ACTA ODONTOLOGICALATINOAMERICANAVol. 31 Nº 2 2018

ISSN 1852-4834 on line versionversión electrónica

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Scientific EditorsEditores CientíficosMaría E. ItoizRicardo Macchi(Universidad de Buenos Aires, Argentina)

Associate EditorsEditores AsociadosAngela M. Ubios(Universidad de Buenos Aires, Argentina)Amanda E. Schwint(Comisión Nacional de Energía Atómica, Argentina)

Assistant EditorsEditores AsistentesPatricia MandalunisSandra J. Renou(Universidad de Buenos Aires, Argentina)

Technical and Scientific AdvisorsAsesores Técnico­CientíficosLilian Jara TracchiaLuciana M. SánchezTammy SteimetzDelia Takara(Universidad de Buenos Aires, Argentina)

Editorial BoardMesa EditorialEnri S. Borda (Universidad de Buenos Aires, Argentina)

Noemí E. Bordoni (Universidad de Buenos Aires, Argentina)

Fermín A. Carranza (University of California, Los Angeles, USA)

José Carlos Elgoyhen (Universidad del Salvador, Argentina)

Andrea Kaplan (Universidad de Buenos Aires, Argentina)

Andrés J.P. Klein­Szanto (Fox Chase Cancer Center, Philadelphia, USA)

Susana Piovano (Universidad de Buenos Aires, Argentina)

Guillermo Raiden (Universidad Nacional de Tucumán, Argentina)

Sigmar de Mello Rode (Universidade Estadual Paulista,Brazil)

Hugo Romanelli (Universidad Maimónides, Argentina)

Cassiano K. Rösing (Federal University of Rio Grande do Sul, Brazil)

PublisherProducción Gráfica y PublicitariaImageGraf / e­mail: info@imagegraf.com.ar

Acta Odontológica Latinoamericana is the officialpublication of the Argentine Division of the InternationalAssociation for Dental Research.

Revista de edición argentina inscripta en el RegistroNacional de la Propiedad Intelectual bajo el N° 284335.Todos los derechos reservados.Copyright by:ACTA ODONTOLOGICA LATINOAMERICANAwww.actaodontologicalat.com

ACTA ODONTOLÓGICA LATINOAMERICANAAn International Journal of Applied and Basic Dental Research

POLÍTICA EDITORIAL

El objetivo de Acta OdontológicaLatinoamericana (AOL) es ofrecer a lacomunidad científica un medio adecuadopara la difusión internacional de los tra­bajos de investigación, realizados prefe­rentemente en Latinoamérica, dentro delcampo odontológico y áreas estrechamen­te relacionadas. Publicará trabajos origi­nales de investigación básica, clínica yepidemiológica, tanto del campo biológi­co como del área de materiales dentales ytécnicas especiales. La publicación de tra­bajos clínicos será considerada siempreque tengan contenido original y no seanmeras presentaciones de casos o series. Enprincipio, no se aceptarán trabajos de revi­sión bibliográfica, si bien los editorespodrán solicitar revisiones de temas departicular interés. Las ComunicacionesBreves, dentro del área de interés de AOL,serán consideradas para su publicación.Solamente se aceptarán trabajos no publi­cados anteriormente, los cuales no podránser luego publicados en otro medio sinexpreso consen timiento de los editores.

Dos revisores, seleccionados por lamesa editorial dentro de especialistas encada tema, harán el estudio crítico de losmanuscritos presentados, a fin de lograr elmejor nivel posible del contenido científi­co de la revista.

Para facilitar la difusión internacional,se publicarán los trabajos escritos eninglés, con un resumen en castellano o por­tugués. La revista publicará, dentro de laslimitaciones presupuestarias, toda infor­mación considerada de interés que se lehaga llegar relativa a actividades conexasa la investigación odontológica del árealatinoamericana.

EDITORIAL POLICY

Although Acta Odontológica Lati ­noamericana (AOL) will accept originalpapers from around the world, the princi­pal aim of this journal is to be an instrumentof communication for and among LatinAmerican investigators in the field of den­tal research and closely related areas.

AOL will be devoted to original articlesdealing with basic, clinic and epidemio­logical research in biological areas or thoseconnected with dental materials and/orspecial techniques.

Clinical papers will be published aslong as their content is original and notrestricted to the presentation of singlecases or series.

Bibliographic reviews on subjects ofspecial interest will only be published byspecial request of the journal.

Short communications which fall with­in the scope of the journal may also besubmitted. Submission of a paper to thejournal will be taken to imply that it pres­ents original unpublished work, not underconsideration for publication elsewhere.

By submitting a manuscript the authorsagree that the copyright for their article istransferred to the publisher if and whenthe article is accepted for publication. Toachieve the highest possible standard inscientific content, all articles will be ref­ereed by two specialists appointed by theEditorial Board. To favour internationaldiffusion of the journal, articles will bepublished in English with an abstract inSpanish or Portuguese.

The journal will publish, within budgetlimitations, any data of interest in fieldsconnected with basic or clinical odonto­logical research in the Latin America area.

Este número se terminó de editar el mes de Octubre de 2018

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CONTENTS / ÍNDICE

Different expression patterns of carbonic anhydrase IX in oral lichen planus and leukoplakiaDiferentes patrones de expresión de la anhidrasa carbónica IX en liquen plano bucal y leucoplasiaMiguel Ángel Pérez, Mariana Silvia Gandolfo, Patricia Masquijo Bisio, María Luisa Paparella, María Elina Itoiz .................................................................................................................................................. 77

Parental perceptions of impact of oral disorders on Colombian schoolchildren’s oral health­ related quality of lifePercepción de los padres del impacto de desordenes orales de escolares colombianos sobre la calidad de vida relacionada con la salud oralShyrley Díaz, María Paula Vélez, Luz Mariela Martínez, Ketty Ramos, Marcelo Boneckër, Saul Martins Paiva, Jenny Abanto .............................................................................................................................. 82

Quality of life related to complete dentureQualidade de vida relacionada à prótese totalAnne C. Alves, Renata V. A. Cavalcanti, Patrícia S. Calderon, Leandro Pernambuco, João C. Alchieri...................................................................................................................................................................... 91

Omega­3 and Omega­6 salivary fatty acids as markers of dietary fat quality: A cross­sectional study in ArgentinaÁcidos grasos salivales Omega­3 y Omega­6 como marcadores de la calidad lipídica de la dieta: un estudio de corte transversal en ArgentinaMaría D. Defagó, Nilda R. Perovic, Mirta A. Valentich, Gastón Repossi, Adriana B. Actis ........................................................................................................................................................................................ 97

Increased interpremolar development with self­ligating orthodontics. A prospective randomized clinical trialMayor desarrollo interpremolar con ortodoncia de autoligado. Estudio clinico prospectivo randomizadoMaría E. Mateu, Sandra Benítez­Rogé, Marina Iglesias, Diana Calabrese, María Lumi, Marisa Solla, Pedro Hecht, Alejandra Folco ...................................................................................................................... 104

Evaluation of apoptosis and osteopontin expression in osteocytes exposed to orthodontic forces of different magnitudesEvaluación de la apoptosis y la expresión de osteopontina en los osteocitos luego de la aplicación de fuerzas ortodóncicas de diferentes magnitudesCarola B. Bozal, Luciana M. Sánchez, Patricia M. Mandalunis, Angela M.Ubios ...................................................................................................................................................................................................... 110

Self­medication in patients seeking care in a dental emergency serviceAutomedicación en pacientes que concurren a un servicio de guardia odontológicaFederico Stolbizer, Daniel F. Roscher, Maria M. Andrada, Lautaro Faes, Carla Arias, Cecilia Siragusa, Silvio Prada, Jonathan Saiegh, Daniel Rodríguez, Ariel Gualtieri, Carlos F. Mendez.................................................................................................................................................................................................................................................... 117

Acta Odontol. Latinoam. 2018 ISSN 1852-4834 Vol. 31 Nº 2/ 2018

ACTA ODONTOLÓGICA LATINOAMERICANAAn International Journal of Applied and Basic Dental Research

Contact us ­ Contactos: Cátedra de Anatomía Patológica, Facultad de Odontología, Universidad de Buenos Aires.M.T. de Alvear 2142­ (C1122AAH) Buenos Aires, Argentina.http://www.actaodontologicalat.com/contacto.htmlactaodontologicalat@gmail.com

ACTA ODONTOLÓGICA LATINOAMERICANA

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Diferentes patrones de expresión de la anhidrasa carbónica IX en liquen plano bucal y leucoplasia

Different expression patterns of carbonic anhydrase IX in oral lichen planus and leukoplakia

Miguel Ángel Pérez1, Mariana Silvia Gandolfo2, Patricia Masquijo Bisio1,2, María Luisa Paparella1, María Elina Itoiz1

1 Universidad de Buenos Aires, Facultad de Odontología, Cátedra de Anatomía Patológica, Argentina

2 Universidad de Buenos Aires, Facultad de Odontología, Cátedra de Clínica Estomatológica, Argentina

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ABSTRACTTumor hypoxia is an important indicator of cancer prognosis.Among the different genes that are up­regulated by hypoxia iscarbonic anhydrase IX, which combines carbon dioxide andwater to form bicarbonate and hydrogen. Although expression ofthis enzyme is very low in normal tissues, carbonic anhydrase IXis overexpressed in several types of cancer. The aim of the presentwork was to analyze carbonic anhydrase IX expression in the twomost frequent potentially malignant oral disorders: oral lichenplanus and oral leukoplakia. Immunohistochemical analysis oforal lichen planus and oral leukoplakia biopsies was performedusing anti­carbonic anhydrase IX antibody. Samples of normalmucosa served as controls. Statistical analysis was performed byFischer’s exact test. The enzyme was detected in the epithelium of

both lesions. The staining was more intense in the basal layer anddecreased towards the surface in oral lichen planus. Conversely,the most intense reaction was observed in the superficial layersin leukoplakia, and staining intensity decreased towards the basalmembrane. No carbonic anhydrase IX expression was seen innormal mucosa samples. Carbon anhydrase IX expression inlichen and leukoplakia epithelia shows that hypoxia plays a rolein the pathogenesis of both lesions. The different distributionpatterns provides further evidence of the different biologicalbehavior of these two entities, which under certain circumstancescan have similar clinical and histological features.

Key words: Carbonic Anhydrase IX; Oral Lichen Planus; OralLeukoplakia.

RESUMENLa hipoxia tumoral es un importante indicador de pronóstico encáncer. Entre los distintos genes que son activados por hipoxia,uno de los principales es la anhidrasa carbónica IX (CAIX), quecombina CO2 con H2O para sintetizar HCO3

­ y H+. Aunque laexpresión de esta enzima es muy baja en tejidos normales, se sobreexpresa en varios tipos de cáncer. La finalidad delpresente trabajo fue analizar la expresión de CAIX en las doslesiones orales potencialmente malignas más frecuentes: el liquen plano y la leucoplasia. Se utilizó una técnica inmuno ­histoquímica con un anticuerpo específico contra CAIX, enbiopsias de liquen plano oral y leucoplasia oral. Se utilizaronmucosas normales como controles. Se realizaron análisisestadísticos utilizando test exacto de Fischer. La identificación

de la enzima fue positiva en el epitelio de ambas lesiones. En loslíquenes la reacción es más intensa en los estratos basales,disminuyendo hacia la superficie. Inversamente, las leucoplasiasmostraron marcación más intensa en estratos superficiales, condisminución hacia la membrana basal. Las mucosas normalesresultaron negativas. La expresión de CAIX en el epitelio delíquenes y leucoplasias indica que la hipoxia juega algún papelen la patogenia de ambas lesiones. El diferente patrón dedistribución es una evidencia más del diferente comportamientobiológico de dos entidades las cuales en ciertas circunstanciaspueden manifestar cuadros clínicos e histológicos semejantes.

Palabras clave: Anhidrasa Carbónica IX; Liquen Plano Oral;Leucoplasia Oral.

INTRODUCTIONTumor hypoxia is an important indicator of cancerprognosis, since it is associated with aggressivegrowth, metastasis and poor response to treatment1.Genes that are up­regulated by microenvironmentalhypoxia through activation of Hypoxia Inducible

Factor­1 (HIF­1) include glucose transporters,glycolytic enzymes, and angiogenic growth factors2.Carbonic anhydrases (CAs), a family of metalloen ­zymes that require Zn2+ as a cofactor, are one of themost important groups. Sixteen isozymes that differin their subcellular localization, catalytic activity

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78 Miguel Ángel Pérez, et al.

and susceptibility to different classes of inhibitorshave been identified. Some of these isozymes are cytosolic (I, II, III, VII and XIII), others aremembrane bound (IV, IX, XII and XIV), two aremitochondrial (VA and VB), and one is secreted insaliva (VI). There are also three acatalytic forms,called CA­related proteins (CARPs): CARP VIII,X and XI. In humans, CAs are present in varioustissues, including the gastrointestinal tract, thenervous system, kidneys, lungs, skin and eyes,among others3, but their expression in these tissuesis very low. The most reactive site was found in thebasolateral surfaces of the crypt enterocytes induodenum, jejunum and ileal mucosa4. These enzymes catalyze the reversible hydration ofcarbon dioxide to bicarbonate

CO2 + H2O ↔ HCO3­ + H+

Membrane­bound CAs, including carbonic anhydraseIX (CAIX), have an extracellular active site and canprovide H+ or HCO3

­ ions formed during catalyticturnover for various physiological processes, such asextracellular acidification. Several isoforms of CAare thus involved in essential cell processes, like res­piration and pH regulation, electrolyte balance, boneresorption, calcification and biosynthetic reactionsrequiring HCO3

­ as a substrate5. CAIX specifically, isimportant because together with its isoform XII, it isinvolved in malignant transfor mation processes6.CAIX is overexpressed in a variety of solid malignanttumors, including renal carcinoma and particularlyclear cell adenocarcinoma7­8, cervical carcinoma9,ovarian carcinoma10, oesophagal carcinoma11, bladdercarcinoma12, non­small cell lung carcinoma13 andmesothelioma14. In addition, overexpression of CAIXhas been found to correlate with greater tumoraggressiveness15.CAIX has several functions in tumor cells. As itextrudes H+ ions to the extracellular environment, itmaintains the latter acidic while making intracellularpH slightly alkaline by incorporating HCO3

­ ions.This combination not only favors tumor cell growthbut also facilitates events such as cell transformation,chromosomal rearrangements, extracellular matrixbreakdown, migration and invasion, protease acti ­vation, and growth factor synthesis16.In addition, both in vitro17 and in vivo18 studies haveshown CAIX to be a useful marker of hypoxia,exhibiting a pattern of expression around regions of

necrosis. This distribution is similar to that ofpimonidazol, the most validated exogenous markerof hypoxia. Few studies have addressed the expressionpattern of CAIX in premalignant lesions. CAIXexpression has been shown to increase in bronchialpremalignant lesions19, in breast20, uterine cervix21

and skin dysplasias22, and in oral leukoplakia23. Theaim of the present work was to study and compareimmunohistochemical expression of CAIX in orallichen planus (OLP) and oral leukoplakia, sinceboth lesions have been classified as potentiallymalignant disorders, but differ greatly in their riskfor malignant transformation.

MATERIALS AND METHODSThe study comprised 37 biopsies corresponding tocases with proven clinical and histopathologicaldiagnosis of OLP (23 cases) and homogenousleukoplakia (14 cases), which were retrieved fromthe archives of the Oral Pathology Department ofthe School of Dentistry, University of Buenos Aires.The study was approved by the Ethics Committeeof the School of Dentistry of the University ofBuenos Aires, Argentina (Res CD 325/02).All diagnoses were established following diagnosticcriteria of the World Health Organization (WHO)24

and modified WHO diagnostic criteria25. Nine of the23 OLP cases were reticular lichen planus, and theremaining cases were the erosive or atrophic type oflichen. Two cases of leukoplakia showed moderatedysplasia. The specimens had been fixed in 10%formalin with PBS and embedded in paraffin. Sevenspecimens of normal oral mucosa obtained duringsurgery of deep­seated lesions were also studied.Endogenous peroxidase was blocked by immersion in0.5% H2O2 in methanol for 30 min. Antigen retrievalwas performed with Citra (Biogenex, Fremont,California, USA) in 3 cycles of 4 minutes each, at 400W microwaves. The sections were then incubatedovernight with the primary antibody, anti­humanCAIX, raised in rabbit (Santa Cruz Biotechnology,Dallas, Texas, USA). Antibody binding sites werevisualized using a streptavidin­peroxidase detectionkit (Kit Multilink, Biogenex, Fremont, California,USA), and incubation in 3,3­diaminobenzidinesubstrate and 1% nickel chloride as intensifier. Asection of a single block of a reactive oral squamouscell carcinoma was included in each staining batch aspositive control. Sections in which the primaryantibody was omitted were used as negative control.

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The percentage of cases showing positive stainingin the different localizations was analyzed usingFisher’s exact test. Values of p <0.05 were consideredstatistically significant.

RESULTSCAIX expression was detected in 16 out of 23 casesof lichen and 12 out of 14 cases of leukoplakia,whereas all normal mucosa samples were negative.Difference in frequency was not satisticallysignificant.OLP and leukoplakia showed different expressionpatterns. In all positive lichen samples, CAIX wasexpressed in the basal layers of the epithelium Aweaker reaction in superficial layers was also seenin 4 cases, whereas the staining was intense in thesuperficial and middle layers of 10 leukoplakiacases and only 4 cases were positive in the basallayers, In addition, CAIX was expressed mainly on the cell membrane (14 out to 16 cases on themembrane only) in lichen planus, but was observedon the membrane (5 cases), in the cytoplasm ( 5 cases),or both localizations (2 cases) in leukoplakia (Fig. 1).Statistical analysis of results showed significantdifferences between leukoplakia and lichen whencomparing localization of staining on the membraneor in the cytoplasm (p < 0.05), and in the layersexpressing CAIX (p < 0.02).The presence or absence of CAIX expression andthe slight differences in staining intensity were notassociated with type of OLP or with the presence orabsence of dysplasia in leukoplakia.

DISCUSSIONThe importance of studies on CAIX expression inmalignant tumors lies mainly in its usefulness as amarker of hypoxia, which in turn influences tumorresponse to radio and chemotherapy1. Overexpressionof CAIX has also been associated with moreaggressive tumor behavior, irrespective of type oftreatment15. Hence, it has been posited that deter ­mination of CAIX could be used as a marker ofmalignancy.The availability of a tumor malignancy markerprompts investigating in which stage of malignanttransformation induction of the gene encoding forexpression of the marker occurs, with the aim toobtain a marker of early cancerization prior toneoplastic development. In this regard, increasedCAIX activity has been demonstrated in premalignant

lung lesions19, breast ductal hyperplasia20, and skinpremalignant lesions22. Increased CAIX activity hasalso been detected in dysplastic epithelium adjacentto oral carcinomas23 and dysplastic epithelium inoral leukoplakia26.Yang et al 27 demonstrated the presence of CAIX inplasma of oral cancer patients, and found increasedexpression of the enzyme and its mRNA not only intumor stroma fibroblasts but also in fibroblasts oforal submucous fibrosis associated with areca nutchewing, a potentially malignant disorder.However, a given molecular change cannot beconsidered in itself indicative of malignant transfor ­mation simply because that same change is moremarked in malignant tumors, but rather a number offactors must come together for the risk of malignanttransformation to increase. Oral leukoplakia carriesan increased risk of cancer development. Accordingto the literature, the annual malignant transformationrate of leukoplakia ranges from 2 to 3%28, whereasthe potentially malignant character of OLP is stillunder discussion, and most authors estimate theannual malignant transformation rate of OLP to beless than 0.7%29. The increased expression of CAIXobserved in oral lichen and leukoplakia in thepresent work is therefore an indicator of aninteresting biological behavior of these lesions,rather than a marker of increased risk of malignanttransformation. Although OLP and leukoplakia are recognized as twodistinct pathological entities, some clinical and

Fig. 1: CAIX immunohistochemical reaction. X 400.A: Biopsy of oral lichen planus. Staining is more intense in cellmembranes of the basal and suprabasal layers of theepithelium. B: Biopsy of leukoplakia. Staining is more intensein the superficial layers, and becomes fainter towards thesuprabasal and basal layers of the epithelium.

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histopathological features may pose differentialdiagnosis. In addition, the etiopathogenesis of bothlesions was not yet fully clarified. Both entitiesundergo hypoxic stress. In fact, Pérez­Sayans et al26

detected CAIX expression in oral leukoplakia; theauthors associated their finding with the premalignantnature of the lesions, since they observed higherexpression in lesions with dysplasia. Ding et al30,found that expression of HIF1­a was higher in RNAisolated from lichen planus than in controls. HIF1­ais the main gene activated by hypoxia, and regulatestranscription of a number of genes, including CAIX.An interesting aspect is the markedly differentexpression pattern of CAIX in the entities studiedhere. CAIX expression was most prominent in thebasal layers of the epithelium, and decreasedtowards the surface layers in lichen planus, whereasin leukoplakia, staining intensity increased from the basal cells towards the granular layer, whereexpression was strongest. It is known that OLP is

an immune process mediated by T lymphocytes thatare present in dense subepithelial infiltrates andcause alteration of the basal keratinocytes31.Hypoxia in the basal layers of the epithelium, asshown by higher intensity of CAIX expression,might mediate T lymphocyte recruitment andcontribute to the onset of the disease. Leukoplakiais mainly triggered by external agents, such assmoking or alcohol consumption, which lead to an acanthotic response of the epithelium. Thisresponse, in turn, separates the epithelial cells fromthe blood supply, inducing higher CAIX expressionin the superficial layers.

