo University – Curitiba – PR – Brazil. 1

Received for publication: November 12, 2012. Accepted for publication: December 19, 2012.

Keywords: resin composites; color; pigmentation.

Abstract Introduction: The color stability of composite resins is a fundamental factor in their clinical behavior. Objective: To evaluate the color stability of composite resins of different colors exposed to a colabased soft drink after different storage periods. Additionally, three methacrylate-based materials and one silorane-based material were evaluated. Material and methods: Specimens of three methacrylatebased materials (Opallis EA3, DA3 and T-Neutral; Filtek Supreme XT A3E, A3D and CT; 4 Seasons A3 Enamel, A3 Dentin and High Value) and one silorane-based material (Filtek P90 A3) were prepared, lightcured for 40 s, and manually polished with Sof-Lex discs. Samples were stored for 1 h, 24 h or 7 days. The color was evaluated by CIELab system before and after immersion for 10 min in a cola-based soft drink. Color variation (∆E) was calculated from individual values of L*, a* and b*, being considered imperceptible when < 1, clinically

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acceptable when ≤ 3.3, and clinically inacceptable when higher than 3.3. Data were evaluated by two-way Anova and Dunnett’s T3 tests (α = 0.05). Results: There were differences among the resins (p < 0.001), with an interaction effect being also observed (p < 0.001). Storage time was not significant (p = 0.246). P90 showed a ∆E smaller than one unit at all studied times. Supreme XT CT and 4 Seasons High Value showed higher ∆E, but not above the critical value of 3.3. The only material that showed ∆E higher than 3.3 was Opallis DA3 after 1 h of storage. Conclusion: The silorane-based composite resin showed smaller ∆E at the times studied.

Introduction The resi n composites have been la rgely accepted as restorative material in Dentistry. Since its appearance, additionally to the use of many monomer types, several types and sizes of filler particles have been tested. These particles are incorporated to increase their physical and chemical properties [7]�� . Despite of the greatest use in Dentistry, it is known to date that most of the composites are based on methacrylates as Bis-GM A, Bis-EM A, UDM A and TEG-DM A [15, 20]�. With the advancement of the composites, the use of these materials started to increase due to the improvement of the physical-chemical and aesthetical properties. Thus, the requirements regarding to the perfect matching between the color of the restorations and teeth became even greater, and, one of the most common reasons to the replacement of the aesthetical restorations is the incorporation of pigments which compromise the aesthetical harmony of the smile [8, �� 30]�. The aesthetical properties are directly related with the degree of the opacity, translucency and opalescent effects. At the long term, factors as color stability, roughness, retention, and surface brightness can determine the success or failure of the restoration �� [3, 12]��. The pigmentation of the resin composite restorations can be caused by several intrinsic and extrinsic factors. Intrinsic factors are those involving the discoloration of the resin itself related to the alterations in the resin matrix and in the interface of the matrix. The intrinsic discolorat ion ca n be a lso at t ributed to t he insufficient polymerization, immersion in liquid solution for longer periods, quality of both the light-curing and the polymers. On the other hand, extrinsic alterations can be attributed to external events, among them, it is the insufficient degree of polymerization, water sorption, heating, and

adsorption of food dyes, such as coffee, tea, and red wine [3, �� 6, 11, 17]�. With the aim of decreasing the polymerization shrinkage and sorption of pigments, siloranebased resin composites have been developed �� [14]��. As advantages, silorane-based resin composites show low polymerization shrinkage, low stress and good insolubility in simulators of biological f luids [9]�� . Additionally, a good color stability in artificial aging tests in which the material is exposed to high doses of ultraviolet light have been reported �� [12]��. It is known that the polymerization process of resin-based materials is continue and can be continued for longer periods, even when lightcured resins are considered [13]�� . Because of this continued resin and because resin composites tend to absorb water at the first hours after its polymerization, it is common to recommend that the patient avoids the consumptions of dyes at the first hours or days. Therefore, the exposure to dyes coming from daily food intake needs to be evaluated. The dyes are present in most of the foods. Beverages, as cola-based soft drinks, black tea, coffee, red wine, and fruit juices seem to produce a greater inf luence on the color of both the teeth �� [31]�� and resin composites [29]�� . It should be highlighted that the consumption of these beverages is highly popular and the inhibition of it s i ngest ion for aest het ica l pu rposes is impracticable to most of the population. This concern has been the reason of studies on other moda lities of aest hetica l treat ments, such a dental bleaching [28]� ��. The aim of this study was to evaluate the color stability of resin composites of different colors exposed to a cola-based soft drink after d i f ferent t i me per iods. Add it iona l ly, t h ree met hacr ylate-based a nd one si lora ne-based material were evaluated.

RSBO. 2013 Apr-Jun;10(2):161-6 – 163

Material and methods Speci mens of t h ree met hacr ylate-ba sed materials (Opallis EA3, DA3 e T-Neutral, FGM, Joinville, Brazil; Filtek Supreme XT A3E, A3D a nd CT 3M Espe, St. Paul, USA; 4 Seasons A3 Enamel, A3 Dentin and High Value, Ivoclar Vivadent, Liechtenstein) and one specimen of a silorane-based material (Filtek P90 A3, 3M Espe, St. Paul, USA) were constructed with the aid of a polytetraf luorethane matrix (10 mm of diameter and 2 mm of thickness). The matrix was placed onto a microscope lamina of 1 mm of thickness, followed by the insertion with resin composite. Then a polyester strip was placed onto the orifice of the matrix and another microscope lamina of the same thickness was positioned onto it. Next the resin composite was light-cured for 40 s with a LED device (Translux Power Blue, Heraeus Kulzer GmbH, Hanau, Germany) with light intensity of 1000 mW/cm2. The device intensity was determined prior to the beginning of the tests with the aid of a radiometer linked to it. The specimens were manually polished with decreasing sequence of polishing discs (Sof-Lex, 3M Espe, St. Paul, USA). A single operator polished all the specimens and each disc was used for 15 s. The specimens were stored into distilled water at 37ºC within dark f lasks for 1 h, 24 h or seven days. After each storage time, the specimens were kept for 10 minutes in a cola-based soft drink. The color was evaluated by the CIE-Lab system with the aid of a spectrophotometer (VITA Easyshade 3D Master, Vita Zahnfabrik, Bäd Sackingen, Germany).

The color variation (∆E) was calculated from the individual values of L*, a* and b*, according to the following formula: ΔE = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2

(1)

In which Δ�� L*, �� Δ�� a* and Δ�� b* represents the differences between the readings of the color parameters obtained from the specimens at the different conditions evaluated. The color va riat ion was considered imperceptible when < 1, clinically acceptable when ≤ 3.3 and unacceptable when higher than 3.3 �� [23, 26]��. The data were evaluated through one-way ANOVA and Dunnett’s T3 test (α = 0.05).

Results The means and standard deviations of the data obtained from the calculation of ∆E can be observed in table I and in figure 1. It was observed differences a mong t he resins (p < 0.001) and an interaction effect among the resins and the storage time (p < 0.001). The time was not significant (p = 0.246). P90 exhibited a ∆E smaller than 1 at all times studied. Supreme XP CT and 4 Seasons High Value showed a higher ∆E, without surpassing the critical value 3.3. The single material presenting a ∆E greater than 3.3 was Opallis DA3 after the 1 h immersion in the dye.

Table I – Means (standard deviations) of ∆E values for the different groups

Opallis

Filtek Supreme XT

4 Seasons

P90

EA3

DA3

TNeutral

A3E

A3D

A3 Enamel

A3 Dentin

High Value

1.04 (0.4)abc

3.85 (2.7)bc

2.31 (0.4)abc

0.67 (0.04)abc

0.45 (0.05)ab

1.78 (1.3)c

0.89 (0.4)abc

0.81 (0.4)abc

0.84 (0.5)c

0.41 (0.1)a

0.75 (0.3)abc

0.70 (0.4)bc

0.81 (0.5)abc

1.39 (0.6)abc

1.27 (0.5)ab

2.01 (1.4)c

2.10 (0.3)abc

0.86 (0.2)abc

2.84 (0.2)c

0.61 (0.2)a

0.91 (0.06)abc

0.58 (0.1)bc

1.32 (0.6)abc

0.99 (0.1)abc

0.56 (0.1)ab

1.67 (0.04)c

0.80 (0.5)abc

1.31 (0.6)abc

2.1 (0.2)c

0.36 (0.3)a

Equal superscript letters indicate no statistically significant differences (p < 0.05)

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Figure 1 – Graphic representation of the results obtained after the calculus of the color variation: A) Opallis; B) Filtek Supreme XT; C) 4 Seasons; D) Filtek P90

Discussion The color stability is an important property which can play a determinant role in the clinical behavior of the resin composite restorations. This aspect is among one of the most used criteria of clinical evaluation in Dentistry: the United States Public Health Service (USPHS). Initially proposed by Ryge [24], this system of evaluation establishes the color and the marginal discoloration as two determinant factors of in vivo longevity of the restorations. ISO 7491:2000 resolution has been commonly employed in the analysis of the color stability of resin-based materials �� [27]�� . According to this resolution, resin composite specimens are exposed to a xenon gas lamp with illuminance of 150 klx for a period of 24 h. The color is evaluated before and after the light exposure through the visual scale or colorimeters. However, this artificial aging

method not even reproduces the clinical conditions, once the restorations performed within the oral cavity are exposed to limited amounts of light with wavelengths at the ultraviolet area. For this reason, this present study assessed the color after the exposure of different resins to a cola-based soft drink, which can be considered as more clinically relevant. When spectrophotometers or colorimeters are utilized for color analysis, one can calculated the color variation (∆E) objectively, through the CIE-Lab color system (Commision Internationale de l’Eclairage, L*, a*, b*). Other systems or color spaces such as Munsell’s could be employed [16]�� . However, the CIE-Lab system is the most employed because it allows the comparison of the results with those of the clinical visualization �� [18]�� . The CIE-Lab system defines the color tridimensionally, because the parameter L* corresponds to the luminosity degree, a* corresponds to red or green (+a* = red and –a* = green) and b* yellow or blue (+b*

RSBO. 2013 Apr-Jun;10(2):161-6 – 165

= yellow and –b* = blue) [16, �� 22, 23]�� . It has been considered that a color variation lower than 1 is not clinically perceptible [26]�� , it is acceptable when it ranges from 1 to 3.3 and unacceptable when it is higher than 3.3 [23]� ��. The color of a resin composite restoration can be affected by chemical differences among the components of the resin phase, such as the monomers and the photo-initiator system [4, �� 10, 25]�� . In this present study, the silorane-based resin exhibited a ∆E smaller than 1 at all time periods studied. These data are in agreement with those of another study evaluating the color stability of a silorane-based resin after the artificial aging with exposure to light and xenon at 150 klx for 192 h [12]�� . One possible explanation for these data can be the different chemical composition of the resin matrix compared with the methacrylate-based resins. It has been reported that silorane-based materials have good chemical stability when exposed to aqueous environment �� [9]�� , in addition to a low solubility and water sorption [19]�� . Conversely to the results of this study, other research assessing the color of a silorane-based resin found a mean ∆E of 18.6 units after 384 h of artificial aging �� [21]�. Together with the silorane-based resin, resins composed of different opacities were evaluated in this present study. The color of a resin composite can be influenced by the features of light transmittance [2]�� . The transmittance means the luminous energy fraction which can surpass a determined material thickness, without being absorbed. It is measured as percentage, relatively to the amount of energy and wavelength of the incident light radiation [5]�� . This optic feature is closely linked to the opacity of the material. Supreme XT CT and 4 Seasons High Value showed the highest ∆E, without surpassing the critical value of 3.3. Despite the fact that only one material displayed a ∆E higher than 3.3 units at a post-polymerization time of only 1 hour, caution must be taken when one interprets the results of this present study. ∆E values higher than 1 were observed many times, meaning that even 24 h to seven days after the polymerization, it can still have color variation capable of being perceived visually if marked exposures to dyes would occur. Thus, after the construction of resin composite restorations it is necessary to instruct the patients that: 1) it is important to keep adequate oral hygiene habits; 2) the esthetics of the restorations can be longer maintained if some restriction of the consumption of food containing dyes were taken [1]� ��.

Conclusion Given the results of this study, it can be concluded that the time was not significant and the silorane-based material showed the smallest ∆E at the time periods studied.

