1 

UNESP – Universidade Estadual Paulista 

“Júlio de Mesquita Filho” 

Faculdade de Odontologia de Araraquara 
 

 

 

 

 

Sâmara Cruz Tfaile Corbi 

 

 

 

Terapia fotodinâmica com ftalocianina de 

zinco tetracarboxi-N-metilglucamina na 

doença periodontal induzida em ratos 
 

 

 

 

 
 
 
 

Araraquara 

2017



 1 

 UNESP – Universidade Estadual Paulista 

“Júlio de Mesquita Filho” 

Faculdade de Odontologia de Araraquara 

 
 

 

Sâmara Cruz Tfaile Corbi 
 

 

Terapia fotodinâmica com ftalocianina de 

zinco tetracarboxi-N-metilglucamina na 

doença periodontal induzida em ratos 
 

 

 

Tese apresentada à Universidade Estadual Paulista 

(UNESP), Faculdade de Odontologia, Araraquara para 

obtenção do título de Doutor em Odontologia, na Área de 

Periodontia. 

 

  Orientadora: 

Profa. Dra. Rosemary Adriana Chiérici Marcantonio 

                           
 

 

 

Araraquara 

2017 



 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

                  Corbi, Sâmara Cruz Tfaile 
            Terapia fotodinâmica com ftalocianina de zinco tetracarboxi-

N-metilglucamina na doença periodontal induzida em ratos / 
Sâmara Cruz Tfaile Corbi.-- Araraquara: [s.n.], 2017 

                84 f. ; 30 cm. 
 
               Tese (Doutorado em Odontologia) – Universidade Estadual 

Paulista, Faculdade de Odontologia 
               Orientadora: Profa. Dra. Rosemary Adriana Chiérici 

Marcantonio 
                                                                       

    1. Doenças     periodontais          2. Fotoquimioterapia                   
3. Microtomografia por raio-X    I. Título 

 
                          Ficha   catalográfica  elaborada   pela  Bibliotecária Ana Cristina Jorge, CRB-8/5036 
                             Universidade Estadual Paulista (Unesp), Faculdade de Odontologia, Araraquara   

                                           Serviço Técnico de Biblioteca e Documentação  
 
 



 2 

Sâmara Cruz Tfaile Corbi 

 
 

 

Terapia fotodinâmica com ftalocianina de 

zinco tetracarboxi-N-metilglucamina na 

doença periodontal induzida em ratos 
 

 

Comissão Julgadora 
 

 

Tese para obtenção do grau de Doutor em Periodontia 

 

 

Presidente e Orientador: Profa. Dra. Rosemary Adriana Chiérici Marcantonio 

2º Examinador: Prof. Dr. Joni Augusto Cirelli 

3º Examinador: Profa. Dra. Raquel Mantuaneli Scarel-Caminaga 

4º Examinador: Prof. Dr. Michel Reis Messora 

5º Examinador: Profa. Dra. Chaine Pavone 

 

 

 

Araraquara, 07 de Dezembro de 2017



 3 

DADOS CURRICULARES  
 

Sâmara Cruz Tfaile Corbi 
 

 

NASCIMENTO    10 de Outubro de 1986, Araraquara/SP 

 

FILIAÇÃO    Sálua Cruz Tfaile 

     Jeferson Luis Corbi 

 

2007/2011     Curso de Graduação em Odontologia pela  

     Faculdade de Odontologia de Araraquara –  

     UNESP  

 

2012/2014     Curso de Pós-Graduação em Odontologia,  

     área de concentração em Periodontia, nível  

     de Mestrado, na Faculdade de Odontologia  

     de Araraquara – UNESP 

 

2014/2017    Curso de Pós-Graduação em Odontologia,  

     área de concentração em Periodontia, nível  

     de Doutorado, na Faculdade de Odontologia 

     de Araraquara – UNESP 



 4 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Dedico este trabalho aos meus pais Sálua e Jeferson e 

 à minha irmã Sâmia, pela inspiração de todo dia e a vontade de vencer na vida. 
 



 5 

Agradeço primeiramente a Deus, por colocar pessoas certas e especiais na minha vida. Pela 

sabedoria, força e amparo. 
 

Aos meus pais Sálua e Jeferson, que me proporcionaram uma vida digna, onde eu pudesse 

crescer, acreditando que tudo é possível, desde que sejamos honestos, íntegros e de caráter e 

tendo a convicção de que desistir nunca seja uma ação contínua em nossas vidas; que sonhar 

e concretizar os sonhos só dependerá da nossa vontade. Em especial, agradeço minha mãe por 

ser um exemplo de vida, guerreira e batalhadora; que me mostra sempre os melhores caminhos, 

que me dá os conselhos certos, que me ensina a ser sensata e que diariamente me faz 

perceber quão abençoada e privilegiada eu sou. 

 

À minha irmã Sâmia, pelo incessante estímulo, incentivo, exemplo, experiência, enorme 

compreensão e companheirismo, com quem compartilhei todos os momentos e que tem sido uma 

constante benção em minha vida. 

 

À amiga e minha orientadora profa. Adriana Marcantonio, a quem dedico admiração, 

gratidão e respeito; quem me ensinou os primeiros passos da pesquisa científica. Pela 

influência, direcionamento, grande amizade, apoio e eterna paciência. E por compartilhar comigo 

seu tempo e conhecimento. 

 

À minha Tia Salma (in memorian), que em vida sempre apoiou as minhas decisões, me 

incentivou a fazer tudo o que eu tinha vontade, mostrou os caminhos do bem e me ensinou que 

bondade e caridade podem vir de pequenos gestos. Agradeço por compartilhar comigo tantos 

sonhos, conversas, conselhos, tempo e risadas. Sinto muito sua falta. 



 6 

 

Agradeço aos meus familiares, em especial aos meus avós Aparecida e 

Geraldo e, minha prima Sabrina Frasnelli e minha Tia Samira, por entenderem 

minha ausência, falta de tempo e as visitas rápidas. Pelo apoio de sempre e o 

incentivo de continuar meu caminho com força de vontade e fé no futuro. 

 

Á minha amiga querida Livia Finoti, a quem tenho grande admiração e 

apreço. Pessoa única, com coração imenso, a irmã mais nova que eu não tive! 

Sempre disposta a me ajudar com paciência e entusiasmo. Me ensinou coisas da 

vida e compartilhou muitos conhecimentos na pós-graduação. Me auxiliou na 

execução da metodologia da análise radiográfica tridimensional do estudo 2, 

juntamente com a amiga Ane Polly, que prontamente me socorreu com soluções 

certeiras para o desenvolvimento da avaliação. 

 

Aos meus amigos do Laboratório de Genética Molecular: Sâmia Corbi, Livia 

Finoti, Suzane Pigossi, Rafael Nepomuceno, Giovana Anovazzi, 

Romerito Lins, Guilherme Braido, Fernanda Coelho, profa. Raquel 

Caminaga e profa. Ticiana Capote pelos momentos de diversão, alegria, risos 

e descontração. 

 



 7 

 

Aos meus amigos de Doutorado: Paula Macedo, Jackeline Tsurumaki, 

Cássio Rocha, Fernanda Florian, Vinícius Paiva, Adriana Cabrera, Tiago 

Fonseca, Lauriê Garcia,  Patricia Maquera, Lélis Nícoli e Elton 

Pichotano, por todos os momentos de aprendizados que passamos juntos. 

 

Aos mestres e amigos: Rubens Spin-Neto, pelos ensinamentos desde 

minha iniciação científica e a quem tenho grande admiração e Guilherme 

Oliveira, pela paciência, explicações, dicas e por sempre estar acessível e à 

disposição para tirar dúvidas e sugerir ideias. 

 

Aos meus amigos do Convescote: Marina Manduca, Alessandro Alavarce, 

Tainara Alves, Aline dos Santos, Aylan Meneghini, Adriano Felicio, 

Bruna Bolsoni, Maria Angélica, Fernando Pavaneli e Paulo Zaccaro, 

pelas tardes de brincadeiras, jogos, gargalhadas e muito conhecimento inútil. 

 

Às minhas amigas do colegial: Mariana Rozatto e Michele Valila, pela amizade 

de sempre, apoio, conselhos, descobertas, cumplicidade e simplesmente por 

estarem na minha vida após tantos anos. 

 



 8 

 

Aos amigos e funcionários que me ajudaram na realização deste trabalho: Mikail, 

no trabalho no biotério; Luana, no escaneamento das imagens do micro-CT; 

Claudinha e Leandro, pela enorme paciência nos procedimentos laboratoriais; 

Isa e Suleima na organização e funcionamento das clínicas de Periodontia. E 

também a Thelma e Toninho. 

 

À Faculdade de Odontologia de Araraquara (UNESP), na pessoa de sua 

Diretora, Profa. Dra. Elaine Maria Sgavioli Massucato, e do Vice-Diretor, 

Prof. Dr. Edson Alves de Campos, pelas condições oferecidas para a 

realização desta pesquisa. 

 

À profa. Janice Rodrigues Perussi, ao prof. Anderson Orzari Ribeiro e 

Cláudia Bernal,  que sintetizaram e cederam o fotossensibilizador ftalocianina 

de zinco tetracarboxi-N-metilglucamina utilizado neste trabalho.  

 

Ao Coordenador atual do Programa de Pós-Graduação em Odontologia, Área de 

Periodontia, Prof. Dr. Joni Augusto Cirelli e ao anterior Prof. Dr. Carlos 

Rossa Jr. e a todos os docentes da disciplina de Periodontia, pela excelente 

formação, dedicação, competência e empenho em suas atividades. 



 9 

 

Aos funcionários da Seção de Pós-Graduação, José Alexandre e Cristiano, 

pela gentileza, paciência e por sempre solucionarem dúvidas! 

 

Aos funcionários da Biblioteca, Ceres, Marley, Eliane, Maria Inês, Ana 

Cristina,  Denise, Laudicélia e Maria Aparecida pela atenção e correção 

desta tese. 

 

À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, 

por conceder bolsa de estudos e à Fundação de Amparo à Pesquisa do Estado 

de São Paulo – FAPESP, pelo auxílio pesquisa e pela utilização do equipamento 

de micrótomografo computadorizado nesta faculdade, processo: 

2009/54080-0. 

 

MUITO OBRIGADA! 
 

