UNIVERSIDADE ESTADUAL DE CAMPINAS Faculdade de Odontologia de Piracicaba
ANA CAMILA BATISTA MEDEIROS DE ASSIS
POTENCIAL CARIOGÊNICO DOS LEITES HUMANO E BOVINO E
SEU EFEITO NA DESMINERALIZAÇÃO DO ESMALTE
CARIOGENIC POTENTIAL OF HUMAN AND BOVINE MILK AND ITS
EFFECT ON ENAMEL DEMINERALIZATION
PIRACICABA 2018
ANA CAMILA BATISTA MEDEIROS DE ASSIS
POTENCIAL CARIOGÊNICO DOS LEITES HUMANO E BOVINO E
SEU EFEITO NA DESMINERALIZAÇÃO DO ESMALTE
CARIOGENIC POTENTIAL OF HUMAN AND BOVINE MILK AND ITS
EFFECT ON ENAMEL DEMINERALIZATION
Dissertação apresentada à Faculdade de Odontologia de Piracicaba, da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestra em Odontologia, na Área de Cariologia.
Dissertation presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Master in Dentistry, in Cariology area.
Orientador: Prof. Dr. ANTÔNIO PEDRO RICOMINI FILHO Coorientador: Prof. Dr. JAIME APARECIDO CURY
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA ANA CAMILA BATISTA MEDEIROS DE ASSIS E ORIENTADA PELO PROF. DR. ANTÔNIO PEDRO RICOMINI FILHO.
PIRACICABA 2018
Agência(s) de fomento e nº(s) de processo(s): CNPq, 163061/2017-3
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159
Assis, Ana Camila Batista Medeiros de,
As76p AssPotencial cariogênico dos leites humano e bovino e seu efeito na
desmineralização do esmalte / Ana Camila Batista Medeiros de Assis. – Piracicaba, SP : [s.n.], 2018.
AssOrientador: Antônio Pedro Ricomini Filho.
AssCoorientador: Jaime Aparecido Cury.
AssDissertação (mestrado) – Universidade Estadual de Campinas, Faculdade
de Odontologia de Piracicaba.
Ass1. Cárie dentária. 2. Leite humano. 3. Sacarose. I. Ricomini Filho, Antônio
Pedro, 1983-. II. Cury, Jaime Aparecido, 1947-. III. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. IV. Título.
Informações para Biblioteca Digital
Título em outro idioma: Cariogenic potencial of human and bovine milks and its effect on
enamel demineralization
Palavras-chave em inglês:
Dental caries Milk, human Sucrose
Área de concentração: Cariologia Titulação: Mestra em Odontologia Banca examinadora:
Antônio Pedro Ricomini Filho [Orientador] Silvia José Chedid
Carolina Patrícia Aires
Data de defesa: 20-04-2018
Programa de Pós-Graduação: Odontologia
UNIVERSIDADE ESTADUAL DE CAMPINAS
Faculdade de Odontologia de Piracicaba
A Comissão Julgadora dos trabalhos de Defesa de Dissertação de Mestrado, em sessão pública realizada em 20 de Abril de 2018, considerou a candidata ANA CAMILA BATISTA MEDEIROS DE ASSIS aprovada.
PROF. DR. ANTÔNIO PEDRO RICOMINI FILHO
PROFª. DRª. SILVIA JOSÉ CHEDID
PROFª. DRª. CAROLINA PATRÍCIA AIRES
A Ata da defesa com as respectivas assinaturas dos membros encontra-se no processo de vida acadêmica do aluno.
DEDICATÓRIA
A Deus que escreveu minha história antes mesmo da minha formação e que durante todos esses anos tem me sustentado com bondade e graça. Guiando meus passos e iluminando meus caminhos. Por mais essa vitória toda minha gratidão.
Aos meus pais, Artur Cézar Medeiros de Assis e Dinorá Batista Medeiros de Assis, aos meus irmãos Ana Clara, Artur Filho, Ana Iza, ao meu sobrinho Pedro Cézar e ao meu marido Diego Figueiredo Nóbrega por serem lugar de descanso, meus melhores amigos, minha referência de valores. A vocês por todo o incentivo e dedicação.
AGRADECIMENTOS
A Deus, pela saúde, pela vida, pela minha família e pelo amparo nos momentos difíceis e por suprir todas as minhas necessidades.
À Universidade Estadual de Campinas, na pessoa do Magnífico Reitor Prof. Dr. Marcelo Knobel.
À Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas, na pessoa do Diretor Prof. Dr. Guilherme Elias Pessanha Henriques.
À Profa. Dra. Cínthia Pereira Machado Tabchoury, coordenadora dos Cursos de Pós-graduação da Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas.
Ao Prof. Dr. Marcelo de Castro Meneghim, coordenador do Programa de Pós-graduação em Odontologia da Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas.
Ao Prof. Dr. Antônio Pedro Ricomini Filho, meu orientador de mestrado. Obrigada pela atenção, paciência, dedicação, competência, disponibilidade e pela intenção de modelar minha evolução profissional.
Aos Profs. Drs. Jaime Aparecido Cury, Cínthia Pereira Machado Tabchoury e Livia Maria Andaló Tenuta da área de Cariologia pelos ensinamentos e por terem participado da minha formação durante a pós-graduação.
À aluna de doutorado Bárbara E. Costa Oliveira, pela atenção, pelos ensinamentos e pela colaboração na realização deste trabalho.
Aos técnicos do laboratório de Bioquímica Oral da FOP-UNICAMP, Waldomiro Vieira Filho e José Alfredo da Silva, pela disponibilidade e pela convivência agradável no dia a dia.
Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico pela bolsa concedida durante o mestrado.
Aos amigos e colegas que fiz e conheci na pós-graduação. Obrigada pelas alegrias, frustrações e sucesso compartilhados.