References 1. Ardu S, Braut V, Gutemberg D, Krejci I, Dietschi D, Feilzer AJ. A long-term laboratory test on staining susceptibility of esthetic composite resin materials. Quintessence Int. 2010 Sep;41(8):695-702. 2. Arikawa H, Fujii K, Kanie T, Inoue K. Light transmittance characteristics of lightcured composite resins. Dent Mater. 1998 Nov;14(6):405-11. 3. Arocha MA, Mayoral JR, Lefever D, Mercade M, Basilio J, Roig M. Color stability of siloranes versus methacrylate-based composites after immersion in staining solutions. Clin Oral Investig. 2012 Sep;20. 4. Asmussen E. Factors affecting the color stability of restorative resins. Acta Odontol Scand. 1983;41(1):11-8. 5. Atkins P, Paula J. Molecular spectroscopy 1: rotational and vibrational spectra. In: Atkins P, Paula J, editors. Atkins’ physical chemistry. 8. ed. Oxford: Oxford University Press; 2006. p. 430-80. 6. Barutcigil C, Yildiz M. Intrinsic and extrinsic discoloration of dimethacrylate and silorane based composites. J Dent. 2012 Jul;40(Suppl 1): e57-63. 7. Darvell BW. Materials science for dentistry. 7. ed. Hong Kong: B W Darvell; 2002. 8. Douglas RD. Color stability of new-generation indirect resins for prosthodontic application. J Prosthet Dent. 2000 Feb;83(2):166-70. 9. Eick JD, Smith RE, Pinzino CS, Kostoryz EL. Stability of silorane dental monomers in aqueous systems. J Dent. 2006 Jul;34(6):405-10. 10. Ferracane JL. Correlation between hardness and degree of conversion during the setting reaction of unfilled dental restorative resins. Dent Mater. 1985 Feb;1(1):11-4. 11. Fontes ST, Fernandez MR, de Moura CM, Meireles SS. Color stability of a nanofill composite: effect of different immersion media. J Appl Oral Sci. 2009 Sep-Oct;17(5):388-91.

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12. 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. 13. Furuse AY, Mondelli J, Watts DC. Network structures of Bis-GMA/TEGDMA resins differ in DC, shrinkage-strain, hardness and optical properties as a function of reducing agent. Dent Mater. 2011 May;27(5):497-506. 14. Guggenberger R, Weinmann W. Exploring beyond methacrylates. American Journal of Dentistry. 2000 Nov;13(Spec No):82D-4D. 15. Ilie N, Hickel R. Resin composite restorative materials. Aust Dent J. 2011 Jun;56(Suppl 1):5966. 16. Joiner A. Tooth colour: a review of the literature. J Dent. 2004;32(Suppl 1):3-12. 17. Kang A, Son SA, Hur B, Kwon YH, Ro JH, Park JK. The color stability of silorane- and methacrylate-based resin composites. Dent Mater J. 2012;31(5):879-84. 18. O’Brien WJ, Hemmendinger H, Boenke KM, Linger JB, Groh CL. Color distribution of three regions of extracted human teeth. Dent Mater. 1997 May;13(3):179-85. 19. Palin WM, Fleming GJP, Burke FJT, Marquis PM, Randall RC. The influence of short and medium-term water immersion on the hydrolytic stability of novel low-shrink dental composites. Dent Mater. 2005;21:852-63. 20. Peutzfeldt A. Resin composites in dentistry: the monomer systems. Eur J Oral Sci. 1997 Apr;105(2):97-116. 21. Pires-de-Souza FC, Garcia LF, Roselino LM, Naves LZ. Color stability of silorane-based composites submitted to accelerated artificial ageing – an in situ study. J Dent. 2011 Jul;39(Suppl 1):e18-24.

22. Rosenstiel SF, Porter SS, Johnston WM. Colour measurements of all ceramic crown systems. J Oral Rehabil. 1989 Sep;16(5):491-501. 23. Ruyter IE, Nilner K, Moller B. Color stability of dental composite resin materials for crown and bridge veneers. Dent Mater. 1987 Oct;3(5):246-51. 24. Ryge G. Clinical criteria. Int Dent J. 1980 Dec;30(4):347-58. 25. Schneider LF, Pfeifer CS, Consani S, Prahl SA, Ferracane JL. Influence of photoinitiator type on the rate of polymerization, degree of conversion, hardness and yellowing of dental resin composites. Dent Mater. 2008 Mar;24(9):1169-77. 26. Seghi RR, Johnston WM, O’Brien WJ. Performance assessment of colorimetric devices on dental porcelains. J Dent Res. 1989 Dec;68(12):1755-9. 27. EN ISO 7491: Dental materials – determination of colour stability. 2000. 28. Téo TB, Takahashi MK, Gonzaga CC, Lopes MGK. Avaliação, após clareamento, da alteração de cor de dentes bovinos imersos em soluções com elevado potencial de pigmentação. RSBO. 2010;7(4):401-5. 29. Tonetto MR, Santezi Neto C, Felício CM, Domingos PAS, Campos EA, Andrade MF. Effect of staining agents on color change of composites. RSBO. 2012;9(3):266-71. 30. Tuncdemir AR, Aykent F. Effects of fibers on the color change and stability of resin composites after accelerated aging. Dent Mater J. 2012;31(5):872-8. 31. Xie P, Lu J, Wan H, Hao Y. Effect of toothpaste containing d-limonene on natural extrinsic smoking stain: a 4-week clinical trial. Am J Dent. 2010 Aug;23(4):196-200.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):167-71

Literature Review Article

Gubernacular cord and canal – does these anatomical structures play a role in dental eruption? Danielly Cunha Araújo Ferreira1 Ana Caroline Fumes1 Alberto Consolaro2 Paulo Nelson-Filho1 Alexandra Mussolino de Queiroz1 Andiara De Rossi1 Corresponding author: Danielly Cunha Araújo Ferreira Departamento de Clínica Infantil, Odontologia Preventiva e Social Faculdade de Odontologia de Ribeirão Preto – USP Av. do Café, s/n. CEP 14040-903 – Ribeirão Preto – SP – Brasil E-mail: daniellycaf@hotmail.com Department of Child Clinics, Preventive and Social Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo – Ribeirão Preto – SP – Brazil. 2 Department of Pathology, School of Dentistry of Bauru, University of São Paulo – Bauru – SP – Brazil. 1

Received for publication: May 22, 2012. Accepted for publication: November 5, 2012.

Keywords: tooth eruption; primary dentition; permanent dentition.

Abstract Introduction: The gubernacular cord is an original structure of the dental lamina, which undergoes apoptosis and their remnants were organized in the form of epithelial islets and strands that lined up, leaving the reduced epithelium of the enamel organ towards the oral mucosa. This structure is located within the gubernacular canal, which can be identified as a small opening in the alveolar region of the lingual or palatal surface of the deciduous teeth. Objective: To conceptualize, identify and assess the possible contribution of the gubernacular cord and canal in the process of tooth eruption. Literature review: A review of literature on Pubmed, Medline and Bireme databases, without datum restriction. Little amount of scientific articles were found, and only 14 studies were identified. The authors addressed the matter succinctly, with little information about these structures, which can play an important role in the process of tooth eruption. Conclusion: The gubernacular cord and canal are anatomical structures located in the alveolar bone crest of the maxilla or mandible,

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Ferreira et al. Gubernacular cord and canal – does these anatomical structures play a role in dental eruption?

behind the deciduous teeth. These structures appear to exhibit the ability to aid the eruption path of the permanent teeth successors. Despite being a relevant subject, few professionals know this structure and its possible role in the process of tooth eruption.

Introduction Tooth eruption is a physiologic process in which a tooth undergoing formation migrates from its development site within the alveolar processes to its functional positions inside the oral cavity [18]. During this process, the participation of different anatomic structures, cells, chemical and molecular mediators occurs and although there have been a great advancement in the scientific knowledge regarding this issue, still today the cellular, molecular and anatomic mechanisms involved in tooth eruption process are not fully understood [17, 28, 29]. Aiming to explain the tooth eruption process, countless theories have been proposed over the years, such as the theory of root growth [18], alveolar bone growth [4, 20], pulpal growth [25], in addition to the theory of the combination of genetic factors [26] and the follicular theory [6, 15], which has been the most accepted nowadays. The follicular theory postulates that the dental follicle is capable of inducing, guiding and coordinating the bone resorption above the crown of a teeth undergoing formation and bone apposition below this same crown, which enables that during the intraosseous eruptive phase of the tooth eruption process the formation of a eruptive path occur and the tooth undergoing formation be passively conducted through this path [6, 15]. The gubernacular cord is a structure composed of conjunctive tissue which link the tooth follicle to the overlying gingiva, showing the function of guiding or directing the course of the tooth eruption. The formation of this structure starts from the remnants cells of the tooth lamina which are organized as a fibrous cord leaving the reduced epithelium of the enamel organ towards the oral mucosa [9, 10]. This structure is located in the alveolar ridge behind the deciduous tooth [7, 12]. In this cord, there is the presence of countless chemical mediators, including EGF (epithelial g row t h factor), a substa nce secreted by t he epithelial cells having the capacity of stimulating

the formation of clasts and consequently a bone resorption, making impossible the filling of the cord area by alveolar bone, always leaving a space surrounding this cord, so-called the gubernacular canal (figures 1 to 3) [8, 9]. Bothe the gubernacular canal and cord are structures very little studied on the scientific literature; however t hey seem to play some role in t he process of tooth eruption. The aim of this study was to�� conceptualize, identify and assess the possible contribution of the gubernacular cord and canal in the process of tooth eruption.

Figure 1 – Dry child skull. Gubernacular canals located in the alveolar bone crest, behind the mandibular deciduous incisors

Figure 2 – Dry child skull. Gubernacular canals located in the alveolar bone crest, behind the mandibular deciduous incisors

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Figure 3 – Dry child skull. Gubernacular canals located in the alveolar bone crest, behind the maxillary deciduous incisors

Material and methods A bibliographic searching was conducted on the following databases Pubmed, Bireme, Medline, Google and Central Library of the University of São Paulo – Campus of Ribeirão Preto, without datum restriction, through using the terms: gubernacular cord, gubernacular canal, gubernaculum dentis, deciduous dentition, permanent dentition and tooth eruption. Only 14 scientific studies were found in all literature researched. Of these studies, ten were written in English, one in Portuguese, one in Japanese, on in Italian, and one in France.

Literature review The first reference to the gubernacular cord and canal occurred in 1778, by an English called John Hunter, which described these structures after observing a connection between the bone ridge of the tooth in development and the gingiva, without little scientific proof [11]. Almost one century later, this observation was supported by microscopic studies conducted by the France histologist Louis-Charles Malassez, in 1887. He described the existence of remnants of the tooth lamina (epithelial cells) and longitudinal fibers that were within a canal connecting the gingiva with the permanent tooth bud [14]. The gubernacular canal, which enables the continuity of the bone ridge of permanent incisors, canines and premolars with the tissue of the overlying gingiva itself, it is filled by the gubernacular cord, which is composed by a fibrous conjunctive tissue containing peripheral nerves, blood and

lymphatic vessels, as well as epithelial cells or cell aggregates coming from the fragmentation of the tooth lamina. Thus, the gubernacular cord is the connection between the conjunctive tissue of the ridge with the oral mucosa [22]. During the pre-eruptive phase of tooth eruption, the tooth buds have special relationship with the growing jaws. At this phase, the gubernacular canal does not undergo perceptive alterations in shape or size [5]. During the eruptive phase, according to the successor tooth moves towards the axial direction to the oral cavity, the gubernacular canal is widened by the local osteoclastic activity, with the aim of accommodating the crown of the permanent tooth undergoing eruption [7]. The tooth moves towards the mucosa, the pericoronary follicle incorporates within its conjunctive tissue the islets and cords of epithelial cells from the gubernacular cord, progressively increasing the epithelial component at this area [9]. The role of the gubernacular cord in the eruption process was investigated Cahill and Marks [6], who observed that the surgical section of this structure in dogs did not alter the eruption rates and the final position of the tooth. A next study, in which the section of the gubernacular cord and the surgical removal of the crown parts of the tooth follicle were executed, enabled to observe the interruption in the tooth eruption process, demonstrating that the tooth follicle has a fundamental role in the physiologic movement of the tooth towards the oral cavity and that if the gubernacular cord has some role in the process of tooth eruption, this is not relevant for the occurrence of the process [5]. According to Hodson [10], the gubernacular cord was not described in association with the deciduous dentition, only with the permanent dentition with the deciduous predecessor. However, according to Scott [27], the permanent molar teeth, which did not have a deciduous predecessor, also have their follicles connected to the oral mucosa by gubernacular cords, so-called by the author “molar gubernacular cords”. Philipsen [23] theorized that the gubernacular cord could have implication in the development of the adenomatoid odontogenic tumor (AOT), because this contains remnants of the tooth lamina. These epithelial remnants are very closer to the crown of the permanent tooth and they can move during the tooth eruption process along with the gubernacular canal and induce AOT formation, because among the many hypotheses for the pathogenesis of this tumor are the remnants of the tooth lamina.