 

 

 

 

 

 



 10 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

“Passarinho quando aprende a voar, sabe mais sobre coragem, que de vôo!” 

(Autor Desconhecido)



 

	

11 

Corbi SCT. Terapia fotodinâmica com ftalocianina de zinco tetracarboxi-N-

metilglucamina na doença periodontal induzida em ratos [Tese de Doutorado]. 

Araraquara: Faculdade de Odontologia da UNESP; 2017. 

 

RESUMO  

A Terapia Fotodinâmica Antimicrobiana (Antimicrobial Photodynamic Therapy – 

aPDT), tem sido utilizada como um tratamento complementar na doença periodontal 

(DP). Ela combina um fotossensibilizador (FS) com uma fonte de luz que induz a 

produção de espécies reativas de oxigênio que elimina células microbianas. O objetivo 

deste trabalho foi avaliar, in vivo, os efeitos da aPDT (com o FS ftalocianina de zinco 

tetracarboxi-N-metilglucamina – 10µg/mL e luz LED vermelho – 655mn, 0,45W de 

potência), coadjuvante a Raspagem e Alisamento Radicular (RAR) e como 

monoterapia, além de verificar as respostas e alterações teciduais da DP induzida em 

ratos, pelas avaliações: microtomográfica, histométrica, estereométrica e histológica. 

Ligaduras foram inseridas nos sulcos dos segundos molares superiores para indução da 

DP. No Estudo 1, as ligaduras permaneceram por 15 dias e foram removidas para 

aplicar os tratamentos e no Estudo 2, as ligaduras foram colocadas por 7 dias e 

continuaram em posição por todo o experimento. 40 animais foram utilizados no Estudo 

1 e distribuídos em 4 grupos: DP (Somente indução da doença, sem tratamento); RAR 

(Indução e tratamento básico periodontal); aPDT (Indução e aplicação da aPDT – FS 

ftalocianina de zinco tetracarboxi-N-metilglucamina e luz LED vermelho); RAR+aPDT 

(Indução, tratamento básico periodontal e aplicação da aPDT). 42 animais foram 

utilizados no Estudo 2 e divididos também em 4 grupos: FS (Tratamento somente com a 

ftalocianina de zinco tetracarboxi-N-metilglucamina); Luz (Tratamento somente com 

irradiação de luz LED vermelha); aPDT (Tratamento com a  terapia fotodinâmica – 

FS+Luz) E DP (Indução da doença, sem tratamento). No Estudo 1, um dia após a 

remoção das ligaduras, foi aplicado os tratamentos e os animais foram eutanasiados nos 

períodos de 7 e 30 dias. No estudo 2, os animais receberam a aplicação das terapias e 

foram eutanasiados nos períodos de 7 e 15 dias. Para ambos os estudos, foi aplicado o 

teste paramétrico ANOVA one way, seguida do pós-teste de Tukey. No Estudo 1: na 

histometria, não foram encontradas diferenças estatísticas; a análise microtomográfica 

mostrou diferenças significantes nos dois períodos entre o grupo DP e RAR+aPDT e em 

7 dias para DP e aPDT na região de furca. Nas proximais não houve diferenças 

significantes e na histologia, mostrou que não houve danos aos tecidos. No Estudo 2, na 



 

	

12 

análise histométrica e radiográfica tridimensional, os resultados não mostraram 

diferenças estatisticamente significantes na região de furca e nas regiões interproximais, 

o perfil inflamatório demonstrou uma tendência a apresentar menor quantidade de 

células inflamatórias no grupo aPDT em 7 dias e na análise histológica não houve 

diferenças entre os grupos, indicando também que as terapias não causaram danos aos 

tecidos. Pode-se concluir que a aPDTcom ftalocianina de zinco tetracarboxi-N-

metilglucamina foi efetiva no controle de perda óssea em DP induzida em ratos e que a 

aplicação da aPDT (com os componentes como monoterapias) e com a preservação da 

ligadura, favoreceu a permanência das bactérias no local e inibiu a ação dos 

tratamentos. 

 

Palavras-chave: Doenças periodontais. Fotoquimioterapia. Microtomografia por raio-

X. 

 

 

 

 

 

 
 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

	

13 

Corbi SCT. Photodynamic therapy with zinc tetracarboxy-N-metylglucamine 

phthalocyanine in induced periodontal disease in rats [Tese de Doutorado]. Araraquara: 

Faculdade de Odontologia da UNESP; 2017. 

 
ABSTRACT 

Antimicrobial Photodynamic Therapy (PDT) is a minimally invasive method consisting 

in the application of a photosensitive dye, which is subsequently stimulated by a light 

source and reacts with oxygen, producing reactive species. The aim of this study was to 

evaluate in vivo, the aPDT effects (with the PS zinc tetracarboxy-N-metylglucamine 

phthalocyanine 10µg/mL, and red LED light with 655nm), as adjuvant treatment to 

Scaling Root and Planing (SRP) and as monotherapy and verify the responses and tissue 

changes after aPDT application in PD-induced rats by microtomographic, histometric, 

stereometric and histological evaluations. Ligatures were placed around the second 

maxillary molars for PD induction. In Study 1, the ligatures were placed for 15 days and 

then they were removed. On the following day the treatments were performed.  In Study 

2, the ligatures were placed for 7 days and remained in position throughout the periods. 

Forty animals were used in Study 1 and they were divided into 4 groups: PD (disease 

induction only, without treatment); SRP (induction and basic periodontal treatment); 

PDT (Induction and application of photodynamic therapy); SRP+PDT (induction, 

application of photodynamic therapy and basic periodontal treatment). Forty-two 

animals were used in Study 2 and they divided into 4 groups: PS (Treatment with zinc 

tetracarboxy-N-metylglucamine phthalocyanine only); Light (Treatment with red LED 

light irradiation only); aPDT (Treatment with photodynamic therapy – PS + Light) and 

PD (Periodontal disease induction, without treatment). In Study 1, the animals were 

euthanized after 7 and 30 days of treatment. In Study 2, the therapies were applied at 

zero period and the animals were euthanized at 7 and 15 days. For both studies, one-

way ANOVA parametric test was applied, followed by Tukey’s post-test. In Study 1, 

concerning histometry data, no statistical differences were observed between groups. 

The microtomographic analysis indicated significant differences in the two periods for 

the PD and SRP+PDT groups and, at 7 days, for the PD and PDT groups in the 

furcation area. No significant differences the interproximal regions were observed. 

Regarding the histological analyses, no tissue damage was observed. In Study 2, the 

three-dimensional radiographic and histometric analyses revealed no statistically 



 

	

14 

different results for the furcation and interproximal regions. The inflammatory profile 

presented a trend of lower amounts of inflammatory cells in the aPDT group at 7 days, 

while the histological analysis indicated no significant differences between the groups, 

indicating that the therapies did not cause tissue damage. Thus, these data indicate that 

PDT using zinc tetracarboxy-N-metylglucamine phthalocyanine was effective in 

maintaining bone loss in PD-induced in rats, although further studies are required to 

further elucidate PDT effects and the application of aPDT and its components as 

monotherapies in PD-induced in rats with the preservation of the ligatures, favored in 

situ bacteria permanence and inhibited treatment action. 

 

Keywords: Periodontal disease. Photochemotherapy. Computadorized 

microtomography. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

	

15 

SUMÁRIO 

1 INTRODUÇÃO.........................................................................16 

  

2  PROPOSIÇÃO..........................................................................24 

 

3  PUBLICAÇÕES........................................................................25 

3.1  Publicação 1...............................................................................25  

3.2  Publicação 2...............................................................................42 

 

4  CONCLUSÃO...........................................................................65 

 

  REFERÊNCIAS........................................................................66 

 

  APÊNDICE................................................................................72 

  Apêndice A – Material e Método.............................................72 

 

 ANEXO......................................................................................83 

Anexo A – Certificado do Comitê de Ética.............................83 

Anexo B – Documentos Comprobatórios................................84 

 

 

 
 

 

 

 

 

 



 

	

16 

1 INTRODUÇÃO 
A Doença Periodontal (DP) é uma doença multifatorial, de caráter inflamatório, 

que acomete os tecidos de suporte do dente e caracteriza-se por inflamação gengival, 

perda do nível de inserção e reabsorção do osso alveolar (American et al.2, 2001; 

Armitage4, 2004). O fator etiológico primário da DP é a presença de microrganismos 

organizados em biofilmes que colonizam a superfície dos dentes. O biofilme é uma 

estrutura complexa composta por microcolônias de bactérias impregnadas em uma 

matrix extracelular que promove proteção celular e facilita a aderência aos dentes 

(Socransky, Haffajee64, 2002; Socransky, Haffajee65, 1991). 

 O padrão ouro no tratamento em Periodontia é a Raspagem e Alisamento 

Radicular (RAR), também conhecido como tratamento básico periodontal, onde a 

remoção mecânica do biofilme e do cálculo é realizada por um profissional em conjunto 

com o controle de placa feito pelo paciente (Tagge et al.72, 1975). No entanto, em 

alguns casos, o debridamento mecânico pode ser falho ao remover alguns organismos 

patogênicos, seja por sua localização no tecido subgengival ou locais de difícil acesso, 

como regiões de furca, bolsas periodontais profundas e anatomias dentárias incomuns 

(Slots61, 2002; Slots, Ting62, 2002). Bonito et al.7 (2005), mostraram que o tratamento 

básico periodontal pode não remover completamente as bactérias patogênicas. Além 

disso, a RAR isolada, reduz temporariamente a infecção bacteriana e pode resultar a um 

retorno aos níveis de pré-tratamento em menos de duas semanas (Adriaens et al.1, 1988; 

Giuliana et al.23, 1997). 

Alguns pacientes continuam apresentando destruição do tecido periodontal após 

a realização da RAR. Estes pacientes, frequentemente, têm fatores de risco associados, 

tal como fumo, diabetes, condições hereditárias e doenças sistêmicas, que são 

acompanhadas por infecção persistente de uma ou mais bactérias periodontais 

patogênicas (Galler19, 2000; Grossi et al.26, 1994; Kuo et al.33, 2008). 

 Assim, para contornar esta temática, o uso de antibióticos sistêmicos tem sido 

indicado. No entanto, a antibioticoterapia tem diminuído ao longo dos anos por causar 

efeitos colaterais, como problemas gastro-intestinais e o aumento de resistência 

bacteriana a estes medicamentos, já que em alguns casos, os pacientes abandonam o 

tratamento antes do término, facilitando assim, a seleção bacteriana (Slots61, 2002; 

Walker76, 1996). 