CONCLUSIONThe different expression patterns of CAIX in orallichen and leukoplakia provides further evidence ofthe differences in the biological behavior of bothentities, and may be a contribution to the study oftheir pathogenic mechanisms.

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FUNDINGThis work was supported by a Grant from the University ofBuenos Aires, UBACyT Program N° 20020170200308BA.

CORRESPONDENCEDr. Miguel Ángel Pérez: M. T. de Alvear 2142, 2º A, ZC: 1122, Buenos Aires, Argentina. valemigue2@yahoo.com.ar

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26. Pérez­Sayans M, Suárez­Peñaranda J, Torres­López M,Supuran C, Gándara­Vila P, Gayoso­Diz P, Barros­AngueiraF, Gallas­Torreira M, et al. The use of CA­IX as a diagnosticmethod for oral leukoplakia. Biotech Histochem. 2015;90:124­131.

27. Yang JS, Chen MK, Yang SF, Chang YC, Su SC, Chiou HL,Chien MH, Lin CW.. Increased expression of carbonicanhydrase IX in oral submucous fibrosis and oral squamouscell carcinoma. Clin Chem Lab Med. 2014; 52:1367­1377.

28. Brouns ER, Baart JA, Karagozoglu KH, Aartman IHA,Bloemena E, Van Der Waal I. Malignant transformation oforal leukoplakia in a well­defined cohort of 144 patients.Oral Dis. 2014; 20:e19­e24. doi: 10.1111/odi.12095. Epub2013 Mar 25.

29. Fitzpatrick SG, Hirsch SA, Gordon SC. The malignanttransformation of oral lichen planus and oral lichenoidlesions: a systematic review. J Am Dent Assoc. 2014;145:45­56.

30. Ding M, Xu JY, Fan Y. Altered expression of mRNA forHIF­1a and its target genes RTP801 and VEGF in patientswith oral lichen planus. Oral Dis. 2010; 16:299­304.

31. Rodrigues Payeras M, Cherubini K, Figueiredo MA,Gonçalves Salum F. Oral lichen planus: focus on etiopatho ­genesis. Arch Oral Biol. 2013; 58:1057­1069.

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RESUMENNo existen estudios que evalúen el impacto de la caries dental(CD), la fluorosis dental (FD) y el trauma dentoalveolar (TDA)sobre la calidad de vida relacionada con la salud bucal (CVRSB)de escolares de Colombia que pertenece a países de hablahispana. El propósito de este estudio fue evaluar el impacto dela caries dental (CD), la fluorosis dental (FD) y el traumadentoalveolar (TDA) sobre la calidad de vida relacionada conla salud bucal de escolares colombianos usando sus padres comoproxies. Los padres de 338 niños y niñas de 6 a 14 años deescuelas públicas y privadas de Cartagena, Colombia,contestaron el Parental­Caregivers Perception Questionnaire(P­CPQ) on child’s OHRQoL adaptado al español colombiano y un cuestionario socioeconómico. Tres examinadores calibra ­dos realizaron la evaluación clínica para CD, FD y TDA. La regresión de Poisson asoció las condiciones clínicas y

socioeconómicas al puntaje total del P­CPQ y sus dominios. Engeneral, el 90,24% de los padres reportaron el impacto oral delos niños sobre la calidad de vida (puntaje P­CPQ total ≥ 1). Lamedia (DE) del P­CPQ fue de 12,49 (14,04). El modelomultivariado ajustado mostró que los niños de escuelas públicasque tenían experiencia de caries dental (RR = 1,28, p = 0,04 yRR = 1,37, p = 0,018, respectivamente) tuvieron mayor probabi ­lidad de experimentar un impacto negativo en las puntuacionestotales del P­CPQ.La CD mostró asociación con la percepción del impacto de lospadres­cuidadores sobre la salud oral de sus hijos en relacióncon la calidad de vida. Sin embargo, FD y TDA no se encon ­traron asociados.

Palabras clave: Caries dental, fluorosis dental, traumatismosde los dientes, calidad de vida, niñez

INTRODUCTIONOral health is currently considered an importantpillar in overall human welfare, with strong influenceon its development1. Everybody without exception

should enjoy good oral health status guaranteeingcertain activities and functions such as chewing,phonation and smiling as a sign of personal image,enabling social interaction, personal and emotional

ABSTRACTThere is no study assessing the impact of dental caries (DC),dental fluorosis (DF) and traumatic dental injuries (TDI) on oralhealth­related quality of life (OHRQoL) in school children fromColombia. The purpose of this study was to assess the impact ofDC, DF and TDI on Colombian schoolchildren´s OHRQoL usingtheir parents as proxies. The parents of 338 children aged 6 to 14years from public and private schools of Cartagena, Colombiaanswered the Parental­Caregiver Perception Questionnaire (P­CPQ) on child’s OHRQoL adapted to Colombian Spanishlanguage and a socioeconomic questionnaire. Three calibratedexaminers performed the clinical assessment for DC, DF andTDI. Poisson regression associated clinical and socioeconomic

conditions to the outcome. Overall, 90.24% of parents reportedchildren’s oral impact (total P­CPQ score ≥ 1). The mean(standard deviation) P­CPQ scores were 12.49 (14.04). Themultivariate adjusted model showed that children from publicschools and who have dental caries experience (RR= 1.28;p=0.04 and RR= 1.37; p= 0.018, respectively) were more likelyto experience negative impact on total P­CPQ scores. DC was found to be associated to parental­caregiverperception of impact on their children’s oral health­relatedquality of life, but DF and TDI were not.

Key words: Dental caries, dental fluorosis, tooth injuries,quality of life, child.

Parental perceptions of impact of oral disorders on Colombian schoolchildren’s oral health- related quality of life

Shyrley Díaz 1, María Paula Vélez 1, Luz Mariela Martínez 1, Ketty Ramos1, Marcelo Boneckër 2, Saul Martins Paiva 3, Jenny Abanto 2, 4

1 Universidad de Cartagena, Facultad de Odontología, Departamento de Odontología Preventiva y Social, Cartagena, Colombia.

2 Universidade de São Paulo, Faculdade de Odontologia, Departamento de Odontopediatria, Sao Paulo, Brazil.

3 Universidade Federal de Minas Gerais, Faculdade de Odontologia, Departamento de Odontopediatria, Minas Gerais, Brazil.

4 Universidade de Sao Paulo, Faculdade de Saúde Pública, Sao Paulo, Brazil.

Percepción de los padres del impacto de desordenes orales de escolares colombianos sobre la calidad de vida relacionada con la salud oral

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development and having positive impact on qualityof life1, 2, throughout their lifetime. There is consensus in the literature regarding thenegative impact of dental caries (DC) on schoolchildren’s Oral Health­Related Quality of Life(OHRQoL) through proxy­reports from parents orcaregivers3­5. Similarly, traumatic dental injuries(TDI) also have been shown to have negative impacton schoolchildren’s OHRQoL6,7. In addition, areview of the literature showed that severe dentalfluorosis (DF) was consistently reported to havenegative effects on OHRQoL8.In spite of thisevidence, to the best of our knowledge, there is nostudy assessing the impact of these oral diseases anddisorders, mainly TDI and DF, on schoolchildren,based on parents’ proxy reports. Parents’ perceptionsconcerning their children’s oral health are importantbecause they may enable clinicians to improvechildren’s oral health, and thereby their OHRQoL9.Moreover, parents are usually the primary decision­makers on matters affecting their children’s healthand healthcare10,11. The parental­caregiver perceptions questionnaire(P­CPQ) is an instrument of the Child Oral HealthQuality of Life Questionnaire (COHQLQ). It wasdeveloped in English in Canadá12 and has beencross­culturally translated. Its validity has beendemonstrated in English in the United Kindgom13,Chinese in China14, Portuguese in Brazil15 andSpanish in Peru16. However, although the Spanishversion of the P­CPQ11 has been validated, it has notyet been tested for Spanish­speaking schoolchildren.The use of the P­CPQ in Spanish­speaking countriessuch as Colombia is essential, considering the highprevalence of DC and DF in schoolchildren, 51%17

and 81% 18, respectively. Prevalence of TDI may havebeen neglected in Colombia, since there is noepidemiological survey of its extent. At present, theassessment of the impact of these oral conditions onColombian schoolchildren’s OHRQoL is unexplored.Therefore, the aim of this study was to assess the impact of DC, DF and TDI on the OHRQoL of 6­ to 14­year­old Colombian schoolchildren in a population­based sample using parents’ proxy­reports.

MATERIALS AND METHODSThis study followed Ethics Guidelines of theDeclaration of Helsinki, Edinburgh amendment 2000,1975 and Resolution 008430 of 1993 the former

Ministry of Health of the Republic of Colombia. Allparents received information regarding the aim of thestudy and signed informed consent forms.

Study population and data collectionA cross­sectional study was performed in 2015 on apopulation­based sample of children aged 6­14years enrolled at public or private schools and livingin Cartagena, Colombia. Cartagena city has anestimated population of 1,001,755 inhabitants,including 215,007 schoolchildren19.Sample size was calculated using a 94.5% prevalenceof oral impact on schoolchildren’s OHRQoL15, with95% confidence interval, a standard error of 3% anda design effect of 1.3. To cover non­response, thesample was increased by 15% to 338 children andtheir parents. Inclusion criteria were children of bothgenders, who had not received dental treatment inthe past 3 months, with no systemic disease and/orneurological disease, with parents/caregivers whowere fluent in Spanish language and who agreed toparticipate in the study.A 2­stage random sampling procedure was adoptedto select the sample. The first stage units were allpublic and private schools in the city. A total fourschools, two public and two private, were randomlyselected [WHO, 1997]. Since the schools were ofdifferent sizes, an equal probability selection method(probability proportional to the size) was used toensure that all children would have the same chanceof being selected [WHO, 1997]. The second stageunits were the 6­ to 14­year­old children enrolled ateach selected school. At the school, the parentswhose children were selected were summoned to beinformed of the study objective, and one of theparents (preferably the one who spent most time withthe child) was invited to answer two structuredquestionnaires in face­to­face interviews: one onsocioeconomic conditions and another on the child’sOHRQoL. Interviews were conducted by two dentalassistants who were blind to the clinical oral exami ­na tions. They were trained in the reading andintonation of each question and the answer optionsin the OHRQoL instruments.Socioeconomic conditions such as parental age,number of children and family income were collectedas discrete quantitative variables, and parental levelof education and family structure were collected asordinal and nominal qualitative variables, respecti ­vely. All these socioeconomic data were then

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84 Shyrley Díaz, et al.

categorized for statistical analysis as follows:child’s age [children (6 – 9 years old), teenager (10– 14 years old)], child’s gender [female, male], typeof school [public, private], age of parents [ ≤ 44years old, > 44 years old], number of siblings [≤ 2children, > 2 children], father’s and mother´seducation [< 10 years, ≥ 10 years], family income[measured in terms of the Colombia minimumwage­CMW, a standard for this type of assessment,which corresponds to approximately US$ 255.4 permonth categorized into ≤ One CMW, ≥ Two CMW],housing ownership[yes, no], household crowding[≤ two members per room, > two members perroom] and type of family [Nuclear family, Non­nuclear family].

OHRQoL instrumentThe parental­caregiver perceptions of Child OralHealth­Related Quality of Life Questionnaire (P­CPQ) was used to assess children’s OHRQoL. Thisinstrument is applied to parents of children aged 6­14 years15. The P­CPQ contains 31 items groupedinto four subscales: oral symptoms (6 items),functional limitations (8 items), emotional well­being (7 items), and social well­being (10 items).The questions are related to the frequency of eventsover the past three months. Answers are recordedon a Likert five­point scale used with the followingresponse options: “Never” = 0, “once / twice” = 1,“sometimes” = 2, “often” = 3, “every day / almostevery day” = 4. The P­CPQ scores are calculated asa simple sum of the response codes. Since there are31 questions, the final score can range from 0 to124, where a higher score indicates a higher degreeof impact of oral conditions on children’s oralhealth­related quality of life according to theirparents. In this study, the P­CPQ was adapted from thePeruvian Spanish version16 to the Colombiancontext. This Peruvian Spanish version was pilot­tested on a convenience sample of 30 parents ofchildren aged 6­14 years. These parents were notincluded in the final sample. Parents suggested thatsome words or expressions should be substituted bysynonyms to facilitate the comprehension of thequestionnaire. Modifications were made accordingthe parents’ comments. A Revision Panel consistingof three postgraduate professors in PediatricDentistry, Research and Family Health areas, allfluent in Spanish, who knew the objectives of the

study and had experience in OHRQoL studies,reviewed the results and in consensus developed theColombian Spanish version of the PCPQ.

Children’s oral examinationFour previously calibrated examiners performed thechildren’s oral examinations. The examiners wereall graduate dentists with experience in previousepidemiological surveys. All examiners underwenttwo 6­hour sessions of training and calibrationexercises with pictures of clinical cases for theclinical study conditions, with a 1­week intervalbetween sessions. Intra­ and inter­examiner reliabilitywas established using all examiners’ assessmentsof 20 children who received dental treatment atDental School of Cartagena University. Thesechildren did not form part of the study sample.Kappa values were calculated for intra­ and inter­examiner for all clinical conditions.Dental caries was assessed according to the WorldHealth Organization criteria and calculated in termsof decayed, missing, and filled teeth in permanentdentition (DMFT) and primary dentition (dmft)11.For most children with mixed dentition, the cariesindex was obtained by the sum of the dmft andDMFT scores. To calculate mean dmft/DMFT index,we added the individual average values and dividedby the total number of children examined. Theprevalence of children affected by the disease wasdescribed using the Knutson index 20: children whohave dental caries or have experienced dental caries(dmft/ DMFT > 0) and children who have never hadexperienced dental caries (dmf/ DMFT = 0). Dental fluorosis (DF) was assessed using thecriteria proposed by Thylstrup­Fejerskov,21 whichis more sensitive than Dean’s index for individualclassification of teeth into ten categories22. A scoreof zero indicates healthy enamel; scores of one tofour indicate spots on the enamel surface, whichincrease as the score increases. Enamel destructionis observed in scores five to nine, where score fiverepresents mottled enamel with holes smaller than2mm in diameter, which are fused in score six toform bands less than 2mm deep. For the currentstudy, DFwas categorized as mild (code 1,2,3),moderate (code 4­6) and severe fluorosis (code 7­9)21.Traumatic dental injury (TDI) was evaluated usingthe criteria proposed by Andreasen et al.23andevaluated as a possible confounding variable, basedon the system adopted by the WHO. It includes

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injuries to hard dental tissues and pulp; injuries tohard dental tissues, pulp and alveolar process; andinjuries to periodontal tissues. TDI data wereanalyzed according to presence of at least one kindof trauma or absence of TDI (tooth present andsound).

Data analysisData were analyzed using STATA 9.0 (Stata Corp,College Station, TX, USA). Descriptive analysesassessed measures of central tendency (mean,standard deviation and observed range) of the totaland individual domain scores of P­CPQ. Poisson regression with robust variance wasperformed to associate domains and total P­CPQscores to oral clinical conditions (DC, DF and TDI)and socioeconomic conditions. Univariate Poissonregression was performed to select variables with ap­value ≤0.20 to enter the final model. Then theselected variables were tested in the adjustedmultivariate model and only remained in the finalmodel if p­value ≤ 0.05. In these analyses, theoutcome was employed as a count outcome, andrate ratios (RR) and 95% confidence intervals (95%CI) were calculated.

RESULTSInternal consistency of the P­CPQ was analyzedusing Cronbach’s alpha coefficient, providingvalues of 0.85 for total P­CPQ scores in the pilottest phase and 0.89 for total P­CPQ scores in thefinal sample size of the study, showing the stabilityof the instrument.As a result of the calibration process, the examinersobtained inter­examiner reliability values ofCohen’s Kappa agreement of 0.87 for DC and 0.92for DF and TDI. For intra­examiner agreement, theexaminers obtained kappa values of 0.89 for DC,0.85 for DF and 0.90 for TDI. A total 370 parents were invited to participate in thestudy, of whom 10 were excluded because they didnot conform to the study inclusion criteria. Of the 360eligible participants, 338 provided signed parentalinformed consent (positive response rate = 93.8%).Table 1 shows the socioeconomic and clinicalconditions of the sample. Overall, most children haddental caries experience (62.4%), whereas DF andTDI were present in 64.5% and 8.9%, respectively.All questionnaires were fully completed without anymissing data, and there were no ‘don’t know’

responses. Most of the questionnaires wereanswered by mothers (79.3%). Overall, 90.24% ofparents reported children’s oral impacts (total P­CPQ score ≥ 1). Table 2 contains the mean, standarddeviation, and the range observed for the total P­CPQ scores and individual domains.Table 3 shows the univariate unadjusted analysis ofclinical and socioeconomic variables associated

Table 1: Sample sociodemographic features (n = 338).

Variables n (%)

Child ageTeenager (10 – 14 years old) 152 (45)Children (6 – 9 years old) 186 (55)

Child genderMale 180 (53.3)Female 158 (46.7)

Type of SchoolPrivate 170 (50.3)Public 168 (49.7)

Age of parents≤ 44 years old 297 (87.9)> 44 years old 41 (12.1)

Number siblings≤ 2 children 205 (60.6)> 2 children 133 (39.4)

Father’s education level≥ 10 years 91 (26.9)< 10 years 247 (73.1)

Mother’s education level≥ 10 years 84 (24.8)< 10 years 254 (75.2)

Family Income≥ Two CSW 263 (77.8)≤ One CSW 75 (22.2)

Household crowding≤ 2 members 239 (79.7)> 2 members 99 (29.3)

Type of familyNuclear family 196 (57.9)Non-Nuclear family 142 (42.1)

Clinical featuresDental CariesAbsence 121 (35.8)Presence 217 (64.2)

Dental FluorosisAbsence 119 (35.4)Presence 217 (64.6)

Traumatic Dental InjuriesAbsence 308 (91.1)Presence 30 (8.9)

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with total and individual domains of the P­CPQ.There was significant association between someindependent variables, total scores, and individualdomains (p < 0.05): oral symptoms and mother’seducation and family income; emotional well­beingdomain and dental caries experience; social well­being domain and children’s education, number ofsiblings; and total P­CPQ and members in familyand dental caries. The multivariate­adjusted model (Table 4) showedthat children from public schools and children whohave dental caries experience (RR= 1.28; p=0.04and RR= 1.37; p= 0.018, respectively) were morelikely to experience a negative impact on total P­CPQ scores. Children whose mothers have aneducational level < 10 years and children who havedental caries experience showed positive andnegative impact on the oral symptoms domain,respectively (RR= 0.75, p= 0.02 and RR= 1.22,p=0.04, respectively). Children who studiedatpublic schools were more likely to experiencenegative impact on the emotional well­being andsocial well­being domains (p<0.05) (Table 4).

DISCUSSIONTo the best of our knowledge, this is the first studyto measure the impact of DC, TDI and DF on theOHRQoL of Colombian schoolchildren fromparents’ proxy­reports using a Spanish version ofthe P­CPQ16. The questionnaire showed semanticequivalence and good understanding by parentsconsidering that Peru and Colombia share manysimilarities in their Spanish language and voca ­bulary. Cronbach’s alpha coefficient was 0.89 forthe total scale, indicating acceptable internalreliability, as values of 0.5 or above are consideredacceptable24. Similar findings were reported for thePeruvian Spanish version16and in the original

English and Brazilian versions12,15, indicating itssatisfactory use for assessing children’s OHRQoLaccording to parents’ perceptions in Colombia.In this study, dental caries was the only oral diseasethat showed negative impact on total P­CPQ scoresin the oral symptoms domain, though it did notaffect other domains. A study in China also foundnegative impact of dental caries using the P­CPQin 12­year­old children25, but in this case, theimpact was on the social and emotional well­beingdomains. There is no study using P­CPQ inColombian and other Spanish speaking countrieslike Perú16, but other instruments in Spanish thatassess OHRQoL according to children andadolescent´s perceptions, such as the ChildPerceptions Questionnaire 11­14 (CPQ11­14)26

report impact of DC on the oral domain. This mayindicate agreement between children and theirparents/caregivers regarding the impact of DC onschoolchildren´s OHRQoL, and it will be importantto explore the matter in future studies. Therefore,even when children and adolescents are able toprovide self­reports, parents/caregivers’ proxyreports should be obtained to provide additionalsupplementary information about the impact ofdifferent oral outcomes on childrens’ OHRQoL27.Both views jointly may offer a more comprehensivebasis for professional clinical decisions. This infor ­mation may also be useful for health authorities inplanning oral healthcare services28. In this study, we found no association betweentraumatic dental injuries and the perception of theimpact of OHRQoL, but this maybe due to the lowprevalence of TDI in the sample, in agreement withAbanto et al.29. Nevertheless, assessment of theimpact of TDI on schoolchildren’s OHRQoL usingCPQ 11­14 shows a negative impact on OHRQoL.Crown discoloration also shows a negative impactusing this scale30. This is another finding that showsdifferent perceptions between children and theirparents/caregivers regarding presence of TDI.Dental fluorosis was not found to be associated withthe perception of impact on OHRQoL, in agreementwith a study performed in Pinheiro Preto, Brazil,but differing from a study in Colombia31 whichreported that children often avoided smilingbecause of the appearance of their teeth due to DF.It should be noted that said study was conducted inan endemic fluorosis zone. In contrast, our resultsshow low prevalence of DF, and the fact that the

Table 2: Mean, standard deviation, possible range, and range observed for overall and for eachP-CPQ11–14 domain scores (n = 338).