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Discussion Despite the first reference to the gubernacular cord and canal have been shown in 1778 by John Hunter, 234 years ago, the existence and function of this structure are still controversial and questioned. According to Hodson [10], these structures are present only in association with the permanent teeth with deciduous predecessor, fact corroborates by Cahill et al. [5] and Philipsen and Reichart [22]. However, Scott [27] suggested that the permanent teeth without deciduous predecessor also displayed gubernacular cord and canal, connecting the bud to the oral mucosa. Notably, the description of the gubernacular cord and canal was only recently incorporated to the pattern of the American texts [3]. The explanation could be based on the fact that, by the omission of this gubernacular canal, the existing theories on the pressure for the eruption recommended by some professor and researchers influenced by the wrong concept that the successor tooth would be stuck by a complete bone coating, could be more easily accepted [5]. Some authors [5, 10, 27] believe that the gubernacular canal and cord have a role in the direction of the teeth, making the eruption easier; while others [6] claimed that these structures do not exert any type of interference. According to Philipsen et al. [23], the gubernacular cord and canal can influence on AOT development, because AOT in about 80% of the cases is located at the region of the permanent incisors and canines, where the gubernacular cord and canal is present [21, 24]. Some authors [13, 19, 30] sympathize with this hypothesis, because rarely AOT is found in association with the deciduous teeth, which could be justified by the absence of the gubernacular cord in the deciduous dentition, according to Hodson [10]. Ide et al. �� [12] questioned that sometimes (4% of the pericoronary lesions) AOT occurs in permanent molars, which did not have according Hodson [10], the gubernacular cord and canal. Notwithstanding, it valid to remember that Scott [27] reported the presence of the “molar gubernacular cords”, which could explain the occurrence of this tumor type in these teeth. Until now, the ability of the dentist to interfere or treat an included or impacted tooth due to eruption failure is limited to surgical or mechanical procedures, such as: the extraction of a deciduous tooth, surgical removal of the alveolar bone, the aid of the mucosal penetration through gingival incision and orthodontic tooth traction [2]. Perhaps by knowing deeply the role of the structures such

the gubernacular cord and canal and its possible role in tooth eruption process, one can develop in the future mechanisms that allow the interference in the eruptive process due to these structures.

Conclusion The gubernacular cord is the structure coming from the tooth lamina that after undergoing apoptosis has its remnants organized in islets and epithelial cords that lined up leaving the reduced epithelium of the enamel organ towards the oral mucosa. The gubernacular cord in located within a alveolar bone scaffold, so-called the gubernacular canal. The existence of both the gubernacular canal and cord is proved in the permanent dentition with deciduous predecessor; however, their existence is not still proved in the deciduous dentition. Concerning to the permanent dentition without deciduous predecessor, some authors defend their existence and others do not. After the careful analysis of the studies, it is observed that still there are divergences on the function of the gubernacular cord and canal in tooth eruption process. Although the tooth eruption process is a very researched issue in the literature, the role of the gubernacular cord and canal in tooth eruption is still very obscure, and further studies are necessary to clarify its real function. Therefore, it is necessary to deepen the knowledge on this issue, which can interfere on the treatment options for impacted or included teeth as well as on the guided direction of tooth eruption.

References 1. Andreasen JO, Andreasen FM. Textbook and color atlas of traumatic injuries to the teeth. 4. �� ed. Copenhagen: Munksgaard; 2001. 2. Assed S. Odontopediatria – bases científicas para a prática clínica. 1. ed. São Paulo: Artes Médicas; 2005. 3. Bhaskar SN. Histologia e embriologia oral de Orban. 10. ed. São Paulo: Artes Médicas; 1986. 4. Brash JC. Growth of the alveolar bone and its relation to the movements of teeth, including eruption. Int J Orthod. 1928;14:196-223. 5. Cahill DR, Marks Jr SC, Wise GE, Gorski JP. A review and comparison of tooth eruption systems

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used in experimentation – a new proposal on tooth eruption. In: Davidovitch Z (Ed.). Biological mechanisms of tooth eruption and root resorption. Alabama: EBSCO Media; 1988. 6. Cahill DR, Marks Jr SC. Tooth eruption: evidence for the central role of the dental follicle. J Oral Pathol. 1980;9:189-200. 7. Cahill DR. Histological changes in the bony crypt and gubernacular canal of erupting permanent premolars during deciduous premolar exfoliation in beagles. J Dent Res. 1974;53:786. 8. Consolaro A. Reabsorções dentárias nas especialidades clínicas. 3. ed. Dental Press; 2012. 9. Consolaro A. Tracionamento ortodôntico: possíveis consequências nos caninos superiores e dentes adjacentes (parte 1). Dental Press J Orthod. 2010 Jul-Aug;15(4):15-23. 10. Hodson JJ. The gubernaculum dentis. Dent Practit. 1971 Aug;21(12):423-8. 11. Hunter J. The natural history of human teeth. 2. ed. Londres; 1778. 12. Ide F, Mishima K, Kikuchi K, Horie N, Yamachika S, Satomura K et al. Development and growth of adenomatoid odontogenic tumor related to formation and eruption of teeth. Head and Neck Pathol. 2011;5:123-32. 13. Kearns GJ, Smith R. Adenomatoid odontogenic tumour: an unusual cause of gingival swelling in a 3-year-old patient. Br Dent J. 1996;181:380-2. 14. Malassez ML. Sur la structure du gubernaculum dentis et la theorie paradentaire. Compte Soc Biol. 1887. 15. Marks Jr SC, Cahill DR. Experimental study in the dog of the non-active role of the tooth in the eruptive process. Arch Oral Biol. 1984;29:31122. 16. Marks Jr SC, Cahill DR. Regional control by the dental follicle of alterations in alveolar bone metabolism during tooth eruption. J Oral Pathol. 1987;16:164-9. 17. Marks Jr SC, Schroeder HE. Tooth eruption: theories and facts. Anat Rec. 1996;245:374-93. 18. Massler M, Shour I. Studies in tooth development: theories of eruption. American

Journal of Orthodontics and Oral Surgery. 1941;27:552-76. 19. Mizukoshi T, Ojima I, Mizuno Y. A case of adenomatoid odontogenic tumor in a 3-year-old boy. Jpn J Stomatol Soc. 1980;29:605-6. 20. O’Brien C, Bhaskar SN, Brodie AG. Eruptive mechanism and movement in the first molar of the rat. J Dent Res. 1958;37:467-84. 21. Philipsen HP, Reichart PA, Siar CH. An updated clinical and epidemiological profile of the adenomatoid odontogenic tumour: a collaborative retrospective study. J Oral Pathol Med. 2007;36:383-93. 22. Philipsen HP, Reichart PA. The development and fate of epithelial residues after completion of the human odontogenesis with special reference to the origins of epithelial odontogenic neoplasms, hamartomas and cysts. Oral Biosci Med. 2004;3:171-9. 23. Philipsen HP, Samman N, Ormiston IW, Wu PC, Reichart PA. Variants of the adenomatoid odontogenic tumor with a note on tumor origin. J Oral Pathol Med. 1992;21:348-52. 24. Rick GM. Adenomatoid odontogenic tumor. Oral Maxillofac Surg Clin N Am. 2004;16:33354. 25. Sandy JR. Tooth eruption and orthodontic movement. Br Dent J. 1992;172:141-9. 26. Sauk JJ. Genetic disorders involving tooth eruption anomalies. In: Davidovitch Z (Ed.). Biological mechanism of tooth eruption and root resorption. Alabama: EBSCO Media; 1988. 27. Scott JH. The development and function of the dental follicle. Br Dent J. 1948 Nov 5;85(9):193-9. 28. Sutton PR. Tooth eruption and migration theories: can they account for the resence of a 13,000 – year-old mesiodens in the vault of the palate? �� Oral Surg Oral Med Oral Pathol. 1985;59:252-5. 29. Ten Cate AR. Erupção dentária. In: Bhaskar SN. Histologia e embriologia oral de Orban. 8. ed. Santa Maria: Artes Médicas; 19�� 78. 30. Unal T, Cetingul E, Gunbay T. Peripheral adenomatoid odontogenic tumor: birth of a term. J Clin Pediatr Dent. 1995;19:139-42.

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Literature Review Article

Ludwig’s angina: diagnosis and treatment Tiago André Fontoura de Melo1 Tiago Rücker1 Marcos Paulo Dias do Carmo1 Luis Eduardo Duarte Irala2 Alexandre Azevedo Salles2 Corresponding author: Tiago André Fontoura de Melo Rua Lucas de Oliveira, n. 336, apto. 206 – Petrópolis CEP 90440-010 – Porto Alegre – RS – Brasil E-mail: tafmelo@gmail.com 1 2

Department of Dentistry, São Leopoldo Mandic (Porto Alegre Unit) – Porto Alegre – RS – Brazil. Department of Dentistry, Lutheran University of Brazil – Canoas – RS – Brazil.

Received for publication: July 20, 2012. Accepted for publication: November 12, 2012.

Keywords: Ludwig’s angina; diagnosis; therapy.

Abstract Introduction: Ludwig’s angina is often an infection of odontogenic origin affecting the soft tissues of the submandibular, sublingual and submental area. Objective: This review aimed to analyze the existing literature regarding the clinical features, applications for diagnosis and treatment modalities of Ludwig’s angina. Literature review: Because it is a disease of rapid evolution, and if not previously identified, may compromise the patient’s general health and even lead to death. Conclusion: Therefore, it is important to identify the correct diagnosis based on careful and complementary clinical examination, together with an effective drug coverage and early surgical intervention to provide greater control of the patient’s health.

Introduction The odontogenic infection is one of the most difficult clinical cases in Dentistry [12]. According to Bross-Soriano et al. [2], Ludwig’s angina is a type of infection of odontogenic origin in more than 70% of the cases.

For Vasconcellos et al. [22] and Jiménez et al. [9], most of the odontogenic infections are of multimicrobial origin because oral cavity is a medium with a normal resident flora very diversified. Such microbiota, at first, lives harmoniously without causing damages to the host. However, when there is an imbalance between the virulence

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of the organism and the conditions of the patient, the infection tends to develop. Ludwig’s angina is a cellulitis frequently located at the area of the mandibular second and third molars involving the submandibular, sublingual and submental spaces. The apexes of these teeth are located just below the insertion of the mylohyoid muscle and consequently they are in close relationship with the submandibular space r [13, 24]. According to Ugboko et al. [21] and Duprey et al. [3], the Ludwig’s angina is an aggressive infectious pattern of fast dissemination, characterized by a swelling area in the oral f loor, tongue, and submandibular region, which in evolution can lead to the patient’s death. Thus, the aim of this literature review study was to exhibit the characteristics, diagnosis and treatment of the Ludwig’s angina.

Literature review Ludwig’s angina The Ludwig’s angina was firstly described in 1836 by Wilhelm Frederick von Ludwig as a cellulitis of fast evolution involving the region of the submandibular gland which is disseminated through anatomic contiguity without tendency towards abscess formation [19]. Among the main etiologic factors of the angina is the tooth infection, for example, a recent tooth extraction, endodontic and periodontal condition and tooth trauma [13]. However, Gulinelli et al. [7] pointed out other factors such as in the cases of submandibular sialadenitis and parapharyngeal or peritonsillar abscesses. According to Soares et al. [18] and Tavares et al. [20], Ludwig’s angina can show a greater susceptibility to occur in subjects with some degree of systemic compromise, such as Aids, glomerulonephritis, diabetes mellitus and aplastic anemia.

The diagnosis of Ludwig’s angina The diagnosis process of the angina is eminently clinical. In physical examination, the patient normally shows a volume increasing hard to palpation in the sublingual, submandibular region bilaterally and submental region, which can extend in many times to the suprahyoid region, leading to

the elevation of the oral floor and the falling of the tongue towards the posterior direction with risk of obstruction of the airways [4, 10]. According to Nogueira et al. [14] and Saifeldeen and Evans [17], the elevation of the tongue is associated with dysphagia, odynophagia, dysphonia and cyanosis, and in all cases the signs and symptoms characteristic of infectious processes are observed: high fever, malaise, anorexia, tachycardia and chills On the other hand, the volume increasing in oral cavity contributes for the appearance of clinical cases of muscle hypertonia. Because of the fast evolution through the anatomic contiguity between the fascial spaces, the knowledge of the head and neck anatomy is essential to understand the clinical presentation and the possible complications of this infection. Thus, as auxiliary diagnosis method, some conventional radiographic tools can be used. For example, through panoramic x-ray, it is possible to identify possible odontogenic sources. Cervical, profile and posterior-anterior radiographs enable to observe the volume increasing in the soft tissues and any deviation of the trachea [4]. Currently, computed tomography is the most complete resource available because through both the axial/coronal cuts and differentiation of the density of soft tissues, it can provide more accurately the dimensions and localization of the infection areas [16]. According to Fogaça et al. [4], the clinical examination is decisive for the diagnosis of Ludwig’s angina; however, it must be added by a complete anamnesis, image examinations and laboratorial tests. The laboratorial tests, such as hemogram, renal function, culture and antibiogram, are also of vital importance to monitor the general state of the patient and to determine the microorganisms involved to define the antimicrobial therapy.