 Na última década, as limitações da terapia periodontal convencional e a 



 

	

17 

problemática da administração de antibióticos, resultaram nas tentativas de introduzir a 

Terapia Fotodinâmica ou Terapia Fotodinâmica Antimicrobiana (do inglês, 

Antimicrobial Photodynamic Therapy – aPDT), como um tratamento adjunto à 

periodontite crônica (Braun et al.9, 2008; de Almeida et al.13, 2008; de Almeida et al.15, 

2007; Meisel, Kocher41, 2005). Além disso, a aPDT tem sido confirmada como sendo 

uma terapia antimicrobiana não antibiótica (Hamblin, Hasan27, 2004; Wilson77, 1993). 

  A aPDT combina luz visível de baixa intensidade (LASER/LED) e um 

fotossensibilizador (FS), corante não tóxico e fotossensível, que na presença de 

oxigênio, produz Espécies Reativas de Oxigênio (EROs) citotóxicos, tal como oxigênio 

singlete (Hamblin, Hasan27, 2004; Huang et al.29, 2012), sendo tóxico para bactérias. 

Assim resumidamente, a aPDT utiliza oxigênio singlete e radicais livres produzidos 

pelo FS ativado por luz, para eliminar bactérias. O processo fotoquímico é iniciado por 

uma fonte de luz de baixa intensidade com um comprimento de onda adequado e, desse 

modo, o FS no estado fundamental absorve luz e resulta em um estado singlete que 

pode perder energia por fluorescência para um estado triplete com longevidade. Este 

ultimo estágio leva à uma reação fotoquímica, que induz oxigênio singlete, radicais 

livres e superóxidos, que são citotóxicos, a causar a morte bacteriana (Kharkwal et al.30, 

2011), como ilustra a Figura 1. Curiosamente, enquanto a aPDT pode eliminar bactérias 

resistente à antibióticos (Maisch37, 2009), não há informações sobre microrganismos 

desenvolvendo resistência a aPDT (Giuliani et al.24, 2010). 

 
Figura 1 – Mecanismo fotoquímico da Terapia Fotodinâmica. 
 

 

 

 

 

 

 

 

  

Fonte: Elaboração própria. Adaptado de Kikuchi et al.31(2015) para o português. 

  



 

	

18 

 Vários estudos tem mostrado que bactérias periodontopatogênicas são 

susceptíveis à aPDT em culturas planctônicas (Bhatti et al.5, 2002; Bhatti et al.6, 1997; 

Chan, Lai11, 2003; Klepac-Ceraj et al.32, 2011; Matevski et al.39, 2003; Nagahara et al.45, 

2013; Soukos et al.70, 1998; Topaloglu et al.74, 2013; Voos et al.75, 2014; Wilson et al.81, 

1993) e biofilms (Klepac-Ceraj et al.32, 2011; Voos et al.75, 2014; Wilson et al.80, 1992; 

Wood et al.83, 1999) utilizando como FS azul de metileno (Chan, Lai11, 2003; Dobson, 

Wilson18, 1992; Wilson et al.81, 1993), azul de metileno com nanopartículas (Klepac-

Ceraj et al.32, 2011), azul de toluidina O (Bhatti et al.5, 2002; Bhatti et al.6, 1997; 

Dobson, Wilson18, 1992; Matevski et al.39, 2003; Wilson et al.81, 1993), ftalocianina 

(Dobson, Wilson18, 1992; Wood et al.83, 1999), hematoporfirina HCl (Dobson, 

Wilson18, 1992), hematoporfirina ester (Dobson, Wilson18, 1992), conjugado de poli-L-

lisina com o FS clorina (e6) (Soukos et al.70, 1998), indocianina verde (Topaloglu et 

al.74, 2013), indocianina com nanoesferas (Nagahara et al.45, 2013) e safranina O (Voos 

et al.75, 2014). Porém, outros estudos tem demonstrado destruição incompleta de 

patógenos orais (Muller et al.44, 2007; O'Neill et al.47, 2002; Qin et al.52, 2008; Soukos 

et al.66, 2003; Soukos et al.67, 2000). 

 A utilização de FS em aPDT, como porfirinas, ftalocianinas e fenotiazínicos 

(azul de metileno e azul de toluidina O) podem afetar tanto bactérias Gram-positivas 

como Gram-negativas por carregarem uma carga positiva (Merchat et al.42, 1996; 

Merchat et al.43, 1996; Wilson et al.78, 1995), sugerindo que a aPDT pode ser útil para 

aplicações orais, especialmente para o tratamento periodontal (Passanezi et al.50, 2015; 

Sgolastra et al.57, 2013; Smiley et al.63, 2015). 

 Bactérias Gram-negativas são amplamente resistentes à muitos FS utilizados na 

aPDT (Malik et al.38, 1992), no entanto, algumas espécies, tal como bactérias 

pigmentadas com cor preta, contém FS naturais e são muito susceptíveis a aPDT. Foi 

demonstrado que o comprimento de onda variando de 380 a 520nm induz uma redução 

tripla de crescimento de Porphyromonas gingivalis, Prevotella intermedia, Prevotella 

nigrescens, e Prevotella melaninogenica em amostras de biofilme dental obtidas de 

pacientes com periodontite crônica (Soukos et al.68, 2005).  

 Além de estudos em bactérias, os efeitos da aPDT têm sido avaliados também 

por vários modelos de ligadura em ratos, com indução de periondontite experimental 

(Carvalho et al.10, 2011; de Almeida et al.15, 2007; Prates et al.51, 2011). A ligadura leva 

ao acúmulo de biofilme, resultando em perda de inserção e e reabsorção de osso 

alveolar em 7 dias (Graves et al.25, 2008). Resultados favoráveis com aPDT como uma 



 

	

19 

terapia adjuvante à RAR têm sido relatados em periodontite experimental em ratos (de 

Almeida et al.13, 2008; de Almeida et al.14, 2008; de Almeida et al.15, 2007; Garcia et 

al.22, 2014). A progressão da periodontite experimental foi substancialmente reduzida 

por aPDT em  análises radiográfica e histológica (de Almeida et al.15, 2007). Resultados 

positivos semelhantes também foram obtidos em áreas de furca (de Almeida et al.14, 

2008; Garcia et al.21, 2013). Os ratos tratados com aPDT exibiram um número reduzido 

de células positivas ao ácido fosfatase resistente a tartarato, fraca imunoreatividade ao 

receptor-ativador do fator nuclear-κB, e forte imunoreactividade da osteoprotegerina 

(Garcia et al.20, 2013; Garcia et al.22, 2014). A eficácia da aPDT também foi confirmada 

na infecção periodontal em um modelo de cão da raça Beagle (de Oliveira et al.17, 2011; 

Sigusch et al.59, 2005). A melhora na cicatrização periodontal, associada à organização 

do colágeno, infiltração de células inflamatórias e perda óssea, com a adição da aPDT 

também tem sido relatada (Prates et al.51, 2011). 

 Um receio para a aplicação clínica da aPDT é a potencial fotocitotoxicidade para 

as células hospedeiras. Contudo, foi demonstrado que as doses de luz necessárias para 

eliminar as bactérias na aPDT são muito mais baixas do que aquelas que são tóxicas 

para queratinócitos e fibroblastos (Soukos et al.69, 1996). De fato, alguns efeitos 

benéficos da PDT foram relatados em células do ligamento periodontal, como inibição 

de mediadores inflamatórios, favorecendo a quimiotaxia celular e a promoção da 

vasodilatação local e a angiogênese (Houreld, Abrahamse28, 2007). Em termos de 

modulação da imunidade inata, a PDT atua sobre neutrófilos e promove migração 

(Tanaka et al.73, 2012). A PDT também inativa citocinas derivadas do hospedeiro, como 

o fator de necrose tumoral-α e a interleucina-1β, para inibir a ativação da E-selectina em 

células endoteliais (Braham et al.8, 2009). A PDT afeta as células apresentadoras de 

antígenos, como macrófagos e células de Langerhans, reduzindo sua capacidade de 

ativar os linfócitos T e enfraquecendo a resposta inflamatória (Seguier et al.56, 2010).  

 Duas vantagens principais são frequentemente citadas para a aPDT em 

comparação à outros tratamentos periodontais: Primeiro: na aPDT, um FS é colocado 

diretamente na bolsa periodontal e pode ser ativado através de uma fibra óptica 

colocada no mesmo local, o que ajuda a evitar danos aos tecidos circundantes (Qin et 

al.53, 2008). Segundo: os efeitos da aPDT são iniciados pela exposição à uma fonte de 

luz e deste modo, as bactérias podem ser erradicadas em um curto período de tempo. 

Assim o desenvolvimento de resistência bacteriana é improvável (Maisch36, 2007). 

Importante ressaltar que a erradicação de biofilmes e a inativação de citocinas 



 

	

20 

inflamatórias por aPDT provou ser eficaz e segura (Kikuchi et al.31, 2015). 

Nas últimas décadas, foram desenvolvidos compostos considerados como a 

segunda geração de corantes, com propósitos diagnóstico e terapêutico. Dentre eles, está 

as ftalocianinas, que são corantes sintéticos semelhantes às porfirinas e estruturalmente 

consideradas azaporfirinas (Spikes71, 1986) e tem sido estudadas e avaliadas (Longo et 

al.35, 2012; Nunes et al.46, 2004; Ribeiro et al.54, 2013). Estes compostos têm uma banda 

de absorção no espectro electromagnético que varia de 650 a 680nm, o que permite uma 

maior penetração da luz nos tecidos (Nunes et al.46, 2004) e as suas propriedades 

fotofísicas dependem da composição, particularmente do íon metálico central. Entre as 

ftalocianinas, a ftalocianina de cloro-alumínio tem sido sugerida por possuir 

propriedades fotofísicas favoráveis para uso em aPDT, uma vez que produz altas 

quantidades de oxigênio singlete (Nunes et al.46, 2004). A eficácia deste 

fotossensibilizador associado à luz LED foi comprovada em um estudo in vitro que 

avaliou o potencial fotodinâmico da ftalocianina de cloro-alumínio diluída em 

nanoemulsão catiônica para inativar as culturas planctônicas e de biofilmes formados 

por Candida albicans (Ribeiro et al.54, 2013). Este FS foi também eficaz para a 

inativação de bactérias em pacientes com lesões cariosas (Longo et al.35, 2012).  Outros 

estudos (Dobson, Wilson18, 1992; Oliveira et al.49, 2006; Sibata et al.58, 2004; Wilson, 

Dobson79, 1993; Wilson et al.81, 1993; Wilson, Pratten82, 1995) têm corroborado a 

eficiência das ftalocianinas como agentes fotossensíveis na eliminação de 

microrganismos periodontopatogênicos com uso em aPDT. E também, as ftalocianinas 

de zinco (FcZn) estão entre os sensibilizadores promissores deste grupo (Anholt, 

Moan3, 1992; Rosenthal55, 1991; Spikes71, 1986).  