À Primeira Igreja Batista de Piracicaba que nos fez como família durante o nosso tempo em Piracicaba. Obrigada pela demonstração de amor quando abrimos nossos corações diante dos anseios, dificuldades e tristezas. Pela fidelidade de vocês à palavra de Deus no andar, no falar, no cuidar. Nós ressignificamos nosso entendimento sobre fé e igreja.
RESUMO
Dentre os açúcares que fazem parte da dieta humana, a lactose, presente no leite e seus derivados, também é metabolizada a ácidos, podendo ocasionar desmineralização do substrato dental. Tendo em vista que o leite humano apresenta maiores concentrações de lactose, tem sido sugerido que este teria um maior potencial cariogênico quando comparado ao leite bovino. No entanto, não há estudo controlado com exposição intermitente do biofilme aos diferentes tipos de leite que sustente esta afirmação. Sendo assim, o objetivo deste estudo foi avaliar in vitro o potencial cariogênico do leite humano e do leite bovino e seu efeito na desmineralização do esmalte, utilizando um modelo de biofilme cariogênico validado. Para tal, foi conduzido um estudo experimental, in vitro e cego. Blocos de esmalte dental bovino (4x7x1 mm) com dureza de superfície conhecida foram imersos em saliva humana para a formação de película adquirida. Biofilmes de Streptococcus
mutans UA159 foram formados utilizando meio ultrafiltrado a base de triptona e extrato
de levedura (UTYEB) suplementado com 0,1 mM de glicose. Os blocos de esmalte e os biofilmes formados sobre eles foram expostos 8 vezes ao dia, por 3 minutos, a um dos 6 tratamentos (n=8): (i) solução de NaCl a 0,9 % (controle negativo); (ii) leite humano (LH); (iii) leite bovino (LB); (iv) solução de lactose a 7 % (controle ativo do LH); (v) solução de lactose a 4,5 % (controle ativo do LB); e (vi) solução de sacarose a 10 % (controle positivo). O meio de cultura foi trocado diariamente, antes e após a realização dos tratamentos, e seu pH foi mensurado a cada troca. Após 120 h de experimento, os biofilmes e os blocos de esmalte foram coletados. As variáveis de resposta avaliadas foram: porcentagem de perda de dureza de superfície (%PDS) dos blocos de esmalte, pH do meio de cultura, contagem de unidades formadoras de colônias (UFC) e quantificação de polissacarídeos extracelulares (PEC), solúveis e insolúveis, na matriz dos biofilmes. Os experimentos foram realizados em duplicata, em dias distintos. Os dados foram analisados por ANOVA two-way e teste de Tukey (α= 5 %). Os valores de %PDS para os grupos LH (7,5 ± 5,0), LB (8,7 ± 6,3), controle ativo do LH (13,3 ± 7,5) e controle ativo do LB (15,3 ± 8,2) não diferiram do controle negativo (7,7 ± 3,1) (p > 0,05), sendo que somente o grupo exposto à sacarose apresentou a maior desmineralização do esmalte (55,1 ± 5,4) (p < 0,05). Apenas o grupo exposto à sacarose apresentou menores valores de pH em todos os tempos após 32 h de crescimento do biofilme (p < 0,05). O grupo de sacarose também foi o
único a mostrar maior contagem bacteriana (p < 0,05) (9,4 ± 0,2; log10) em
comparação com o grupo controle negativo (7,8 ± 0,6; log10). Os PEC solúveis e
insolúveis foram formados apenas no biofilme exposto à sacarose, 19,5 ± 10,8 e 164,0 ± 12,6 μg/biofilme, respectivamente. Os resultados sugerem que tanto leite humano quanto leite bovino não apresentam potencial cariogênico em termos de estrutura do biofilme e não são capazes de provocar desmineralização do esmalte em comparação com a sacarose.
ABSTRACT
Among sugars used in the human diet, lactose, present in milk and its derivatives, can also be metabolized to acids, which may lead to tooth demineralization. Considering that human milk contains higher lactose concentrations, it has been suggested that it could be more cariogenic than bovine milk. However, up to date there is no controlled study with intermittent biofilm exposure to the different types of milk that supports this statement. Therefore, the aim of this study was to evaluate the cariogenic potential of human and bovine milk and its effect on enamel demineralization, using a validated in vitro cariogenic biofilm model. Bovine enamel slabs (4x7x1 mm) with known surface hardness were immersed in human saliva for the formation of acquired pellicle. Streptococcus mutans UA159 biofilms were formed using ultrafiltered tryptone-yeast extract broth (UTYEB) supplemented with 0.1 mM glucose. Enamel blocks and the biofilms formed on them were exposed 8 times a day for 3 minutes to one of the following treatments (n = 8): (i) 0.9 % NaCl solution (negative control); (ii) human milk (HM); (iii) bovine milk (BM); (iv) 7 % lactose solution (HM active control); (v) 4.5 % lactose solution (BM active control); and (vi) 10 % sucrose solution (positive control). During the biofilm growth, the culture medium was changed twice daily (before and after the treatments) and the pH was measured. On the morning of the 5th day (120 h) the enamel blocks and biofilms were collected. The response variables evaluated were: percentage of enamel surface hardness loss (% SHL), pH of the culture medium, counts of colony forming units (CFUs) and quantification of soluble and insoluble extracellular polysaccharides (PEC) in the biofilm matrix. The experiments were performed in duplicate on different days. Data were analyzed by two-way ANOVA and Tukey's test (α = 5 %). The %SHL was higher in the group exposed to sucrose (55.1 ± 5.4) (p < 0.05), while the groups HM (7.5 ± 5.0), BM (8.7 ± 6.3), HM active control (13.3 ± 7.5) and BM active control (15.3 ± 8.2) did not differ from the negative control group (7.7 ± 3.1) (p > 0.05). Regarding pH, only the sucrose group had lower pH values at all times after 32 h of biofilm growth (p < 0.05). The sucrose group was also the only one to show higher bacterial counts (p < 0.05) (9.4 ± 0.2; log10) than the negative
control group (7.8 ± 0.6; log10). Soluble and insoluble EPS were formed only in the
biofilm exposed to sucrose (19.5 ± 10.8 and 164.0 ± 12.6 μg/mg of biofilm, respectively). The results suggest that both human and bovine milk have no cariogenic
potential in terms of biofilm structure and are not able to cause enamel demineralization when compared to sucrose.