Treatment approaches for Ludwig’s angina Ludwig’s angina is a severe condition once it has a fast evolution that can put the patient’s life at risk because either the obstruction of the airways secondary to the sublingual and submandibular swelling or at a latest stage of the process, the dissemination of the infection that could lead to mediastinitis, necrotizing fasciitis or sepsis [9]. Thus, the treatment concentrates around four attitudes: maintenance of the airways, incision and

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drainage, antimicrobial therapy and elimination of the infectious site [11]. The maintenance of the airways must be a priority in the treatment of the patient, since the main cause of death at a first moment is the asphyxia due to obstruction. The patients must be rigorously followed-up regarding signs and symptoms of airway obstructions, such as stridor and use of accessory muscle of breathing. The control of the airways can be executed through endotracheal intubation or tracheostomy. The endotracheal intubation is not recommended because of some factors such as the risk of not planned extubation with difficult of re-intubation due to the swelling and possibility of leading the infection to other sites through the rupture of pustules during intubation. Consequently, the tracheostomy has been indicated for the most severe cases and has the risks inherent to any surgical procedure and some difficult of execution due to the loss of the anatomic references because of the swelling [15]. The stage of incision and drainage is indicated for the decompression of the fascial spaces involved and suppuration evacuation. The execution of multiple incisions could be necessary. The location and size of the incision will depend on the anatomic spaces involved by the infection. Normally, it is necessary the separation of the superficial lobes of the submandibular gland and divulsion of the milo-hyoid muscles to decompress the fascial spaces [24]. Freire-Filho et al. [5] �� recommended the surgical drainage associated with the antimicrobial therapy in the cases of Ludwig’s angina completely developed to avoid the previous dissemination to the internal anatomic spaces. Ludwig’s angina is a mixed polymicrobial i n fe c t i on (a e ro b e s a nd a n a e ro b e s), w h i c h normally colonizes the oropharyn x and it is commonly to find streptococcus, staphylococcus, bacteroids, pseudomonas, B melaninogenicus and peptostreptococcus [1, 23]. Thus, the use of the antimicrobial therapy of broad spectrum is primordial to treat the disease [6]. Penicillin G at high doses through endovenous route associated with metronidazole is generally t he a nt i m icrobia l t herapy of choice i n t he emergence treatment of Ludwig’s angina. The use of cephalosporin, erythromycin or clindamycin is an alternative antimicrobial therapy for patients allergic to penicillin, and this antimicrobials should be employed for the specific microorganism present in the infection [14].

Additionally, some authors as Hutchison and James [8] have associated corticosteroids aiming to reduce the swelling of the upper airways.

Conclusion Ludwig’s angina, because of its fast evolution and aggressive power of dissemination, assumes a character of emergence treatment when diagnosed to prevent the swelling of the fascial tissues and the obstruction of the upper airways T he re fore, it i s i mp or t a nt t he c or re c t identification of the diagnosis based on careful clinical and complementary examination together with an effective drug coverage and an early surgical intervention to provide a higher control of the patient’s health.

References 1. Barakate MS, Jensen MJ, Helmli JM, Graham AR. �� Ludwig’s angina: report of a case and review of management issues. Ann Otol Rhinol Laryngol. 2001;110(5):453-6. 2. Bross-Soriano D, Arrieta-Gómez JR, PradoCalleros H, Schimelmitz-Idi J, Jorba-basave S. Management of Ludwig’s angina with small neck incisions: 18 years experience. Otolaryngol Head Neck Surg. 2004;130(6):712-7. 3. Duprey K, Rose J, Fromm C. Ludwig’s angina. Int J Emerg Med. 2010;3(3):201-2. 4. Fogaça PFL, Queiroz EA, Kuramochi MM, Vanti LA, Correa JDH. Angina de Ludwig: uma infecção grave. Rev Port Estomatol Cir Maxilofac. 2006;47(3):156-7. 5. Freire-Filho FWV, Freire EF, Melo MS, Pinheiro DP, Cauby AF. Angina de Ludwig: relato de caso. Rev Bras Cir Periodontia. 2003;1(3):190-6. 6. Furst IM, Ersil P, Caminiti M. A rare complication of tooth abscess – Ludwig’s angina and mediastinitis. J Can Dent Assoc. 2001;67(6):324-7. 7. Gulinelli JL, Esteves JC, Queiroz TP, Ricieri CB, Garcia Júnior IR. Angina de Ludwig. �� ROBRAC. 2007;16(42):1-9. 8. Hutchison IL, James DR. New treatment for Ludwig’s angina. Br J Oral Maxillofac Surg. 1989;27(1):83-4. 9. �� Jiménez Y, Bagán JV, Murillo J, Poveda R. Odontogenic infections. �� Complications. Systemic

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manifestations. �� Med Oral Patol Oral Cir Bucal. 2004;9(143-7):139-43.

17. Saifeldeen K, Evans R. Ludwig’s angina. Emerg �� Med J. 2004;21(2):242-3.

10. Leite Segundo AV, Pinheiro RTA, Inaoka SD, Rocha Neto A, Lago CAP. Angina de Ludwig decorrente de exodontias de molares inferiores: relato de caso clínico. Rev �� Cir Traumatol BucoMaxilo-Fac. 2006;6(4):23-8.

18. Soares LP, Oliveira MG, Beltrão RC, Silva TSN, Beltrão GC. Angina de Ludwig associada à presença de corpo estranho em região sublingual. RFO. 2004;9(2):23-6.

11. Little C. Ludwig’s angina. Dimens Crit Care Nurs. 2004;23(4):153-4. 12. Mariano RC, Melo WM, Mariano LCF, Magnago LR. Tratamento de abscesso dentoalveolar em paciente com alcoolismo. Rev Odontol Univ São Paulo. 2007;19(3):341-6. 13. Martins L, Rocha RCA, Santos KCP, Marcucci M, Costa C, Oliveira JX. Angina de Ludwig – considerações sobre conduta e relato de caso. Rev Inst Ciênc Saúde. 2009;27(4):413-6. 14. Nogueira JSE, Silva CC, Brito KM. Angina de Ludwig: relato de casos clínicos. Rev Bras Cir Periodontia. 2004;2(5):6-14. 15. Potter JK, Herford AS, Ellis E. Tracheotomy versus endotracheal intubation for airway management in deep neck space infections. J �� Oral Maxillofac Surg. 2002;60(4):349-54. 16. Rodrigues AF, Vitral RWF. Aplicação da tomografia computadorizada na Odontologia. Pesq Bras Odontoped Clín Integr. 2007;7(3):317-24.

19. Srirompotong S, Art-Smart T. Ludwig’s angina: a clinical review. �� Eur Arch Otorhinolaryngol. 2003;260(7):401-3. 20. Tavares SSS, Tavares GR, Cavalcanti MOA, Carreira PFS, Cavalcante JR, Paiva MAF. Angina de Ludwig: revisão de literatura e relato de caso. Rev �� Cir Traumat Buco-Maxilo-Facial. 2009;9(3):9-14. 21. Ugboko V, Ndukwe K, Oginni F. Ludwig’s angina: an analysis of sixteen cases in a suburban Nigerian tertiary facility. African �� J Oral Health Sciences. 2005;2(1-2):16-23. 22. Vasconcellos BCE, Cauás M, Albert DGM, Nascimento GJF, Holanda GZ. Disseminação de infecção odontogênica através das fácias cervicais profundas – relato de caso clínico. Rev �� Cir Traumat Buco-Maxilo-Facial. 2002;2(1):21-5. 23. Zaleckas L, Rasteniene R, Rimkuviene J, Seselgvte R. Retrospective analysis of cellulitis of the floor of the mouth. �� Stomatologija. 2010;12(1):23-7. 24. Zanini FD, Stefani E, Santos JC, Perito LS, Kruel NF. Angina de Ludwig: relato de caso e revisão do manejo terapêutico. Arq Catarin Med. 2003;32(4):21-3.

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Literature Review Article

Microbiological aspects of endoperiodontal lesion Cristiane Tokunaga1 Bruno Monguilhott Crozeta1 Mariangela Schmitt Bonato1 Beatriz Serrato Coelho1 Flares Baratto-Filho1 Flávia Sens Fagundes Tomazinho1 Corresponding author: Flávia Sens Fagundes Tomazinho Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: flavia.tomazinho@gmail.com 1

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

Received for publication: October 14, 2012. Accepted for publication: December 12, 2012.

Keywords: microbiology; Endodontic; periodontal pocket.

Abstract Introduction: The endoperiodontal lesion occurs when a tooth undergoing endodontic disease is united to a periodontal lesion with apical progression. Many times, the differential diagnosis between the endodontic and periodontal disease can be of difficult execution and the correct diagnosis and planing of the treatment is of main importance for a good prognosis Objective: To identify the main microorganisms within the lesion of endodontic and periodontal origin and correlate them with the endoperiodontal lesion. Literature review: The search strategy comprised the electronic studies of databases such as PubMed and Cochrane on the microbiology of the endodontic and periodontal systems through employing the following keywords: microbiology, endodontics, periodontal pocket. Results: There were similarities in the endodontic and periodontal microflora. However, the number of microorganisms within the cross infection is limited, including Bacteroides, Eubacteria, Fusobacteria, spirochaetes, Wolinella. The bacterias forming the red complex are closely related to the severity of the periodontal disease and can also participate in the pathogenesis of the periradicular abscesses. Conclusion: There are many communication routes between the periodontium and pulpal tissue, therefore the contamination from um tissue to another can occur, existing a microbiological interrelationship between these tissues.

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Introduction The endodontic and periodontal systems are closely related and the disease occurrence in one of the tissues can be diffused to another [16]. The differential diagnosis between endodontic and periodontal disease can be difficult in many times and the correct diagnosis and treatment planing is of main importance for a good prognosis [16]. There are many communication routes between these two tissues, as follows: the apical foramen, lateral and accessory canals, cavo inter-radicular canal and the dentinal tubules [2, 5, 1]. It is common that the microorganisms reach the pulp through the carious lesion; the bacterias have access to the pulp when the distance between the dentine and the border of the carious lesion is smaller than 0.2 mm [3]. Other route is summarized as the infection of microorganisms from the gingival sulcus which can reach the pulp through the lateral canals or the apical foramen. These accesses are available for the microorganisms during dental prophylaxis and more significantly as the result of the migration of the epithelial insertion in the establishment of the periodontal pockets [11]. The endoperiodontal lesion occurs when a tooth undergoing endodontic disease is united with a periodontal lesion with apical progression [16]. In most of the cases, the clinical and radiographic examinations did not allow the identification of the initial cause of the lesion [5]. For Bender and Seltzer [2], the endoperiodontal syndrome can be started by the pulp or periodontal disease and it expresses both the periodontal and pulpal signs. According to Singh [16], there are four types of endoperiodontal lesion that are classified according to the pathogenicity in: endodontic lesion, periodontal lesion, combined true lesion and iatrogenic lesions. The clinical signs and symptoms can be both the pulpal and periodontal manifestations and it can present pain to horizontal and vertical percussion, suppuration through the gingival sulcus or fistula presence and presence of periodontal pocket. Concerning to the radiographic signs, bone resorptions, forming periodontal pockets and periapical resorptions have been noted [5]. This literature review aimed to identify the main microorganisms within the lesion of endodontic and periodontal origin and correlate them to the true endoperiodontal lesion.

Literature review The pulpal disease can play a significant role in the beginning, progression and healing of the

periodontal disease, as well as the periodontal disease can be the causing agent of the pulpal pathology. This is possible because the pulpal tissue and the periodontium are inter-related. The apical foramen and the accessory root canals can transmit the endodontic infection to the periodontium and vice-versa [14]. The endoperiodontal lesion can be defined as the inflammatory or degenerative involvement of the pulp together with a clinical periodontal pocket in the same tooth [2]. This type of lesion shows a varied pathogenesis and it can vary from simple to relative complex lesion [16]. In most of the lesions involving the pulpal-periodontal complex the bacteriological etiology dictates the clinical course of the disease and, therefore, its treatment planing [19]. By knowing the difficult in the determination and origin of the endoperiodontal lesion, DahlĂŠn [3] conducted a study on the microbiology of the dental abscesses and endoperiodontal lesions. The author affirmed that while a tooth is vital, the lesion should be diagnosed as periodontal lesion and should be treated as that. However, if the tooth is not vital, it can be difficult to determine whether the lesion is primarily endodontic or periodontal. If a periodontal problem exists surrounding the non-vital tooth, one should always consider the hypothesis of an endodontic infection. The microbiology of the combined endoperiodontal lesion reflects the microbiota of the endodontic and periodontal lesion separated. Endoperiodontal lesions cannot have a single microbiological profile. Kerekes and Olsen [7] affirmed that similarities in the endodontic and periodontal microflora of an intact tooth without periapical lesion would indicate that the infection between the root canal and periodontal pocket could occur and that, considering the great variety of microorganisms colonizing the periodontal pocket versus the microorganisms recovered from necrotic pulps of infected teeth, the number of species participating in such cross infection should be limited, including the anaerobes, such as Fusobacterium, Eubacterium, spirochaetes, Selenomonas, Peptostreptococcus and Wolinella. In a study of clinical case report [18], a male patient with 28 years-old, searched the Columbia University because of tooth pain. He was diagnosed with localized aggressive periodontitis. The right maxillary second premolar showed a probing depth between 4 and 10 mm. It did not present either caries or restorations; sensitivity to palpation and percussion were observed. After the endodontic opening, a sample of the subgingival plaque and exudate within the root canals was gathered

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at the first appointment, and at the next two appointments the endodontic samples were gathered. The “checkerboard” DNA analysis revealed the presence of most microorganisms selected for the periodontal pocket as: P. gingivalis, P. intermedia, P. nigrescens, A. actinomycetemcomitans, F. nucleatum, T. denticola, P. micros, S. intermedius, S. noxia, S. oralis, V. parvula, Cochracea, A. naeslundii and S. sanguis. From the sample of the root canal obtained at the first appointment were found: P. gingivalis, P. intermedia, P. nigrescens, Cochracea, A. naeslundii and S. sanguis. None bacterial iDNA was found in the root canal which had not been found in the periodontal pocket. At the second appointment only the DNA of Actinomyces naeslundii was found, and at the third appointment none bacterial DNA was found. At the 10 month appointment, the patient did not exhibit symptoms and there was the radiographically gain of the supporting tissue. Kurihara et al. �� [8], by analyzing microscopically the microbiological and immunologic aspects of the endoperiodontal lesions through the collections of the root canal content and of the deepest portion of the periodontal pocket, observed a significant difference in the microflora of the root canal and periodontal pocket. While the periodontal pocket exhibited a great variety of species, the root canal was limited to gram-positive coccus, including Peptostreptococcus and Streptococcus, or grampositive rods, such as Actinomyces and Rothia. Trope et al. [17] studied in dark field microscope the microflora of the exudate of periodontal and endodontic abscesses of two clinical cases. The analysis with dark field microscope was used to determine the percentage of spirochaetes in the exudate of the periodontal pocket and of the fistula. The authors concluded that the percentage of spirochaetes was mostly found in the lesions of periodontal origin. By studying the etiology of the endoperiodontal infections, Laevi et al. [9] concluded in a literature rev iew study that different clinica l forms of periodont it is were associated w it h different microorganisms and that different endodontic pathogens were associated with different periradicular diseases. In chronic peri-radicular lesions would be involved: Bacterioides, Treponema, Prevotella, Porphyromonas, Fusobacterium, Peptostreptococcus, Streptococcus, Eubacterium, Actinomyces and Campylobacter. In acute periradicular lesions: Porphyromonas, Treponema, F u s o b a c t e r i u m , B a c t e r o i d e s, P re v o t e l l a , Streptococcus and Peptostreptococcus �� were involved.