 A ftalocianina (Figura 2) é um macrociclo simétrico composto por quatro 

unidades iminoisoindol com uma cavidade central de tamanho suficiente para acomodar 

vários íons metálicos e, este metal central possui influência considerável em sua 

propriedade fotossensibilizadora (Figura 3) (Anholt, Moan3, 1992; Rosenthal55, 1991; 

Spikes71, 1986). O nome ftalocianina vem de uma combinação do prefixo phthal, 

originalmente do grego naphtha (óleo de rocha), para enfatizar a associação com seus 

vários precursores derivados do ácido ftálico, e o grego cyanine (azul) (Mckeown40, 

1998). 

 

 

 



 

	

21 

 

Figura 2 – Estrutura química da ftalocianina. 
 

 

 

 

 

 

Fonte: Adaptado de de Melo16 (2014). 

 

Figura 3 – Estrutura química da ftalocianina acomodando o íon metálico zinco. 

 

 

 

 

 

 

 

Fonte: de Melo16 (2014). 

 

Um fato importante é quanto à hidrofobicidade que alguns fotossensibilizadores 

apresentam, pois essa propriedade em meio aquoso leva à auto-agregação e em muitos 

casos a uma subsequente precipitação, o que reduz drasticamente a capacidade do 

composto de gerar oxigênio singlete (Simplicio et al.60, 2002). Desse modo é necessário 

que o princípio fotoativo apresente-se solúvel em meio aquoso para possível aplicação 

clínica. 

 Uma estratégia interessante para aumentar a solubilidade em meio aquoso de 

alguns FS envolve a formação de espécies supramoleculares hidrofílicas. Uma 

supramolécula é definida como uma espécie química constituída por duas ou mais 

moléculas unidas por interações intermoleculares. Nesse sentido a química 

supramolecular utiliza uma abordagem centrada na associação de moléculas, visando a 

obtenção de uma determinada propriedade ou funcionalidade (de Melo16, 2014). 

A meglumina, conhecida também como N-metilglucamina (Figura 4) é um 

aminocarboidrato derivado da glicose capaz de formar espécies supramoleculares 



 

	

22 

binárias hidrofílicas com compostos que possuam em sua estrutura átomos de 

hidrogênio ácidos(de Melo16, 2014). 

 
Figura 4 – Estrutura química da meglumina. 

 

 

 

Fonte: de Melo16 (2014). 

 

A formação dos compostos de meglumina envolve como condição uma reação 

ácido-base em que o hidrogênio ácido é transferido ao grupo amina do 

aminocarboidrato. Para viabilizar a formação de espécies supramoleculares entre a 

meglumina e as ftalocianinas, estas primeiramente são funcionalizadas com grupos 

carboxílicos como a ftalocianina de zinco tetracarboxilada (FcZnTc, Figura 5) (de 

Melo16, 2014). 
 
Figura 5 – Ftalocianina-Zn-tetracarboxilada. 

 

 

 

 

 

 

Fonte: de Melo16 (2014). 

 

Posteriormente à inserção dos grupos ácidos, a reação com o aminocarboidrato 

pode ser conduzida produzindo espécies mais solúveis em água. Esse procedimento 

permite a inserção de até quatro moléculas de meglumina como é observado na Figura 6 

para a ftalocianina de zinco tetracarboxi-N-metilglucamina (FcZnTcG). A presença de 

vários grupos hidroxila na supramolécula fornece os sítios onde as ligações de 

hidrogênio são estabelecidas aumentando a hidrofilicidade das ftalocianinas (de Melo16, 

2014). 

 

 
 
 

	 	 	

Zinc(tetracarboxy)phthalocyanine		 	 Zinc(tetracarboxy)phthalocyanine-N-methylglucamine	



 

	

23 

Figura 6 – Ftalocianina-Zn-tetracarboxi-N-metilglucamina. 
 

 

 

  

 

 

 

Fonte: de Melo16 (2014). 

 

O espectro de absorção das ftalocianinas em solução consiste de duas bandas 

principais centradas em torno de 350 nm e 670 nm. A Figura 7 ilustra o espectro de 

absorção molecular da ftalocianina de zinco tetracarboxilada.  

 

Figura 7 – Espectro de absorção molecular da ftalocianina de zinco tetracarboxilada em DMSO. 
 

 

 

 

 

 

 

 

Fonte: de Melo16 (2014). 

 

Na literatura atual, há uma considerável quantidade de estudos em humanos e 

animais avaliando a aPDT com agentes fotossensibilizadores tradicionais (azul de 

metileno e azul de toluidina O), obtendo resutados satisfatórios utilizando diferentes 

metodologias. Os estudos não possuem padronização nos parâmetros de luz, dos tipos e 

concentrações dos fotossensibilizadores, tempos empregados, entre outros fatores que 

influenciam a ação da terapia. Alémd isso, existe uma escassez de estudos sobre 

corantes recém descobertos/descritos (como as ftalocianinas) e seu potencial na 

eliminação de microrganismos que mostra sua efetividade nas respostas e alterações 

teciduais da aplicação da aPDT na DP induzida em ratos. 

 

	 	 	

Zinc(tetracarboxy)phthalocyanine		 	 Zinc(tetracarboxy)phthalocyanine-N-methylglucamine	



 

	

24 

2 PROPOSIÇÃO 

Os objetivos deste estudo foram: 

 

Objetivos gerais 

Ambos os estudos tiveram como intuito observar perda óssea alveolar, verificar 

o grau de inflamação gengival e analisar características histológicas 

periodontais, utilizando o FS na sua forma mais solúvel, ftalocianina de zinco 

tetracarboxi-N-metilglucamina.  

 

Objetivos Específicos 

Publicação 1. Efeitos da aPDT como terapia associada ao tratamento da DP 

experimentalmente induzida em ratos. 

 

Publicação 2. Respostas e alterações teciduais utilizando os componentes da 

aPDT como monoterapias também na DP induzida. 

 
 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



 

	

25 

3 PUBLICAÇÕES 

3.1 Publicação 1 
 

 

Evaluation of Antimicrobial Photodynamic Therapy Effects 
Using Phthalocyanine-Glucamine Photosensitizer as an 

Adjunct Therapy in the Treatment of Induced Periodontal 
Disease in Rats* 

 

 

 

 

 

 

 

 

Sâmara C. T. Corbi, Paula D. Macedo, Janice R. Perussi, 

Anderson O. Ribeiro, Rosemary A. C. Marcantonio 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
*Artigo submetido à revista Archives of Oral Biology em 08/11/2017 (Anexo B)  
Manuscript ID: AOB-D-17-00667 



 

	

26 

 

Evaluation of Antimicrobial Photodynamic Therapy Effects 
Using Phthalocyanine-Glucamine Photosensitizer as an 

Adjunct Therapy in the Treatment of Induced Periodontal 
Disease in Rats 

 

 

 

Sâmara Cruz Tfaile Corbi (SCT, Corbi) – PhD student in Periodontics1; email: 

sa_tfaile@yahoo.com.br 

Paula Delello Macedo (Macedo, PD), PhD student in Periodontics1; email: 

paula_mac2@yahoo.com.br 

Janice Rodrigues Perussi (Perussi, JR), Associate Professor in Chemistry2; 

e-mail: janice@iqsc.usp.br 

Anderson Orzari Ribeiro (Ribeiro, AO), Adjunct Professor from UFABC3, 

email: anderson.ribeiro@ufabc.edu.br 

Rosemary Adriana Chiérici Marcantonio (Marcantonio, RAC), Associate 

Professor in Periodontics1; email: adriana@foar.unesp.br  
 

1Sao Paulo State University (UNESP), School of Dentistry, Diagnosis and 

Surgery Department, Araraquara, SP, Brazil 
2Sao Paulo University (USP), Chemistry and Molecular Physics Department, 

Sao Carlos, SP, Brazil 
3Federal University of ABC (UFABC), Centre for Natural Sciences and 

Humanities, Santo Andre, SP, Brazil 
 

 

 

*Corresponding author: 

Rosemary Adriana C. Marcantonio, PhD 

Department of Diagnosis and Surgery 

Sao Paulo State University – School of Dentistry at Araraquara (UNESP) 

CEP 14801-903, Araraquara, SP, Brasil 

Tel: +55 16 3301-6376 

E-mail: adriana@foar.unesp.br 



 

	

27 

ABSTRACT 

Antimicrobial Photodynamic Therapy (PDT) is a minimally invasive method consisting 

in the application of a photosensitive dye, which is subsequently stimulated by a light 

source and reacts with oxygen, producing reactive species. The aim of the present 

study was to evaluate in vivo tissue responses to PDT using phthalocyanine-glucamine 

and red LED light in the treatment of induced periodontal disease (PD) in rats, through 

microtomographic, histometric and histological evaluations. Ligatures were placed into 

the sulcus of the second maxilarry molars for PD induction. The animals were divided 

into 4 groups: PD (disease induction only, without treatment); SRP (induction and basic 

periodontal treatment); PDT (Induction and application of photodynamic therapy); 

SRP+PDT (induction, application of photodynamic therapy and basic periodontal 

treatment). The ligatures were removed after 15 days and treatments were performed 

the following day. The animals were euthanized after 7 and 30 days of treatment. As all 

data were normaly distributed (Kolmogorov-Smirnov), the parametric ANOVA test was 

applied, followed by the Tukey test. Concerning histometry data, no statistical 

differences were observed between groups. The microtomographic analysis indicated 

significant differences in the two periods for the PD and SRP+PDT groups and, at 7 

days, for the PD and PDT groups in the furcation area. No significant differences the 

interproximal regions were observed. Regarding the histological analyses, no tissue 

damage was observed. These data indicate that PDT using phthalocyanine-glucamine 

was effective in maintaining bone loss in PD-induced in rats, although further studies 

are required to further elucidate PDT effects.  