P-CPQ Mean(SD) Range Observed

Oral symptoms 3.88 (3.5) 0-20

Functional limitations 3.43 (4.17) 0-24

Emotional well-being 2.09 (3.90) 0-30

Social well-being 3.09 (6.15) 0-48

Total Score 12.49 (14.04) 0-90

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86 Shyrley Díaz, et al.

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25

Vol. 31 Nº 2 / 2018 / 82-90 ISSN 1852-4834 Acta Odontol. Latinoam. 2018

Quality of life and oral disorders in schoolchildren 87

AOL­2­2018:3­2011 25/10/2018 15:03 Página 87

Tab

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: Mu

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Acta Odontol. Latinoam. 2018 ISSN 1852-4834 Vol. 31 Nº 2 / 2018 / 82-90

88 Shyrley Díaz, et al.

DF variable was dichotomized may explain thedifferent findings.Our study found significant associations between P­CPQ scores, its domains and some socioeconomicconditions such as children who study at public schoolswith poor OHRQoL in emotional and social domainsand mother’s education with better OHRQoL in oraldomains. This is similar to the findings in a syste ­matic review assessing the impact of parentalsocioeconomic status and home environment on chil­dren’s OHRQoL, which reports that children from anarea with high deprivation had poorer OHRQoL thanchildren from areas with low and medium categoriesof deprivation32,33. It also reports that higher educa­tional level of the mother and father predicted betterOHRQoL in children, but observed the mother’s education – and not the father’s education – wassigni ficantly related to OHRQoL scores.34 Severalstudies also report that attending a poor school andmaternal level education are meaningful aspects forchildren’s oral health. In such conditions, someparents and their children do not have enough oppor­tunities to access dental services or to purchase oralhygiene articles29,30, which may explain the impacton social and emotional domains. Most families inColombia live in areas with high deprivation and donot have oral health education or services to promotegood OHRQoL. It is important to foster the role ofschools in reducing health disparities among chil­dren35. With regard to mother’s low level of educationhaving positive impact on the oral symptoms domainof P­CPQ, one study reports how Latina mothers inthe USA with higher educational status did not per­ceive cavities as more serious36,37. Previousqualitative studies describe how Latina mothersreported they did not perceive that dental decay wasa condition that affected young children38,39. This mayexplain the positive impact in our study. Thus, it isimportant identify specific cultural beliefs that runcounter to optimal oral health in young children.This study is the first to test the P­CPQ in Spanish­speaking schoolchildren and indicates the need todesign public policies in oral health providingcomprehensive care to children and their parents andredirecting efforts towards providing proper treatment,reducing the prevalence of dental caries and improvingquality of life. This study also contributes to publichealth by providing understanding of the psychosocialimpact of DC, DF and TDI. In addition, the first use ofthe Spanish version P­CPQ has important implicationsfor research and practice. In this regard, the first use of

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ACKNOWLEDGMENTSWe thank Beverly Hills School and John F. Kennedy School inCartagena­Colombia, for allowing us to conduct this study andthe children’s parents for cooperating with the necessary infor­mation. We also thank David Madrid for helping us to evaluatethe children clinically.

FUNDINGNone

CORRESPONDENCEDr. Shyrley Díaz Cárdenas, Facultad de Odontología,Departamento de Odontologia preventiva y socialCampus de la Salud, Zaragocilla, Cartagena, Colombia sdiazc@unicartagena.edu.co

REFERENCES1. Vadiakas G. Case definition, aetiology and risk assessment

of early childhood caries (ECC): a revisited review. Eur ArchPaediatr Dent 2008;9:114­125.

2. Petersen PE. The World Oral Health Report 2003:continuous improvement of oral health in the 21st century—the approach of the WHO Global Oral Health Programme.Community Dent Oral Epidemiol 2003;31:3­23.

3. Vetter TR, Bridgewater CL, McGwin G Jr. An observationalstudy of patient versus parental perceptions of health­relatedquality of life in children and adolescents with a chronic paincondition: who should the clinician believe? Health QualLife Outcomes 2012;10:85. doi: 10.1186/1477­7525­10­85.

4. Kumar S, Kroon J, Lalloo R, Johnson NW. Validity andreliability of short forms of parentalcaregiver perception andfamily impact scale in a Telugu speaking population ofIndia.Health Qual Life Outcomes 2016;14:34. doi: 10.1186/s12955­016­0433­7.

5. Do LG, Spencer A. Oral health­related quality of life ofchildren by dental caries and fluorosis experience. J PublicHealth Dent 2007;67:132­139.

6. Bomfim RA, Herrera DR, De­Carli AD.Oral health­related quality of life and risk factors associated withtraumatic dental injuries in Brazilian children: A multilevelapproach.Dent Traumatol 2017;33:358­368.

7. Freire­Maia FB, Auad SM, Abreu MH, SardenbergF, Martins MT, Paiva SM Pordeus IA, Vale MP. Oral Health­Related Quality of Life and Traumatic Dental Injuries inYoung Permanent Incisors in Brazilian Schoolchildren: AMultilevel Approach. PLoS One 2015;10(8):e0135369. doi:10.1371/journal.pone.0135369. eCollection 2015.

8. Chankanka O, Levy SM, Warren JJ, Chalmers JM. Aliterature review of aesthetic perceptions of dental fluorosisand relationships with psychosocial aspects/oral health­related quality of life. Community Dent Oral Epidemiol2010;38:97­109.

9. Antunes LA, Luiz RR, Leão AT, Maia LC. Initial assessmentof responsiveness of the P­CPQ (Brazilian Version) to

describe the changes in quality of life after treatment fortraumatic dental injury. Dent Traumatol 2012;28:256­262.

10. Cafferata GL, Kasper JD. Family structure and children’suse of ambulatory physician services. Med Care 1985; 23:350­360.

11. Hickson GB, Clayton EW. Parents and their children’sdoctors. Handb Parent; Pract Issues Parent. 2002;(5): 438.URL: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.457.3309&rep=rep1&type=pdf

12. Jokovic A, Locker D, Stephens M, Kenny D Tompson B,Guyatt G. Measuring parental perceptions of child oral health­related quality of life. J Public Health Dent 2003; 63:67­72.

13. Marshman Z, Rodd H, Stem M, Mitchell C, Robinson PG.Evaluation of the Parental Perceptions Questionnaire, acomponent of the COHQoL, for use in the UK. CommunityDent Health 2007;24:198­204.

14. McGrath C, Pang HN, Lo ECM, King NM Hägg U,Samman N. Translation and evaluation of a Chineseversion of the Child Oral Health­related Quality of Lifemeasure. Int J Paediatr Dent. 2008;18:267­274.

15. Goursand D, Paiva SM, Zarzar PM, Pordeus IAGrochowski R, Allison PJ.Measuring parental­caregiverperceptions of child oral health­related quality of life:Psychometric properties of the brazilian version of the P­CPQ. Braz Dent J 2009;20:169­174.

16. Albites U, Abanto J, Bönecker M, Paiva SM Aguilar­Gálvez D, Castillo JL.Parental­Caregiver Perceptions ofchild oral health­related quality of life (P­CPQ):Psychometric properties for the Peruvian Spanishlanguage. Med Oral Patol Oral Cir Bucal 2014;19: e220­e224. doi:10.4317/medoral.19195 http://dx.doi.org/doi:10.4317/medoral.19195

17. Diaz­Cardenas S, Gonzalez­Martinez F. The prevalence ofdental caries related to family factors in schoolchildrenfrom the city of Cartagena in Colombia. Rev. Salud pública(Bogotá) 2010;12:843­851.

18. Ramírez­Puerta BS, Franco­Cortés ÁM, Ochoa­AcostaEM. Dental fluorosis in 6­13­year­old children attending

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Quality of life and oral disorders in schoolchildren 89

the P­CPQ in a Spanish­speaking country enablescomparisons with other cultural and ethnic groupsaround the world, and provides support for public oralhealth programs and dental care services for this agegroup. It also provides additional information to theresults of a previous study on preschool children.40

CONCLUSIONSAccording to parents’ proxy­reports, the presenceof dental caries and attending public schools have anegative impact on the OHRQoL of Colombianschoolchildren, whereas mothers with educationallevel < 10 year has a positive impact.

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public schools in Medellín, Colombia. Rev Salud pública(Bogotá) 2009;11:631­640.

19. Velez Trujillo D SC. Plan Sectorial de Educacion Ahora SiCartagena 2013­2015. Educacion, La gran Estrategia parala inclusion social [Internet]. Cartagena; 2014. Availablefrom: http://www.sedcartagena.gov.co/attachments/article/1667/PLAN_EDUCATIVO_AHORA_SI CARTAGENA.pdf

20. Knutson JW. An index of the prevalence of dental caries in school children. Pub Hlth Rep 1944;59:253­263.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2017044/pdf/pubhealthreporig01509­0001.pdf

21. Thylstrup A, Fejerskov O. Clinical appearance of dentalfluorosis in permanent teeth in relation to histologic changes.Community Dent Oral Epidemiol 1978;6:315­328.

22. Rozier RG. Epidemiologic indices for measuring theclinical manifestations of dental fluorosis: overview andcritique. Adv Dent Res 1994;8:39­55.

23. Andreasen JO, Andreasen FM. Textbook and color atlas oftraumatic injuries to the teeth. Copenhagen: Munksgaard1993;216­256.

24. Cronbach LJ. Coefficient alpha and the internal structureof tests. Psychometrika 1951;16:297­334.

25. Li YJ, Gao YH ZY. The impact of oral health status on theoral health­related quality of life (OHRQoL) of 12­year­olds from children’s and parents’ perspectives. CommunityDent Health 2014;31:240­244.

26. Abanto J, Albites U, Bönecker M, Martins­Paiva S CastilloJL, Aguilar­Gálvez D.Cross­cultural adaptation andpsychometric properties of the child perceptionsquestionnaire 11­14 (CPQ11­14) for the peruvian spanishlanguage. Med Oral Patol Oral Cir Bucal 2013;18: e832­8.doi:10.4317/medoral.18975 http://dx.doi.org/doi:10.4317/medoral.18975

27. Jokovic A, Locker D, Guyatt G. How well do parents knowtheir children? Implications for proxy reporting of childhealth­related quality of life. Qual Life Res. 2004;13:1297­1307.

28. Ferreira MC, Goursand D, Bendo CB, Ramos­Jorge MLPordeus IA, Paiva SM.Agreement between adolescents’and their mothers’ reports of oral health­related quality oflife. Braz Oral Res 2012;26:112­118.

29. Abanto J, Tsakos G, Paiva SM, Carvalho TS RaggioDP, Bönecker M.Impact of dental caries and trauma onquality of life among 5­ to 6­year­old children: Perceptionsof parents and children. Community Dent Oral Epidemiol2014;42:385­394.

30. Pulache J, Abanto J, Oliveira LB, Bonecker M Porras JC.Exploring the association between oral health problems andoral health­related quality of life in Peruvian 11­ to 14­year­old children. Int J Paediatr Dent 2016; 26: 81­90.

31. Tellez M, Santamaria RM, Gomez J, Martignon S. Dentalfluorosis, dental caries, and quality of life factors amongschoolchildren in a Colombian fluorotic area. CommunityDent Health 2012;29:95­99.

32. Nurelhuda NM, Ahmed MF, Trovik TA, Åstrøm AN.Evaluation of oral health­related quality of life amongSudanese schoolchildren using Child­OIDP inventory.Health Qual Life Outcomes. 2010; 23; 8:152 doi: 10.1186/1477­7525­8­152.

33. Foster Page LA, Thomson WM, Ukra A, Farella M. Factorsinfluencing adolescents’ oral health­related quality of life(OHRQoL). Int J Paediatr Dent. 2013; 23:415­423.

34. Alwadi MAM, Vettore MV. Are school and home environ ­mental characteristics associated with oral health­relatedquality of life in Brazilian adolescents and young adults?Community Dent Oral Epidemiol 2017;45:356­364.

35. Ji Y, Wang Y, Sun L, Zhang Y Chang C1.The MigrantParadox in Children and the Role of Schools in ReducingHealth Disparities: A Cross­Sectional Study of Migrant andNative Children in Beijing, China. Dalal K, ed. PLoS One.2016; 26;11(7):e0160025. doi: 10.1371/journal.pone.0160025.eCollection 2016.

36. Wilson AR, Mulvahill MJ, Tiwari T. The Impact ofMaternal Self­Efficacy and Oral Health Beliefs on EarlyChildhood Caries in Latino Children. Front Public Health2017; 28;5:228. doi: 10.3389/fpubh.2017.00228. eCollection2017.

37. Finlayson TL, Beltran NY, Becerra K. Psychosocial factorsand oral health practices of preschool­aged children: aqualitative study with Hispanic mothers. Ethn Health2017;11:1­19.

38. Hoeft KS, Barker JC, Masterson EE. Urban Mexican­American mothers’ beliefs about caries etiology in children.Community Dent Oral Epidemiol 2010; 38:244­255.

39. Cortés DE, Réategui­Sharpe L, Spiro Iii A, García RI.Factors affecting children’s oral health: perceptions amongLatino parents. J Public Health Dent 2012 Winter; 72:82­89.

40. Díaz S, Mondol M, Peñate A, Puerta G, Boneckër M,Martins Paiva S, Abanto J. Parental perceptions of impactof oral disorders on Colombian preschoolers’ oral health­related quality of life. Acta Odontol Latinoam. 2018;31:38­44.

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RESUMOO edentulismo completo caracteriza­se pela perda total dosdentes permanentes, resultando em alterações estéticas,estruturais e funcionais, podendo impactar negativamente naqualidade de vida, sendo minimizado através da reabilitaçãocom a prótese dentária. O objetivo deste estudo foi comparara qualidade de vida relacionada à saúde oral em pacientes coma prótese original completamaxilar e mandibular, três mesesapós instalação da nova prótese e dois anos depois daconfecção da nova prótese total bimaxilar removível. Nesseestudo compara tivo longitudinal, participaram 15 voluntários,com faixa etária entre 50 e 82 anos, de ambos os sexos, quebuscaram tratamento no Departamento de Odontologia daUniversidade Federal do Rio Grande do Norte, para aconfecção de novas próteses. Utilizou­se a versão brasileira

do Oral Health Impact Profile para pacientes edêntulos(OHIP­EDENT) para avaliar a qualidade de vida. A análisedos dados foi realizada de forma descritiva e analítica com ostestes de Friedman e Wilcoxon, com nível de significância de5%.Em relação aos domínios do OHIP­EDENT, verificou­sediferença para desconforto e incapacidade mastigatória entrea avaliação inicial e após dois anos. Nos domínios Dor edesconforto orofacial, Incapacidade Psicológica e Incapa ­cidade Social, não ocorreram diferenças entre os períodosavaliados. Foram observados indicadores de melhora naqualidade de vida dos pacientes, no domínio desconforto eincapacidade mastigatória entre a avaliação e a 2 anos.

Palavras­chave: Sistema estomatognático; Edêntulo; Prótesetotal; Qualidade de vida.

INTRODUCTIONComplete edentulism is defined as a complete lossof permanent teeth1, and is very common worldwidein the elderly population2 aged 651 to 74 years2,3.

It also characterizes a reality of the Brazilianpopulation assessed in the latest epidemiologicalsurvey conducted in 2010 by the Ministry of Health.Survey results showed that 15.4% of the older adult

ABSTRACTFull edentulism is characterized by the complete loss ofpermanent teeth, resulting in aesthetic, structural and functionalchanges which can negatively impact quality of life, and whichare minimized through rehabilitation with complete dentures.The aim of this study was to compare oral health­related qualityof life in patients with complete original dentures three monthsafter installation of new dentures and two years after fabricationof new complete removable dentures. In this longitudinalcomparative study, 15 volunteers of both genders, aged 50 to82 years, who sought treatment at the Department of Dentistryof the Federal University of Rio Grande do Norte, participatedin the preparation of new dentures. The Brazilian version of theOral Health Impact Profile for edentulous patients (OHIP­

EDENT) was used to evaluate quality of life. Data analysis wasperformed descriptively and with hypothesis testing using theFriedman and Wilcoxon tests with 5% significance level. Inrelation to the OHIP­EDENT domains, there was a differencefor chewing discomfort and inability to chew between baselineand two years. However, there was no difference between theevaluated periods in the areas of pain and orofacial musclediscomfort, psychological inability and social disability.Improvement indicators in patient quality of life were observedin the area of discomfort and inability to chew between baselineand 2 years.

Key words: Stomatognathic System; Mouth Edentulous, MeshPubmed; Complete Denture; Quality of life.

91

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Quality of life related to complete denture

Anne C. Alves1, Renata V. A. Cavalcanti2, Patrícia S. Calderon3, Leandro Pernambuco4, João C. Alchieri5

1 Universidade Federal do Rio Grande do Norte, Brasil.2 Universidade Federal do Rio Grande do Norte, Departamento de Fonoaudiologia, Brasil.

3 Universidade Federal do Rio Grande do Norte, Departamento de Odontologia, Brasil.

4 Universidade Federal da Paraíba, Departamento de Fonoaudiologia, Brasil.

5 Universidade Federal do Rio Grande do Norte, Departamento de Psicologia, Brasil.

Qualidade de vida relacionada à prótese total

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92 Anne C. Alves, et al.

population was edentulous and needed completedentures4. The 2013 National Health Survey foundthat 11% of individuals aged over 18 years werecompletely edentulous, with a higher proportionamong women aged 60 years or older. Althoughcomplete loss of teeth is not necessarily a part of the natural aging process, age is one of the mostprominent factors5. Other common factors arebiological processes such as tooth decay, periodontaldisease, trauma and oral cancer, and non­biologicalfactors including dental procedures, the quest forhealthcare, and socioeconomic and cultural factors1.Edentulism outcomes include changes to functional,neuromuscular and physiological6 levels. Functionalcapacity includes chewing and speech, psychologicalstatus includes self­esteem and satisfaction withappearance, and social aspects involve pain anddiscomfort related to oral health7­9. Over time, thetotal loss of teeth leads to atrophy of supportstructures and loss of muscle tone, which haveadverse effects on facial aesthetics5 and also involvechewing, swallowing and speech functions10. Thus,proper oral function is not only associated with theability to perform jaw movements and physiologicalparameters, but also with comfort and aesthetics,which can affect the quality of life8.The changes caused by total loss of teeth can beminimized through rehabilitation with dentalprostheses, which is the most economical andcommon treatment2,8,9,11. Its purpose is to restorethe harmony of the stomatognathic system andgeneral health9. Acceptance of complete denturesrequires psychosocial and functional adaptation, aprocess that can be influenced by patient expecta ­tions12 and perceptions7, which may involve qualityof life. As defined by the World Health Organization,the term quality of life refers to “an individual’sperception of their position in life in the culturalcontext and systems values in which they live andin relation to their goals, expectations, standardsand concerns“13.Oral health­related quality of life thus plays acrucial role in the process of prosthetic rehabili ­tation, which includes functional, psychologicaland social aspects7. The aim of this study wasto compare oral health­related quality of life with the previous dentures to oral health­relatedquality of life three months and two years afterfabrication and fitting of the new complete removabledentures.

MATERIALS AND METHODSA descriptive, comparative longitudinal study wasapproved by the Research Ethics Committee of theOnofre Lopes University Hospital under number578.993. All participants were informed regardingstudy objectives and procedures, which weredescribed in the Informed Consent Form, withpatient consent and signature requested. Participantswere selected among individuals who visited theDepartment of Dentistry of the Federal University ofRio Grande do Norte to have conventional completedentures made. Inclusion criteria were individualswho had been fully edentulous for over a year andconventional complete upper and lower denture usersneeding new dentures. Participants who had motordifficulties, evident cognitive deficiencies ineveryday actions or pathological changes of thealveolar edges were excluded.The instrument used to assess the impact of quality oflife was the Brazilian version of the Oral HealthImpact Profile for edentulous patients (OHIP­EDENT), validated by Souza et al.12. Quality of lifewas evaluated in three stages: during the use of theold dentures, and after three months and two yearsof using the new dentures. The sample consisted of15 individuals of both genders. It should be noted thatmuch of the sample was lost over the course of theassessments, and the number of participants wasreduced from 36 original participants to 22, andfinally to 15 individuals. The OHIP­EDENT is aninventory consisting of 19 questions grouped into foursubscales described by Souza et al.14, emphasizing“pain and discomfort in orofacial muscles”, “masti ­catory discomfort and inability”, “psychologicaldiscomfort and inability” and “social inability”. Theoptions for the answers are: never, sometimes andalmost always, which are assigned the scores of “0”,“1” and “2”, respectively. Higher scores representworse oral health­related quality of life.Statistical analysis was performed with SPSSversion 20.0 for Windows. The Shapiro­Wilktest was used to evaluate the normality of datadistribution, finding that the variables did not havenormal distribution. The Friedman test was used toverify whether the sample showed any significantdifference between the three periods evaluated bymeans of multiple comparisons. The post hocanalysis was performed using therection in order toidentify in which periods there were differences. A5% (p ≤ 0.05) significance level was considered.