In adult periodontitis, the microorganisms involved were: Actinobacillus actinomycetencomitans, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Eikenella corrodens, B acteroides forsythu s, Peptost reptococcu s micros, Selenomonas spp, Treponema denticola, spirochaetes and Wolinella recta. Johnson et al. [6] �� analyzed the protheolytic activity expressed Bacillus pumilus isolated from the endodontic and periodontal lesion. The aim was to identify the isolated Bacillus which had been obtained from the root canals demanding endodontic therapy and from periodontal pockets with severe marginal periodontitis and to determine whether these Bacillus showed extracellular protheolytic activity. Also, by using an in vitro test to determine whether any activity could degrade the substrates that would be pathophisiologically relevant regarding to the production of the endodontic and periodontal lesion. The isolated Bacillus was identified by gram confirmation, cellular morphology, and patterns of carbohydrate formation. The screening for the production of the extracellular protheolytic activity for all strains was executed with a proteinase substrate. Extracellular activities were observed. The strains of B. pumillus isolated from endodontic and periodontal lesions exhibited extracellular activities degrading elastin, collagen and other substrates. These activities can be virulence factors contributing for the tissue damage in apical periodontitis and severe marginal periodontitis. In a study conducted by Drucker et al. [4], the endodontic was analyzed regarding to the hypothesis that particular species could be associated with individual signs and symptoms in endodontic infection, such as P. intermedia, P. melaninogenica, Fusobacterium necrophorum and Peptostreptococcus. The statistical analyses emphasized interesting associations between the individual clinical features and individual bacterial species. The authors concluded that combinations of different bacterial groups were not normally associated with the clinical features. However, some combinations were strongly associated with pain, swelling and moist root canal, such as the association of Prevotella with Peptostreptococcus. Rôças et al. [12], aiming to identify the bacterias from the red complex (P. gingivalis, T. denticola and B. forsythus) within root canals, analyzed 50 single-rooted teeth with carious lesions, necrotic pulp and radiographic evidence of peri-radicular bone loss. Of these, 25 teeth exhibited mild pain to percussion, and of these ten were diagnosed with acute peri-radicular abscess. The other cases were

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asymptomatic and the teeth did not show periodontal pockets greater than 4 mm. Thus, they concluded that at least a member of the red complex was found in 66% of the cases (33 out of 50). In a study conducted by Saito et al. [13] P. gingivalis and T. forsythia were quantified through PCR molecular method in real time in primarily endodontic infections. Thirty-two patients requiring endodontic treatment were selected and the teeth used showed necrotic pulp, most due to large carious lesions. There is a coexistence of P. gingivalis and T. forsythia in the cases selected. However, none statistical difference in the relative levels of both microorganisms was observed between the symptomatic and asymptomatic groups. Selcuk and Ozbek [15] analyzed the presence of the red complex in acute peri-radicular abscesses through using the PCR method in real time. Samples were gathered from 32 patients diagnosed with acute peri-radicular abscess, all showing carious lesion, necrotic pulp and radiographic evidence of periapical bone loss. None tooth selected showed periodontal pockets greater than 4 mm. The samples were gathered with the aid of sterile disposable syringe for aspiration of the purulent collection. The DNA of the samples were extracted and analyzed. At least one member of the red complex was found in 84% of the cases. T. denticola, P.gingivalis and T. forsythia were found in 65.6%, 43.7% and 40.6% of the cases, respectively. The red complex was detected in 15.6% of the samples from the purulent collection of the peri-radicular abscesses. In an in vivo study conducted by Lin et al. [10], with the aim of detecting periodontal pathogenic bacterias in persisting periapical lesions, 24 patients who had been referred to surgical endodontic treatment were selected. All patients showed asymptomatic persisting apical periodontitis with absence of fistula and radiographic evidence of periapical bone loss and endodontic treatment. The samples were gathered from the periapical lesions during the surgery with the aid of sterile paper points. The technique of DNA hybridization was carried out with a kit of periodontal pathogenic bacterias. The lesion was also curetted for microbiological analysis. At least one periodontal pathogen was found in seven of 24 cases when DNA hybridization technique was employed. The bacterial species found were: T. denticola (three cases), P. gingivalis (three cases), T. forsythensis (four cases), P. intermedia (one case) and A. actinomycetemcomitans (three cases). None periodontal pathogenic bacteria were found in other samples and periodontal pathogenic bacteria was found in the anaerobial cultures

performed for the same species. Also, none sign and symptom could be correlated with the presence of these bacterias.

Discussion According with the study of Zehnder [18], all bacterias found within root canals were present in the periodontal pocket (P. gingivalis, P. intermedia, P. nigres cen s, A . a ct inomycetencomitan s, Cochracea, A. naeslundii and S. sanguis); however, the periodontal pocket presents a greater variety of microorganisms which was similar to the findings of the study of Kerekes and Olsen [7], who affirmed that existed similarities between the endodontic and periodontal microflora of an intact tooth (Fusobacterium, Eubacterium, spirochetes, Selenomonas, Peptostreptococcus and Wolinella). Notwithstanding, according to Kurihara et al. [8], there was a difference between the microflora from the root canal and from the periodontal pocket because coccus and rods predominate within root canals while spirochaetes and rods predominate within periodontal pockets. Trope et al. [17] determined through dark field microscopy a greater percentage of spirochaetes in lesions of periodontal origin. On the other hand Laevi et al. [9] affirmed that it would exist the presence of spirochaetes within the infected root canal, mainly Treponema denticola, which is a periodontal pathogen. Trope et al. �� [17] claimed that the differentiation between the endodontic and periodontal abscess sometime is quite difficult, mainly when the vitality test cannot be used. These authors studied the microflora of the endodontic and periodontal exudate through using dark field microscopy and observed difference in the counting of spirochaetes. They concluded that in the periodontal abscesses the counting of spirochaetes ranged from 30 and 60%, while in the endodontic abscesses ranged from 0 and 10%. Kurihara �� et al. [8] �� concluded that the periodontal pocket exhibited a great variety of species while the root canal was limited to grampositive coccus, including Peptostreptococcus and Streptococcus, or gram-positive rods, such as Actinomyces and Rothia. The studies conducted by Rôças et al. [12] and Selcuk and Ozbek [15] suggested that the red complex (T. denticola, B. forsythus and P. gingivalis) separately, in pair, or in triplet is closely related with the severity of the periodontal disease. In the study performed by Rôças et al. [12], none sample of purulent collection from peri-radicular abscesses exhibited the red complex; on the other

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hand, in the study of Selcuk and Ozbek [15], the red complex was found in 15.6% of the purulent samples from the peri-radicular abscesses. Selcuk and Ozbek [15] affirmed that the members of the red complex can participate in the pathogenesis of the acute peri-radicular abscess; however, further studies are necessary to elucidate the mechanisms of pathogenicity of the red complex. By analyzing the endodontic microbiota regarding to the association of individual signs with symptoms in the endodontic infection, some species could be correlated. According to Drucker et al. [4], species of Prevotella, found both in root canals and periodontal pockets were associated with pain; Bacteroides, Fusobacterium and Fusobacterium necrophorum were associated with the purulent exudate. Notwithstanding, the combinations of different groups of bacterias are not normally associated with clinical features, as demonstrated in the study of Lin et al. [10], in which none sign and symptom could be related with the presence of the bacterias found. The most important in the treatment is the correct diagnosis, which it is reached by a careful evaluation of the history, examinations and special tests, such as the pulpal vitality test and radiographic examination [2, 16]. The following-up of the clinical signs, symptoms, tests and radiographic findings plays an important role in determining which treatment should be executed first: endodontic or periodontal [2].

Conclusion Based on the literature evaluated, it can be concluded that there are many communication routes between the periodontium and the pulpal tissue and contamination may occur from one tissue to another. The microbiological relationship between the two systems can also be evidenced and the bacterias within periodontal pockets and root canals are similar. A correct diagnosis, considering the inter-relationship between the periodontium and the radicular pulp is essential for the election of the treatment type with a good prognosis.

References 1. Anele JA, Silva BM, Baratto-Filho F, Haragushiku G, Leonardi DP. Prevalência de foraminas e canais acessórios em região de furca e assoalho pulpar e sua influência na etiologia da lesão endoperiodontal. Odonto. �� 2010;18(35):106-16.

2. Bender IB, Seltzer S. The effect of periodontal disease on the pulp. Conference on the Biology of the Human Dental Pulp. 1972;33(3):458-74. 3. Dáhlen G. Microbiology and treatment of dental abscesses and periodontal-endodontic lesions. Periodontol 2000. 2002;28:206-39. 4. Drucker DB, Gomes BPFA, Lilley JD. Role of anaerobic species in endodontic infection. �� Clin Infect Dis. 1997;25(Suppl 2):220-1. 5. Fagundes CF, Storrer CM, Sousa AM, Deliberador TM, Lopes TR. Lesões endoperiodontais – considerações clínicas e microbiológicas. �� RSBO. 2007 Aug;4(2):54-60. 6. Johnson BT, Shaw LN, Nelson DC, Mayo JA. Extracellular proteolytic activities expressed by Bacillus pumilus isolated from endodontic and periodontal lesions. J Med Microbiol. 2008 Feb;57:643-51. 7. Kerekes K, Olsen I. Similarities in the microfloras of root canals and deep periodontal pockets. Endod Dent Traumatol. 1990;6:1-5. 8. Kurihara H, Kobayashi Y, Francisco IA, Isoshima O, Nagai A, Murayama Y. A microbiological and immunological study of endodontic-periodontic lesions. J Endod. 1995 Dec;21(12):617-21. 9. Laevi A, Vrani E, Zuli I. Etiological findings in endodontic-periodontal infections. Bosn J Basic Med Sci. 2004 Feb;4(1):57-61. 10. Lin S, Sela G, Sprecher H. Periopathogenic bacteria in persistent periapical lesions: an in vivo prospective study. J Periodontol. 2007 May;78(5):905-8. 11. Narayanan LL, Vaishnavi C. Endodontic microbiology. J Conserv Dent. 2010 OctDec;13(4):233-9. 12. Rôças IN, Siqueira JF, Santos KRN, Coelho AMA. �� “Red complex” (Bacteroides forsythus, Porphyromonas gingivalis, and Treponema denticola) in endodontic infections: a molecular approach. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;91(4):468-71. 13. Saito D, Coutinho LL, Saito CPB, Tsai SM, Höfling JF, Gonçalves RB. Real-time polymerase chain reaction quantification of Porphyromonas gingivalis and Tannerella forsythia in primary endodontic infections. J Endod. 2009 Nov;35(11):1518-24. 14. Sartori S, Silvestri M, Cattaneo V. Endoperiodontal lesion. J Clin Periodontol. 2002;29:781-3.

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15. Selcuk MO, Ozbek A. Real-time polymerase chain reaction of “red complex” (Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola) in periradicular abscesses. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2010 Nov;110(5):670-4. 16. Singh P. Endo-perio dilemma: a brief review. Dent Res J. 2011;8(1):39-47. 17. Trope M, Rosenberg E, Tronstad L. Darkfield microscopic spirochete count in the differentiation

of endodontic and periodontal abscesses. J Endod. 1992 Feb;18(2):82-6. 18. Zehnder M. Endodontic infection caused by localized aggressive periodontitis: a case report and bacteriologic evaluation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2001;92(4):440-5. 19. Zehnder M, Gold SI, Hasselgren G. Pathologic interactions in pulpal and periodontal tissues. J Clin Periodontol. 2002;29:663-71.