Keywords: Periodontal diseases. Photochemotherapy. X-ray microtomography 

 

INTRODUCTION 

Periodontal disease (PD) is a multifactorial inflammatory disease that develops 

when the equilibrium between the host response and microbial challenge is altered 

(Andersen, Loebel, Hammond, & Wilson, 2007). This condition is clinically 

characterized by inflammation, bleeding on probing and pronounced loss of insertion, 

whose primary etiological factor is constituted by oral biofilm bacteria (de Almeida, 

Garcia, & Theodoro, 2006; de Almeida et al., 2007; Qin et al., 2008).  

Scaling Root and Planing (SRP) is aimed at the removal of this oral biofilm, 

enabling health reinstatement, tissue regeneration and lessening clinical signs of 

inflammation (Qin et al., 2008). However, in some cases, this therapy does not seem to 

be able to repair or maintain periodontal health, which may lead to the permanence or 

recolonization of microorganisms (Drisko, 1998). 



 

	

28 

Studies have proposed the use of local and systemic antibiotics associated with 

SRP to aid in bacterial combat (Vergani, Silva, Vinholis, & Marcantonio, 2004). 

However, the regular and indiscriminate use of these drugs can cause several side 

effects, in addition to possible bacterial resistance (de Almeida et al., 2006; de Almeida 

et al., 2007; Jori et al., 2006; Pfitzner, Sigusch, Albrecht, & Glockmann, 2004; Qin et 

al., 2008). Therefore, the relevance of a search for a new technique or therapy 

compatible with conventional mechanical treatments is clear, allowing for greater 

efficiency in periodontal treatment. 

Photodynamic Therapy (PDT), also called Antimicrobial Photodynamic Therapy 

(aPDT), because it affects microorganisms, is a new and thriving clinical treatment that 

basically employs the combination of the triad: oxygen, light source, and a 

photosensitizing agent (PS) (Maisch, 2007). Each of these isolated factors is not 

capable of causing damage, but, when combined, produce lethal cytotoxic agents that 

can selectively kill cells (Sharman, Allen, & van Lier, 1999). 

The mechanism of action of PDT occurs due to the excitation of a non-toxic 

photosensitive dye (PS) which, when irradiated by visible light at a frequency resonant 

with the level of optical absorption of said substance, transfers energy to the 

surrounding molecules, generally molecular oxygen (O2), and produces highly reactive 

species (ROS), such as free radicals and singlet oxygen and the latter, can modify the 

structures of plasma membranes or even DNA (Jori et al., 2006) and can also cause 

cell death through various mechanisms, including lipid peroxidation, inhibition of the 

enzymatic system, protein agglutination and reactions with other biological systems 

(Andersen et al., 2007). In periodontics, the success in eliminating microorganisms by 

PDT indicates that is as an adequate adjunct therapy in the fight against localized 

infections (Jori et al., 2006).  

In vitro (Klepac-Ceraj et al., 2011) and in vivo (Andersen et al., 2007; de 

Almeida et al., 2007; Qin et al., 2008; Sigusch, Pfitzner, Albrecht, & Glockmann, 2005) 

studies have analyzed PDT effects on both periodontopathogenic and non-

periodontopathogenic bacteria exposed to several PS and different wavelengths, 

resulting in an effective antimicrobial action of up to 99%. Clinically, a reduction in 

pocket depth, clinical insertion level and bleeding has been observed, thus 

documenting the effectiveness of PDT as a adjunct therapy in the treatment of PD (Qin 

et al., 2008). However, despite the benefits of this technique, some studies reveal 

difficulties in reaching microorganisms located in the deeper layers of oral biofilms. 

Even so, PDT produces higher bacterial death when compared to the use of systemic 

antibiotics (Fontana et al., 2009). 

 Phthalocyanines (FC) are a promising group of second generation 



 

*Para metodologia completa, ver Apêndice 1. #Aprovação do Comitê de Ética, ver  Anexo A. 

29 

photosensitizers for PDT. They comprise organic compounds whose structure includes 

a ring formed by eight carbon atoms and eight nitrogen atoms joined by conjugated 

double bonds (Ryskova & Slezak, 2010). As a rule, FC display effective tissue 

penetration, since their most adequate light absorption region is between 600nm and 

800nm (Ogunsipe et al., 2008). FC show high selectivity, low phototoxicity and are 

resistant to chemical or photochemical degradation (Garcia & Bentley, 2003). Their 

photophysical properties are strongly influenced by the presence and nature of the 

central metal ion, directly reflecting on the life time of the triplet excited state of 

metallophthalocyanines (Bonnet, 1995), providing a significant amount of singlet 

oxygen species that are able to remain in the triplet excited state for a longer period of 

time (Ryskova & Slezak, 2010).  

 Scarce studies are available on PS and their potential in eliminating 

microorganisms, mainly in dentistry, when applying PDT in the treatment of PD, as well 

as their placement associated with conventional periodontal therapy. In this context, 

the aim of the present study was to evaluate in vivo tissue responses after PDT using a 

phthalocyanine-glucamine as an adjunct therapy in the treatment of PD-induced in rats 

by means of three-dimensional radiographic (µCT), histometric and histological 

analyses. 

 

MATERIAL AND METHODS*  

Ethics comittee 

This project was approved by the Ethics Committee on Animal Experimentation 

(No. 07/2012)#. 

 

Samples 

Forty rats (Rattus norvegicus) of the albinus variation, Holtzman, adults, 

weighing between 300-330g were used. The animals were kept in plastic boxes, 5 

animals per box, and treated with water and food ad libitum before and during the 

whole experimental period. The animals were maintained in an environment with 

controlled light, humidity and temperature. 

 

Periodontal disease induction 

 The animals were anesthetized with a combination of ketamine (ketamine 

hydrochloride - Francotar 3% - Virbac do Brasil Ind. e Com. Ltda.) and xylazine 

(xylazine hydrochloride - Virbaxyl 2% - Virbac do Brasil Ind. e Com. Ltda.) at 

0,08mL/100g and 0,04mL/100g body weight, respectively. The PD-induced 

hemimaxilae were chosen randomly (right or left). The ligatures were inserted in the 



 

 

30 

subgingival region, into the sulcus and around the second maxillary molars using no. 

24 cotton threads. The ligatures were removed after 15 days, and the treatments were 

performed one day after in each group (Figure 1). 

 

Photodynamic Therapy 

Photosensitizer preparation 

Phthalocyanine-glucamine was prepared from a stock solution of zinc-

tetracarboxy-phthalocyanine at 1,1mg/mL in DMSO and subsequently diluted in 

phosphate buffered saline (pH=7.2) to a final concentration of 10µg/mL. Soon after the 

preparation, the PS was stored in light-protected poliproylene tubes maintained in a 

refrigerated environment until use. For application, a blunt tip syringe containing 0,2mL 

of the PS was inserted into the gingival sulcus. The solution was  applied around the 

entire tooth and, after 10 minutes of incubation time, light irradiation was performed. 

 

Light source 

 The light source used to activate the phthalocyanine-glucamine PS 

corresponded to a wavelength of 655 nm, 0,45W power, 0,47W/cm2 power density and 

170,52J/cm2 dose (red LED, 11mm diameter, DMC Equipamentos Ltda, São Carlos, 

Brazil), coinciding with the maximum absorption band of phthalocyanine-glucamine. 

The LED light was placed on the occlusal surface of the teeth and irradiation was 

maintained for 6 minutes. 

 

Experimental groups 

On the day after ligatures removal, the animals were randomly divided and 

treated according to their group (5 animals/group/period): 

• PD Group (PD): disease induction only, without treatment. 

• Scaling Root and Planing Group (SRP): SRP performed with specific curettes 

(Gracey mine Five 5-6, HuFriedy). 

• Photodynamic Therapy Group (PDT): Application of the PS followed by LED 

application. 

• SRP+PDT Group (SRP+PDT): SRP (same as the SRP group) followed by PDT 

(same as the PDT group). 

 

The animals were euthanized with an anesthetic overdose at 7 and 30 days after 

the treatments. The hemimaxillae were removed and fixed in 4% paraformol for 48h. 

Subsequently, the samples were washed in running water for 24h and placed in 70% 

alcohol, where they remained until the computerized microtomograph scanning. 



 

 

31 

 

Three-dimensional Radiographic Analysis (µCT)  

The samples were scanned by means of X-ray beam scanning in a 

computerized microtomography system (Skyscan 1176, Aatselaar, Belgium, 2003). 

The parameters of the equipment were set as follows: Al 0.5mm filter; Voxel size: 

17.48µm; Voltage 50KV and electric current 500µA. After scanning, the 3D images for 

each sample were obtained through the equipment software (NRecon 1.6.1.5 - 

SkyScan N. V., Belgium, 2003). 

The images were rotated and repositioned in a standard orientation the 

Dataviewer software (SkyScan 1176, Aartselaar, Belgium, 2003) and a contrast 

threshold (ranging from 59 to 255) was established to distinguish mineralized tissues 

using the CTan/CTvol software (Skyscan 1176, Aatselaar, Belgium, 2003). The regions 

of interest (ROI) were positioned by measuring 3 regions of the second maxillary molar; 

a 1.26x1.15mm2 furcation area and the mesial and distal proximal areas 

(1.26x0.56mm2) from the cement-enamel junction (Figure 2). The data were expressed 

as a percentage of volume of bone tissue of each region. 

 

Histological processing 

After scanning, the samples were placed in a 7% EDTA solution, pH 7,2 (Synth, 

São Paulo, Brazil), buffered with sodium phosphate for decalcification. Following 

laboratory procedures, the samples were then included in paraffin. Semi-serial sections 

were made along the axis of the tooth, at 4µm thickness. For each hemimaxillae, 

approximately 30 sections were obtained, divided into slides containing 3 sections 

each. 

 

Histometric analyses 

 For this analysis, a blind and calibrated examiner (Pearsons’ Correlation, 

r=0.99) selected 2 slides from each hemimaxillae. The furcation area was delimited 

according to the methodology reported by da Silva et al., (2008).  