SUMÁRIO
1. INTRODUÇÃO ...12 2. ARTIGO: CARIOGENIC POTENTIAL OF HUMAN AND BOVINE MILKS AND ITS EFFECT ON ENAMEL DEMINERALIZATION …...…………..………...15
3. CONCLUSÃO ...30 REFERÊNCIAS ... 31
ANEXO: Comprovante de aceite do projeto pelo Comitê de Ética em Pesquisa FOP-UNICAMP ... 34
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1 INTRODUÇÃO
Cárie é uma doença biofilme-açúcar dependente, que se caracteriza pela desmineralização progressiva e localizada do substrato dental por ácidos orgânicos produzidos por bactérias, quando da exposição frequente a carboidratos fermentáveis (Fejerskov e Kidd, 2015). Na dieta humana há diversos carboidratos fermentáveis que estão presentes na alimentação da população e que podem ser metabolizados pelas bactérias, a exemplo de glicose e frutose, monossacarídeos, lactose e sacarose, dissacarídeos, e amido, como principal polissacarídeo (FAO 1998).
Dentre estes carboidratos, a sacarose, a qual é utilizada para adoçar alimentos, é reconhecida como o açúcar mais cariogênico da dieta humana (Cury at al., 2000). A sacarose é um dissacarídeo composto de monômeros de glicose e frutose (Glc(α1↔2β)Fru). Além de ser facilmente metabolizada a ácidos pelas bactérias, a sacarose pode ser substrato para a produção de polissacarídeos extracelulares (PEC) (Rølla et al.,1989). Enzimas bacterianas, denominadas de frutosiltransferases (FTFs) e glucosiltransferases (GTFs), sintetizam os PEC (Rølla et al.,1989; Koo et al., 2013), ao quais se acumulam na matriz do biofilme. FTFs utilizam moléculas de frutose para a síntese de polissacarídeos do tipo frutanos, enquanto as GTFs utilizam moléculas de glicose para a síntese de polissacarídeos do tipo glucanos.
A presença dos PEC na matriz do biofilme, em especial os PEC do tipo glucanos insolúveis, composto por ligações do tipo α1→3, alteram a estrutura do biofilme contribuindo para maior cariogenicidade do mesmo. Estes PEC insolúveis favorecem a adesão de micro-organismos à estrutura dental e ao biofilme (Rølla, 1989; Bowen e Koo, 2011), bem como, aumentam a porosidade do biofilme dental (Dibdin e Shellis, 1988), criando um microambiente altamente cariogênico (Paes Leme et al., 2006). Ao mesmo tempo que o biofilme mais poroso possibilita a retenção de carboidratos em seu interior para serem metabolizados, também dificulta a remoção e neutralização dos ácidos produzidos pelas bactérias (Koo et al., 2013).
Além da sacarose, a lactose é outro carboidrato frequentemente consumido pela população, e também pode ser metabolizado pelas bactérias do biofilme, podendo provocar desmineralização do substrato dental (Birkhed et al, 1993; Aires et al., 2002; Bowen e Lawrence 2005). A lactose é o principal carboidrato presente no leite e derivados de leite (Crisà, 2003), os quais são amplamente consumidos pela população, desde o leite humano, logo após o nascimento, bem como o leite bovino,
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durante toda a vida até a velhice. A lactose é também um dissacarídeo, no entanto é composto de monômeros de galactose e glicose (Gal(β1→4)Glc). Diferentemente da sacarose que é rapidamente metabolizada, a lactose requer um conjunto de enzimas para a metabolização do substrato para obtenção de energia (Zeng et al., 2010a). Apesar de apresentar um processo de metabolização diferenciado e não ser substrato para a síntese de PEC, a presença de lactose na dieta, nas diferentes formas de leite, tem sido relacionado com cárie.
A relação entre o consumo frequente de leite, seja humano ou bovino, e o desenvolvimento de cárie é ainda assunto controverso na literatura. Estudos sugerem que tanto leite humano quanto bovino possam propiciar o desenvolvimento de cárie dental, se consumido com frequência e mantido por longos períodos na boca (Thomson et al., 1996; Birkhed et al, 1993; Feldens et al., 2010). Ao contrário, outros estudos sugerem que o leite humano possa não ter potencial cariogênico quando é fonte exclusiva de carboidratos (Araújo et al, 1997; Erickson e Mazhari, 1999; Neves et al., 2016).
O leite humano é considerado mais cariogênico que o leite bovino (Rugg-Gunn et. al, 1985; Thomson et al., 1996; Bowen e Lawrence 2005), sobretudo devido a maior concentração de lactose (Darke, 1976; Crisà, 2003). O leite humano apresenta uma concentração média de lactose de 7%, enquanto o leite bovino possui 4,5% (Darke, 1976; Crisà, 2003). Adicionalmente, as menores concentrações de cálcio, fósforo e proteínas no leite humano (Darke, 1976; Crisà, 2003) também favoreceriam maior desmineralização dental.