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RESULTSThe final sample consisted of 13 females (86.67%)and 2 males (13.33%). Average age was 63.73years, standard deviation 7.67, with minimum age50 and maximum age 82 years. Average time ofedentulism was 26.6 years with standard deviation12.8. The usage time of the dentures was catego ­rized into equal to or less than 5 years (4 ­ 26.67%),and more than 5 years (11 ­ 73.33%). The distri ­bution of OHIP­EDENT areas between periodsevaluated is shown in Table 1.The area of masticatory discomfort and inabilityshowed a difference between baseline and twoyears. There was no difference between theevaluated periods in the areas of orofacial musclepain and discomfort and psychological inability andsocial inability (Table 2).

DISCUSSIONAverage age above 60 years agrees with datareported in other studies9, 15, 16. Higher prevalenceof females has also been reported in otherstudies16,17, which may be explained by the fact thatwomen are more concerned about caring for theiroral health and seek treatment more often than mendo18. For quality of life, we found no significantdifference for discomfort and chewing inabilitybetween baseline and 2 years, highlighting theimprovement in patient quality of life.The analysis categorized the average time ofdenture usage, with up to 5 years being used tounderstand the main morphological and functionalchanges referred to edentulous individuals. Sixtypercent of our sample used the dentures for overfive years. The literature indicates that the quality

Table 1: Distribution of OHIP-EDENT areas between the evaluated periods. Natal 2017.

Areas Median Q25 - Q75

Evaluation 6.00 5.00 - 7.00

Orofacial muscle 3 months 5.00 3.00 - 6.00

Pain and Discomfort 2 Years 2.00 2.00 - 6.00

Evaluation 5.00 1.00 - 7.00

Masticatory 3 months 2.00 2.00 - 4.00

Discomfort and inability 2 Years 1.00 0.00 - 3.00

Evaluation 1.00 0.00 - 7.00

3 months 0.00 0.00 - 3.00

Psychological inability 2 Years 0.00 0.00 - 1.00

Evaluation 0.00 0.00 - 2.00

3 months 0.00 0.00 - 0.00

Social inability 2 Years 0.00 0.00 - 0.00

Table 2: Comparison of OHIP-EDENT areas in three evaluated periods. Natal 2017.

Areas n Evaluation 3 months 2 Years

Orofacial muscle 15 6.00 ABC 5.00 BC 2.00 C

Pain and Discomfort

Masticatory 15 5.00 EF 2.00 FG 1.00 G

Discomfort and inability

Psychological inability 15 1.00 IJK 0.00 JK 0.00 K

Social inability 15 0.00 MON 0.00 NO 0.00 O

The groups of letters on the medians represent multiple comparisons of the Friedman test (p<0.05). The medians or pairs of median values with different letters indicate that there are significant differences between the corresponding medians after Bonferroni correction (p<0.0167).

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94 Anne C. Alves, et al.

of dentures may decrease over time, and individualsmay present problems in mastication15 after thefourth15 and fifth9 year of use, thereby making itnecessary to replace the dentures, which is inaccordance with our data.The main reasons for the change in denturesobserved in this study and in agreement with otherstudies relates to lack of stability and retention11.These factors can be explained by the periodof bone loss which results in the first year after toothextraction, when the bone may lose up to 25% ofits width and approximately 4 mm in height due tothe continuous process of bone resorption11. Adecline in the alveolar ridge of approximately 1mmper year is expected over the years, being four timeshigher in the mandible than in the jaw9. Resorptioncan cause poor adaptation of the resin base fordental prostheses, making them slightly loosearound the residual bone edge. This factor and theproblems caused by the reduced vertical dimensioncause great discomfort when eating, which cansignificantly reduce the efficiency of chewing,thereby influencing patient nutrition as a result offood not being crushed, thus reducing nutrientabsorption5.Measuring oral health­related quality of life enablesevaluation of patient’s subjective perception of theircondition and constitutes a key factor for clinicalpractice, which is complemented by physicalindicators19, with the aim of improving theunderstanding and therapeutic direction of theprofessionals involved14. It should be noted that therehabilitation success of dentures is based on theopinion of the individual, emphasizing denturestability, comfort, speech, ease of removal forcleaning, chewing and aesthetics20. The literaturesuggests the OHIP­EDENT with psychometricproperties is a specific instrument to assess anychanges in clinical aspects and the quality of life ofedentulous individuals after prosthetic treatment14.The results of this study showed differences in the

OHIP­EDENT subscales for masticatory discomfortand inability between baseline and 2 years, repre ­senting an improvement in quality of life with theuse of the new dentures. Masticatory discomfort andinability are improved by correct intermaxillarypositioning, tooth anatomy, the shape and fit of thebase of the dentures on the supporting tissues, gene ­rating comfort5. The difference in the masticatoryinability index can be improved by the restoration

of the vertical dimension of occlusion, correctcentric occlusion11, restoration of the cusps forcrushing food, improved chewing efficiency andaesthetics. Moreover, new functional molding fromthe edge provides greater retention and stability,resulting in patient comfort with the new dentures5.In agreement with our findings, other studies have shown that a substitution for new denturessignificantly improves quality of life5,7,16,17,21.Goiato et al.5 evaluated the quality of life andpatient perception before and after insertion of anew prosthesis in 60 patients with an interval ofthree months between evaluations, also using theOHIP­EDENT and resulting in significant impacton patient quality of life in all areas. However, incontrast to our study, they analyzed each issue inisolation. Komagamine et al.16 determined factorsrelated to self­assessment of dentures through theOHIP­EDENT and the masticatory performancebefore and after replacement of the dentures usinga sample of 93 individuals, reporting that denturestability and retention provided better appearanceand quality of life in edentulous patients, althoughthey did not identify differences in mastication. InIndia17, differences were observed after one monthand six months of denture fitting when compared topre­treatment. In relation to quality of life andgender, it was noted that women showed statisticallysignificant differences in relation to men.With regard to whether simplified and conventionalmanufacturing techniques of the dentures wasrelated to oral health­related quality of life, onerandomized study found no significant differencesat three and six months8. Cardoso et al.18 evaluatedoral health­related quality of life and the efficiencyof chewing in patients rehabilitated with mandibularoverdentures of two implants and conventionaldentures, finding that treatment with two mandibularimplants on the dentures provides better efficiencyin chewing and quality of life when compared withconventional prostheses.It is interesting to note that while functionalmodifications provide a great deal of satisfaction topatients, facial appearance should also be taken intoconsideration. As stated by Nordenram et al.22

regarding facial appearance, edentulous individualslive in a constant state of anxiety, and in some casesof self­recrimination for neglecting their oral healthin the past and also worry about the perception ofothers. In addition, the appearance of premature

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ACKNOWLEDGMENTSThe authors are grateful to the Department of Dentistry for itssupport and encouragement of research and development.

FUNDINGNone

CORRESPONDENCEDr.Anne da Costa Alves Rua do Calcário, 100 – apt.203 – Lagoa Nova ­ Natal, RN –59076­240 Brazile­mail: annecalves@hotmail.com

REFERENCES1. Felton DA. Edentulism and comorbid factors. J Prosthodont

2009; 18:88­96.2. Lemos MMC, Zanin L, Jorge MLR, Flório FM. Oral health

conditions and self­perception among edentulous individualswith different prosthetic status. Braz J Oral Sci 2013; 12:5­10.

3. Cousson PY, Bessadet M, Nicolas E, Veyrune JL, et al.Nutritional status, dietary intake and oral quality of life inelderly complete denture wearers. Gerodontology 2012;29:685­692.

4. Brasil. Projeto SB Brasil 2010. Condições de saúde bucal dapopulação brasileira 2010: Resultados principais. In: Minis ­tério da Saúde. Brasília; 2011. p. 54­55.

5. Goiato MC, Bannwart LC, Moreno A, Dos Santos DM, et al.Quality of life and stimulus perception in patients’ rehabilitatedwith complete denture. J Oral Rehabil 2012; 39:438­445.

6. Rosa LB, Bataglion C, Siéssere S, Palinkas M, et al. Biteforce and masticatory efficiency in individuals with differentoral rehabilitations. Open Journal of Stomatology 2012;2:21­26.

7. Shigli K, Hebbal M. Assessment of changes in oral health­related quality of life among patients with complete denturebefore and 1 month post­insertion using Geriatric OralHealth Assessment Index. Gerodontology 2010; 27:167­173.

8. Regis RR, Cunha TR, Della Vecchia MP, Ribeiro AB, et al.A randomised trial of a simplified method for complete

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Complete denture and quality of life 95

aging22 is emphasized as a result of the loss of teethcausing a decrease in the lower third of the face,protrusion of the jaw, sharp nasolabial folds, arcuatecheeks, depressed labial commissures, and thin,drawn lips23. Changes in the orofacial muscles withthe required prosthetic rehabilitation provide betterself­esteem and confidence through rejuvenatedappearance.The use of dentures improves oral functions;however, denture adaptation deserves specialattention because there are morphological andfunctional changes which may hamper denture fitand stability. The adaptation process seems to relateto the characteristics of the dentures, as well as theorofacial myofunctional situation due to the actionof the muscular forces applied, which destabilizethe dentures10.During the adjustment period, there may befunctional difficulties such as lack of intelligibilityin speech articulation, lack of saliva control,reduced mandibular and labial movements by virtueof the repositioning of teeth, refurbishment of thedenture palate, and restoration of the verticaldimension of occlusion24. In addition to the aspectsmentioned above, another important factor is thepositioning and movement of the tongue, whichoperates in the functions of speech, mastication anddeglutition. Furthermore, when people receive theirnew dentures, they may present tongue interpositionin the production of dentilingual phonemes, sothe aid of tongue contra movement for the retention

and stability of jaw dentures through tactilesensation25 should also be considered.Concerning masticatory function, there is adecrease in perceptual and reduced or inaccuratesensorineural information which complicates themasticatory pattern organization because the textureof food is not accurately perceived as it is insubjects with teeth10. In addition to uncoordinatedmovements, a reduction in muscle strength for theincision and grinding of food occurs23, as well aslow masticatory efficiency6,10,26. A factor to beconsidered for the functional limitations describedabove refers to the time the evaluation wasperformed at approximately three months, and theadaptation process may occur in up to six months10.A limitation of this study is small sample size. Itwas noted that the adaptation period of conven ­tional complete dentures can cause discomfort dueto morphofunctional modifications, thereforemyofunctional therapy is a possible treatment toassist in balanced performance of oral functions. Regarding the sample and methodology employed,differences in discomfort and chewing inabilitybetween the initial evaluation and 2 years intowearing the dentures were confirmed, demonstratingan improvement in patient quality of life. This studyemphasizes the importance of assessing relevantaspects of oral health­related quality of life ofcomplete denture users. Further research should beconducted with larger samples and longer studyperiods.

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denture fabrication: patient perception and quality. J OralRehabil 2013; 40:535­545.

9. Palac A, Bitanga P, Capkun V, Kovacic I. Association ofcephalometric changes after 5 years of complete dentureswearing and oral health­related quality­of­life. ActaOdontol Scand 2013; 71:449­456.

10. Cavalcanti RVA, Bianchini EMG. Verificação e análisemorfofuncional das características da mastigação emusuários de prótese dentária removível. Rev CEFAC. 2008;10:490­502.

11. Bilhan H, Erdogan O, Ergin S, Celik M, et al. Complicationrates and patient satisfaction with removable dentures. JAdv Prosthodont 2012; 4:109­115.

12. Souza RF, Patrocinio L, Pero AC, Marra J, et al. CompagnoniMA. Reliability and validation of a Brazilian version of theOral Health Impact Profile for assessing edentulous subjects.J Oral Rehabil 2007; 34:821­826.

13. Group TW. The World Health Organization quality of lifeassessment (WHOQOL): position paper from the WorldHealth Organization. Social Science and Medicine. 1995;41:1403­1409.

14. Souza RF, Leles CR, Guyatt GH, Pontes CB, et al. Explo ­ratory factor analysis of the Brazilian OHIP for edentuloussubjects. J Oral Rehabil 2010; 37:202­208.

15. Ribeiro JA, de Resende CM, Lopes AL, Mestriner W Jr, etal. Evaluation of complete denture quality and masticatoryefficiency in denture wearers. Int J Prosthodont 2012;25:625­630.

16. Komagamine Y, Kanazawa M, Kaiba Y, Sato Y, et al.Association between self­assessment of complete denturesand oral health­related quality of life. J Oral Rehabil 2012;39:847­857.

17. Sivakumar I, Sajjan S, Ramaraju AV, Rao B. Changes inOral Health­Related Quality of Life in Elderly Edentulous

Patients after Complete Denture Therapy and Possible Roleof their Initial Expectation: A Follow­Up Study. JProsthodont 2015; 24:452­456.

18. Cardoso RG, Melo LA, Barbosa GA, Calderon PD, et al.Impact of mandibular conventional denture and overdentureon quality of life and masticatory efficiency. Braz Oral Res2016; 30:102­109.

19. Jenei Á, Sándor J, HegedqsC, Bágyi K,et al. Oral health­related quality of life after prosthetic rehabilitation: alongitudinal study with the OHIP questionnaire. HealthQual Life Outcomes 2015; 13:99­106.

20. Albaker AM. The oral health­related quality of life inedentulous patients treated with conventional completedentures. Gerodontology 2013; 30:61­66.

21. Viola AP, Takamiya AS, Monteiro DR, Barbosa DB. Oralhealth­related quality of life and satisfaction before andafter treatment with complete dentures in a Dental Schoolin Brazil. J Prosthodont Res 2013; 57:36­41.

22. Nordenram G, Davidson T, Gynther G, Helgesson G, et al.Qualitative studies of patients’ perceptions of loss of teeth,the edentulous state and prosthetic rehabilitation: a systematicreview with meta­synthesis. Acta Odontol Scand 2013; 71:937­951.

23. Felício CM, Cunha CC. Relações entre condições miofun ­cionais orais e adaptação de próteses totais. PCL: Revibero­am de prótese clín. e Lab. 2005;7:195­202.

24. Rosa RR, Berretin­Félix G. Speech and dental prothesis:integrative review. Distúrb Comun 2015; 27:174­181.

25. Chen YF, Yang YH, Lee JH, Chen JH, et al. Tongue supportof complete dentures in the elderly. Kaohsiung J Med Sci2012; 28:273­278.

26. Farias Neto A, Mestriner Junior W, Carreiro Ada F. Mastica ­tory efficiency in denture wearers with bilateral balancedocclusion and canine guidance. Braz Dent J 2010; 21:165­169.

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RESUMENEl uso de biomarcadores salivales está en continua expansión.El objetivo de este estudio fue analizar la asociación entrefuentes alimentarias de ácidos grasos (AG) n­3 y n­6 y susconcentraciones salivales como marcador de ingesta.Parti ci paron 79 voluntarios sanos. Se aplicó un cuestionariovalidado de frecuencia de consumo alimentario y el softwareInterfood v.1.3 para su procesa miento. Las muestras saliva ­les se recogieron según estándares internacionales y sedeterminó el perfil de AG salivales por cromatografíagaseosa. Se desarrolló un modelo de regresión lineal múltipleajustado por sexo, edad, índice de masa corporal, valorenergético total, actividad física, consumo de tabaco yalcohol para analizar la asociación entre el perfil de AGsalivales y la ingesta de alimentos fuente de AG n­3 y n­6.

Las concentraciones salivales del AG alfa­linolénico (ALA)18:3 n­3 se asociaron positivamente con la ingesta de nueces(β=0.05, IC 95% 0.02­0.07, p=0.04), mientras que lasconcentraciones salivales de ácido linoleico (AL) 18:2 n­6 yaraquidónico (AA) 20:4 n­6 se asociaron con el consumo deaceites ricos en n­6 (β=0.80, 95% IC 0.06­0.09 p=0.03) y decarnes rojas, fiambres y embutidos y vísceras, (β=0.40, IC 95%0.30­0.50, p=0.02). De acuerdo a estos resultados, las concen ­traciones salivales de AG n­3 y n­6 se relacionan a la ingestade sus alimentos fuente. El monitoreo de la ingesta lipídica através de biomarcadores salivales constituye un aporte a laepidemiología nutricional y a la prevención y tratamiento depatologías vinculadas a la ingesta de grasas.

Palabras clave: ácidos grasos, saliva, dieta, biomarcadores.

INTRODUCTIONThe relationship between diet and the risk ofdeveloping chronic diseases has been documentedin recent decades. Nutritional epidemiology, focusing

on dietary intake, involves the development ofseveral tools to estimate calories, proteins,carbohydrates, fats, vitamins, minerals, etc., suchas diet records, dietary recalls and food frequency

ABSTRACTThe use of saliva for analyzing biological compounds hasrecently been expanded. The aim of this study was to analyzethe correlation between specific dietary sources of n­3 and n­6fatty acids (FA) and their salivary levels to evaluate their roleas intake markers. Seventy­nine healthy volunteers were included. A validatedfood frequency questionnaire was used for data collection andInterfood v.1.3 software was employed to quantify food intake.Salivary samples were collected following internationalstandards and FA profile was determined by gas liquid­chromatography. Multiple linear regression analyses wereperformed for dependent variables (salivary FA profile) todetect independent associations with n­3 and n­6 FA foodsource intake, adjusted by age, gender, body­mass index, total

energy intake, regular exercise, alcohol intake and smoking.Salivary concentrations of alpha­linolenic acid (ALA) 18:3 n­3were significantly associated with nuts intake (β=0.05, 95% CI0.02­0.07, p=0.04). Salivary concentrations of linoleic acid(LA) 18:2 n­6 and arachidonic acid (AA) 20:4 n­6 wereassociated with the intake of n­6 vegetable oils and red meat,cold meat and viscera (β=­0.80, 95% CI 0.06­0.09 p=0.03;β=­0.40, 95% CI 0.30­0.50, p=0.02, respectively).This study supports the hypothesis that salivary concentrationsof n­3 and n­6 FA are related to food intake. Monitoring dietaryFA though salivary markers is relevant for nutrition epidemiologyand for prevention and management of several diseases relatedto fat intake.

Key words: fatty acids, diet, saliva, biomarkers.

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Omega-3 and Omega-6 salivary fatty acids as markers of dietary fat quality: A cross-sectional study in Argentina

María D. Defagó1,2, Nilda R. Perovic2, Mirta A. Valentich1, Gastón Repossi1, Adriana B. Actis1,3

1 Universidad Nacional de Córdoba, Instituto de Investigaciones en Ciencias de la Salud (INICSA), CONICET, Córdoba, Argentina.

2 Universidad Nacional de Córdoba, Facultad de Ciencias Médicas Escuela de Nutrición, Córdoba, Argentina.

3 Universidad Nacional de Córdoba, Facultad de Odontología, Cátedra B de Anatomía, Córdoba, Argentina.

Ácidos grasos salivales Omega-3 y Omega-6 como marcadores de la calidad lipídica de la dieta: un estudio de corte transversal en Argentina

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98 María D. Defagó, et al.

questionnaires (FFQ). The FFQ is one of the mostcommonly used methods because it provides insightinto the regular dietary intake of a population overtime and is relatively cheap, quick and easy to use1.Although this progress in dietary recall tools ispromising and cost­effective, the methods forassessing dietary intake are still not without intakeerror, a commonly cited research limitation. Thecombination of different methods (e.g. adminis ­tration of different questionnaires and assessmentof biomarker levels) could reduce respondentburden and reporting bias2,3.Blood is the most commonly used biological fluid forbiomarker determination and is sensitive to dietaryintake. Urine is a good indicator of hydrosolublecompound intake, since their concentration in urinedepends on the nutrient saturation degree in tissues4,5.The use of saliva for analyzing biological compoundsto evaluate nutritional status has recently beenexpanded6­8. Collecting saliva is non­invasive, morecomfortable than venipuncture and requires nospecial equipment, and it is easily stored prior toanalysis9­11.Fatty acid (FA) intake is reflected in a wide varietyof biological tissue samples, such as serum andsubcutaneous adipose tissue12,13. A previous studyshowed a significant increase of α­linolenic acid18:3 n­3 (ALA) in saliva from vegetarians or semi­vegetarians compared to people who consumed amixed diet rich in animal products, who hadsignificantly higher levels of salivary arachidonicacid 20:4 n­6 (AA)14. This finding could be relatedto the significant intake of food rich in n­3 essentialfatty acids (EFA), such as soy, dried fruits,sunflower and corn oils. Competition between metabolic pathways may lead to changes in FA composition independent ofdietary content. It is reasonable to expect that the best markers of dietary intake would be FA thatcannot be endogenously synthetized such as trans,linoleic acid 18:2 n­6 (LA) and ALA. Other FAswhose synthesis depends on EFA intake includeeicosapentaenoic acid 20:5 n­3 (EPA) anddocosahexaenoic acid 22:6 n­3 (DHA), also presentin fish and seafood15. EFA are important compoundswhich play a role in the complex biological processof inflammation. EFA and their metabolites areknown to have pro­ and anti­inflammatory actionsand to regulate gene expression, enzyme activity,immune response and gluconeogenesis16.