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Case Report Article

Apicoectomy after conventional endodontic treatment failure: case report Lorena Oliveira Pedroche1 Neisiana Barbieri1 Flávia Sens Fagundes Tomazinho1 Luciene Miranda Ulbrich1 Denise Piotto Leonardi1 Stephanie Martins Sicuro1 Corresponding author: Lorena Oliveira Pedroche Rua Alferes Ângelo Sampaio, n. 1.166 CEP 80420-160 – Curitiba – PR – Curitiba E-mail: lo.pedroche@gmail.com 1

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

Received for publication: December 4, 2012. Accepted for publication: December 19, 2012.

Keywords: apicoectomy; retrofilling; apical surgery; endodontic failure; apical barrier.

Abstract Introduction: Paraendodontic surgery is a safe and adequate alternative when teeth are not responding to conventional treatment and endodontic re-treatment. It must only be applied in specific situations. Endodontic treatment failures can be related to: extraradicular infections such as periapical actinomycosis; to foreign body reactions that can be caused by endodontic material extrusion; to endogenous cholesterol crystal accumulation in apical tissues; and unresolved cystic lesion. Paraendodontic surgery comprehends a set of procedures recommended in periapical diseases treatment, when traditional endodontic therapy does not obtain favorable outcomes. Objective: To report a clinical case where an apicoectomy was indicated due to failure in conventional endodontic treatment. Case report and Conclusion: Clinical case report of a tooth with unsatisfying conventional endodontic treatment history, due to lack of treatment in fourth root canal and an unsuccessful apicoectomy, since the lesion and the fistula had persisted. It was chosen to retreat tooth #26 and perform a new apicoectomy in the mesiobuccal root. The treatment was successful due to absence of fistula and painful symptoms and due to periapical bone repair.

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Introduction T rad it iona l endodont ic t re at ment a i ms to eliminate bacteria from root canal system a nd establish effective barriers against root recontamination [9]. To achieve success, cleaning, shaping and filling of the entire root canal system are considered essential steps in endodontic therapy. Failure factors in root canal conventional treatment are frequently related to presence of residual bacteria (persistent infection) or reinfection in a previously disinfected canal (secondary infection) [29]. Endodontic treatment failures can be related to: extraradicular infections such as periapical actinomycosis [31]; to foreign body react ions t hat ca n be caused by endodont ic material extrusion [22]; to endogenous cholesterol crystal accumulation in apical tissues [20]; and unresolved cystic lesion [21]. Thus, success relies on different factors and is verified through clinical and radiographic evaluations during follow up [2022, 29, 31]. Teeth treated in conventional ways, which presents persistent periapical lesion, may have ret reat ment as first t herapeut ic a lternat ive. Especially, accidents during conventional treatment may have negative effect over success, contributing to infection establishment in inaccessible apical areas, requiring surgical intervention [7, 35]. Parendodontic surgery comprehends a set of procedures recommended in periapical diseases treatment, when traditional endodontic therapy does not obtain favorable outcomes. Periapical surgery indications are: root canal obliteration impeding endodontic instrumentation access to apical region; endodontic material apical extrusion impeding radiolucent lesions repair and/or causing clinical symptoms; unsuccessful endodontic treatment and retreatment impossibility due to prothesis; root perforation impeding root canal hermetic sealing. The surgery goal is periapical lesion removal and the apical third sealing, allowing soft and hard tissue regeneration [13, 33, 43]. Parendodontic surgery is a widely studied procedure. According to the meta-analysis of Tsesis et al. [32], paraendodontic surgery success rate is 91.6%, while failure rate is 4.7%. However, its prognosis is influenced by several factors, such as: different surgical procedures and materials, clinical and radiographic evaluation, demography, systemic conditions, local quality factors, for example, the involved teeth and their anatomy, conventional treatment or previous root canal retreatment and restoration quality [5, 8, 32].

Thus, this study aims to report a clinical case of an apicoectomy indicated due to conventional endodontic treatment failure.

Case report Patient, R. J. F., female, Caucasian, 53 yearsold, attended to the Positivo University Dentistry clinic complaining about pain in tooth #26, which presented a history of endodontic treatment and parendodontic surgery (apicoectomy). After clinical and radiographic examination, an unsatisfying endodontic treatment was confirmed in the referred tooth. It was observed that the fourth root canal was not treated. There was a radiographic image suggesting a persistent injury in the periapical region of the mesiobuccal root. The patient was submitted to tomography (figures 1, 2, 3 and 4) and endodontic retreatment was recommended. Ten months after endodontic treatment completed, we observed the reappearance of fistula. Hence, the patient was submitted again to apicoectomy of the mesiobuccal root canal of tooth #26.

Figure 1 â&#x20AC;&#x201C; Preoperative tomography cut showing the palatal root treated

Figure 2 â&#x20AC;&#x201C; Preoperative tomography cut showing the untreated second mesiobuccal root canal

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Figure 3 – Preoperative tomography cut showing second mesiobuccal canal and the persistent lesion

After apical resection, apical surface planing and finishing was performed using multi bladed drills with 5 and 10 blades (KOMET, Santo André, São Paulo, Brazil) and spherical diamond drill size #6 (KOMET, Santo André, São Paulo, Brazil), counterclockwise. The flap was repositioned and fixed with moderated digital pressure and moist gauze. Suturing was made with silk thread 4.0 (Ethicon Johnson, São Paulo, São Paulo, Brazil), performing simple stitches. The postoperative medication prescribed to the patient, was Amoxicillin 500mg every 8 hours for 7 days in therapeutic regime, tramadol 50mg every 6 hours for 3 days and dipyrone 500mg for analgesia every 6 hours during 3 days. To oral hygiene, it was prescribed 0.12% chlorhexidine solution twice a day. The patient returned after 7 days for suture removal. After 7 months, a new computed tomography was requested to follow-up the case (figures 5 and 6). In this examination, the successful of the apicoectomy surgery was proven due to healing and lack of fistula (figures 7, 8 and 9).

Figure 4 – Preoperative tomography cut showing the distal-buccal root canal treated

To perform the surgical procedure, supraperiosteral and subperiosteral anesthetic techniques were adopted. The topical anesthetic used was benzocaine (DFL, Jacarepaguá, Rio de Janeiro, Brazil) and the local anesthetic was 4% articaine with epinephrine 1:100.000 (DFL, Jacarepaguá, Rio de Janeiro, Brazil) because its anesthetic salt has the biggest anesthesia potential and duration. Three anesthetic tubes was used. Neumann’s incision was chosen , starting from the mesial surface of tooth #24 to the distal surface of tooth #27, with the aid of a scalpel blade size #15 (BD, São Paulo, São Paulo, Brazil). The periapical exposure must be satisfactory, to make the injury visible. For this purpose, spherical burs size #6 and #4 were utilized (KOMET, Santo André, São Paulo, Brazil) to access the mesiobuccal root canal. Apical curettage was performed using lucas curette size #85 and #86 (Hu-Friedy, Rio de Janeiro, Rio de Janeiro, Brazil). Then, 2 mm were cut from root apical area, with the aid of size #702 (KOMET, Santo André, São Paulo, Brazil). Surgical site irrigation was executed with water for injection.

Figure 5 – Postoperative following tomography cut showing mesiobuccal root canal after apicoectomy demonstrating lesion absence

Figure 6 – Postoperative following tomography cut showing lesion absence

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Figure 7 – Postoperative following tomography cut showing good healing

Figure 8 – Tomography cut showing good healing

Figure 9 – Tomography cut showing good healing

Discussion Endodontic surgery is a surgical procedure which consists in the excision of pathological

periapical tissue from root surface (including apical accessory canals), and, lastly, canal or canals sealing against pathologic agents, thus reaching the goal of creating the best conditions to the tissue health, regeneration and creation of new tooth structural support. Among the most adopted surgical methods to solve difficulties, accidents and complications of conventional endodontic treatment, are: curettage with apical planing, apicoectomy, apicoectomy with retrofilling, apicoectomy with retroinstrumentation and canal retrofilling and filling simultaneous to surgery [23]. Apicoectomy consists in the surgical removal of tooth apical portion. It can be indicated in several clinical situations: periapical lesions persistent to conventional treatment, perforations, fractured instruments, apical delta removal and external absorption presence [30, 15, 16]. In this clinical case, the chosen treatment was apicoectomy with curettage and planing, because it was found that the filling was well compacted and then it was chosen not to apply retrograde filling. Apical portion was cut in 45º related to tooth long axis [6]. Despite some authors [10] advocate that the larger the cut angle the larger will be dentinal tubules exposure, this inclination degree was needed to allow total root surface exposure, aiming to facilitate the operative procedures. The chosen drill to cut was size #702 tapered bur, which makes cutting easier through all root extension. The apical cut performed at 3 mm leads to the reduction of the lateral canal in 93%, without the need of largest resections, such as 4 mm. However, we chose to just perform the cut at 2 mm, because the tooth had already suffered an earlier apicoectomy. Leonardi et al. [11] stated that several factors can influence apicoectomy success, such as: the root region where the apicoectomy is done; the drill type employed or laser execution, as well as the cut angle. It is important to obtain the cut surface as regular as it can. The apical cut must involve anatomical variations such as the presence of isthmuses and accessory canals, because they act as a reservoir for bacteria and necrotic pulp tissue, which can lead to treatment failure. After apicoectomy, it must be observed whether the filling material is not displaced, using a microscope, because failures may be invisible with unaided eyes. To pathological tissue removal, lucas curettes size #85 and #86 were employed. Periapical curettage aims to remove pathological tissue in a lesion at the apical level of a tooth or foreign bodies

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at periapical region.This must be followed by apical planing. This is necessary, because the cement covering the root apical portion is reabsorved due to periapical lesion [28]. Lastly, it is very important to perform the apical cut surface finishing, which can be made with manual or mechanical instruments, such as steel files and drills, to promote neoformed cement deposition over the sectioned apical dentine [1]. In this clinical case, multibladed drills with 5 and 10 blades and spherical bur size #6 were employed, counterclockwise. This aims to the planing and sealing of dentinal tubules that were exposed to promote repair. To complement dia g nosis a nd g uide t he treatment, pre and postoperative tomography were applied. Computed tomography has better detection performance in periapical lesions [4, 12, 18, 24], root canal system evaluation [13, 17], reabsorption evaluation, and endodontic surgery planning when compared with conventional radiography. It has the advantage of overcoming conventional radiography limitations, such as anatomical image overlaying and geometric distortion [2, 26]. Seven months after, it was perceived the absence of fistula, painful symptoms and periapical bone repair, data that confirms a successful procedure. By the means of computed tomography, the radiolucent image, it is interpreted as fibril healing.

Conclusion The surgical technique applied in this case, apicoectomy, was appropriate. This was proven by both clinical results and computed tomography.

References 1. Bramante CM, Torres M, Morais IG, Bernardineli N, Garcia RB. Avaliação da superfície apical após apicectomia e alisamento com instrumentos manuais e mecanizados. J �� Bras Endod. 2003. 2. Cotton TP, Geisler TM, Holden DT, Schwartz SA, Schindler WG. Endodontic application of cone-beam volumetric tomography. J Endod. 2007;33:1121-32. 3. Eskandarinezhad M, Ghasemi N. Nonsurgical endodontic retreatment of maxillary second molar with two palatal root canals: a case report. J Dent Res Dent Clin Dent Prospect. 2012;6(2):75-8. �� 4. Estrela C, Bueno MR, Leles CR, �� Azevedo B, Azevedo JR�� . Accuracy �� of cone beam computed tomography and panoramic and periapical radiography for detection of apical periodontitis. J Endod. 2008;34:273-9.

5. Friedman S. Considerations and concepts of case selection in the management of post-treatment endodontic disease (treatment failure). Endod Topics. 2002;1:54-78. 6. Gilhe PA, Figdor D, Tyas MJ. Apical dentin permeability end microleakage associated if root end ressection end retrograde filling. J Endod. 1994;20:22-6. 7. Gorni FG, Gagliani MM. The outcome of endodontic retreatment: a 2-yr follow-up. J Endod. 2004;30:1-4. 8. Gutmann JL, Harrison JW. Surgical endodontics. Blackwell Scientific Publications. 1991:362-8. 9. Kim S, Kratchman S. Modern endodontic surgery concepts and practice: a review. J Endod. 2006(32):601-23. 10. Kim S, Pécora G, Rubinsten R, Dorscher-Kim J. Microsurgery in endodontics. �� W.B. Saunders Company; 2001. 172 p. 11. Leonardi DP, Fagundes FS, Haragushiku GA, Tomazinho PH, Baratto-Filho F. Cirurgia parendodôntica: avaliação de diferentes técnicas para a realização da apicectomia. RSBO. 2006. 12. Lofthag-Hansen S, Huumonen S, Grondahl K, Grondahl HG. Limited cone-beam CT and intraoral radiography for the diagnosis of periapical pathology. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007;103:114-9. 13. Matherne RP, Angelopoulos C, Kulild JC, Tira D. Use of cone-beam computed tomography to identify root canal systems in vitro. J Endod. 2008;34:87-9. 14. Martos RR, Lagares DT, Cosano LC, Figallo MRS, Egea JJS, Perez JLG. Evaluation of apical preparations performed with ultrasonicdiamond and stainless steel tips at different intensities usinga scanning electron microscope in endodontic surgery. Med Oral Patol Oral Cir Bucal. 2012. 15. Matsura SJ. A simplified root-end filling technique using silver amalgam. J Mich St Dent Assoc. 1962;44:40-1. 16. Messing JJ. The use of alamgam in endodontics surgery. J Br Endod Soc. 1967;1:34-6. 17. Michetti J, Maret D, Mallet JP, Diemer F. Validation of cone beam computed tomography as a tool to explore root canal anatomy. J Endod. 2010;36:1187-90.