O maior potencial cariogênico do leite humano é principalmente suportado por estudos que avaliaram períodos prolongados de exposição aos dois tipos de leite (Rugg-Gunn et al, 1985; Thomson et al., 1996;Prabhakar et al., 2010). Tendo o leite humano maior concentração de lactose, consequentemente menores valores de pH foram obtidos, pois a exposição ao leite foi contínua, e não intermitente como ocorre na boca. Tendo em vista que a exposição contínua aos leites não mimetiza condição clínica real, seria importante a realização de estudos com modelos de biofilme cariogênico que mimetizassem a exposição intermitente do biofilme. Dessa maneira, por meio da exposição intermitente aos diferentes tipos de leite seria possível o melhor entendimento do potencial cariogênico do leite humano e bovino durante o desenvolvimento do biofilme dental.
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Estudos sugerem que tanto leite humano quanto bovino propiciam o desenvolvimento de cárie dental, se consumido com frequência e mantido por longos períodos na boca (Rugg-Gunn et al, 1985; Birkhed et al, 1993; Thomson et al., 1996). Em relação ao leite humano, estudos epidemiológicos mostraram relação entre cárie e consumo de leite humano, quando este é feito após os 12 meses de idade até os 24 meses de vida (Chaffee et al. 2014; Tham et al., 2015; Victora et al., 2016; Peres et al., 2017). No entanto, estes estudos apresentam fatores de confundimento que impossibilitam esta avaliação de causa e efeito, o que poderia ser observado em estudo laboratorial controlado. Dentre os fatores de confundimento citados [Tham et al. 2015, Peres et al. 2018] está a alimentação das crianças. Em crianças a partir dos 12 meses de vida, a amamentação não é mais fonte exclusiva de nutrição e outros alimentos contribuiriam para o desenvolvimento de cárie.
Tendo em vista que não há na literatura estudo controlado que mimetize a exposição intermitente do biofilme aos diferentes tipos de leite, o que mostraria como o leite humano e o leite bovino podem contribuir para o desenvolvimento de cárie dental, o objetivo deste estudo será avaliar in vitro o potencial cariogênico do leite humano e do leite bovino e seu efeito na desmineralização do esmalte utilizando modelo de biofilme cariogênico.
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2 ARTIGO
Cariogenic potential of human and bovine milks and its effect on enamel demineralization
ABSTRACT
The cariogenicity of milk is a controversial issue and in addition human milk (HM) has been considered more cariogenic than bovine milk (BM). Therefore, we evaluated the effect of HM and BM on biofilm composition and enamel demineralization using a cariogenic biofilm model. Streptococcus mutans UA159 biofilms (n=8) were grown on human saliva-coated bovine enamel slabs of known surface hardness (SH). The biofilms were grown in UTYEB medium (pH 7.0) and were exposed 8x/day to one of the following treatments: (i) 0.9% NaCl solution (negative control), (ii) human milk (HM), (iii) bovine milk (BM), (iv) 7% lactose solution (HM - active control), (v) 4.5% lactose solution (BM - active control), or (vi) 10% sucrose solution (positive control). The culture medium was changed twice daily, before and after the treatments, and the medium pH was analyzed. After 120 h of formation, at the begging of the 6th day, the
biofilms were collected to evaluate CFU counts and extracellular polysaccharides, soluble and insoluble. Enamel slabs were used to quantify the percentage of enamel SH loss (%SHL). Data were analyzed by ANOVA one-way, followed by Tukey’s test (α=5%). The %SHL values for the groups HM (7.5 ± 5.0), BM (8.7 ± 6.3), HM - active control (13.3 ± 7.5) and BM - control (15.3 ± 8.2) did not differ from the negative control (7.7 ± 3.1) (p>0.05). Only the sucrose group presented lower pH values than the negative control for all time points after 32 h of biofilm growth (p<0.05). The group exposed to sucrose was also the only to show higher (p<0.05) bacterial counts (9.4 ± 0.2; log10) in comparison to the negative control group (7.8 ± 0.6). Soluble and insoluble
EPS were only formed in the biofilm exposed to sucrose, 19.5 ± 10.8 and 164.0 ± 12.6 µg/biofilm, respectively. and sucrose presented the highest enamel demineralization (55.1 ± 5.4) (p<0.05). In conclusion, the findings suggest that both human milk and bovine milk have no cariogenic potential in terms of biofilm structure and they are not able to provoke enamel demineralization in comparison to sucrose.
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INTRODUCTION
The relationship between frequent consumption of milk, whether human or bovine, and the development of caries is still a controversial issue in the literature. Studies have suggested that both human and bovine milk can promote the development of caries, if it is frequently consumed and maintained in the mouth [Rugg-Gunn et al., 1985; Birkhed et al., 1993; Thomson et al., 1996]. On the other hand, other studies suggest that human milk may not have a cariogenic potential when it is an exclusive source of carbohydrate [Araújo et al, 1997; Erickson and Mazhari, 1999; Neves et al., 2016].
The human milk is considered more cariogenic than bovine milk [Rugg-Gunn et al., 1985; Thomson et al., 1996; Bowen and Lawrence 2005], mainly due to the higher concentration of lactose, which is almost twice as high the concentration in bovine milk [Rugg-Gunn et. al, 1985; Thomson et al., 1996; Bowen and Lawrence 2005]. Additionally, lower concentrations of calcium, phosphorus and proteins are found in human milk [Darke, 1976; Crisà, 2003], which could also favor to higher dental demineralization.
The higher cariogenic potential of human milk is mainly supported by studies that evaluated prolonged periods of exposure to human and bovine milks [Rugg-Gunn et al., 1985; Thomson et al., 1996; Prabhakar et al., 2010]. As the human milk has a higher concentration of lactose, consequently lower pH values were obtained, since the milk exposure was continuous, not intermittent, as occurs in the mouth. The use of a cariogenic biofilm model that mimics the intermittent biofilm exposure to the different types of milk could contribute to a better understanding of the cariogenic potential of human and bovine milk during the dental biofilm development.