Measurements were taken using the ImageJ Launcher imaging software, version 

1.48b (National Institutes of Health, USA), evaluating the following areas: 

• Furcation area: the area was defined, a 1000µm-zone under the furcation limited 

by the roots. Furcation and bone area were measured, thus obtaining the 

percentage of bone present in the furca region of each histological section.  

• Interproximal region (mesial and distal): a linear measurement of the cement-

enamel junction up to the top of the bone crest was performed, thus obtaining 

bone loss values. 



 

 

32 

 

Histological analyses  

 Using a DIASTAR (Leica Reichert & Jung products, Germany) optical 

microscope with 4,0-10,0-fold objective and 10 ocular magnifications, the images were 

captured and sent to a microcomputer with the aid of a DXC-1107A/107AP video 

camera (Sony Electronics Inc, Japan). The inflammatory reactions of the connective 

tissue, bone resorption processes and tissue neoformation in each experimental group 

were evaluated by an experienced, blind and calibrated examiner for the experimental 

groups. 

 

Statistical analyses 

The experimental data were tabulated using the Microsoft Excel for Mac 2011 

software (Apple Inc, USA) and analyzed statistically with the aid of the GraphPad Prism 

6.0 software (GraphPad Inc, USA). 

The data were evaluated applying the central point theorem, to verify if their 

arrangement respected a normal distribution, using the Kolmogorov-Smirnov test. As 

all data were normally distributed, the parametric ANOVA (One Way) test was applied 

to verify the existence of statistical differences between the groups. Tukey’s post-hoc 

test was subsequently applied, in order to detect differences among groups. For 

comparisons between the treatment periods, the ANOVA (Two Way) parametric test 

was applied. All tests were applied with a 95% confidence interval. 

 

RESULTS 

Three-dimensional radiographic analysis (µCT)  

The images of this analysis are displayed in Figure 3. Regarding the furcation 

analysis, a statistically significant difference was observed between the PD and PDT 

groups concerning the percentage of bone tissue volume in the furca region of the 

second maxilarry molars of the hemimaxillae treated within the 7-day period (*p <0.05), 

as well as between the PD and SRP+PDT groups (*p <0.05). During the 30-day period, 

a statistical difference between the PD and SRP+PDT groups was observed (&p<0.05). 

The PD group displayed a lower percentage of bone volume when compared to the 

experimental groups in both periods, indicating bone loss. These data are displayed in 

Figure 3.1. On the other hand, no statistically significant differences concerning the 

percentage of bone tissue volume was observed in the proximal analysis. These data 

are displayed in Figure 3.2. 

 

 



 

 

33 

Histological analyses 

 In this analysis, a blind examiner (P.A.O.) evaluated the images and verified 

that the periodontal area of the animals of the PD group displayed several 

morphological alterations. At 7 days, an intense inflammatory process was observed in 

the interdental gingiva, which often appeared ulcerated; the gingival epithelium was 

adjacent to the surface of the acellular cement, in other words, located apically to the 

cement-enamel junction. A gradual but evident decrease in the inflammatory process 

was observed in subsequent periods. The SRP, PDT and SRP+PDT groups presented 

the same histological characteristics found in the PD group, but with additional 

migration of the epithelium, inflammatory processes, loss of the alveolar process and 

bone resorption in the furca region, albeit less pronounced. Thus, histological 

descriptions were similar for all experimental groups. The images illustrate the 

periodontal tissues of each treatment during the evaluated periods (Figure 4). 

 

Histometric analysis 

All data were statistically the same for the bone area in the furcation region 

(Figure 4.1) and for bone loss in the interproximal regions (Figure 4.2) of the second 

maxillary molars treated in the experimental groups. 

 

DISCUSSION 

The aim of this study was to evaluate in vivo tissue responses to PDT using the 

phthalocyanine-glucamine PS as an adjucant therapy in the treatment of PD-induced in 

rats.  

It is known that PDT action is related to the experimental model used, in this 

case, ligature-induced periodontitis in rats, which has been widely applied to 

investigate PDT effects on PD (de Almeida et al., 2007; Garcia et al., 2011; Qin et al., 

2008). Since PD is a multifactorial and polymicrobial disease, ligature placement may 

be the most representative method for its induction for periodontal treatment evaluation 

(Gaspersic, Stiblar-Martincic, Osredkar, & Skaleric, 2003). The study conducted by 

Graves et al. (2008) demonstrated that the ligature model is simple, versatile and 

inexpensive, and, when applied to rats, allows for considerable information of the 

immune system, due to the availability of a wide range of genetically modified strains 

and immunochemistry and cellular reagents.  

In addition to the experimental model, the applied PS should also be evaluated, 

since the formation of aggregates between PS molecules can hinder the process of 

reactive oxygen species generation. Such aggregates tend to be formed as the 

concentration of the photosensitizing agent is increased or when the hydrophobic agent 



 

 

34 

is in an aqueous medium (da Silva et al., 2009). In the present study, a PS 

concentration that would be effective in PD treatment was used.  

Light irradiation time should also be taken into account when evaluating PDT 

effects. This concerns the maximum amount of light possible at the maximum 

absorption of the PS, without significant thermal effects. In this context, an efficient 

heat dissipation system is required, in order to minimize the thermal effects of the heat 

generated by the light source on the irradiated tissue and its surroundings. For this 

reason, a LED light was used. Finally, the availability of essential oxygen must also be 

considered, to obtain a successful therapy. 

The histological results observed herein indicated no differences between the 

groups in the evaluated periods, demonstrating that PDT did not cause tissue damage. 

Kaestner et al. (2003) applied zinc phthalocyanine to the skin of mice and 

demonstrated satisfactory responses regarding absorption, since the dye penetrates 

only to the epidermis, and not the dermal layers, which are rich in blood capillaries, in 

topical applications. Therefore, this dye should pose no risks regarding either 

photosensitivity or phototoxicity.  

de Almeida et al. (2007) evaluted the effects of PDT on the evolution of PD-

induced in rats by histological and radiographical methods. The PDT group in that 

study was treated with methylene blue, irradiated with a low intensity laser and 

displayed lower bone loss compared to the control group at 5 and 15 days, with no 

statistical difference at 30 days. After 15 days, the histological results indicated a 

statistically significant difference in the extent of the inflammatory reaction in the 

gingival tissue. The authors concluded that PDT can, thus, reduce periodontal bone 

destruction in a transient manner. In the following year, de Almeida et al. (2008) 

developed an experiment, also in rats, with the purpose of histologically evaluating the 

influence of PDT applied with methylene blue on bone loss in furcation areas. The PDT 

group showed significantly lower bone loss compared to the other groups in a 7-day 

period. After 15 days, the dye-only and PDT groups presented significantly lower bone 

loss when compared to the other groups. 

The histometric results of the present study indicated no statistically significant 

differences between the groups in the evaluated periods. However, the PDT group 

maintained the same pattern of bone loss when compared to the SRP group, in both 

the interproximal and furcation areas. The results of the three-dimensional radiographic 

analysis indicate that, at the 7 and 30 day evaluations, the PDT group presented higher 

bone volume in the interproximal and furcation regions when compared to the other 

groups. The PD group exhibited higher bone loss at all periods, but was statistically 

significant at the furcation region at 7- and 30-day evaluations. The results of these 



 

 

35 

analyses, evaluating the same regions, differ due to the concepts of each method. 

Traditionally, quantitative histological techniques are considered the standard in 

the evaluation of trabecular and cortical bone architecture. Although histological 

analyses provide unique information on cellularity and dynamic indices of bone 

remodeling, they display limitations regarding the evaluation of bone microarchitecture, 

since the evaluated structural parameters are derived from stereometry of only a few 

2D sections, generally assuming that the underlying structure is overlaid on the image 

(Parfitt et al., 1987). In comparison, 3D high-resolution imaging techniques, such as 

three-dimensional radiography directly measure bone microarchitecture without relying 

on stereological models. Introduced by Feldkamp and colleagues (Feldkamp, 

Goldstein, Parfitt, Jesion, & Kleerekoper, 1989) in the late 1980s, micro-CT has now 

become the gold standard for the evaluation of bone morphology and microarchitecture 

in mice and other small ex vivo animal models. 

Some animal studies (Qin et al., 2008; Sigusch et al., 2005) have demonstrated 

that PDT is effective in reducing periodontopathogenic microorganisms, while others 

(de Almeida et al., 2008; Garcia et al., 2011) have indicated that, when PDT is 

combined with conventional periodontal treatment, alveolar bone loss is lower when 

compared to the isolated intervention. One of the factors that may affect PDT results in 

PD is the presence of gingival fluid, blood and inflammatory exudate (Matevski et al., 

2003), as these conditions may reflect or absorb light, promoting a "protective effect" 

for the bacteria present in the area. This may explain the results observed herein, 

although not significant, of the lower amount of bone tissue formed in the SRP+PDT 

group, since scraping prior to the application of PDT may have generated bleeding that 

inhibited the PS from acting on the microorganisms. 

The PDT mechanism of action seems to be very clear, so there is no dispute 

about its bactericidal capacity. Studies are unanimous in affirming that the excitation of 

the photosensitizer caused by the light source triggers the appearance of molecules 

with toxic effects on the microorganisms (Sigusch et al., 2005). Several studies 

(Pfitzner et al., 2004; Qin et al., 2008; Sigusch et al., 2005) have proven the high rate 

of microorganism destruction in samples submitted to PDT. On the other hand, certain 

bacterial species, such as Aggregatibacter actinomycetemcomitans, are more resistant 

to this therapy when compared to others, such as Porphyromonas gingivalis and 

Fusobacterium nucleatum (Pfitzner et al., 2004). Gram-positive bacteria are generally 

susceptible to photoinactivation, while gram-negative bacteria are often resistant 

(Soukos & Goodson, 2011). This resistance is attributed to the outer membrane of 

these organisms, which acts as a barrier to the penetration of the PDT-produced 

compounds. The use of a cationic dye shows greater photodynamic activity against 



 

 

36 

gram-negative bacteria, since the positive charge promotes an electrostatic bond to the 

external surface of the cell, inducing an initial damage that favors dye penetration 

(Merchat, Spikes, Bertoloni, & Jori, 1996). 