We hypothesized that FA salivary levels may beaffected by dietary sources of FA. The main aim ofthis study was to analyze the correlation betweenspecific dietary sources of n­3 and n­6 FA andsalivary levels of EFA and derivatives, to evaluatetheir role as intake markers.

MATERIALS AND METHODS Study participantsA cross­sectional study was carried out involvingnon­probability sampling of 79 male and femaleadults aged eighteen to eighty years who visited two hospitals in Córdoba (Privado and Córdoba),Argentina for a routine check­up. Subjects on specialdiets and those with digestive and/or metabolicdysfunction (such as celiac disease, lactose into ­lerance or diabetes) were excluded. All participantsprovided informed consent to participate in thisstudy. The study complies with the HelsinkiDeclaration and was carried out according to theguidelines for the protection of the volunteers’rights. It was approved by the Institutional EthicsCommittees (Córdoba, Argentina).

Dietary assessmentParticipants were interviewed by two well­trainedprofessional nutritionists. A validated qualitative andquantitative FFQ was used to collect data. It includedquestions related to 257 types of food and drinksconsumed, with emphasis on food sources of fat17.This instrument includes questions on intake ofwhole and low­fat dairy products, cheese, eggs, beef,poultry, pork, fish, canned fish, seafood, viscera,sausages, vegetables, tomato derivatives, herbs,fruits, nuts, legumes, cereals, bakery products,animal fat, oils, seasonings, sugar, confectionery,pastry, beverages (alcoholic and non­alcoholic),snacks, ice cream and soy products. The frequencyreferences used were the number of times each itemwas consumed per month, per week or per day andnever. Portion size was described as small, mediumor large using a photographic guide to help subjectsunderstand these definitions18.Food data were processed using a software packagecalled Interfood v.1.3, to produce information ingrams/day of each food item consumed19. Thissoftware is an open­source program that has threebasic components: dietary intake FFQ; a databaseof common foods and their composition considering131 substances (macro­ and micronutrients and

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phytochemicals); and a relational database that linksthe FFQ data with the food database.

Collection of saliva samplesSaliva was collected from 8:00 to 10:00 a.m. after aminimum of six to eight hours fasting. With the subjectleaning forward, unstimulated but spontaneouslyflowing whole saliva was collected in sterile plastictest tubes (5 mL or more). Prior to saliva collection,the study subjects did not consume any drinks andwere at rest with no previous physical activity, toothbrushing and/or oral rinse9. Samples were frozen at ­20ºC until they were processed by centrifuging at80xg at 5ºC for 30 minutes. The supernatant wasanalyzed.

Fatty acids analysisTotal salivary lipids were extracted with chloroform­methanol 2:1 according to Folch’s method and FAwere methylated by sodium methoxide20,21. SalivaryFA was separated, identified and quantified using acapillary column (BPX 70.30 m long, 0.25 mm ID,0.25 um film, Phenomenex, Torrance, USA) in a gaschromatograph (Clarus 500 Perkin Elmer, Waltham,USA). Oven temperature began at 170ºC, rising1.2ºC/min up to 240ºC, with a total chromatographicrun time of 30 minutes22. The free FA were identi ­fied using commercial standards. Values, expressedas a percentage of detected FA, corresponded to themean of two chromatographic runs on each sample.

Assessment of other variablesCertified personnel following standardized proce ­dures took all additional measurements. Bodyweight and height were measuredusing a profes sional mechanicalscale equipped with calibratedstadiometer. Participants were inunderwear and without shoes.Body mass index (BMI) wascalculated as weight in kilogramsdivided by the square of height inmeters (kg/m2). Other variablessuch as physical activity andalcohol and tobacco use were alsoevaluated.

Statistical analysisBaseline demographic characte ­ristics were calculated as means and

standard deviations for continuous variables or asnumbers and percentages for categorical variables.Gender differences in variables of lifestyle status,food intake and salivary FA concentrations werecompared by Wilcoxon’s test for continuousvariables and Fisher’s test for categorical variables.Multiple linear regression analyses were performedfor dependent variables (salivary FA profile) todetect independent associations with n­3 and n­6FA food source intake in grams/day (fish andseafood, nuts, vegetables oils rich in n­6, and redmeat, cold meat and viscera), adjusted by age,gender, BMI, total energy intake, regular exercise,alcohol intake and smoking. All tests were two­sided and significance was considered at p<0.05.All analyses were performed with Stata® statisticalsoftware package (v.11.0).

RESULTSTable 1 shows the basic characteristics of the studysubjects. Mean age was 57.7 years for men and 49.2years for women. 84.7% of men and 72.2% ofwomen were current drinkers and 57.9% of men and41.1% of women were current smokers. Averagebody mass index (BMI) was similar in men and women (27.7 and 25.5 kg/m2, respectively).Regular physical activity was low in both men andwomen (47.3% and 45.1%, respectively). Calorieintake was 2678 ± 105 kcal/day for men and 2468± 387 kcal/day for women.Dietary fat source profile is shown in Figure 1.Overall, the foods most often consumed were dairyproducts, red meat, confectionary and stuffed pasta.Regarding specific dietary n­6 FA food source

Fig. 1: Fat dietary source intake, grams per day.

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Diet and salivary fatty acids 99

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intake, sunflower, soybean and corn oils were theoils most frequently consumed (oil average 21 ± 3g/day) followed by red meat (84 ± 12 g/day). Totalintake of red meat, viscera and cold meats was 120± 21 g/day). Regarding n­3 FA food source, fish,seafood and derivatives intake was 11 ± 5 g/day.

Hake, mackerel, dorado and tuna were the mostcommon fish species in the diet. Nuts intake (5± 2g/day) included mainly walnuts and peanuts. Non­significant statistical differences were found forfood intake, alcohol and tobacco use and physicalactivity by gender (p>0.05). Eighteen salivary FAs were analyzed by gaschromatography. Table 2 shows salivary FAcomposition. Non­significant statistical differenceswere found between men and women (p>0.05).The results of multivariate analyses are shown inTable 3. Salivary concentrations of ALA 18:3 n­3were significantly associated with the intake of nuts(β=0.05, 95% CI 0.02­0.07, p=0.04). On the otherhand, salivary concentrations of LA 18:2 n­6 andAA 20:4 n­6 were associated with vegetable oilsand with red meat, cold meat and viscera intakes,respectively (β=0.80, 95% CI 0.06­0.09 p=0.03;β=0.40, 95% CI 0.30­0.50, p=0.02, respectively).

DISCUSSION This study showed a significant correlationbetween salivary ALA 18:3 n­3, LA 18:2 n­6 andAA 20:4 n­6, and dietary sources in a healthy adult

Table 1: Characteristics of study subjects.

Men Women(n=37) (n=42) p-value

Age (years) 57.7 ± 12.4 49.2 ± 15.8 0.16

BMI 27.7 ± 3.2 25.5 ± 1.2 0.36

Energy intake 2678 ± 105 2468 ± 387 0.68(Kcal/day)

Current smoking (%) 57.9 41.1 0.27

Current alcohol 84.7 72.2 0.14drinking (%)

Regular physical 47.3 45.1 0.88activity (%)

p-values for sex differences are based on t tests for continuous variables and chi-square tests for categorical variables. Values are mean ± standard deviation for continuous variables and number (percentage) for categorical variables.

Table 2: Salivary fatty acid profile*.

FAs

4:0

6:0

12:0

14:0

16:0

16:1 n-7

18:0

18:1 n-9

18:2 n-6

Men

5.8

± 0.9

4.3

± 1.2

3.1

± 1.8

2.3

± 0.6

22.2

± 4.2

2.2

± 0.5

13.5

± 3.9

11.2

± 2.7

13.4

± 4.4

Women

3.1

± 1.1

2.8

± 0.9

4.2

± 0.7

4.1

± 1.1

23.6

± 5.6

3.1

± 1.1

15.4

± 4.2

8.3

± 1.2

15.5

± 3.8

Total

4.4

± 1.3

3.5

± 1.3

3.6

± 0.9

3.6

± 0.7

22.5

± 3.8

2.7

± 0.6

14.7

± 2.2

9.2

± 2.1

14.1

± 3.1

p-value

0.69

0.20

0.42

0.22

0.40

0.36

0.45

0.44

0.66

FAs

18:3 n-3

20:0

20:3 n-3

20:4 n-6

20:5 n-3

22:0

22:5 n-3

22:6 n-3

24:1 n-9

Men

3.9

± 1.2

1.8

± 0.8

1.5

± 0.3

5.2

± 0.9

1.3

± 0.4

1.7

± 0.4

2.6

± 1.0

2.2

± 0.4

1.8

± 0.6

Women

4.4

± 0.4

0.5

± 0.1

2.3

± 0.7

6.3

± 2.1

0.9

± 0.3

0.9

± 0.2

1.8

± 0.4

1.8

± 0.6

1.1

± 0.3

Total

4.1

± 0.4

1.1

± 0.5

1.8

± 0.4

5.6

± 1.1

1.0

± 0.3

1.4

± 0.5

2.2

± 0.6

2.1

± 0.7

1.5

± 0.6

p-value

0.20

0.37

0.43

0.41

0.46

0.19

0.80

0.22

0.52

*Values are expressed as mean percentage (± standard deviation) of total detected fatty acids.

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population. Although the presence of FA in adiposetissue, serum or plasma is accepted as a reliablebiomarker of their intake in food, there is stillscanty literature referring to the correlation betweendietary and salivary lipids23,24. Salivary LA 18:2 n­6 and ALA 18:3 n­3 would beexpected to have the strongest association withdietary intake because they cannot be synthetizedendogenously. In our study, nut intake, a source of n­3 FA, was associated with 18:3 n­3 sali ­vary levels. Nuts are natural foods rich inunsaturated FA; most nuts contain substantialamounts of monounsaturated FA, while walnuts areespecially rich in both n­6 and n­3 FA25. Althoughthere is no evidence about nuts intake and salivarylipids, frequent consumption of nuts is related withlower concentrations of inflammation markers andis also recommended by the WHO Guideline onSugar Intake for Adults and Children for dentalhealth professionals26­28. The American HeartAssociation promotes the consumption of fourservings (30­40 gram per portion) per week of nuts,seeds or legumes29. The study population showsinadequate intake of nuts according recommenda ­tions, but even though it was low, it was reflectedin the salivary n­3 profile. On the other hand, wefound no association between fish intake andsalivary levels of long chain n­3 FAs due to the verylow fish intake recorded, which was considerablylower than current guidelines (100 grams, twice aweek). In this respect, other authors have shown a correlation between fish intake and plasma n­3 FA30,31.Our results showed significant association betweenoil intake –mainly sunflower oil, followed bysoybean and corn oils– and salivary LA 18:2 n­6.Previous studies have reported high intake ofsunflower oil in Argentina32. The aforementionedoils are the main sources of 18:2 n­6 FA33,34. Theeffects of 18:2 n­6 on health and disease are stillcontroversial. An imbalance in the n­3:n­6 ratio,produced by an increased n­6 consumption or areduced n­3 intake, may promote the endogenoussynthesis of inflammatory molecules35.Significant association was observed between redmeat intake, cold meat and viscera and AA 20:4 n­6salivary concentrations. The presence of this FA inhuman saliva as related to diet and inflammatoryconditions has been previously demonstrated 10,36.Westernized diets characterized by high intake of

red meat and processed foods have been related toinflammation biomarkers such as leukotrienes­2and thromboxanes­4, which are associated withcardiovascular diseases and certain types of cancer37. EFA metabolites, derived by lipoxygenase andcyclooxygenase enzymatic pathways, have beenquantified in human mixed saliva as well as insaliva fractions. The level of free AA and thequantitative hydroxyeicosatetraenoic acid (HETE)act as markers for the inflammatory processesoccurring in the oral mucosa and in saliva inresponse to the development of squamous cellcarcinoma38. In addition, pro­inflammatory lipidmediator precursors have been detected as potentialmarkers for aggressive periodontitis39. In this sense,monitoring dietary FAs such as LA and ALA andtheir metabolites is a relevant strategy in theprevention and management of several fat intake­related diseases, not for only oral diseases, but alsocardiovascular disorders, diabetes and tumors suchas prostate and mammary, among others. Secretion of saliva plays an important role innumerous significant biological processes. Althoughsome salivary components are well characterized,data for characterizing salivary lipids and theirfunctions is scarce and controversial. However, the

Table 3: Linear regression analysis (ß coefficient) by food intake.

Food intake ß 95% Conf. p-valueInterval

Fish and seafood

20:5 n-3 0.006 0.002-0.001 0.65

22:5 n-3 -0.0003 -0.0001-0.001 0.41

22:6 n-3 0.001 0.0004-0.002 0.59

Nuts

18:2 n-6 0.02 0.008-0.07 0.36

18:3 n-3 0.05 0.02-0.07 0.04

Vegetable oils high inn-6 FA

18:2 n-6 0.80 0.06-0.09 0.03

20:4 n-6 0.02 0.01-0.05 0.19

Red meat, cold meat and viscera

20:4 n-6 0.40 0.30-0.50 0.02

ß: coefficient in multivariable model adjusted for age, sex, smoking status, body mass index, energy intake, alcohol consumption and physical activity.

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FUNDINGThis work was supported by a grant from the Secretaría deCiencia y Tecnologia, Universidad Nacional de Córdoba,Argentina, Number RHCS 2472/10.

CORRESPONDENCEDra. María Daniela Defagó Instituto de Investigaciones en Ciencias de la Salud (INICSA),CONICET. Enrique Barros esq. Enfermera Gordillo, Ciudad Universitaria,Córdoba (5000), Argentina. danieladefago@hotmail.com

REFERENCES 1. Ortiz­Andrellucchi A, Sánchez­Villegas A, Doreste­Alonso

J, de Vries J, de Groot L, Serra­Majem L. Dietary assessmentmethods for micronutrient intake in elderly people: asystematic review. Br J Nutr 2009; (102 Suppl 1):S118­149.

2. Naska A, Lagiou A, Lagiou P. Dietary assessment methodsin epidemiological research: current state of the art andfuture prospects. F1000 Res 2017; 6:926. doi: 10.12688/f1000research.10703.1. eCollection 2017.

3. Jenab M, Slimani N, Bictash M, Ferrari P, Bingham SA.Biomarkers in nutritional epidemiology: applications,needs and new horizons. Hum Genet 2009; 125: 507­525.

4. Corella D, Ordovás JM. Biomarkers: background, classifi ­cation and guidelines for applications in nutritionalepidemiology. Nutr Hosp 2015; 31(Suppl 3):177­188.

5. Blanck HM, Bowman BA, Cooper GR, Myers GR, MillerDT. Laboratory issues: use of nutritional biomarkers. J Nutr2003; 133:888S­894S.

6. Karjalainen S, Sewón L, Söderling E, Larsson B, JohanssonI, Simell O, Lapinleimu H, Seppänen R.Salivary cholesterolon healthy adults in relation to serum cholesterol concentrationand oral health. J Dent Res 1997; 76:1637­1643.

7. Morzel M, Truntzer C, Neyraud E, Brignot H, DucoroyP, Lucchi G, Canlet C, Gaillard S, et al. Associationsbetween food consumption patterns and saliva compo ­sition: Specificities of eating difficulties children. PhysiolBehav 2017; 173:116­123.

8. Defagó MD, Valentich MA, Actis AB. Lipid characte ­rization of human saliva. J Calif Dent Assoc. 2011; 39:874­880.

9. Hofman LF. Human saliva as a diagnostic specimen. J Nutr2001;131:1621S­1625S.

10. Kaufman E, Lamster IB. The diagnostic applications ofsaliva. A review. Crit Rev Oral Biol Med 2002; 13:197­212.

11. Lawrence HP. Salivary markers of systemic disease:noninvasive diagnosis of disease and monitoring of generalhealth. J Can Dent Assoc 2002; 68:170­174.

12. Astorg P, Bertrais S, Laporte F, Arnault N, EstaquioC, Galan P, Favier A, Hercberg S.Plasma n­6 and n­3polyunsaturated fatty acids as biomarkers of their dietaryintakes: a cross­sectional study within a cohort of middle­aged French men and women. Eur J Clin Nutr 2008;62:1155­1161.

13. Hodson L, Skeaff CM, Fielding BA. Fatty acid compositionof adipose tissue and blood in humans and its use as abiomarker of dietary intake. Prog Lipid Res 2008; 47:348­380.

14. Actis AB, Perovic NR, Defagó D, Beccacece C, EynardAR. Fatty acid profile of human saliva: a possible indicatorof dietary fat intake. Arch Oral Biol 2005; 50:1­6.

15. Burdge GC. Metabolism of alpha­linolenic acid in humans.Prostaglandins Leukot Essent Fatty Acids 2006; 75:161­168.

16. Das UN. Essential fatty acids and their metabolites as mod­ulators of stem cell biology with reference to inflammation,cancer, and metastasis. Cancer Metastasis Rev 2011; 30:311­324.

17. Perovic NR, Defagó MD, Aguinaldo A, Joekes S, Actis AB.Validación y reproducibilidad de un cuestionario de fre ­cuencia de consumo alimentario para valorar la ingesta delípidos y fitoquímicos Fac Cien Med Univ Nac Cordoba2015; 72:69­77.

18. Vázquez MB, Witriw AM. Modelos visuales de alimentosy tablas de relación peso/volumen. 1st ed. Buenos Aires:Argentina, 1997.

19. Defagó MD, Perovic NR, Aguinaldo CA, Actis AB.Desarrollo de un programa informático para estudiosnutricionales. Rev Panam Salud Publica 2009; 25:362­366.

20. Folch J, Lees M, Sloane Stanley GH. A simple method forthe isolation and purification of total lipids from animaltissues. J Biol Chem 1957; 226:497­508.

21. Cantellops D, Reid AP, Eitenmiller RR, Long AR.Determination of lipids in infant formula powder by directextraction methylation of lipids and fatty acid methyl esters(FAME) analysis by gas chromatography. J AOAC Int1999; 82:1128­1139.

qualitative and quantitative content of salivary lipidsmay change in pathological states (e.g. cysticfibrosis and diabetes), and according our results, dueto nutritional exposure40. Thus, salivary lipids couldbe used for validating dietary measurement or asmarkers of food consumption. Some limitations of the present study must beconsidered. It is cross­sectional design and thereforewe cannot infer causality from these results. In

addition, these findings cannot yet be generalizeddue to the sample size and the fact that a particularpopulation was analyzed. In conclusion, the present study supports thehypothesis that salivary concentrations of n­3 andn­6 FA are related to the intake of certain foods.Further studies are needed to confirm these resultsand to determine the causal direction of thisrelationship.

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22. Tomita Y, Miyake N, Yamanaka S. Lipids in human parotidsaliva with regard to caries experience. J Oleo Sci 2008;57,115­121.

23. Arab L, Akbar J. Biomarkers and the measurement of fattyacids. Public Health Nutr 2002; 5:865­871.

24. Øverby NC, Serra­Majem L, Andersen LF. Dietaryassessment methods on n­3 fatty acid intake: a systematicreview. Br J Nutr 2009; (102 Suppl 1):S56­63.

25. Ros E, Mataix J. Fatty acid composition of nuts—implications for cardiovascular health. Br J Nutr 2006; (96Suppl 2):S29­S35.

26. Rajaram S, Connell KM, Sabaté J. Effect of almond­enriched high­monounsaturated fat diet on selected markersof inflammation: a randomised, controlled, crossover study.Br J Nutr 2010; 103:907­912.

27. Ros E. Nuts and novel biomarkers of cardiovasculardisease. Am J Clin Nutr 2009; 89:1649S­1656S.

28. Moynihan P, Makino Y, Petersen PE, Ogawa H. Implicationsof WHO Guideline on Sugars for dental health professionals.Community Dent Oral Epidemiol 2018; 46:1­7.

29. Van Horn L, Carson JA, Appel LJ, Burke LE, EconomosC, Karmally W, Lancaster K, Lichtenstein AHet al.Recommended dietary pattern to achieve adherence to theAmerican Heart Association/American College ofCardiology (AHA/ACC) Guidelines: a scientific statementfrom the American Heart Association. Circulation. 2016;134:e505­e529.

30. Wilk JB, Tsai MY, Hanson NQ, Gaziano JM, Djoussé L.Plasma and dietary omega­3 fatty acids, fish intake, andheart failure risk in the Physicians’ Health Study. Am J ClinNutr 2012; 96:882­888.

31. Lucas M, Proust F, Blanchet C, Ferland A, Déry S, AbdousB, Dewailly E. Is marine mammal fat or fish intake moststrongly associated with omega­3 blood levels among the

Nunavik Inuit? Prostaglandins Leukot Essent Fatty Acids2010; 83:143­150.