In addition, recent epidemiological studies have shown a relationship between caries and breastfeeding, mainly when the human milk consumption occurs later than 12 months of age [Chaffee et al., 2014; Tham et al., 2015; Victora et al., 2016; Peres et al., 2017; Peres et al., 2018]. However, these studies may present confounding factors, even reported by the authors [Tham et al., 2015; Peres et al., 2017; Peres et al., 2018], being not possible to evaluate the cause and effect and conclude the role of human milk and caries development. Therefore, controlled laboratory studies are necessary to understand the mechanism underlaying this process.
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Considering the need of controlled laboratory studies and that there is no study that mimics the intermittent biofilm exposure to different types of milk, which could show how human milk and bovine milk may contribute to caries development, the aim of this study was to evaluate the cariogenic potential of human milk and bovine milk during biofilm development and its effect on enamel demineralization using a cariogenic biofilm model.
MATERIAL AND METHODS Experimental design
An experimental in vitro, randomized and blinded study was performed using a validated cariogenic biofilm model [Ccahuana-Vásquez and Cury, 2010].
Streptococcus mutans UA159 biofilms were formed on saliva-coated enamel slabs
using ultrafiltered tryptone-yeast extract broth (UTYEB; pH 7.0) medium containing 0.1 mM glucose. The biofilms were exposed 8 times a day to one of the treatments (n = 8): (i) 0.9% NaCl solution (negative control), (ii) human milk, (iii) bovine milk, (iv) 7% lactose solution (active control of human milk), (v) 4.5% lactose solution (active control of bovine milk), and (vi) 10% sucrose solution (positive control). The culture medium was changed twice daily, and the pH was measured as an indicator of biofilm acidogenicity. After 120 h of formation, biofilms and enamel slabs were collected. The response variables evaluated were the pH of the culture medium, the percentage of enamel surface hardness loss (%SHL), the counts of viable microorganisms in the biofilms, and the quantification of extracellular polysaccharides, soluble and insoluble, in the biofilm matrix. Two independent experiments were performed, and the data were statistically analyzed by one-way ANOVA followed by Tukey's HSD Test, considering enamel slab as a statistical block (α=5%). Due to the use of human saliva to form the acquired pellicle, and human milk as a treatment under investigation, the study was submitted and approved by the Research and Ethics Committee of the Piracicaba Dental School, University of Campinas (protocol number 67729717.2.0000.5418).
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Preparation of enamel slabs
The enamel slabs were obtained from bovine incisors [Ccahuana-Vásquez and Cury, 2010]. The crowns were sectioned using a low-speed diamond blade to obtain the slabs. The enamel surfaces were ground with aluminum oxide abrasive papers (grits number 400, 600 and 1200) and polished with 1 μm diamond paste on polishing cloth using a grinder machine (Phoenix Beta, Buehler, USA). The enamel surface hardness (SH) was determined using a Knoop indenter (Future-Tech FM, Kawasaki, Japan). Three indentations spaced 100 μm from each other were performed with 50 g load for 5 s. The slabs with intra-variability higher than 10% were excluded and the selected slabs were randomized into the groups. The slabs were coupled to metallic holders and placed in 24-well culture plates in vertical position. Before the biofilm assay, the slabs were sterilized by exposure to ethylene oxide [Fernández et al., 2016].
Treatments - human and bovine milk and solutions
The human milk used in the study was donated by the Human Milk Bank of the Fornecedores de Cana de Piracicaba Hospital at Piracicaba city, São Paulo state, Brazil. The human milk donated was the surplus volume that would not be used to feed babies and would be discarded. The milk was donated in coded bottles, being not possible to identify the donors. The milk was pooled, pasteurized (60ºC, 30 min) and volumes of 50 mL was stored in polypropylene tubes at -80ºC. To verify the pasteurization process, aliquots of 50 μL was platted on Columbia blood agar (CBA) plates, which were incubated under aerobic, microaerophilic (10% CO2) and anaerobic
(10% CO2, 10% H2, 80% N2) growth conditions at 37ºC for 72 hours. After
pasteurization, no human milk presented bacterial growth.
The whole bovine milk (brand name Paulista) used was purchased from a local supermarket at Piracicaba city, São Paulo state, Brazil. The bovine milk was pasteurized by Ultra High Temperature (UHT) process, as most of the bovine milk consumed by the population. UHT pasteurization process aims to inactivate all bacteria, being the milk free of viable micro-organisms. To verify the UHT pasteurization process, the bovine milk was also evaluated for the presence of bacterial growth as previously described. No bovine milk purchased presented bacterial growth.
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In addition to human and bovine milk, solutions of 0.9% NaCl, 7% lactose, 4.5% lactose, and 10% sucrose were also prepared in ultrapure water and autoclaved at 121ºC, 1 atm for 15 min.
Human saliva and acquired pellicle
Fresh stimulated human whole saliva was collected from the same two healthy donors for each experiment. Salivary flow was stimulated by chewing a plastic paraffin film and the saliva was collected in 50 mL polypropylene tubes on ice for 30 minutes. The collected saliva was pooled, diluted 1:1 with adsorption buffer containing a final concentration of 1.0 mmol/L phenylmethylsulfonylfluoride (PMSF), centrifuged (10,000
g, at 4ºC, 5 min), filtered (0.22 μm) and used immediately [Ccahuana-Vasquez and
Cury, 2010]. The enamel slabs were immersed into the prepared human saliva and incubated at 37ºC for 30 min to form the acquired pellicle prior to bacterial cell adhesion.