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18. Nakata K, Naitoh M, Izumi M, Inamoto K, Ariji E, Nakamura H. Effectiveness of dental computed tomography in diagnostic imaging of periradicular lesion of each root of a multirooted tooth: a case report. J Endod. 2006;32:583-7.

27. Rigolone M, Pasqualini D, Bianchi L, Berutti �� E, Bianchi SD�� . Vestibular �� surgical access to the palatine root of the superior first molar: ‘‘low-dose cone-beam’’ CT analysis of the pathway and its anatomic variations. J �� Endod. 2003;29:773-5.

19. Nakata K, Naitoh M, Izumi M, �� Ariji E, Nakamura H�� . Evaluation �� of correspondence of dental computed tomography imaging to anatomic observation of external root resorption. J �� Endod. 2009;35:1594-7.

28. Rosa Neto JJ. Estudo em microscopia eletrônica de varredura do ápice radicular e do limite de obturação em dentes portadores de lesão periapical crônica [dissertação]. Faculdade de Odontologia de Araraquara; 1997.

20. Nair PN. Cholesterol as an aetiological agent in endodontic failures: a review. Aust Endod J. 1999;25:19-26.

29. Siqueira Jr JF. Reaction of periradicular tissues to root canal treatment: benefits and drawbacks. �� Endod Topics. 2005;10:123-47.

21. Nair PN, Pajarola G, Schroeder HE. Types and incidence of human periapical lesions obtained with extracted teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1996;81:93-102.

30. Tanzilli JP, Raphael D, Moodnik RM. A comparison of the marginal adaptation of retrograde techniques: a scanning electron microscopic study. O Surg O Med O Pathol. 1980;50:74-80.

22. Nair PN, Sjögren U, Krey G, Sundqvist G. Therapy-resistant foreign body giant cell granuloma at the periapex of a root-filled human tooth. �� J Endod. 1990;16:589-95.

31. Tronstad L, Barnett F, Cervone F. Periapical bacterial plaque in teeth refractory to endodontic treatment. Endod Dent Traumatol. 1990;6:73-7.

23. Orso VA, Sant’ana FM. Cirurgia parendodôntica: quando e como fazer. Rev Fac Odontol Porto Alegre. 2006.

32. Tsesis I, Faivishevsky V, Kfir A, Rosen E. Outcome of surgical endodontic treatment performed by a modern technique: a meta-analysis of literature. J Endod. 2009;35:1505-11.

24. Patel S, Dawood A, Mannocci F, Wilson R, Pitt Ford T. Detection of periapical bone defects in human jaws using cone beam computed tomography and intraoral radiography. Int Endod J. 2009;42:507-15. 25. Patel S, Dawood A, Wilson R, Horner K, Mannocci F. The detection and management of root resorption lesions using cone beam computed tomography: an in vivo investigation. Int Endod J. 2009;42:831-8. 26. Patel S. New dimensions in endodontic imaging: part 2 – cone beam computed tomography. Int �� Endod J. 2009;42:463-75.

33. Von Arx T, Gerber C, Hardt N. Periradicular surgery of molars: a prospective clinical study with a one-year follow-up. Int Endod J. 2001;34:520-5. 34. Zerbinati LPS, Tonietto L, Moraes JFD, Oliveira MG. Assessment of marginal adaptation after apicoectomy and apical sealing with Nd: YAG laser. Photomedicine and Laser Surgery. 2012;30(8):444-50. 35. Wu MK, Dummer PM, Wesselink PR. Consequences of and strategies to deal with residual post-treatment root canal infection. Int Endod J. 2006;39:343-56.

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Case Report Article

Hidden caries’ challenge diagnosis: case report Fernanda Mara de Paiva Bertoli1 Bruno Marques da Silva1 Mariana Dalledone1 Estela Maris Losso1 Corresponding author: Estela Maris Losso Rua Professor Pedro Viriato Parigot de Souza, n. 5.300 – Campo Comprido CEP 81280-330 – Curitiba – PR – Brasil E-mail: lossoem@gmail.com

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

Received for publication: December 1, 2012. Accepted for publication: December 20, 2012.

Keywords: dental caries; bitewing radiograph; diagnosis.

Abstract Introduction: Hidden caries is a term used to describe occlusal dentine caries that is missed on a visual examination, but is large and demineralised enough to be detected by another exam for example, radiographs. Case report: This article reports a case of large dentine caries, which presented as to be a small pit-and-fissure carious lesion on the occlusal surface of the right mandibular permanent first molar in a 10-year-old girl. The treatment included root canal treatment and the sealing of the cavity with composite resin. Conclusion: Careful visual examination, with cleaning and drying of teeth, associated to bitewing radiographs may improve occlusal caries detection. Dentists should examine bitewing radiographs carefully for proximal caries and occlusal demineralization. Radiographs are an effective method of caries diagnosis that can avoid large destruction and allows less invasive treatment.

Introduction Hidden (or occult) caries is a term used to describe a carious lesion seen in dentine through bitewing radiograph that clinically, the occlusal enamel appears healthy or slightly demineralized [3, 10, 11]. They are found in pit or fissure lesions that develop through

tiny enamel defects, progressing under seemingly intact tooth structure. The etiology of hidden caries is still unknown, although some authors believed to be related to the massive use of fluorides, which act in the re-mineralization of occlusal enamel lesions, masking their evolution [3, 4, 9, 13, 15, 17]. However, Hashizume et al. [6] determined the prevalence of

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clinically undetected occlusal dentine caries (hidden caries) in a group of 8-10 year-old children before and after public water supply and dentifrice fluoridation and the results indicated that fluoride is not responsible for the increase in hidden occlusal caries [6]. Many dental practitioners find hidden caries when they start to intervene operatively into what they suspect is a small carious lesion, revealing instead an extended carious lesion that is well into dentin [4]. Dental caries management demands detection of carious lesions at an early stage. In the last 30 years the diagnosis of occlusal caries became more complex and many authors believe that it is possibly due to the increase in the use of fluoride [3, 14]. It has been widely reported that the prevalence of occlusal caries is significantly underestimated by clinical examination alone [6], and there is no “gold standard” in the caries detection [12]. Besides that, complex occlusal fissure morphology, lesion extension and nature of the lesion can lead to misdiagnosis, and frequently dentine lesions are detected on bitewing radiographs [9]. Nevertheless this type of examination is not totally effective and recent studies have been done and other methods, beyond the traditional (visual inspection aided by compressed air, tactile examination with a dental explorer and radiographic examination) in caries detection have been developed. The most current methods and devices are: alternating current impedance spectroscopy technique (ACIST), computer-aided radiography (CAR), dental digital radiography (DDR), digital imaging fiber-optic transillumination (DIFOTI), DIAGNOdent, diagora image plate system (DIPS), diode laser fluorescence (DLF), electrical conductance fixed frequency (ECFF), endoscope filtered fluorescence (EFI), qualitative light-induced laser fluorescence (QLF), visualix highdefinition imager and intra-oral sensor technology

[1, 14, 17]. However, dentists seem to be resistant in adopting these new caries detection and treatment modalities [18].

Case report A 10-year-old girl was referred to pediatric dentistry clinic at Positivo University with complaint of toothache. No relevant medical history was reported during t he a na mnesis. A fter toot h prophylaxis, the clinical examination showed that she was at mixed dentition with caries cavities in the teeth #55 and #65. A large pit and demineralization in the occlusal surface was observed in teeth #36 and #46, although these teeth appeared healthy (figure 1). Bitewing radiograph revealed rizolysis of teeth #55 and #65 and a large radiolucent area in the coronal dentin of the tooth #36 affecting the pulp tissue (figures 2 and 3).

Figure 1 – Clinical view of left mandibular permanent first molar

Figure 2 – Initial bitewing radiograph revealing a carious lesion in the left permanent mandibular first molar and the rizolysis of the primary maxillary right second molar

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Figure 3 – Periapical radiography showing the presence of an extensive radiolucent area affecting the coronal pulp tissue of the left mandibular permanent first molar��

First of all, the patient was anesthetized, and rubber dam isolation was performed. Access to the carious lesion was made using a diamond bur in a high-speed with water irrigation. Dentine caries removal was completed by hand and rotary instruments and the pulp tissue was exposed and removed (figure 4). The root canals were cleaned, dried and filled (figure 5). The coronal cavity was restored with glass ionomer cement (figure 6).

Figure 4 – A) Rubber dam isolation; B) Total caries lesion removed and pulp exposure

Figure 5 – Root canals filled

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Figure 6 – Immediate restoration with glass ionomer cement performed in the left mandibular permanent first molar

Discussion According to the National Institutes of Health, Consensus Development Conference Statement 2001, dental caries is now defined as ‘‘an infectious, communicable disease resulting in the destruction of tooth structure by acid-forming bacteria found in dental plaque, an intraoral biofilm, in the presence of sugar” [5]. The most cariogenic organisms are adherent streptococci such as Streptococcus mutans that starts the process, S. sobrinus, and the bacillus Lactobacillus [10], among other factors as changes in salivary components and exposure to fluorides. In the case reported, the tooth affected was the mandibular permanent first molar, that according to DeJean et al. [3] is the most common tooth affected by hidden caries, because is the first permanent tooth to erupt and it has many pit and fissures [3]. Therefore, one may conclude that adopting the implementation of bitewing radiographs in clinical practice is an important auxiliary tool for diagnosing hidden caries, even in “caries-free” patients [3, 7, 9]. Because of the low attenuation of radiation in the demineralized zone, the typical radiographic appearance of caries is a radiolucent area; however, 40% to 60% of tooth decalcification is required to produce the radiographic image. An initial or incipient lesion may not be seen radiographically [16]. Although the incidence of caries is decreasing, mainly in developed countries, occlusal pit and fissure is still present and it is well accepted that bitewing radiograph has additional benefit in the detection of non-cavitated and small cavitated lesions [7, 11, 14]. It has been postulated that the incidence of hidden caries is increasing due to the use of fluoride,

which reduces or makes absent the demineralization characteristic of the carious lesion of enamel, hidding the caries progression into dentine [3, 4, 14]. The detection rate of such lesions will depend upon the prevalence of caries in the population and the frequency with which bitewing radiographic examinations are performed. In the present case, radiographs were properly used for the diagnosis of hidden caries, which seemed clinically like a slight enamel demineralization around de occlusal pit, as related by Candido et al. [2]. Early and accurate diagnosis of occlusal caries enables successful prevention and minimal restorative techniques intervention, ending the common evolution from occlusal restorations to cusp restorations, crowns, and Endodontics or even tooth loss [2].

References 1. Barnes CM. Dental hygiene participation in managing incipiente and hidden caries. Dent Clin N Am. 2005 Oct;49(4):795-813. 2. Candido LA, Oliveira TM, Sakai VT, Silva SMB, Abdo RCC, Machado MAAM. �� The importance of radiographic diagnosis of incipiente occlusal caries – case report. Odontologia �� Clín Científ. 2008 Jan/ Mar;7(1):87-91. 3. DeJean KS, Caldas LD, Gois DN, Souza CS. Hidden �� dental caries: a study of diagnosis and prevalence. ClipeOdonto – UNITAU. 2009 Dec;1(1):7-13. 4. Dennison JB, Hamilton JC. Treatment decisions and conservation of tooth structure. Dent Clin N Am. 2005 Oct;49(4):825-45.

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5. Diagnosis and Management of Dental Caries Throughout Life. NIH Consensus Statement Online. 2001;18(1):1-24.

primary dentition with and without bitewing radiography. Australian Dental Journal. 2009 Mar;54(1):23-30.

6. Hashizume LN, Mathias TC, Cibils DM, Maltz M. Effect of the widespread use of fluorides on the occurrence of hidden caries in children. Int J Paediatr Dent. 2012 Feb;20:1-5.

12. Nyvad B. Diagnosis versus detection of caries. Caries Res. 2004 May-Jun;38(3):192-8.

7. Hopcraft MS, Morgan MV. Comparison of radiographic and clinical diagnosis of approximal and occlusal dental caries in young adult population. Community Dent Oral Epidemiol. 2005 Jun;33(3):212-8. 8. Latta MA, Naughton WT. Bonding and curing considerations for incipient and hidden caries. Dent Clin N Am. 2005 Oct;49(4):889-904. 9. Mestriner SF, Pardini LC, Mestriner WJ. Impact of the bitewing radiography exam inclusion on the prevalence of dental caries in 12-year-old students in the city of Franca, São Paulo, Brazil. J �� Appl Oral Sci. 2006 Jun;14(3):167-71.