32. Navarro A, Muñoz SE, Lantieri MJ, Fabro EA, EynardAR.Composition of saturated and unsaturated fatty acidsof foods frequently consumed in Argentina. Arch LatinoamNutr 1997; 47:276­281.

33. Matthaus B, Ozcan MM. Fatty acid and tocopherol contentsof several soybean oils. Nat Prod Res 2014; 28:589­592.

34. Alezones J, Avila M, Chassaigne A, Barrientos V. Fattyacids profile characterization of white maize hybrids grownin Venezuela. Arch Latinoam Nutr 2010; 60:397­404.

35. Marangoni F, Novo G, Perna G, Perrone Filardi P, Pirelli S,Ceroti M, Querci A, et al. Omega­6 and omega­3 polyunsa ­turated fatty acid levels are reduced in whole blood ofItalian patients with a recent myocardial infarction: theAGE­IM study. Atherosclerosis 2014; 232:334­338.

36. Elabdeen HR, Mustafa M, Szklenar M, Rühl R, AliR, Bolstad AI.Ratio of pro­resolving and pro­inflammatorylipid mediator precursors as potential markers foraggressive periodontitis. PLoSOne 2013; 8:e70838.

37. Kim E, Coelho D, Blachier F. Review of the associationbetween meat consumption and risk of colorectal cancer.Nutr Res 2013; 33:983­994.

38. Prasad S, Tyagi AK, Aggarwal BB. Detection ofinflammatory biomarkers in saliva and urine: Potential indiagnosis, prevention, and treatment for chronic diseases.Exp Biol Med (Maywood) 2016; 241:783­799.

39. Elabdeen HR, Mustafa M, Szklenar M, Rühl R, AliR, Bolstad AI.Ratio of pro­resolving and pro­inflammatorylipid mediator precursors as potential markers foraggressive periodontitis. PLoS One 2013; 8:e70838.

40. Matczuk J, Żendzian­Piotrowska M, Maciejczyk M, KurekK. Salivary lipids: A review. Adv Clin Exp Med 2017;26:1021­1029.

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RESUMENEn las arcadas dentarias comprimidas, con apiñamientodentario de leve a moderado, es posible obtener ganancia deespacio que favorezca la ubicación de las piezas en la fasede alineación por remodelación alvéolo­dentaria de lasarcadas. El objetivo de este trabajo fue comparar las diferencias pre y posttratamiento en la etapa de alineación de los grupos A: bracketsautoligables pasivos, (Sistema Damon) y grupo B: bracketsconvencionales (Filosofía de Roth). Se evaluaron las variacionesen el diámetro transversal, antes y después de alinear ortodón ­cicamente los dientes, utilizando modelos de estudio.Participaron 24 pacientes de ambos sexos, entre 13 y 36 años conapiñamiento dentario moderado (entre 4 y 6 mm) tratados en laCátedra de Ortodoncia de la Facultad de Odontología, Universidadde Buenos Aires. Los pacientes fueron distribuidos al azar en dos

grupos de 12 pacientes cada uno. No se realizaron exodonciashasta finalizar la alineación. Se midieron los modelos pre y postalineación.La distancia entre primeros premolares superiores aumentó en elgrupo A más que en el B (p=0.008) y entre caninos fue mayor enel grupo B, con diferencia estadísticamente significativa (p< 0,01).En pacientes con apiñamiento leve a moderado, la expansión conambos sistemas permite la alineación dentaria por aumento deldiámetro transversal de las arcadas fundamentalmente en el áreapremolar. El mayor desarrollo transversal se produce en el áreapremolar para ambas técnicas y es significativamente mayor conortodoncia autoligable.En ortodoncia convencional, aumenta significativamente ladistancia intercanina en comparación con la autoligable.

Palabras clave: arcos dentales ­ ortodoncia; desarrollo.

INTRODUCTIONSelf­ligating brackets were introduced in 1946 byStolzenberg1 and the Damon system used in this studywas introduced in 19962,3. Different authors havehighlighted advantages of the self­ligating systemcompared to conventional brackets: reduction in

treatment time and greater comfort for the patient4,5

shorter consultations, longer intervals betweenappointments6­8. Pandis et al. 2008 conducted aclinical trial comparing dental changes during theinitial stages of alignment without extractions in lowerjaw, finding no statistically significant difference9.

ABSTRACTIn compressed dental arches with mild to moderate crowding,space can be gained during the alignment phase to helpimprove tooth location.The aim of this study was to compare transverse measurementsbefore and after the alignment stage in two groups: Group Atreated with passive self­ligating brackets, (Damon System)and Group B with conventional brackets (Roth philosophy).The change in transverse diameter between teeth was measuredon dental casts taken before and after orthodontic alignment.Twenty four patients of both sexes aged 13 to 36 years, withmoderate tooth crowding (4 to 6 mm), were treated. They all receivedtreatment at the Department of Orthodontics, School of Dentistry,University of Buenos Aires. Patients were distributed randomly into

two groups of 12. No extraction was performed until the end of thealignment. Dental casts were measured before and after alignment.The distance between upper first premolars increased more inGroup B (p = 0.008), and the distance between canines was higherin Group B, with statistically significant difference (p < 0.01).Both systems enable tooth alignment by increasing transversediameter of the arches in patients with mild to moderate crowding.The greatest transversal development occurs in the inter­premolar area in both techniques and is significantly higher withself­ligating orthodontics.Inter­canine distance increases significantly with conventionalorthodontics compared to self­ligating orthodontics.

Key words: dental arches ­ orthodontics; development.

Increased interpremolar development with self-ligating orthodontics. A prospective randomized clinical trial

María E. Mateu1, Sandra Benítez-Rogé1, Marina Iglesias1, Diana Calabrese1, María Lumi1, Marisa Solla1, Pedro Hecht2, Alejandra Folco1

1 Universidad de Buenos Aires, Facultad de Odontología, Cátedra de Ortodoncia.2 Universidad de Buenos Aires, Facultad de Odontología, Cátedra de Biofísica y Bioestadística.

Mayor desarrollo interpremolar con ortodoncia de autoligado. Estudio clinico prospectivo randomizado

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Our team compared the gingival response in patientstreated with conventional and self­ligating bracketsand found that orthodontic treatment increasedbacterial plaque and gingival inflammatory responsein both groups without statistically significantdifferences between groups10. Moreover, adequatebasic therapy can maintain gingival and periodontalhealth. These results are consistent with recentstudies published by Kaklamanos et al.11

A review of the literature published by Miles12 andHerradine13 compared conventional with self­ligating treatment, finding that both treatmentsachieved alignment with a combination of dentalarch expansion, with less incisive flaring when self­ligating treatment was used. Celar14 published a systematic review in 2013 comparing initial pain, number of consultations and treatment time,concluding that further prospective studies arerequired to define whether there are any differencesbetween the techniques. Reddy et al. reported in2014 that self­ligating bracket treatments are more efficient regarding space closure than theconventional systems and that alignment occursthrough lower incisor inclination15.Recently published studies quantify the increaseand bone loss by vestibular and lingual / palatal withcone beam tomography in orthodontic patients.Transverse changes in dental arches have beenanalyzed in plaster study casts16 and in digitalmodels17.

Our hypothesis is that the greatest gain in space withthe self­ligating system occurs in the lateral areasof the arches, at the level of the premolars, with noalteration of inter­canine or inter­molar distance.Our goal is to compare differences in the distancebetween canines, first and second premolars andfirst and second molars in both arches before andafter alignment using both techniques.

MATERIAL AND METHODSThis project was approved by the BioethicsCommittee of the school of Dentistry of theUniversity of Buenos Aires, and follows theprinciples of the Declaration of Helsinki. Patientssigned informed consent to participate in thisprospective clinical trial.

PatientsInclusion criteria: Patients 13 to 36 years old of both sexes, with permanent dentition, with little

development in their dental arches, moderate dentalcrowding (­4 to ­6 mm of dental discrepancy).Exclusion criteria: patients with joint disorders,untreated caries or periodontal disease. Patientswith mixed or temporary dentition. Patients withdiastemas, teeth retained or absent except thirdmolars.Treatment plan: Patients were assigned randomlyto treatment with one of the techniques. Treatmentplans were made after diagnosis, which was basedon cephalometric analysis, study of plaster casts andclinical analysis. Extractions were determined fromthe clinical analysis of facial aesthetics, sagittalproblems, open bite, dental protrusion and dentaldiscrepancy. When extractions were required, theywere performed after the alignment stage.

Experimental designPatients were treated by calibrated professionalsfrom the Orthodontic department, with unifiedcriteria. The sample was distributed into two groupsof 12 patients each.Group A: 12 patients of both sexes, 13 to 34 yearsold, were treated with the Damon System (lowfriction). Damon II brackets (A Company), nickel­titanium­copper archwires (Damon Cooper­Ni­Ti.014and 0.016), Damon format.Group B: 12 patients of both sexes, 13 to 36 yearsold, were treated with a straight wire technique (withfriction). Roth brackets (A Company, Synthesis),nickel­titanium archwires (Ni­Ti .012, .014 and.016), True Arch format and elastomeric ligature(Fig.1).The treatments followed the protocol of each tech­nique. Routine diagnostic studies were performed(Panoramic Rx, Tele­Rx profile, cephalometric

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Fig. 1: The archwires used for the treatment with Damon Systempresent Damon format, which is the same for both jaws (a).Treatments performed with Roth’s Philosophy True Arch usedarchwires with different shapes for upper (b) and lower jaw (c).

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analysis, articulator mounting, clinical examinationand dental study casts). The dental arch was con­sidered aligned when it was possible to place therectangular archwire. During the initial stage, noextractions or interproximal wear were performed.Operational definitions of variables: Pre­ and post­alignment dental casts were studied.

Dental cast study The expansion of the dental arches in transversedirection was measured in upper and lower jaws.Measurements were performed taking as a guidethe projection on the gingiva of the palatal sulcusof the first or second upper molars and the lingualgroove of the lower molars, the projection on thegingiva of the tip of the palatine cusp of the firstand second upper and lower premolars, and theprojection on the gingiva of the cingulum of theupper and lower canines.

Measurements were taken with a digital caliper(Mitutoyo Digimatic NTD12­6” C; Mitutoyo Corp.,Tokyo, Japan). Five measurements per jaw weretaken on each patient for transverse analysis. Thecasts were measured by three calibrated operatorswho were blinded to the groups (pre­ and post­treatment, identified with numbers)18 (Fig. 2).Measurements were compared using descriptivestatistics of mean, SD and 2­tailed Student’s T­testfor unpaired samples.

RESULTSAnalysis of dental casts: transversal variationTable 1 shows the averages and Standard Deviations(SD) of the variation in the distance betweencontralateral teeth of 24 patients at the end of thealignment stage taking into account all transversemeasurements.

Statistical analysisThe following descriptive statis­tics were performed: average,SD and Student T Test for two­tailed unpaired samples forcomparison between samples.Comparative analyses were per­formed by jaws, by techniquesand by teeth. Student’s T­testshowed statistically significantdifferences in the distancebetween first upper premolars,which was greater with self­lig­ating brackets (p=0.008). At thelevel of the canines, there wasgreater expansion with the con­ventional technique (p< 0.01).The rest of the comparisonsshowed no statistically signifi­cant difference. Consideringboth maxillaries, the sum of thedistances between all contralat­eral teeth evaluated increasedwith the alignment by an average1.3mm in group A (self­ligating)and 1.1mm in Group B (conven­tional); with no statisticallysignificant difference (p = 0.19)(Fig. 3).

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Fig. 2: A: Measurement with digital caliper of the transverse distance betweenhomologous teeth in upper jaw casts. B: Transverse measurements performed pre­ andpost­alignment in upper jaw casts.

Fig. 3: Bar chart considering both jaws. Difference in the development of the archeswith self­ligating (Damon System) (n= 12) and Roth Technique (n = 12) (p = 0.19).

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DISCUSSIONThe Damon system uses self­ligating bracketsand heat­activated copper­Ni­Ti archwires whichare the same size for both jaws, whereas the Rothsystem uses True Arch Ni­Ti archwires, with theupper archwire being larger than the lower. Ourhypothesis is that these two different orthodon­tic systems produce different responses in thetissues.In the study subjects, who presented mild tomoderate discrepancy, transversal development wasslightly higher for the self­ligating system, althoughwith no statistically significant difference. Thiswould indicate that an archwire larger than thedental arch does not produce an expansion greaterthan needed when its composition is superelastic,as is the case of heat­activated copper­nickel­titanium, which exerts a very low force that can becontrolled by the musculatureA paper published in 2014 by Atik et al19 comparedthe two treatment systems taking into account theposition of the incisors, the changes in transversedimensions in the upper arch, inclination changesin upper teeth, the clinical periodontal parametersand the intensity of pain in patients with a Class Imalocclusion by studying pre­ and post­treatmentcasts and cephalometry in patients with dentalcrowding. The study concluded that there was no difference between the two systems relative tothe position of the incisors, changes in the dentalarch, transverse dimension, periodontal clinicalparameters or intensity of pain. We believe that the

same outcomes can be achieved with both systemsbecause a Quad­Elix expander was used prior to theconventional treatment, enabling treatment withoutextractions. We understand that arches behavedifferently according to whether they are treatedwith passive self­ligating system or conventionalsystem.

Analysis of transverse dimension between teeth The increase in transverse diameter is mainly theresult of variations in the shape of the arches. Incompressed dental arches with moderate crowding,light arch wires can be used during the alignmentphase to gain space and help improve tooth location.Franchi et al. (2006) compared the use of low­friction ligature instead of self­ligating brackets,reporting a similar increase in upper inter­molarwidth in comparison to that obtained by conven ­tional ligation with elastomers. The study showed 4 degrees of vestibular molar inclination andconcluded that this may imply that the molarexpansion observed with self­ligating brackets isrelated to displacement or inclination of the molarsrather than to bodily movement or basal maxillaryexpansion20.

In the present study, the evaluation of the distancebetween contralateral teeth used anatomicallandmarks as reference: insertion of the teeth at thepalatal or lingual level. This explains why thetransverse increase is smaller in millimeters thanwhen the increase is measured at the crown level.

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Table 1: Variation in the distance between contralateral homologous teeth in alignment treatment.

VARIATION MAXILLARY DAMON SYSTEM n=12 CONVENTIONAL ROTH n=12 TEST T UNPAIRED

BETWEEN AVERAGE DS AVERAGE DS p

CANINES UPPER 0,217 2,458 0,81 1,6476 *< a 0,001

1PMs UPPER 2,47 1,111 2,077 1,674 *0.008

2PMs UPPER 1,741 1,538 1,434 1,301 0,614

1Ms UPPER 0,268 1,556 -0,227 0,957 0,357

2Ms UPPER -0,7 1,127 -0,296 2,33 0,594

CANINES LOWER 0,416 1,637 1,903 0,923 *0,011

1PMs LOWER 1,744 2,394 1,879 1,177 0,862

2PMs LOWER 1,66 1,847 1,111 2,016 0,493

1Ms LOWER -0,09 1,45 -1,07 1,645 0,135

2Ms LOWER 0,055 1,465 -0,535 1,206 0,292

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Inter­canine distanceFor inter­canine distance, the increase was slight:0.2mm + 2.4 with Damon system and 0.8 mm + 1.6with Roth. In the lower jaw, average variation was0.4mm + 1.6 with the self­ligating system and1.9mm + 0.9 with the Roth system. Differenceswere statistically significant in both cases.

Premolars and molarsFor premolars and molars, the average increase in transverse diameter was 2.47 mm + 1.11 for the self­ligating group and 2.07 mm + 1.67 for theRoth group, with statistically significant difference(p = 0.008). Vajaria et al included the measurement of thedistance between premolars to compare the Damonsystem with straight­wire appliance, and foundincreased distances between canines and premolarsfor both groups, though with no significant differencein transverse level between groups21. Almeida et al studied the changes produced withboth types of treatment only in the lower arch16,finding no significant increase between premolars.However, in said study the measurements weretaken at the cusps, which are anatomical landmarksdifferent from those evaluated in the current studyor in the study by Fleming et al22.Pandis et al. in 2009, carried out a comparativestudy between straight­wire appliance and self­ligating, finding that mandibular crowding wascorrected through incisive flaring and expansion ofthe arches. However, they recorded measurementsbetween canines and molars, without taking intoaccount the inter­premolar distance, which in ourstudy is the distance that varies the most and differssignificantly between techniques9.

In the individual evaluation of the response in thearches with the self­ligating system, it was foundthat the greatest modification in the transversediameter of upper and lower arches occurs at thelevel of the inter­premolar distance, in agreementwith papers recently published by Cattaneo et al17.They evaluated digital casts of patients treated withpassive self­ligating and active orthodontics;finding a statistically significant increase at thelevel of premolars. Lineberger et al 23 used digitalcasts to study 25 patients treated with self­ligatingbrackets compared to untreated controls, and founda 2 ­ 2.2 mm increase in transverse diameter at thelevel of premolars and an increase in torque.In the molar sector, the differences found wereminimal. There was even reduction between upperand lower first and second molars with the conven ­tional technique, and between second upper molarand first lower molar with the self­ligating technique,with no statistically significant difference betweentechniques.Vajaria et al found a significant increase betweenmolars in the treatment with Damon System, but themeasurements were made using dental landmarks,so the results may be due to the vestibular inclinationof the crowns21.

In patients with mild to moderate crowding, transversedevelopment with conventional and self­ligatingtreatments enable dental alignment, in both cases byincreasing transverse diameter.Most of the transverse development occurs in thepremolar area with both techniques and is significantlygreater with self­ligating orthodontics. Inter­caninedistance increases significantly more for both jawswith conventional than with self­ligating orthodontics.

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ACKNOWLEDGMENTSWe thank Orthodontika Company for its cooperation withorthodontic materials and orthodontists Pelizardi C and Her­rera del Corral MA.

FUNDINGThis study was supported by grants from Buenos Aires Univer­sity: 2008­2010 Program (code O 408), 2010­2012 Program(code 0020090200390) and 2012­2014 Program (code CO22).

CORRESPONDENCEDr,Alejandra Folco Ramón Castro 2128, Olivos, Buenos Aires, Argentinaale.folco@hotmail.com

REFERENCES1. Stolzenberg J. The efficiency of the Russell attachment. Am J

Orthod Oral Surg. 1946; 32:572­582. 2. Damon DH. The rationale, evolution and clinical application

of the self­ligating bracket. Clin Orthod Res 1998; 1: 52­61.

3. Damon DH. The Damon low friction bracket: a biologicallycompatible straight­wire system. J Clin Orthod 1998; 32:670­680.

4. Eberting JJ, Straja SR, Tuncay OC. Treatment time, outcome,and patient satisfaction comparisons of Damon and conventionalbrackets. Clin. Orthod.Res. 2001;4: 228­234.

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5. Harradine NW. Self­ligating brackets and treatmentefficiency. Clin Orthod Res, 2001; 4:220­227

6. Shivapuja PK, Berger J. A comparative study of conven ­tional ligation and self­ligation bracket systems, Am JOrthod Dentofacial Orthop. 1994; 106:472­480.

7. Berger J, Byloff FK. The clinical efficiency of self­ligatedbrackets, J Clin Orthod., 2001; 35:304­308.

8. Turnbull NR, Birnie DJ. Treatment efficiency of conven ­tional vs. self­ligating brackets: effects of arch wire sizeand material. Am J Orthod Dentofacial Orthop. 2007;131:395­399.

9. Pandis N, Polychronopoulou A, Makou M, Eliades T.Mandibular dental arch changes associated with treatmentof crowding using self­ligating and conventional brackets.Eur J Orthod. 2010; 32:248­253.

10. Folco AA, Benítez­Rogé SC, Iglesias M,Calabrese D,Pelizardi C, Rosa A, Brusca MI, Hecht P, Mateu ME.Gingival response in orthodontic patiens. Comparativestudy between self­ligating brackets. Acta Odontol. Latinoam.2014: 27:120­124.

11. Kaklamanos EG, Mayreas D, Tsalikis L, Karagiannis V,Athanasiou AE. Treatment duration and gingival inflammationin Angle’s Class I malocclusion patients treated with theconventional straight­wire method and the Damon technique:a single­centre, randomized clinical trial. J Orthod. 2017;44:75­81.

12. Miles PG. Self­ligating brackets in orthodontics: Do theydeliver what they claim? Aust Dent J. 2009; 54:9­11.

13. Harradine NW. Self­ligating brackets: where are we now?J Orthod., 2003; 30:262­273.

14. Celar A, Schedberger M, Dorfler P, Bertl M. Systematicreview on self­ligating vs. conventional brackets: initialpain, number of visits, treatment time. J Orofac Orthop.2013; 74:40­51.

15. Reddy VB, Kumar TA, Prasad M, Nuvvula S, Patil RG,Reddy PK. A comparative in­vivo evaluation of the alignment

efficiency of 5 ligation methods: A prospective randomizedclinical trial. Eur J Dent. 2014; 8:23­31.

16. Almeida MR, Futagami C, Conti ACCF, Oltramari NavarroPVP, Navarro RL. Dentoalveolar mandibular changes withself­ligating versus conventional bracket systems: A CBCTand dental cast study. Dental Press J Orthod. 2015; 20:50­57.