Biofilm assay
Biofilms were formed on enamel slabs using the reference strain Streptococcus
mutans UA159 (ATCC 700610) [Ajdić et al., 2002] as previously described by
Ccahuana-Vasquez and Cury [2010]. The bacterial adhesion was performed with UTYEB supplemented with 1% sucrose under incubation at 37ºC, 10% CO2, for 8 h
(Figure 1). In the adhesion phase, the UTYEB was strongly buffered (10x higher than the usual phosphate concentration) to avoid pH drop and enamel demineralization [Costa Oliveira et al., 2017]. After the bacterial adhesion, the biofilms were always maintained in UTYEB medium containing 0.1 mM glucose (salivary basal concentration). After a fasting period of 16 h the biofilms were exposed to episodes of “feast” and “famine” comprised of 8 daily exposures to the treatments: (i) 0.9% NaCl solution (negative control), (ii) human milk, (iii) bovine milk, (iv) 7% lactose solution (active control of human milk), (v) 4.5% lactose solution (active control of bovine milk), and (vi) 10% sucrose solution (positive control) at predetermined times (08:00, 09:30, 11:00, 12:00, 13:30, 15:00, 16:00 and 17:30 h) for 3 min. After the cariogenic challenge, enamel slabs were rinsed 3 times in 0.9% NaCl solution and transferred back into the culture media. The culture media was changed twice daily, before the first challenge and after the last one. At beginning of the 6th day, after 120 h of growth,
20
the biofilms were collected. The enamel slab was inserted in a microcentrifuge tube containing 1.0 mL of saline solution and the biofilm harvest was performed by sonication at 7 watts for 30 s [Aires et al., 2008]. The slabs were used to evaluate enamel demineralization and the suspension was used for biofilm analyses.
Figure 1: Flow chart of the biofilm assay from the bacterial adhesion phase till the biofilm collection. The timeline shows the fasting and the cariogenic challenge periods.
Biofilm analyses
An aliquot of 100 μL of the biofilm suspension was ten-fold serially diluted in saline solution until 1:107. Two drops of 20 μL of each dilution were plated on
Todd-Hewitt (THB) agar plates, incubated at 37ºC, 10% CO2 for 48 h and the colony forming
units (CFU) were counted [Tenuta et al., 2006]. The extraction of extracellular polysaccharides, soluble (S-EPS) and insoluble (I-EPS), was performed as described by Aires et al. [2008] from an aliquot of 400 μL of the sonicated biofilm suspension. The amount of total carbohydrates was quantified by the phenol sulfuric method [Dubois et al., 1956] using glucose as standard.
Determination of culture medium pH
The culture medium pH was used as an indicator of biofilm acidogenicity. The pH was evaluated twice a day at each culture medium change. The pH measurement was performed using a pH microelectrode (Accumet; Cole-Parmer, USA) coupled to a pH meter (Procyon SA-720, Olímpia, Brazil) calibrated with pH standards of 4.0 and
21
7.0, done directly inside the wells, just after the medium change [Fernández et al., 2016].
Determination of enamel demineralization
The enamel slabs were used to evaluate the enamel demineralization after the biofilm collection. The SH was again performed by three indentations 100 μm apart from the initial SH measurement as previously described. The percentage of surface hardness loss (% SHL) was calculated by the formula: (baseline SH – SH after assay × 100)/baseline SH [Cury et al., 2000].
Statistical analysis
Data were analyzed by one-way ANOVA followed by Tukey's HSD Test. The statistical analysis was done using SAS software (SAS Institute Inc., version 8.01, Cary, N.C., USA) employing a significance level fixed at 5%. Assumptions of homogeneity of variances and normal distribution of errors were checked for all tested response variables using the Kolmogorov-Smirnov test. Data that violated the assumptions were transformed to square-root
22
RESULTS
The %SHL values (Fig. 2) for the groups HM (7.5 ± 5.0), BM (8.7 ± 6.3), HM - active control (13.3 ± 7.5) and BM - control (15.3 ± 8.2) did not differ from the negative control (7.7 ± 3.1) (p>0.05).
Figure 2: Percentage of surface hardness loss (%SHL) of enamel slabs according to the treatments (NaCl 0.9%, Human milk, Bovine milk, Lactose 7%, Lactose 4.5% and Sucrose 10%). Distinct capital letters indicate significant statistically differences (p < 0.05) among groups (Mean ± SD; n = 8).
Only the sucrose group presented lower pH values (Fig. 3) than the negative control for all time points after 32 h of biofilm growth (p<0.05).
Figure 3: pH values of the culture medium according to the treatments (NaCl 0.9%, Human milk, Bovine milk, Lactose 7%, Lactose 4.5% and Sucrose 10%) and biofilm development time (h) as an indicator of biofilm acidogenicity. Asterisks indicate statistically significant difference of sucrose group from the other treatments at the evaluated time points (p < 0.05). (Mean ± SD; n = 8).
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The group exposed to sucrose was also the only to show higher (p<0.05) bacterial counts (9.4 ± 0.2; log10) in comparison to the negative control group (7.8 ±
0.6) (Fig. 4).
Figure 4: CFU counts per biofilm (Log10(CFU)/biofilm) according to the treatments (NaCl 0.9%,
Human milk, Bovine milk, Lactose 7%, Lactose 4.5% and Sucrose 10%). Distinct capital letters indicate significant statistically differences (p < 0.05) among groups (Mean ± SD; n = 8).
Soluble and insoluble EPS (Fig. 5) were only formed in the biofilm exposed to sucrose, 19.5 ± 10.8 and 164.0 ± 12.6 µg/biofilm, respectively.
Figure 5: Amount per biofilm (μg/biofilm) of extracellular polysaccharides, soluble (S-EPS) and insoluble (I-EPS) according to the treatments (NaCl 0.9%, Human milk, Bovine milk, Lactose 7%, Lactose 4.5% and Sucrose 10%). Distinct capital letters indicate significant statistically differences (p < 0.05) among groups (Mean ± SD; n = 8).