Although this study did not directly evaluate PDT effects on microorganisms, 

Persson et al. (2001) demonstrated that, when a decrease in the amount of bacteria 

present in periodontitis or peri-implantitis is observed, tissue repair or re-

osseointegration is subsequently observed. In vitro (Klepac-Ceraj et al., 2011) and in 

vivo(de Almeida et al., 2006; de Almeida et al., 2008; Qin et al., 2008; Sigusch et al., 

2005) studies have shown favorable results using PDT principles. A study performed in 

humans (Andersen et al., 2007) verified that PDT was effective in PD treatment, 

reducing clinical insertion level and probing depth. Thus, this therapy has been 

proposed as a complementary alternative, especially in areas of difficult access to 

manual instruments, such as furcation regions, concavities and deep pockets (de 

Almeida et al., 2007). This therapy may also be an alternative method to reduce the 

use of antibiotics, avoiding the development of resistant organisms (Maisch, 2007).  

As mentioned previously, the limitations of this study are due to the fact that 

different variables were present, such as the experimental animal model, dye 

concentration, tissue retention period, time for biological response, irradiation time, light 

energy and wavelength, pH of the site (tissue/tooth/interface), presence of exudate, 

blood and gingival fluid and frequency and mode of application of the dye, all of which 

may influence biological responses to PDT (de Almeida et al., 2006). Thus, further 

studies evaluating these factors are required in order to better understand the 

photoinactivation action of the local microorganisms. 

 

CONCLUSIONS 

 With the results obtained herein, we can conclude that PDT applied with 

phthalocyanine-glucamine as the PS, both as a monotherapy and associated to SRP, 

is effective in bone loss control in PD-induced rats, similar to the conventional 

mechanical treatment. Thus, phthalocyanine-glucamine may be considered a 

promising photosensitizer. However, further in vitro and in vivo studies are required to 

elucidate PDT action and the effect of this PS as an adjunct therapy in the treatment of 

PD. 

 

ACKNOWLEDMENTS 

We would like to thank Capes and CNPq for financial support and Prof. Dr. 

Anderson O. Ribeiro from the Federal University of ABC for synthesize the solution of 

the photosensitizer phtalocyanine-glucamine used in this study. 



 

 

37 

CONFLICT OF INTEREST AND SOURCES OF FUNDING STATEMENT 

The authors declare that there are no conflicts of interest. 

REFERENCES 
Andersen, R., Loebel, N., Hammond, D., & Wilson, M. (2007). Treatment of periodontal disease 

by photodisinfection compared to scaling and root planing. The Journal of Clinical 
Dentistry, 18, 34-38.  

Bonnet, R. (1995). Photosensitizers of the porphyrin and phthalocyanine series for 
photodynamic therapy. Chemical Society Reviews, 24, 19-33.  

da Silva, A. R., Inada, N. M., Rettori, D., Baratti, M. O., Vercesi, A. E., & Jorge, R. A. (2009). In 
vitro photodynamic activity of chloro(5,10,15,20-tetraphenylporphyrinato)indium(III) 
loaded-poly(lactide-co-glycolide) nanoparticles in LNCaP prostate tumour cells. Journal 
of Photochemistry and Photobiology B: Biology, 94, 101-112.  

da Silva, V. C., Cirelli, C. C., Ribeiro, F. S., Leite, F. R., Benatti Neto, C., Marcantonio, R. A., & 
Cirelli, J. A. (2008). Intrusion of teeth with class III furcation: a clinical, histologic and 
histometric study in dogs. Journal of Clinical Periodontology, 35, 807-816.  

de Almeida, J. M., Garcia, V. G., & Theodoro, L. H. (2006). Terapia fotodinâmica: uma opção na 
terapia periodontal. Arquivos do Centro de Estudos do Curso de Odontologia da 
Universidade Federal de Minas Gerais, 42, 199-210.  

de Almeida, J. M., Theodoro, L. H., Bosco, A. F., Nagata, M. J., Oshiiwa, M., & Garcia, V. G. 
(2007). Influence of photodynamic therapy on the development of ligature-induced 
periodontitis in rats. Journal of Periodontology, 78, 566-575.  

de Almeida, J. M., Theodoro, L. H., Bosco, A. F., Nagata, M. J., Oshiiwa, M., & Garcia, V. G. 
(2008). In vivo effect of photodynamic therapy on periodontal bone loss in dental 
furcations. Journal of Periodontology, 79, 1081-1088.  

Drisko, C. H. (1998). The use of locally delivered doxycycline in the treatment of periodontitis. 
Clinical results. Journal of Clinical Periodontology, 25, 947-952; discussion 978-949.  

Feldkamp, L. A., Goldstein, S. A., Parfitt, A. M., Jesion, G., & Kleerekoper, M. (1989). The direct 
examination of three-dimensional bone architecture in vitro by computed tomography. 
Journal of Bone and Mineral Research, 4, 3-11.  

Fontana, C. R., Abernethy, A. D., Som, S., Ruggiero, K., Doucette, S., Marcantonio, R. C., & 
Soukos, N. S. (2009). The antibacterial effect of photodynamic therapy in dental plaque-
derived biofilms. Journal of Periodontal Research, 44, 751-759.  

Garcia, F. S., & Bentley, M. V. (2003). Phthalocyanine dyes: importance and future directives 
for use in photodynamic therapy of cancer. Trends in Photochemistry & Photobiology, 
10, 77-82.  

Garcia, V. G., Fernandes, L. A., Macarini, V. C., de Almeida, J. M., Martins, T. M., Bosco, A. F., 
& Theodoro, L. H. (2011). Treatment of experimental periodontal disease with 
antimicrobial photodynamic therapy in nicotine-modified rats. Journal of Clinical 
Periodontology, 38, 1106-1114.  

Gaspersic, R., Stiblar-Martincic, D., Osredkar, J., & Skaleric, U. (2003). Influence of 
subcutaneous administration of recombinant TNF-alpha on ligature-induced 
periodontitis in rats. Journal of Periodontal Research, 38, 198-203.  

Graves, D. T., Fine, D., Teng, Y. T., Van Dyke, T. E., & Hajishengallis, G. (2008). The use of 
rodent models to investigate host-bacteria interactions related to periodontal diseases. 
Journal of Clinical Periodontology, 35, 89-105.  

Jori, G., Fabris, C., Soncin, M., Ferro, S., Coppellotti, O., Dei, D., & Roncucci, G. (2006). 
Photodynamic therapy in the treatment of microbial infections: basic principles and 
perspective applications. Lasers in Surgery and Medicine, 38, 468-481.  

Kaestner, L., Cesson, M., Kassab, K., Christensen, T., Edminson, P. D., Cook, M. J., & Jori, G. 
(2003). Zinc octa-n-alkyl phthalocyanines in photodynamic therapy: photophysical 
properties, accumulation and apoptosis in cell cultures, studies in erythrocytes and 



 

 

38 

topical application to Balb/c mice skin. Photochemical and Photobiological Sciences, 2, 
660-667.  

Klepac-Ceraj, V., Patel, N., Song, X., Holewa, C., Patel, C., Kent, R., & Soukos, N. S. (2011). 
Photodynamic effects of methylene blue-loaded polymeric nanoparticles on dental 
plaque bacteria. Lasers in Surgery and Medicine, 43, 600-606.  

Maisch, T. (2007). Anti-microbial photodynamic therapy: useful in the future? Lasers in Medical 
Science, 22, 83-91.  

Matevski, D., Weersink, R., Tenenbaum, H. C., Wilson, B., Ellen, R. P., & Lepine, G. (2003). 
Lethal photosensitization of periodontal pathogens by a red-filtered Xenon lamp in vitro. 
Journal of Periodontal Research, 38, 428-435.  

Merchat, M., Spikes, J. D., Bertoloni, G., & Jori, G. (1996). Studies on the mechanism of 
bacteria photosensitization by meso-substituted cationic porphyrins. Journal of 
Photochemistry and Photobiology B: Biology, 35, 149-157.  

Ogunsipe, A., Durmusß, M., Atilla, D., Gürek, A. G., Ahsen, V., & Nyokong, T. (2008). 
Synthesis, photophysical and photochemical studies on long chain zinc 
phythalocyanines. Synthetic Metals, 158, 839-847.  

Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H., Meunier, P. J.,  & 
Recker, R. R. (1987). Bone histomorphometry: standardization of nomenclature, 
symbols, and units. Journal of Bone and Mineral Research, 2, 595-610.  

Persson, L. G., Berglundh, T., Lindhe, J., & Sennerby, L. (2001). Re-osseointegration after 
treatment of peri-implantitis at different implant surfaces. An experimental study in the 
dog. Clinical Oral Implants Research, 12, 595-603.  

Pfitzner, A., Sigusch, B. W., Albrecht, V., & Glockmann, E. (2004). Killing of 
periodontopathogenic bacteria by photodynamic therapy. J Periodontol, 75, 1343-1349.  

Qin, Y. L., Luan, X. L., Bi, L. J., Sheng, Y. Q., Zhou, C. N., & Zhang, Z. G. (2008). Comparison 
of toluidine blue-mediated photodynamic therapy and conventional scaling treatment for 
periodontitis in rats. Journal of Periodontal Research, 43, 162-167.  

Ryskova, L., & Slezak, R. (2010). Photodynamic antimicrobial therapy. Central European 
Journal of Biology, 5, 400-406.  

Sharman, W. M., Allen, C. M., & van Lier, J. E. (1999). Photodynamic therapeutics: basic 
principles and clinical applications. Drug Discovery Today, 4, 507-517.  

Sigusch, B. W., Pfitzner, A., Albrecht, V., & Glockmann, E. (2005). Efficacy of photodynamic 
therapy on inflammatory signs and two selected periodontopathogenic species in a 
beagle dog model. Journal of Periodontology, 76, 1100-1105.  

Soukos, N. S., & Goodson, J. M. (2011). Photodynamic therapy in the control of oral biofilms. 
Periodontology 2000, 55, 143-166.  

Vergani, S. A., Silva, E. B., Vinholis, A. H., & Marcantonio, R. A. (2004). Systemic use of 
metronidazole in the treatment of chronic periodontitis: a pilot study using clinical, 
microbiological, and enzymatic evaluation. Brazilian Oral Research, 18, 121-127.  

 
Figure1 – Experimental design of the present study. 
	

	

	 	

	

	

	



 

 

39 

 
Figure 2 – µ-CT  images obtained of the second maxillary molar region, where the ROIs were 
obtained. A: Furcation region with a 1.26x1.15mm2 area; B: proximal regions (mesial and distal) 
with a 1.26x0.56mm2 area from the cement-enamel junction. 
	