17. Cattaneo PM, Treccani M, Carlsson K, Thorgeirsson T,Myrda A, Cevidanes LH, MelsenB. Transversal maxillarydento­alveolar changes in patients treated with active andpassive self­ligating brackets: a randomized clinical trialusing CBCT­scans and digital models. Orthod CraniofacRes. 2011; 14:222­233.

18. Folco A, Benítez­Rogé S, Calabrese D, Iglesias M, LumiM, Hetch P, Mateu ME. Method for evaluation of transversedimension in treatment with self­ligating orthodontictreatment. A comparative study. Acta Odontol. Latinoam.2017;30:124­128.

19. Atik E, Ciğer S. An assessment of conventional and self­ligating brackets in Class I maxillary constriction patients.Angle Orthod. 2014; 84:615­622.

20. Franchi L, Baccetti T, Camporesi M, Lupoli M. Maxillaryarch changes during leveling and aligning with fixedappliances and low­friction ligatures. Am J OrthodDentofacial Orthop. 2006; 130:88­91.

21. Vajaria R, Begole E, Kusnoto B, Galang MT, Obrez A.Evaluation of incisor position and dental transverse dimen ­sional changes using the Damon system. Angle Orthod.2011; 81:647­652.

22. Fleming PS, Di Biase AT, Sarri G, Lee RT. Comparison ofmandibular arch changes during alignment and levelingwith 2 preadjusted edgewise appliances. Am J OrthodDentofacial Orthop. 2009; 136:340­347.

23. Lineberger MB, Franchi L, Cevidanes LH, Huanca GhislanzoniLT, McNamara JA Jr. Three­dimensional digital cast analysisof the effects produced by a passive self­ligating system.Euro J Orthod.2016;38: 609­614.

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RESUMENHasta el momento no se ha dilucidado la respuesta temprana invivo de los osteocitos a la aplicación de fuerzas de diferentesmagnitudes sobre el hueso. El objetivo de este estudio fueexaminar la respuesta temprana de la aplicación de una fuerzaortodóncica muy liviana (FL: 0,16 N) y muy fuerte (FF: 2,26 N)durante una hora sobre la expresión de apoptosis y osteopontina(OPN) en los osteocitos del hueso alveolar, en ratas. Resultados:FL: en comparación con el grupo control, mostraron un aumentosignificativo en la expresión de OPN y una disminuciónsignificativa en el número de osteocitos TUNEL­positivos. FF: encomparación con el grupo control, mostraron un aumento

significativo en la expresión de OPN y una disminución signi ­ficativa en el número de osteocitos TUNEL­positivos. Nuestrosresultados muestran que los osteocitos responden muy tempranoa la aplicación de fuerzas de tensión y presión de diferentesmagnitudes, y la aplicación de fuerzas disminuye el número deosteocitos apoptóticos y aumenta la expresión de OPN. Estosresultados permiten concluir que los osteocitos se activanrápidamente cuando se los somete a fuerzas aplicadas localmente,ya sean estas fuerzas de presión o tensión, livianas o fuertes.

Palabras clave: osteocito, fuerzas mecánicas, mecanotrans ­ducción, ortodoncia apoptosis, osteopontina.

INTRODUCTIONMechanosensation and transduction in osteocytesenables efficient exchange of physical and chemicalsignals among cells1­3, mainly the signals that generatewhen bone is subjected to mechanical stress, as is thecase of orthodontic forces applied to teeth4.Orthodontic tooth movement exerts different typesof forces which have a different biomechanicaleffect: pressure and tension. It is assumed that theenvironmental changes in periodontal tissue duringorthodontic tooth movement influence alveolar bone

mechanically, acting on osteocyte activity andosteocyte network communication during theadaptive process5­11. Studies on alveolar osteocytesshowed that application of tension forces during 48hours was found to result in expression of connexin43 protein5, whereas application of pressure forcesup to 24 hours resulted in a progressive increase inosteocytes undergoing apoptosis up to 1 day post­application, and a peak in the proportion of necroticosteocytes and empty lacunae at 2 and 4 daysrespectively6. Results obtained using an experimental

ABSTRACTThe in vivo response of osteocytes to different force magnitudessoon after they are applied remains to be elucidated. The aimof this study was to examine the early effects of applying a verylight (LF: 0,16 N) and a very strong (SF: 2,26 N) orthodonticforce during one hour on apoptosis and osteopontin (OPN)expression on alveolar bone osteocytes, in rats. Results: LF:compared to the control group, they showed a significantincrease in OPN expression, and a significant decrease in thenumber of TUNEL­positive osteocytes. SF: compared to thecontrol group, they showed a significant increase in OPNexpression and a significant decrease in the number of TUNEL­

positive osteocytes. Our results show that osteocytes respondvery early to the application of tension and pressure forces ofdifferent magnitudes, and application of forces decreases thenumber of apoptotic osteocytes and increases OPN expression.These results allow concluding that osteocytes activate rapidlywhen subjected to locally applied forces, whether these forcesbe pressure or tension, light or strong forces. Grants: UBACyT 200201301002270 BA and School of Dentistry,University of Buenos Aires.

Key words: osteocytes, mechanical stress, mechanotransduction,orthodontic tooth movement, apoptosis, osteopontin.

Evaluation of apoptosis and osteopontin expression in osteocytes exposed to orthodontic forces of different magnitudes

Carola B. Bozal, Luciana M. Sánchez, Patricia M. Mandalunis, Angela M.Ubios

Universidad de Buenos Aires, Argentina, Facultad de Odontología, Cátedra de Histología y Embriología.

Evaluación de la apoptosis y la expresión de osteopontina en los osteocitos luego de la aplicación de fuerzasortodóncicas de diferentes magnitudes

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model that combined both types of forces showedDMP­1 (dentin matrix protein­1) expression toincrease after 6 hours and peak between days 3 and 7post­application7 and MEPE (matrix extracellularphosphoglycoprotein) expression to increase at 3 days8. According to reports in the literature, thepercentage of osteopontin (OPN) positive osteocytes9

and of connective tissue growth factor (CTGF)mRNA expressing osteocytes10 also increased 12hours after applying a force. Our previous studiesshowed that both the tensile and compressive forcesexerted by orthodontia induced early changes inosteocytes and their lacunae, which manifested as anincrease in lacunar volume and changes in lacunarshape and orientation, with an increase in canalicularwidth and increase in the length of cytoplasmicprocesses11.

A study on in vitro response of osteoblastic cells tovarying rates of fluid shear stress found that nitricoxide (NO) production was linearly dependent onthe fluid shear stress rate, showing that strain rate(determined by the frequency and magnitude) is animportant parameter for cell activation to stress12.However, in vivo osteocyte response to differentmagnitudes of orthodontic pressure and tensionforces in terms of apoptosis and OPN expressionhas not been studied to date. The aim of this studywas to examine the early effects of applying a verylight (LF: 0,16 N) and a very strong (SF: 2,26 N)orthodontic force during one hour, on apoptosis andosteopontin (OPN) expression on alveolar boneosteocytes, in rats.

MATERIALS AND METHODSExperimental Tooth MovementTwenty four nine­week­old male Wistar rats, 220 gaverage body weight, were divided into groups ofeight as follows: control group (C), a second groupsubjected to a 0,16N (16 gf) orthodontic forceduring 1 h (LF: light force) and a third groupsubjected to a 2,26 N (230 gf) orthodontic forceduring 1 h (SF: strong force). The appliance used toinduce experimental tooth movement in the ratsconsisted of two stainless steel bands cemented tothe upper first molars with a bracket welded to itspalatal aspect through which a stainless steel wirespring was threaded7,13. Square section stainlesssteel wire (edgewise arch) was used to constructtwo springs designed to generate a 2,26 N (SF) and

a 0,16 N (LF) respectively, towards the palatalaspect of the alveolus, exerting a compresssionforce on the palatal site and a tensile force on thebuccal site of the same alveolus (Fig. 1). The animals were anesthetized by intraperitonealinjection of 50 mg/kg and 20 mg/kg body weight of5% ketamine and 2% xylazine respectively prior toperforming the procedures, and euthanized by etheroverdose 1 h after applying the force. All experiments were conducted in keeping with “TheNational Institutes of Health Guidelines for the Careand Use of Laboratory Animals (NIH Publication 85­23, Rev 1985)”. The experiment was approved by theEthics Committee of the School of Dentistry,University of Buenos Aires (Res CD 015/14).

Tissue PreparationMaxillae were resected and fixed in 4% formaldehydein 0.2 M sodium phosphate buffer (PBS) at 4 ºCduring 48 hours. The tissues were decalcified in 10%ethylenediaminetetraacetate (EDTA) pH 7.4 at 4 ºCfor 60 days, and processed for embedding in paraffin.Five micrometer­thick bucco­palatine orientedsections were obtained at the level of the mesial rootof the first upper molar. The specimens were orientedin the microtome under a stereoscopic microscope;they were sectioned to visualize the most axial planeof the mesial root in which the root canal and the apexare completely open, in a longitudinal orientation; thestudy sections were obtained at this level.

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Fig. 1: Experimental orthodontic model. (a) Orthodonticsprings: two different springs made of 0.016´´ X 0.016´´stainless steel wire. Altering the diameter of the loop and thelength of the spring allowed obtaining springs that exertedthe force magnitudes used here, i.e. LF: 0,16N and SF: 2,26N.(b) Orthodontic appliance: two stainless steel bands werecemented to the upper first molars of experimental rats. Eachband had a bracket welded to its palatal aspect through whicha stainless steel wire spring was threaded. Because thesprings exert force toward the palatal aspect of the alveolus,they exert a tension force on the periodontal aspect of thebuccal wall, and a pressure force on the periodontal aspectof the palatal wall.

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Detection of DNA fragmentation by TUNEL(transferase­mediated biotin­dUTP nick end­labeling)The deparaffinized sections were treated with 20µg/mL proteinase K (DakoCytomation, Carpinteria,CA, USA) in 10 mM Tris­HCL buffer, pH 7.4, atroom temperature (RT) for 20 min, and thenincubated with 0.30% H2O2 in methanol at RT for 30min to block endogenous peroxidase activity. Afterrinsing with distilled water, the sections wereincubated with TdT containing biotin­16­UTP buffer,pH7.2 (Chemicon, Temecula, CA, USA) at 37ºC for90 min and then incubated with anti­digoxigeninantibody (Chemicon, Temecula, CA, USA), reactedwith 3,3­diaminobenzidine (DAB) (Biogenex, SanRamon, CA, USA) and counterstained with methylgreen. Sections of mammary gland after weaningwere used as positive controls.

OPN Immunohistochemistry The sections were incubated in 0.30% H2O2 inmethanol for 30 min, followed by a wash in 10 mMphosphate­buffered saline (PBS) (pH7.2) during 10min and incubated with 1% bovine serum albumin(BSA) (Sigma Chemical Co., St Louis, MO, USA)at RT for 30 min to block nonspecific binding of theantibody. The anti­OPN primary antibody (anti­ratOPN monoclonal antibody (Akm2A1:sc21742),Santa Cruz Biotechnology Inc., California, USA)was diluted 1:3000 in 10 mM PBS containing0.10% BSA and 0.05% Tween 20 (Sigma­Aldrich,St. Louis, MO, USA). The sections were incubatedwith the primary antibody at 4 ºC for 18 h. Forcontrol experiments, sections were incubated withnon­immune rabbit IgG in place of the primaryantibody. The primary antibody was detected usingthe avidin­biotin­peroxidase complex (ABC kit)(Biogenex, San Ramón, CA, USA) followinginstructions on the data sheet and reacted with DAB(Biogenex, San Ramón, CA, USA). The sectionswere counterstained with hematoxylin. Humanbone marrow sections were used as positive control.

The number of TUNEL­positive osteocytes, thenumber of OPN­expressing osteocytes, and theproportion of OPN­expressing bone matrix weredetermined in an area measuring 100 µm wide andcovering the full length of the periodontal aspect ofboth the buccal and the palatal walls of the alveolus.The immunolabeled sections were photographed

using a 40X objective and imported into imageanalysis software for quantification. The number ofTUNEL or OPN­positive osteocytes, defined asosteocyte cell bodies exhibiting brown staining, andthe number of TUNEL or OPN­negative osteocytes,defined as osteocyte cell bodies exhibiting (methyl)green or hematoxylin staining respectively, werecounted on each section. The percentage of immuno ­labeled­positive osteocytes was calculated as thenumber of positive cells divided by the total numberof osteocytes (positive and negative).

Statistical analysisResults are shown as the mean ± standard deviation(SD). Data were compared using one­way analysisof variance (ANOVA) and Dunnett post­hoc test.Values of p <0.05 were considered statisticallysignificant.

RESULTS Histological observations At the experimental time points studied herein, thewidth of the periodontal ligament (PDL) wasnarrower on the palatine wall of the alveolus(pressure strain side) and wider on the buccal wallof the alveolus (tension strain side) in experimentalgroups compared to the control. This compressionand stretching of the PDL was even along the fulllength of the corresponding wall and no hyalinizedtissue was evident in any of the cases (Figure 2).

TUNEL detection of apoptosis. A significant decrease in the number of TUNEL­positive osteocytes was observed on both thetension and pressure strain sides in the light­force(82.22% and 64.3% decrease respectively) and thestrong­force (74.5% and 53.8% respectively) groups,compared to controls. The number of TUNEL­positive osteocytes was lower on the tension side inboth experimental groups (Fig. 3).

OPN Immunohistochemistry The number of OPN­expressing osteocytes wasfound to increase significantly on both the tensionand pressure strain sides in both experimentalgroups compared with controls (148% and 172.7%increase corresponding to the light force and 117.6%and 116% increase in the case of the strong force).In addition, the proportion of OPN­expressing bonematrix was found to increase significantly on both

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Fig. 2: Histological observations.Microphotographs of the mesial rootof the first molar of a control rat (A)and experimental rats (B: LF; C: SF),showing the areas on the buccal side(tension strain side) and on the palatalside (pressure strain side) where weperformed the immunohistochemicaldeterminations. H­E,10X. White andblack bars show stretching (B: buccalside) and compression (P: palatalside) of the PDL respectively occurredevenly along the full length of thecorresponding wall. H­E, 10X.

Fig. 3: Immunohistochemical analysis of apoptosis using TUNEL.Tension (A) and pressure (B) strain sides. The figure shows thatthe percentage of TUNEL+ osteocytes decreased significantly onboth the pressure and tension strain sides in animals underorthodontic forces compared to controls. Values are expressed asmean ± SD. *Statistically significant difference between controland experimental groups, p<0.05. SF: strong force, LF: lightforce, C: control. Representative microphotographs of paraffin­embedded sections immunostained for DNA fragmentation(brown) and counterstained with methyl green to show the numberof osteocytes undergoing apoptosis.

Fig. 4: OPN immunostaining. Tension (A) and pressure (B) strainsides. The figure shows the percentage of OPN+ osteocytes and thepercentage of OPN+ mineralized matrix. The percentage of OPN+osteocytes increased significantly on both the pressure and tensionsides in groups subjected to orthodontic forces compared to thecontrol group; the increase was greater in those subjected to LF. Thepercentage of OPN+ mineralized matrix also increased significantlyon both the pressure and tension sides in groups subjected toorthodontic forces compared to the control group. Values areexpressed as mean ± SD. *Statistically significant differencesbetween control and experimental groups, p<0.05. SF: strong force,LF: light force, C: control. Representative microphotographs ofparaffin­embedded sections immunostained for OPN (brown) andcounterstained with hematoxylin to show the overexpression of theprotein both inside the cell and in the bone mineralized matrix.

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the tension and pressure strain sides in both experi ­mental groups compared with controls (81.66% and82.12% increase in the case of the light force and96.85% and 100.16% increase in the strong forcegroup) (Fig. 4).

DISCUSSIONThis is the first study to evaluate the early in vivoresponse of alveolar osteocytes to different forcemagnitudes applied using an experimental modelof orthodontic tooth movement. In this regard, thedesign of the spring used in this study allowedcomparing the effect of a very light and a verystrong force on both the tension and pressure strainsides. The orthodontic model used in the presentstudy allows adapting the shape of the spring inorder to vary the magnitude of the force andtherefore analyze the biological effect of forcemagnitude on the cells involved in the adaptiveremodeling process. In the present study, osteocytes exhibited an earlyresponse to both tension and pressure forces ofdifferent magnitudes applied to bone. Specially, asignificant decrease in the number of TUNEL­positive osteocytes was observed both in the lightand strong force groups, with a marked diminutionon tension strain sides. In other study6, alveolarbone subjected to orthodontic forces exhibited agradual increase in TUNEL positive osteocytes onthe pressure side after 3h, peaking at 24 h. It isnoteworthy however, that the authors encounteredhyalinization in the periodontal ligament (PDL) onthe pressure side where they performed thedeterminations, and they suggested that hyalinizedPDL would impair transport systems resulting inischemia or hypoxia, which would trigger osteocytecell death. Conversely, though bearing in mind thedifferences between ours and their studies asregards the experimental conditions, our resultsshowed neither an increase in the number ofTUNEL­positive osteocytes nor hyalinization. It istherefore possible that since the mechanical stressin our experiment does not induce hyalinization of the PDL not impairing nutritional supply,osteocytes are prevented from entering apoptosis.In addition, it is known that forces exert anantiapoptotic effect. Shear stress reverses apoptosisof endothelial cells induced by different stimuli14.Moreover, shear stress upregulates the expressionof integrins, and potential mechanotransducers

located at the cell surface15. Various integrinsprevent apoptosis16,17 and it has been suggested thatantiapoptotic integrin signaling involves theactivation of the extracellular signal–regulatedkinase (ERK) 1/218. Several in vitro studies haveshown that mechanical stimulation inhibitsosteocyte apoptosis caused by serum­starvation19,dexamethasone20, and TNF­α21. Osteocytes detectfluid shear stress inside the lacuno­canalicularnetwork via integrins22­24, and NO production isinitiated immediately12. NO prevents apoptosis ofendothelial cells25 and could be thought to have thesame effect on osteocytes. NO production in vitrowas found to be linearly dependent on fluid shearstress rate12, and this may account for the differences,though not significant, in the percentage of thedecrease in apoptosis observed between the forcemagnitudes used in the present study. Anotherresponse of osteocytes to mechanical loading is theimmediate release of several growth factors26. Bybinding to cell­surface receptors, growth factorsactivate signaling pathways involving Bcl­2 familymembers that suppress apoptosis. In an unloadedmodel, apoptosis of osteocytes is associated with atransient decline in integrin and Bcl­2 survivalprotein levels27. Moreover, it has been demonstratedthat mechanical stimuli preserve osteocyte viabilityvia activation of ERKs and new gene transcriptionin vitro20 and prevent glucocorticoid­inducedapoptosis in MLO­Y4 osteocytic cells28. Even, amore recent study shows that mechanical stimulationfor just 10 min is sufficient to trigger survivalsignaling in osteocytic cells29. In our work,mechanical reversion of osteocyte apoptosis wasobserved in osteocytes of groups subjected toorthodontic forces. It is therefore possible that NOproduction inside the lacuno­canalicular system,ERKs activation and autocrine effect of mechanical­induced growth factors secretion may be some ofthe mechanisms involved in protecting osteocytesfrom apoptosis, when subjected in vivo to mechanicalstress exerted by orthodontic forces under theexperimental conditions used herein. Based on the above, we conclude that forces within thephysiological range seem to be an important survivalfactor for osteocytes at the experimental time pointsused in this study. Further in vivo studies must beconducted in order to confirm our observation thatmechanical stimulation prevents, or at least delaysosteocytes from entering apoptosis, triggering a

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response that mediates activation of the remodelingprocess induced by the mechanical loads.In our study, the potential association between forcemagnitude and OPN expression was evaluated onboth the pressure and tension strain sides afterapplying orthodontic forces, showing a significantincrease in OPN expression in all cases, and highestvalues in osteocytes subjected to the light force.OPN is considered to play an important role in boneremodeling since it is thought to promote or regulatethe chemotaxis and attachment of osteoclasts to thebone surface during bone resorption30. Studiesreported in the literature found that osteocyteslocated near resorption sites expressed OPN duringphysiological tooth movement31, and almost allosteocytes expressed OPN 48 h after applying anorthodontic force, prior to osteoclast recruitment tothe bone surface9. We found that OPN expressionalso increased significantly in the mineralized bonematrix of bone under stress, suggesting that thisOPN was synthesized by pre­existing matureosteocytes and not by osteocytes recently includedin the matrix, or by active osteoblasts located on the bone surface. This phenomenon suggests clear activation of osteocytes associated withcommunication via the lacuno­canalicular network

inside the mineralized bone matrix. Interestingly, wealso found that OPN expression also increasedsignificantly in the tension side, where boneformation is stimulated. Accordingly with ourresults, Morinobu et al showed that OPN expressionwas enhanced during bone formation under tensilestress to calvarial sutures suggesting that thepresence of OPN is one of the positive factors for osteoblastic bone formation in the suture under mechanical stress32. Our results demonstratethat tension and pressure forces of physiologicalmagnitude applied in vivo seem to activate mechano ­transduction and to promote the remodeling processvia early expression of osteocyte OPN.It is noteworthy that both pressure and tension forceswere found to generate immediate osteocyte response.The marked decrease in apoptosis, and the significantincrease in OPN expression observed soon after apply­ing different forces (as regards type and magnitude)allow concluding that osteocytes activate very rapidlywhen subjected to locally applied forces in vivo,whether these forces be pressure or tension, light orstrong forces, and lends support to the role of osteo­cytes as mediators of the activation of bone resorptionand formation, which appear as the final response tothe application of orthodontic forces.