24
DISCUSSION
In the present study, neither human milk nor the bovine was able to provoke enamel demineralization during the biofilm development. The exposure to the different types of milk were not able to cause significant pH drop in the culture medium, showing that both, human and bovine milk, had no cariogenic potential. The enamel demineralization was only observed when the biofilm was exposed to sucrose, which was metabolized by bacteria, causing significant pH drop in the culture medium.
The enamel demineralization data contrast with other studies [Rugg-Gunn et al., 1985; Thomson et al., 1996; Prabhakar et al., 2010] mainly due to the period that the dental substrate was exposed to the milk, which varied from 5 nights of periods of 8 h [Thomson et al., 1996] to 12 weeks [Prabhakar et al., 2010]. The period that the biofilm is immersed in the milk, human or bovine, is extremely relevant. Longer periods of exposure to milk favor the bacteria to maintain lactose metabolization, leading to continuous acid production, causing enamel demineralization. Considering that, we used a validated cariogenic biofilm model [Ccahuana-Vásquez and Cury, 2010] to assess the enamel demineralization, mimicking the intermittent biofilm exposure as it occurs in the mouth.
Our study showed that the biofilm exposed to human or bovine milk, or to the lactose solutions, were not able to demineralize the enamel slabs (Fig. 2). Only the biofilms exposed to sucrose solution provoked enamel demineralization (p<0.05). These data were supported by the pH values observed in the culture medium (Fig. 3), which showed similar bacterial metabolization pattern among the milk and lactose solutions groups, differing only from the sucrose group. The difference in the amounts of acids produced and released into the culture medium can be explained by the nutritional source provided to bacteria and the biofilm structure (bacterial cell and extracellular matrix).
Although lactose can be fermented by oral bacteria, including the bacterium
Streptococcus mutans used in the biofilm model, it is not as easily fermented as
sucrose [Zeng et al., 2010a]. Lactose is not the preferred source of nutrition for energy when compared to other carbohydrate sources, such as glucose, fructose, sucrose and mannose [Zeng et al., 2010a; 2010b]. The bacteria require the expression of lac operon genes to metabolize lactose, and also galactose, which is produced by lactose break down [Zeng et al., 2010a]. Therefore, the bacteria exposed to lactose, from milk or active control solutions, could not be able to produce as much acids as the bacteria
25
exposed to sucrose. The lower efficiency using lactose, when compared to sucrose, could also affected the biofilm growth. Biofilms exposed to milk or lactose solutions presented similar CFU counts when compared to the negative control (NaCl 0.9% solution) (p<0.05), but lower counts than the exposed to sucrose (p>0.05) (Fig. 4).
Besides the nutritional source, the biofilm structure is also relevant for the amounts of acids produced and released into the culture medium. Different from sucrose, lactose is not substrate for bacterial enzymes to produce EPS (Fig. 5). In human diet, only sucrose is substrate for EPS synthesis. S. mutans glucosyltransferases (GTF) enzymes, mainly GTFB and GTFC, are responsible for the synthesis of insoluble EPS (I-EPS), glucans with α1→3 linkages [Rølla, 1989; Bowen and Koo, 2011]. I-EPS favor bacterial adhesion on the enamel surface and on the already-adhered cells, which could also explain the higher bacterial counts in the biofilm exposed to sucrose (Fig. 4). In addition, the I-EPS synthesized modify the biofilm matrix tridimensional organization, mainly enhancing its porosity [Dibdin and Shellis, 1988; Rølla, 1989; Cury et al., 2000; Bowen and Koo, 2011]. The porous matrix favors carbohydrate diffusion and retention, which maintains the bacterial metabolization, increasing the acid production near enamel surface [Dibdin and Shellis, 1988]. Moreover, I-EPS makes difficult acid removal from the modified matrix, increasing enamel demineralization. Therefore, the higher demineralization observed only for sucrose group is due to the sum of these two factors, the substrate that is easily metabolized, and to the modified biofilm matrix rich in I-EPS that maintain an acid environment.
The enamel demineralization for the groups exposed to human or bovine milk, or the lactose solutions as active control, was statistically similar (p<0.05). However, it possible to observe a tendency of higher %SHL (Fig. 2) for the human and bovine milk active control, lactose solutions of 7% and 4.5%, respectively. This tendency could be explained by the protective components present in the milk, as calcium, phosphate and proteins [Darke, 1976; Reynolds 1987; Rose 2000]. The main protein is casein, a phosphoprotein that can represent up to 80% of total proteins. Casein contains a phosphoseryl cluster sequence, which provides a negatively charged region that works as calcium-binding site [Rose 2000]. Consequently, casein stabilizes high calcium phosphate concentration in milk, avoiding precipitation. The presence of casein in the human and bovine milk may favored lower %SHL due to the increase of calcium phosphate concentration in biofilm and the acid-buffering capacity also.
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Therefore, our results strengthen that breastfeeding can be safely provided to children, without fear of caries development. Although epidemiological studies have shown a relationship between caries and breastfeeding [Chaffee et al., 2014; Tham et al., 2015; Victora et al., 2016; Peres et al., 2017], they are not able to conclude that the human milk was the only source of feeding that favored caries development. All those studies showed that breastfeeding duration longer than 12 months [Tham et al. 2015], 18 months [Tham et al. 2015], or 24 months [Chaffee et al. 2014; Peres et al. 2017] increased the caries risk, however confounding factors were reported by the authors. Consequently, it is not possible to establish a cause-effect relationship between milk exposure and caries development. Therefore, our study clearly shows that when human or bovine milk are the only source of nutrition, they are not cariogenic for enamel.