13. Pereira RF, Gomes RH, Volpato LE. Occult �� caries lesion: restoration using the occlusal matrix technique. �� Rev INPEO de Odontologia. 2008 Jul;2(1):1-76. 14. Strassler HE, Porter J, Serio CL. �� Contemporary treatment of incipient caries and the rationale for conservative operative techniques. Dent Clin N Am. 2005 Oct;49(4):867-87. 15. Thompson VP, Kaim JM. Nonsurgical treatment of incipient and hidden caries. Dent Clin N Am. 2005 Oct;49(4):905-21. 16. Yang J, Dutra V. Utility of radiology, laser fluorescence, and transillumination. Dent Clin N Am. 2005 Oct;49(4):739-52.

10. Mount JH. Defining, classifying, and placing incipient caries lesions in perspective. Dent Clin N Am. 2005 Oct;49(4):701-23.

17. Zanardo A, Rego MA. Occlusal caries diagnosis in permanent teeth: an in vitro study. Ciênc Odontol Bras. 2003 Jul-Sep;6(3):50-7.

11. Newman B, Seow WK, Kazoullis S, Ford D, Holcombe T. Clinical detection of caries in the

18. Zandoná AF, Zero DT. Diagnostic tools for early caries detection. JADA. 2006 Dec;137(12):1675-84.

ISSN: Electronic version: 1984-5685 RSBO. 2013 Apr-Jun;10(2):193-7

Case Report Article

Importance of the comparative anatomy in Forensic Anthropology – case report Rhonan Ferreira da Silva1, 2, 3 Solon Diego Santos Carvalho Mendes3 Décio Ernesto de Azevedo Marinho1, 3 Ademir Franco do Rosário Júnior4 Marco Aurélio Guimarães5 Corresponding author: Rhonan Ferreira da Silva Instituto Médico-Legal Avenida Atílio Correa Lima, n. 1.223 – Cidade Jardim CEP 74425-030 – Goiânia – GO – Brasil E-mail: rhonanfs@terra.com.br. 1 2 3 4 5

School of Dentistry, Federal University of Goiás – Goiânia – GO – Brazil. School of Dentistry, Campus Flamboyant, Paulista University – Goiânia – GO – Brazil. Institute of Legal Medical of Goiânia, Forensic Police of Goiás – Goiânia – GO – Brazil. Forensic Dentistry, Department of Oral Health Sciences, Katholieke Universiteit Leuven – Leuven – Belgium. School of Medicine of Ribeirão Preto, University of São Paulo – Ribeirão Preto – SP – Brazil.

Received for publication: December 2, 2012. Accepted for publication: December 20, 2012.

Keywords: Forensic Dentistry; Forensic Anthropology; comparative anatomy.

Abstract Introduction: In forensic sciences, reconstructive victim profile is a commonly used procedure to provide individual data in cases of complex human identifications. In forensic anthropology, valuable data are obtained from skeletal and dental analysis such as gender, age, ancestry, stature, and differentiation between human and nonhuman remains. Objective: To highlight the relevance of comparative anatomy analysis to differentiate human and non-human remains. Case report: Four bone fragments and one tooth were found on a potential crime scene, and were submitted to forensic examinations. The examinations revealed non-human anthropological remains. Additionally, the analyzed bones and tooth were classified as animal remains, specifically from a domestic dog (Canis lupus familiares). Conclusion: In this context, it is relevant to be trained and aware of the usefulness of comparative anatomy into the forensic anthropology routine in order to perform complete and accurate examinations.

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Introduction

Case report

The Forensic Anthropology can be conceptualized as the practical application to Law of a set of knowledge of general Anthropolog y aiming to respond to both the questions regarding to the forensic identity and the judiciary or police identity [4]. In other words, it is the branch of the forensic sciences that studies the anatomic particularities of the men, dead or alive, intact or fragmented aiming to determine the biological profile (species, age, gender, ancestry, manual skills, and height), as well as the cause and nature of the death to solve legal questions [14]. In the alive person, the Forensic Anthropology can be adequately applied in investigations of the gender determination in complex cases (such is the presence of ambiguous genitalia) [5] an in the investigations of age estimative, such as in the cases of doubts regarding to the age of criminal responsibility of subjects committing crimes [12]. In dead individuals, the anthropological examinations have been performed to determine the biological profile in skeletonized, decomposing, mutilated and charred bodies attempting to decrease an universe of search for human identification in addition to contribute for the determination of the causa mortis, identification of the instrument or modus operandi or other circumstances that caused the death. Concerning to the determination of the biological profile, there is specifically a greater difficult in responding the following question whether “the material is or is not of human origin” when the sample analyzed is fragmented, damage or incomplete. For this purpose, and depending on the type of the material referred (bone, tooth, blood, hair etc.), a macroscopic or microscopic analysis can be attempted to search either the normal characteristics of the human being or any animal species [9-11], or even the execution of the chemical reactions in the sense of characterizing that determined material is or is not human [2, 5, 13]. Generally, teeth and bones are the non-human materials most referred to the laboratories of the Forensic Anthropology and the use of comparative anatomy techniques can be extremely useful for the inclusion or exclusion of a material of human specimen. In this context, the present study aimed to report a forensic case in which it is demonstrated the importance of the application of techniques of comparative anatomy for the differentiation between human and non-human remains, when a biological material of bone and dental nature is found in a presumed crime scene of concealment of corpse.

In the middle of 2005, fragments of a skeleton were found at a desert place which initially would be of a missing person victim of homicide. Aiming to know whether this aforementioned material was of human origin (from Homo sapiens species), the material was referred for forensic examination in the Section of the Forensic Anthropology and Forensic Dentistry of the Forensic Institute of Goiânia (GO). In a preliminary analysis, it was possible to observe that the material was composed by five pieces: four bones and one tooth (figure 1). Three bone pieces were long, compatible with the upper or lower extremity showing no fractured points. The smallest bone was fractured with irregular aspect compatible with a vertebra fragment. The tooth referred had one root with a curve crown/root and a flattening at mesial-distal direction.

Figure 1 – Material sent for forensic examination: four bone fragments and one tooth

By analyzing the morpholog y of the bone pieces, especially the epiphyses and the areas of muscle origin and insertion associated to their dimensions (reduced) and lack of evident fusion points (growth centers) between the epiphyses and diaphysis, it was concluded that this material was of non-human origin, belonging to an adult animal of medium size. By comparing the tooth referred for examination with a permanent mandibular human canine tooth, it was possible to observe that the tooth did not

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have a similar shape and dimensions with a human canine tooth, fact that also excluded this material from human origin (figure 2).

Figure 2 – Tooth sent for analysis (A) compared with a human canine tooth (B)

Attempting to determine the animal specimen from which the pieces had been referred for examination, they were assessed in the Department of Morphology of the Federal University of Goiás. By comparing the anatomic characteristics of both the bones and the tooth, it was possible to observe the compatibility between the forensic material with bones (tibia and femur) and canine tooth of a domestic dog (Canis lupus familiares), of medium size (figures 3 and 4).

Figure 3 – Comparison between the examined femur (A) and the tibia (B) with the skeleton of the domestic dog

Figure 4 – Tooth sent for analysis compared with a canine tooth of a domestic dog

Discussion The Forensic A nt h ropolog y t h roug h t he knowledge of the normal anatomic particularities associated with other forensic techniques normally is capable of providing adequate explanations for the police procedures when there are doubts regarding the origin of certain mortal remains or biological vestiges found in crime scenes. Considering the scope of the police investigation in which normally the search for a missing person is being carried out, the most important question to be answered is: Is the material found in the crime scene of human origin? The response “yes” or “no” usually is enough for the investigations. However, some authors of Forensic Medicine and Anthropology use the following expressions: diagnosis of the species [7], investigation of the animal species [2, 5, 13] or determination of the species [3]. Such expressions, when disposed in the form of question to be responded at forensic environment, need a more detailed analysis of the material referred because it is understood that the identification of the animal species is relevant, even when it is a non-human material, as in the cases of environmental crimes involving the death of animals. In the case present here, it was possible to identify with absolute certainty that the material examined was not of human origin. Also, the morphological compatibility between the material referred and the part of the skeleton of a dog of medium size was obtained.

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However, it was not possible to establish through comparative anatomy, the race of the animal (subspecies). By comparing the taxonomic classifications of the man (Homo sapiens) and domestic dog (Canis lupus familiaris), it is emphasized the necessity in most of the police investigations of the differentiation between human and non-human material because both species analyzed are only coincident up to their class (Mammalia) (table I). Depending on the species to be examined, such

as some primates (gorillas and chimpanzees), the deepening of the forensic examinations should really be made possible, once there is morphological similarities in some bones and mainly in genetics between the species when DNA examination is conducted. Genetically, the similarities can be of such magnitude that Wildman et al. [15] proposed that the chimpanzees (Pan troglodytes) had been included in genus Homo, due to the coincidence of about 99.4% of the genomes from this species with the genomes of the men.

Table I – Comparison of the taxonomy classification among the domestic dog, gorillas and chimpanzees and men

Domain Kingdom Phylum Class Order Family Subfamily Tribe Genus Species Subspecies

Domestic dog Eukaryota Animalia Chordata Mammalia Carnivora Canidae Caninae Canini Canis Canis lupus Canis lupus familiaris

Gorillas Eukaryota Animalia Chordata Mammalia Primates Hominidae Homininae Gorillini Gorilla Gorilla gorilla Gorilla gorila gorilla

Thus, the use of the comparative anatomy with forensic purpose is an important stage for the differentiation of human bone/tooth material from non-human biological evidences because the dentition and the human skeleton has unique characteristics of its relief and constitution that in most times makes possible to affirm absolutely that a given piece studied is or is not of the human species. Additionally, the comparative material is very practical, of low cost for the forensic services, normally requiring a multi-institutional cooperation for the access of the collections of animal anatomy available in the higher education institutions. In cases of absence of institutional partnership, atlas of bone animal anatomy can be employed [1, 6, 8], even with little literature on the recording of the anatomic particularities of animals raised or natives of Brazil.

Chimpanzees Eukaryota Animalia Chordata Mammalia Primates Hominidae Homininae Hominini Pan Pan troglodytes Pan troglodytes troglodytes

Men Eukaryota Animalia Chordata Mammalia Primates Hominidae Homininae Hominini Homo Homo sapiens –

examination and the differentiation between human and non-human bone/dental remains, it is fundamental that the Forensic Anthropology experts master the knowledge on human anatomy and had the possibility of proper access to the departments of animal morphology or anatomy to investigation the species, when necessary.

Acknowledgment We thank the professors of the Department of Morphology of the Federal University of Goiás by the support in the conduction of this present forensic case

References

Conclusion

1. Adams BJ, Crabtree PJ. Comparative �� skeletal anatomy – a photographic atlas for medical examiners, coroners, forensic anthropologists and archeologists. Totowa: �� Humana Press; 2008.

Considering the potential of forensic information that can be obtained in the comparative anatomic

2. Arbenz GO. Medicina legal e antropologia forense. Rio de Janeiro: Atheneu; 1988.

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3. Couto RC. Perícias em Medicina e Odontologia legal. Rio de Janeiro: Medbook; 2011. 4. Croce D, Croce Júnior D. Manual de Medicina legal. 8. ed. São Paulo: Saraiva; 2012. 5. França GV. Medicina legal. 9. ed. Rio de Janeiro: Guanabara Koogan; 2011. 6. France DL. Human and nonhuman bone identification – a color atlas. Boca Raton: CRC Press; 2009. 7. Hércules HC. Medicina legal – texto e atlas. Rio �� de Janeiro: Atheneu; 2005. 8. Hillson S. Mammal bones and teeth – an introduction guide to methods of identification. London: Left Coast Press; 1999. 9. Martiniaková M, Grosskopf B, Omelka R, Vondrakova M, Bauerova M. Differences among species in compact bone tissue microstructure of mammalian skeleton: use of a discriminant function analysis for species identification. J Forensic Sci. 2006;51(6):1235-9. 10. Oliveira RN, Silva RHA, Boldrini SC. �� Pericial analysis of a dental element found inside food used for human consumption. �� J Forensic Legal Med. 2008;15:269-73.

11. Ramalho AS, Daruge E, Cruz BV, Francesquini MA, Pereira SDR, Francesquini Junior L et al. La importancia pericial del estudio comparativo histomorfológico del hueso humano y de otros géneros. Rev ADM. 2003;60(5):173-9. 12. Silva RF, Marinho DEA, Botelho TL, Caria PHF, Berzin F, Daruge Júnior E. Estimativa da idade por meio de análise radiográfica dos dentes e da articulação do punho: relato de caso pericial. Arq Cent Estud Curso Odontol Univ Fed Minas Gerais. 2008;44(2):93-8. 13. Vanrell JP. Odontologia legal e Antropologia forense. 2. ed. Rio de Janeiro: Guanabara Koogan; 2009. 14. Velho JA, Geiser GC, Espíndula A. Ciências forenses – uma introdução às principais áreas da criminalística moderna. Campinas: Millennium; 2012. 15. Wildman DE, Uddin M, Liu G, Grossman LI, Goodman M. Implications of natural selection in shaping 99.4% nonsynonymous DNA identity between humans and chimpanzees: enlarging genus Homo. Proc Natl Acad Sci USA. 2003;100(12):7181-8.

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