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AKNOWLEDGEMENTThe careful technical assistance of Vet. Marianela Lewicki andHt. Mariela Lacave is acknowledged.

FUNDINGThis work was supported by Grants UBACyT 20020130100270from the University of Buenos Aires and School of Dentistry,University of Buenos Aires.

CORRESPONDENCEDr. Carola B. BozalMarcelo T. de Alvear 2142 1ºAC1122AAH Buenos Aires, Argentinae­mail: carolabozal@yahoo.com

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9. Terai K, Takano­Yamamoto T, Ohba Y, Hiura K, SugimotoM, Sato M, Kawahata H, Inaguma N, Kitamura Y, NomuraS. Role of osteopontin in bone remodeling caused bymechanical stress. J Bone Miner Res 1999; 14:839­849.

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10. Yamashiro T, Fukunaga T, Kobashi N, Kamioka H,Nakanishi T, Takigawa M Takano­Yamamoto T. Mechanicalstimulation induces CTGF expression in rat osteocytes. JDent Res 2001; 80:461­465.

11. Bozal CB, Sánchez LM, Mandalunis PM, Ubios AM. Histo­morphometric study and three­dimensional reconstructionof the osteocyte lacuno­canalicular network one hour afterapplying tensile and compressive forces. Cells TissuesOrgans 2013; 197:474­483.

12. Bacabac RG, Smit TH, Mullender MG, Dijcks SJ, Van LoonJJWA, Klein­Nulend J. Nitric oxide production by bonecells is fluid shear stress rate dependent. Biochem BiophysRes Commun 2004; 315:823­829.

13. Bozal CB, Labate L, Ubios AM. Estandarización delmétodo para el estudio de fuerzas ortodóncicas de magnitudconocida en maxilares de rata. Ortodoncia 2006; 69:32­37.

14. Dimmeler S, Haendeler J, Rippmann V, Nehls M, ZeiherAM. Shear stress inhibits apoptosis of human endothelialcells. FEBS Lett 1996; 399:71­74.

15. Urbich C, Walter DH, Zeiher AM, Dimmeler S. Laminarshear stress upregulates integrin expression: role inendothelial cell adhesion and apoptosis. Circ Res 2000;87:683­689.

16. Zhang Z, Vuori K, Reed JC, Ruoslahti E. The alpha 5 beta1 integrin supports survival of cells on fibronectin and up­regulates Bcl­2 expression. Proc Natl Acad Sci USA 1995;92:6161­6165.

17. Scatena M, Almeida M, Chaisson ML, Fausto N, NicosiaRF, Giachelli CM. NF­kappaB mediates alphavbeta3integrin­induced endothelial cell survival. J Cell Biol 1998;141:1083­1093.

18. Dimmeler S, Assmus B, Hermann C, Haendeler J, ZeiherAM. Fluid shear stress stimulates phosphorylation of Aktin human endothelial cells: involvement in suppression ofapoptosis. Circ Res 1998; 83:334­341.

19. Bakker A, Klein­Nulend J, Burger E. Shear stress inhibitswhile disuse promotes osteocyte apoptosis. BiochemBiophys Res Commun 2004; 320:1163­1168.

20. Plotkin LI, Mathov I, Aguirre JI, Parfitt AM, ManolagasSC, Bellido T. Mechanical stimulation prevents osteocyteapoptosis: requirement of integrins, Src kinases and ERKs.Am J Physiol Cell Physiol 2005; 289:C633­C643.

21. Tan SD, Kuijpers­Jagtman AM, Semeins CM, BronckersALJJ, Maltha JC, Von Den Hoff JW, Everts V, Klein­Nulend J. Fluid shear stress inhibits TNFα­induced osteocyteapoptosis. J Dent Res 2006; 85:905­909.

22. Miyauchi A, Gotoh M, Kamioka H, Notoya K, Sekiya H,Takagi Y, Yoshimoto Y, Ishikawa H, Chihara K, Takano­Yamamoto T, Fujita T, Mikuni­Takagaki Y. AlphaVbeta3integrin ligands enhance volume­sensitive calcium influxin mechanically stretched osteocytes. J Bone Miner Metab2006; 24:498­504.

23. Wang Y, Mcnamara LM, Schaffler MB, Weinbaum S. A modelfor the role of integrins in flow induced mechanotransductionin osteocytes. Proc Natl Acad Sci USA 2007; 104:15941­15946.

24. Phillips JA, Almeida EA, Hill EL, Aguirre JI, Rivera MF,Nachbandi I, Wronski TJ, Van Der Meulen MC, Globus RK.Role for beta1 integrins in cortical osteocytes during acutemusculoskeletal disuse. Matrix Biol 2008; 27:609­618.

25. Kim YM, Talanian RV, Billiar TR. Nitric oxide inhibitsapoptosis by preventing increases in caspase­3­like activityvia two distinct mechanisms. J Biol Chem 1997; 272:31138­31148.

26. Smalt R, Mitchell FT, Howard RL, Chambers TJ. Mechano ­transduction in bone cells: induction of nitric oxide andprostaglandin synthesis by fluid shear stress, but not bymechanical strain. Adv Exp Med Biol 1997; 433: 311­314.

27. Dufour C, Holy X, Marie PJ. Skeletal unloading inducesosteoblast apoptosis and targets alpha5beta1­PI3K­Bcl­2signaling in rat bone. Exp Cell Res 2007; 313:394­403.

28. Kitase Y, Barragan L, Jiang JX, Johnson ML, BonewaldLF. Mechanical induction of PGE2 in osteocytes blocksglucocorticoid­induced apoptosis through both the β­catenin and PKA pathways. J Bone Miner Res 2010; 25:2657­2668.

29. Gortazar AR, Martin­Millan M, Bravo B, Plotkin LI,Bellido T. Crosstalk between caveolin­1/extracellularsignal­regulated kinase (ERK) and β­catenin survivalpathways in osteocyte mechanotransduction. J Biol Chem2013; 22:288:8168­8175.

30. Sodek J, Ganss B, Mckee MD. Osteopontin. Crit Rev OralBiol Med 2000; 11:279­303.

31. Takano­Yamamoto T, Takemura T, Kitamura Y, Nomura S.Site­specific expression of mRNAs for osteonectin, osteocalcin,and osteopontin revealed by in situ hybridization in ratperiodontal ligament during physiological tooth movement.J Histochem Cytochem 1994; 42:885­896.

32. Morinobu M, Ishijima M, Rittling SR, Tsuji K, YamamotoH, Nifuji A, Denhardt DT, Noda M. Osteopontin expressionin osteoblasts and osteocytes during bone formation undermechanical stress in the calvarial suture in vivo. J BoneMiner Res 2003; 18:1706­1715.

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RESUMENLos objetivos del presente trabajo fueron: Determinar quéporcentaje de pacientes que concurrió por primera vez alServicio de Urgencias de la Facultad de Odontología de laUniversidad de Buenos Aires consumió medicamentos paraaliviar o tratar su dolencia. Determinar qué porcentaje depacientes fueron automedicados, y cuáles fueron los medica ­mentos más utilizados. Determinar si existe relación entre laautomedicación y el nivel de estudio y entre la automedicación yla presencia de cobertura médica. Se realizó un estudioobservacional y transversal. Se relevaron 567 historias clínicasde pacientes que concurrieron entre marzo 2015 y septiembre

2016 y se valoraron los siguientes parámetros: sexo, edad,origen de la consulta, medicación, dosis, intervalo, duración, eindicación. Se indagó el nivel educacional alcanzado y laexistencia de cobertura médica. Se calcularon intervalos deconfianza al 95% para porcentajes mediante el método score deWilson. Se realizaron análisis inferenciales mediante la pruebaChi­cuadrado (ᵪ2). Se fijó un nivel de significación del 5%. El 85% (n=481) de los pacientes había consumido al menos

un medicamento. El 77% (n=372) de los pacientes estabaautome dicado. Los medicamentos más utilizados fueron anti­inflamatorios no esteoroideos (61%), antibióticos (34%) yglucocorticoides (2%). No se encontró asociación entre la

ABSTRACTThe aims of this work were: To determine what percentage offirst­time patients to the Dental Emergency Department at theSchool of Dentistry of Buenos Aires University had takenmedications to relieve or treat their condition. To determinewhat percentage of these had used self­medication, and whichwere the most frequently taken medicines. To determine whetherthere is an association between self­medication and educationallevel, and between self­medication and whether the patient hashealth coverage. This was an observational, cross­sectionalstudy which reviewed 567 clinical histories of patients whovisited the Dental Emergency Department from March 2015 to September 2016. The following parameters were assessed:sex, age, reason for consultation, medication, dose, interval,duration and indication. Patients’ educational level and whetherthey had health coverage were ascertained. Confidenceintervals of 95% were calculated for percentages using theWilson score method. Inferential analyses were performed usingthe Chi­square test (ᵪ2). Significance level was set at 5%.Eighty five percent (85%,.n=481) of the patients had taken atleast one medication; 77% (n=372) had used self­medication.

The most frequently used medicines were non­steroid anti­inflammatory drugs (61%), antibiotics (34%) and glucocorticoids(2%). No association was found between self­medication andpatients’ having health coverage (ᵪ2=13; p=0.08). No significantassociation was found between educational level and self­medication (ᵪ2=10; p=0.22). Nevertheless, the lowestpercentages of self­medication were found in subjects withcomplete university studies (77%; CI95: 60% to 89%), while thehighest percentages were found in subjects with incompleteprimary education (89%; CI95: 69% to 97%), complete primaryeducation (92%; CI95: 82% to 96%) and incomplete secondaryeducations (90%; CI95: 84% to 94%).High levels of self­medication were found in the study population. Although no association was found between educational level and self­medication behavior, the percentage of self­medication was higher among patients with lower educational levels. Thehigh level of self­medication highlights the importance ofconducting campaigns to raise awareness about the adequate useof medicines.

Key words: self­medication, emergency service, hospital, dentistry.

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Self-medication in patients seeking care in a dental emergency service

Federico Stolbizer1, 2, Daniel F. Roscher1, Maria M. Andrada1, Lautaro Faes2, Carla Arias2, Cecilia Siragusa1, 2, Silvio Prada2, Jonathan Saiegh2, Daniel Rodríguez2, Ariel Gualtieri 3, Carlos F. Mendez4, 5

1 Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Cirugía y Traumatología Bucomaxilofacial II. Buenos Aires. Argentina.

2 Universidad de Buenos Aires. Facultad de Odontología. Servicio de Emergencias. Buenos Aires. Argentina.

3 Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Biofísica y Bioestadística. Buenos Aires. Argentina.

4 Universidad de Buenos Aires. Facultad de Odontología. Cátedra de Farmacología. Buenos Aires. Argentina.

5 CONICET-Universidad de Buenos Aires. Instituto de investigaciones Biomédicas (INBIOMED). Buenos Aires. Argentina.

Automedicación en pacientes que concurren a un servicio de guardia odontológica

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INTRODUCTIONAccording to the World Health Organization, self­medication is the selection and use of medicines byindividuals to treat self­recognized diseases orsymptoms1,2.Acute dental pain is, in many cases, invalidating andis often described as one the most severe a personcan suffer in everyday life. In dentistry, patients oftenrecur to self­medication to reduce dental pain, mainlyby taking analgesics and sometimes includingantibiotics3. This practice has numerous conse ­quences, including an increased risk in untowardeffects, drug­drug interactions, increased bacterialresistance to antibiotics, masking of underlyingpathologies and/or conditions and reduction of theefficacy of treatment due to inadequate or insufficientuse of medicines4.Self­medication today is still relevant to publichealth worldwide, with 8% to 13% prevalence inEuropean and North American populations. Thishigh frequency may be attributed to the availabilityof medicines to society and sometimes to poorcoverage in healthcare services5.In Argentina, a survey conducted by the ArgentinePharmaceutical Confederation found that out of1500 respondents in the cities of Buenos Aires andCórdoba, 82% take over­the­counter medicines andmore than half of the individuals are unaware of theuntoward effects they can cause. It is estimated that11% of all cases of chronic kidney failure are aconsequence of the use of analgesics and 40% ofhigh digestive hemorrhage cases can be attributedto acetylsalicylic acid and other non­steroid anti­inflammatory drugs (NSAIDs)6.To date, no study has been published in Argentinareporting the extent of self­medication in situationsof urgent dental conditions. We aim here to studythe prevalence of self­medication in patientsseeking dental care at the Dental EmergencyService of the School of Dentistry at the Universityof Buenos Aires. We also aimed to determine a

possible association between self­medication andtype of health coverage and/or patient’s educationallevel.

MATERIALS AND METHODSA total of 567 clinical histories of patients whospontaneously visited the Dental EmergencyDepartment of the School of Dentistry at theUniversity of Buenos Aires seeking dental care fromMarch 2015 to September 2016 were analyzed.Exclusion criteria were patients younger than 16years, patients consulting for esthetic reasons andpatients failing to provide written informed consentto participate in the study. Before case resolution,clinical histories were prepared, type of urgencydetermined, and patients were asked to declare any prior care or medicine they have taken whetherprescribed or self­indicated. Patients on medicationwere asked to indicate type of medicine, origin oftreatment, dose, route of administration, interval and duration. Self­perceived improvement of patient condition, total duration and/or possiblediscontinuation of the treatment was also recorded.The sample consisted of 381 males (67.2%) and 186females (32.8%) with median age 34 and a rangebetween 16 to 89 years.Confidence intervals of 95% were calculated forpercentages using the Wilson score method.Inferential analyses were performed using the Chi­square test (ᵪ2). Significance level was set at 5%.

RESULTSOf recorded cases, 85% (n=481; CI95: 82% to 88%)had taken at least one medicine, while 15% (n=109;CI95: 12% to 18%) declared no medication relatedto the dental episode prior to consultation (ᵪ2=275;p<0.05). Of total medicated patients, 77% (n = 372;CI95: 73% to 81%) recurred to self­indication,while 23% (n=109; CI95: 19% to 27%) had takenmedicines under professional prescription (82%dentists, 18% physicians).

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automedicación y la presencia de cobertura médica (ᵪ2=13;p=0,08). No se encontró asociación significativa entre el nivelde estudios y la automedicación (ᵪ2=10; p=0,22). Sin embargo,los sujetos con estudio universitario completo presentaron elmenor porcentaje de automedicación (77%; IC95: 60% a89%), mientras que los mayores porcentajes se encontraron ensujetos con primario incompleto (89%; IC95: 69% a 97%),primario completo (92%; IC95: 82% a 96%) y secundarioincompleto (90%; IC95: 84% a 94%).

Se encontraron niveles elevados de automedicación en lapoblación estudiada. Si bien no se observó asociación entrenivel educativo y la conducta de automedicación, fue mayor elporcentaje de automedicación en pacientes con menor niveleducativo. La alta presencia de automedicación refuerza laimportancia de realizar campañas de concientización sobre elconsumo adecuado de medicamentos.

Palabras clave: Automedicación, servicio de urgencia, odontología.

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No significant differences (ᵪ2=1.55; p=0.21) wereobserved when data was analyzed by gender, with85 (325/381) and 84% (156/186) medication ratesfor males and females, respectively.Among the 567 urgencies treated, the reason forconsultation was primarily endodontic in 60% ofincluded cases (n=340; CI95: 56% to 64%), infectiousin 16% (n=92; CI95: 13% to 19%), post­surgicalproblems in 12% (n=67; CI95: 9% to 15%),periodontal in 5% (n=31; CI95 4% to 8%),temporomandibular joint disorders in 1% (n=5; CI95:0% to 2%), and miscellaneous reasons in 6% (n=32;CI95: 4% to 8%). Fifty­six percent (n=269; CI95:51% to 60%) of patients had taken one medicine, 36%(n=173; CI95: 32% to 40%) had taken 2, and 8%(n=39; CI95: 6% to 11%) had taken three or more. Self­medicated patients used medicines from avariety of therapeutic classes (ᵪ2=667; p<0.05). Themost frequently used category was NSAIDs (61%;CI95: 57% to 64%), followed by antibiotics (34%;CI95: 31% to 38%), glucocorticoids (2%; CI95: 1%to 3%), and others (3%; CI95: 2% to 5%).Among NSAIDs, the most frequently used wasibuprofen (45%; CI95: 41% to 50%), followed byketorolac (33%; CI95: 28% to 37%), diclofenac(11%; CI95: 8% to 14%), and others 11%; CI95:9% to 15%) (Table 1).

A wide range of antibiotics was used. Among self­medicated patients, the most frequent antibiotic wasamoxicillin (80%, CI95: 75% to 85%), followed byamoxicillin/clavulanate association (10%; CI95:7% to 14%), azithromycin (6%; CI95: 4% to 10%),cefalexin (2%; CI95: 1% to 5%), and others (2%;CI95: 1% to 5%) (Table 2).The rate of self­medication was also studied interms of type of health coverage presented bypatients. As shown in Fig.1, when analyzed againsthealth coverage, a trend that did not reachedstatistical significance (ᵪ2=3.18; p=0.08) towards ahigher use of medicines without prescription wasdetected in patients lacking health insurance.No significant association was found either betweeneducational level and self­medication (ᵪ2=10; p=0.22).Nevertheless, the percentage of subjects withcomplete university studies who used self­medicationwas lower (77%; CI95: 60% to 89%), as compared tosubjects with incomplete primary education (89%;CI95: 69% to 97%), complete primary education(92%; CI95: 82% to 96%) and incomplete secondaryeducation (90%; CI95: 84% to 94%) (Fig. 2).

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Table 1: Frequency of NSAID use in the self-medicated group.

Drug %

Ibuprofen 45%

Ketorolac 33%

Diclofenac 11%

Acetaminophen 5%

Flurbiprofen 1%

Clonixin 4%

Piroxicam 1%

Total 100%

Table 2: Frequency of antibiotic use in the self-medicated group.

Drug %

Amoxicillin 80%

Amoxicillin / Clavulanate 10%

Azithromycin 6%

Cephalexin 2%

Others 2%

Total 100%Fig. 1: Percentage of self­medicated patients and medicalcoverage.

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DISCUSSIONSelf­medication is a very common habit in LatinAmerica. We report here that 77% of the patientssurveyed at the Dental Emergency Service of theSchool of Dentistry at the University of BuenosAires incurred in self­medication. Our results agreewith those presented by Ramay, et al that reported79% self­medication with antibiotics in suburbanareas and 77% in central areas of Guatemala City9.Other investigators also reported similar results inBolivia13, with 72% and Peru14, 15 with 81 and 69%prevalence of self­medication, respectively. Evenhigher numbers for self­medication were reportedamong university students in Brazil11 and medicalstudents in Cuenca, Ecuador12 while a 41% preva ­lence was informed in Mexico10. Differences inresults may be accounted by the type and size ofpopulation surveyed and institution in which thestudy was performed, among other variables. Ourstudy included patients who spontaneously soughtattention by the Emergency ward at a DentalUniversity Hospital in central Buenos Aires,Argentina. Our study surveyed also all types ofmedications related to the dental condition affectingthe sampled individuals and is, thus, not limited toa single class of therapeutic agents.Prescription legislation in Argentina is extensiveand current laws were originally established in the1960s. The fact that 70% of the medicines thatenrolled patients declare to use in our study require

prescription, shows that neither patients norpharmacies comply with the enforced regulation.Analgesics were most frequently used by patients,as reported in other studies 4,7,10,12­15. Although notstatistically significant, a lower prevalence of self­medication was detected among patients withcomplete university studies, something that pointsout to a possible relationship between instructionand a more responsible use of medication.Some of the causes of these high figures in LatinAmerica might be deficient healthcare services,long waiting times at urgent care service providers,cost of medical care, media advertising and the role of pharmacists and healthcare professionals.Many patients are unaware of the consequences ofself­medication, which include increased risk ofuntoward reactions to drugs, drug­drug interaction,increase in bacterial resistance, masking of under ­lying pathologies and/or conditions and reductionin the efficacy of treatment as a result of inadequateor insufficient use of medicines.

CONCLUSIONStrategies should be developed to ensure thatphysi cians, dentists, nurses and pharmacists makerational use of medicines. Other effective strategiesmay be campaigns to raise awareness among the population regarding the consequences of self­medi cation and improved access to primaryhealthcare centers.

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Fig. 2:Educational level for allpatients in the study.

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FUNDINGThis study was supported, in part, by a grant form Universidadde Buenos Aires (UBACYT 20020150100172BA) to CFM.

CORRESPONDENCEDr. Federico StolbizerCátedra de Cirugía y Traumatologia Bucomaxilofacial IIFaculatad de OdontologíaMarcelo T. de Alvear 2142(C1122AAH) Buenos Aires, Argentina fstolbizer@gmail.com

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