It is important to emphasize that human milk is recommended as an exclusive nutrition source for children up to 6 months of age [WHO 2008], and after that, other foods are present in their diet, which may contribute to caries development, mainly foods containing sucrose as a sweetener source. After 6 months, up to 2 years and beyond breastfeeding is just a complementary source of nutrition. The frequent presence of sucrose in children’s diet may change the biofilm structure and the bacterial composition due to the acid environment, favoring the enamel demineralization. In addition to the consumption of foods containing sucrose, children with poor oral hygiene practices would also be more susceptible to have caries.
Even though lactose is not easily fermented [Zeng et al., 2010a], Birkhed et al. [1993] showed that some oral bacteria, including S. mutans and other streptococci, may be adapted to this carbohydrate source, which could be evaluated in future studies using this biofilm model. Another issue to be evaluated, simulating an in vitro experiment, is the role of sucrose when it is consumed by children that are breastfed, which could further investigate the mechanism underlaying the relationship between breastfed children after 12 months of age and caries development. In summary, our findings suggest that both human milk and bovine milk have no cariogenic potential in terms of biofilm structure and they are not able to provoke enamel demineralization in comparison to sucrose.
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30
3 CONCLUSÃO
Os resultados do presente trabalho demonstram que tanto leite humano quanto leite bovino não apresentam potencial cariogênico em termos de estrutura do biofilme e não são capazes de provocar desmineralização do esmalte em comparação com a sacarose.
31
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Araujo FB, Cury JA, Araujo DR, Velasco LFL. Study in situ cariogenicity of human milk: clinical aspects. Rev ABO Nac 1997; 4: 42–44.
Birkhed D, Imfeld T, Edwardsson S. pH changes in human dental plaque from lactose and milk before and after adaptation. Caries Res. 1993;27(1):43-50.
Bowen WH, Lawrence RA. Comparison of the cariogenicity of cola, honey, cow milk, human milk, and sucrose. Pediatrics. 2005 Oct;116(4):921-6.
Bowen WH, Koo H. Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res. 2011;45(1):69-86.
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Chaffee BW, Feldens CA, Vítolo MR. 2014. Association of long-duration breastfeeding and dental caries estimated with marginal structural models. Ann Epidemiol. 24(6):448–454.
Cury JA, Rebelo MA, Del Bel Cury AA, Derbyshire MT, Tabchoury CP. Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res. 2000 Nov-Dec;34(6):491-7.
Dibdin GH, Shellis RP. Physical and biochemical studies of Streptococcus mutans sediments suggest new factors linking the cariogenicity of plaque with its extracellular polysaccharide content. J Dent Res, v.67, n.6, p.890-895, 1988.
Darke SJ. Human milk versus cow’s milk. J Hum Nutr 1976;30:233-8.
Erickson PR, Mazhari E: Investigation of the role of human breast milk in caries development. Pediatr Dent 1999; 21: 86–90.
* De acordo com as normas da UNICAMP/FOP, baseadas na padronização do International Committee of
32
Food and Agriculture Organization of the United Nation, acessado em 29/11/2016. (http://www.fao.org/faostat/en/#data)
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Feldens CA, Giugliani ER, Vigo Á, Vítolo MR. Early feeding practices and severe early childhood caries in four-year-old children from southern Brazil: a birth cohort study. Caries Res. 2010;44(5):445-52.
Koo H, Falsetta ML, Klein MI. The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm. J Dent Res. 2013 Dec;92(12):1065-73
Neves PA, Ribeiro CC, Tenuta LM, Leitão TJ, Monteiro-Neto V, Nunes AM, Cury JA. Breastfeeding, Dental Biofilm Acidogenicity, and Early Childhood Caries. Caries Res. 2016;50(3):319-24.
Paes Leme AF, Koo H, Bellato CM, Bedi G, Cury JA. The role of sucrose in cariogenic dental biofilm formation-new insight. J Dent Res. 2006 Oct;85(10):878-87.
Peres KG, Nascimento GG, Peres MA, Mittinty MN, Demarco FF, Santos IS, Matijasevich A, Barros AJD. Impact of Prolonged Breastfeeding on Dental Caries: A Population-Based Birth Cohort Study. Pediatrics. 2017 Jul;140(1). pii: e20162943. Peres KG, Chaffee BW, Feldens CA, Flores-Mir C, Moynihan P, Rugg-Gunn A. Breastfeeding and Oral Health: Evidence and Methodological Challenges. J Dent Res. 2018 Mar;97(3):251-258.
Prabhakar AR, Kurthukoti AJ, Gupta P. Cariogenicity and acidogenicity of human milk, plain and sweetened bovine milk: an in vitro study. J Clin Pediatr Dent. 2010 Spring;34(3):239-47.
Rølla G. Why is sucrose so cariogenic? The role of glucosyltransferases and polysaccharides. Scand J Dent Res. 1989; 97:115-119.
Rugg-Gunn AJ, Roberts GJ, Wright WG. Effect of human milk on plaque pH in situ and enamel dissolution in vitro compared with bovine milk, lactose, and sucrose. Caries Res. 1985;19(4):327-34.
33
Tham R, Bowatte G, Dharmage SC, Tan DJ, Lau MXZ, Daí X, Allen KJ, Lodge CJB.Breastfeeding and the risk of dental caries: a systematic review and meta-analysis. Acta Pædiatrica.2015;104:62-84.
Thomson ME, Thomson CW, Chandler NP. In vitro and intra-oral investigations into the cariogenic potential of human milk. Caries Res. 1996;30(6):434-8.
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Zeng L, Das S, Burne RA. Utilization of lactose and galactose by Streptococcus mutans: transport, toxicity, and carbon catabolite repression. J Bacteriol. 2010 May;192(9):2434-44.
34
ANEXO 1: Comprovante de aceite do projeto pelo Comitê de Ética em Pesquisa