	

	

	

	

	

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

A B 



 

 

40 

Figure 3 – Photograph of the 3D models obtained from the scanning of the hemimaxillae in the 
µ-CT, palatal view, indicating the analyzed regions (distal, mesial and furcation) of the 
experimental groups: A, B, C and D (7 day period) and E , F, G and H (30 day period); In 
sequence, the PD, SRP, PDT and SRP+PDT groups. The graphs display the means and 
standard deviations of the percentage of bone volume (%) of the furcation area (3A) and 
proximal regions (3B), of the second maxillary molar of the treated hemimaxillae of the 
experimental groups. *, # ,& indicate statistically significant differences between groups. 



 

 

41 

Figure 4 – Photomicrographs of the sagittal sections of rat hemimaxillae of the experimental 
groups, in the 7 and 30 day periods, at 4x magnification, showing the alveolar process (AP) 
formed by irregular bone trabeculae, located between the 1st (1M) and 2nd (2M) and between 
the 2nd (2M) and 3rd (3M) molars. The cervical portion of the alveolar process (AP) is located 
near the middle and apical third of the 2M roots. Reabsorption of alveolar bone (AB) was 
observed in the furcation region. At 10x magnification, the gingival epithelium (GE) is located 
apically at the cement-enamel junction (CEJ), and the gingival mucosa exhibits an inflammatory 
process (IP) evident in the PD group and the destroyed interdental papilla. In the other groups, 
the inflammatory process (IP) is less pronounced than the PD group. CEJ, cement-enamel 
junction; GE, gingival epithelium; D, dentin; P, Tooth pulp; PL, periodontal ligament; AB, 
alveolar bone of the furcation region. HE. X10 - Bar: 80. x4 - Bar: 330. Graph 4A displays the 
means and standard deviation of the bone area in square millimeters (mm2) of the furcation 
region and figure 4B displays the means and standard deviations of bone linear loss in 
millimeters (mm) of the proximal regions of the second maxillary molar of the treated 
hemimaxillae of the experimental groups. 
 



 

 

42 

3.2 Publicação 2 

 

Tissue responses of aPDT with phthalocyanine-
tetracarboxyglucamine photosensitizer in ligature-induced 

periodontal disease in rats* 
 

 

 

 

 

 

 

 

 

Sâmara C. T. Corbi, Paula D. Macedo, Janice R. Perussi, 

Anderson O. Ribeiro, Rosemary A. C. Marcantonio 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*Artigo a ser submetido à revista Jornal of Applied Oral Science. 



 

 

43 

Tissue responses of aPDT with phthalocyanine-
tetracarboxyglucamine photosensitizer in ligature-induced 

periodontal disease in rats 

 

 

 

Sâmara Cruz Tfaile Corbi (SCT, Corbi) – PhD student in Periodontics1; email: 

sa_tfaile@yahoo.com.br 

Paula Delello Macedo (Macedo, PD), PhD student in Periodontics1; email: 

paula_mac2@yahoo.com.br 

Janice Rodrigues Perussi (Perussi, JR), Associate Professor in Chemistry2; 

e-mail: janice@iqsc.usp.br 

Anderson Orzari Ribeiro (Ribeiro, AO), Adjunct Professor from UFABC3, 

email: anderson.ribeiro@ufabc.edu.br 

*Rosemary Adriana Chiérici Marcantonio (Marcantonio, RAC), Associate 

Professor in Periodontics1; email: adriana@foar.unesp.br  
 

1Sao Paulo State University (UNESP), School of Dentistry, Diagnosis and 

Surgery Department, Araraquara, SP, Brazil 
2Sao Paulo University (USP), Chemistry and Molecular Physics Department, 

Sao Carlos, SP, Brazil 
3Federal University of ABC (UFABC), Centre for Natural Sciences and 

Humanities, Santo Andre, SP, Brazil 
 

 

 

 

*Corresponding author: 

Rosemary Adriana C. Marcantonio, PhD 

Department of Diagnosis and Surgery 

Sao Paulo State University – School of Dentistry at Araraquara (UNESP) 

CEP 14801-903, Araraquara, SP, Brasil 

Tel: +55 16 3301-6376 

E-mail: adriana@foar.unesp.br 

 

 



 

 

44 

ABSTRACT 

Antimicrobial Photodynamic Therapy (aPDT) has been used as an adjuvant treatment 

in periodontal disease (PD). This technique combines a photosensitizer (PS) with a 

light source to induce the production of reactive oxygen species (ROS), eliminating 

microbial cells. The aim of this study was to evaluate in vivo responses and tissue 

changes after aPDT application (with the PS phthalocyanine-tetracarboxyglucamine - 

10µg/mL, and red LED light with 655nm) in PD-induced rats by microtomographic, 

histometric, stereometric and histological evaluations. Ligatures were placed around 

the second maxillary molars in both sides for PD induction for 7 days and remained in 

position throughout the experiment. Forty-two animals were divided into 4 groups: PS 

(treatment with phthalocyanine-tetracarboxyglucamine only); Light (Treatment with red 

LED light irradiation only); aPDT (Treatment with photodynamic therapy – PS + Light) 

and PD (periodontal disease induction, without treatment). At the baseline, the animals 

were treated and euthanized at 7 and 15 days. A one-way ANOVA parametric test was 

applied, followed by Tukey’s post-test. The three-dimensional radiographic and 

histometric analyses revealed no statistically different results for the furcation and 

interproximal regions. The inflammatory profile presented a trend of lower amounts of 

inflammatory cells in the aPDT group at 7 days, while the histological analysis indicated 

no significant differences between the groups, indicating that the therapies did not 

cause tissue damage. Thus, the application of aPDT and its components as 

monotherapies in PD-induced in rats with the preservation of the ligatures, favored in 

situ bacteria permanence and inhibited treatment action. 

Keywords: Periodontal diseases, photochemotherapy, X-ray microtomography. 

 

INTRODUCTION 

Periodontal Disease (PD) is an oral infection affecting the gingiva, cementum, 

periodontal ligament and alveolar bone, and its primary etiological factor is the 

presence of pathogenic bacteria in biofilm, which adhere to the teeth surface of 

susceptible host. Pathogens release enzymes, endotoxins and cytotoxic factors that 

can, in turn, trigger inflammation mechanisms and cause various changes in the 

periodontium36.  

The standard therapy for PD treatment is Scaling Root and Planing (SRP)56, 

which aims to remove microorganisms found on all teeth in both the supra and 

subgingival region. SRP benefits can be verified by decreases in marginal bleeding, 

bleeding at probing and probing depth, as well as stabilization of clinical insertion 

levels9. These clinical improvements are due to well-executed SRP, which influences 

the amount and composition of the oral microbiota11. However, this therapy does not 



 

 

45 

seem to be responsive to mechanical treatment in some sites, which makes it less 

effective 

To overcome this difficulty, some therapeutic alternatives have been applied, 

such as the use of local and systemic antibiotics. However, it is known that this type of 

treatment has undesirable side effects and contributes to the development of bacterial 

resistance to drugs29, 35, 72. Thus, alternative methods that target bacterial reduction, 

such as the use of lasers and associated dyes, known as Antimicrobial Photodynamic 

Therapy (aPDT), could be a complementary feature to conventional periodontal 

treatment. 

PDT has emerged in recent years as a new, noninvasive, therapeutic modality 

for the treatment of various bacterial, fungal, and viral infections27. This therapy is 

defined as an oxygen-dependent photochemical reaction that occurs after the 

activation of a light-mediated photosensitizing compound, leading to the generation of 

cytotoxic reactive oxygen species (ROS), predominantly singlet oxygen44. This therapy 

can be topically applied in the periodontal pocket, thus avoiding overdoses. Other 

aPDT advantages include, reducing the likelihood of side effects associated with the 

systemic administration of antimicrobial agents and minimizing bacterial resistance22, 73. 

Several in vitro8, 10, 21, 31, 37, 41, 59, 70, 74 and in vivo experimental studies20, 24, 30, 61, 62 

have obtained satisfactory results through aPDT, applying protocols comprising 

different types of dyes and wavelengths to eliminate several types of oral bacteria, 

such as periodontopathogenic and non-pathogenic bacteria. 

In the last decades, certain compounds with diagnostic and therapeutic 

purposes, considered second-generation dyes, have been developed, such as 

phthalocyanines, which are synthetic dyes similar to porphyrins and structurally 

classified as azaporphyrins34, 55, 67. The name phthalocyanine originates from a 

combination of the prefix phthal, originally from the Greek word naphtha (rock oil), to 

emphasize the association with its various precursors derived from phthalic acid, and 

the Greek word cyanine (blue)38. Phthalocyanines are symmetric macrocycles 

composed of four iminoisoindole (Figure 1A) units with a central cavity that 

accommodates different metal ions, such as chlorine, aluminum and zinc. When 

inserted, these metals present considerable influence on photosensitizing properties 

(Figure 1B)3, 57, 67. These compounds display an absorption band in the electromagnetic 

spectrum, ranging from 650 to 680nm, which allows greater light penetration into 

tissues40, and their photophysical properties depend on their composition, particularly 

that of the central metal ion. Among phthalocyanines, a chloro-aluminum 

phthalocyanine has been suggested as displaying photophysical properties favorable 

for use in aPDT, as it produces high amounts of singlet oxygen40. The effectiveness of 



 

 

46 

this photosensitizer associated with LED light was confirmed in an in vitro study that 

evaluated the photodynamic potential of a chloro-aluminum phthalocyanine diluted in a 

cationic nanoemulsion to inactivate planktonic cultures and biofilms formed by Candida 

albicans55. This PS was also shown to be effective for bacteria inactivation in patients 

with carious lesions34. Other studies16, 47, 63, 74-76 have supported the efficiency of 

phthalocyanines as photosensitive agents in the elimination of periodontopathogenic 

microorganisms with applications in aPDT. Another phthalocyanine routinely applied as 

a PS is zinc phthalocyanine (FCZn)3, 57, 67.  

The hydrophobicity of some PS is an important fact, since this property in 

aqueous media leads to molecule self-aggregation and, in most cases, to a subsequent 

precipitation, drastically reducing the ability of the compound to generate singlet 

oxygen64. Thus, the photoactive principle must be soluble in aqueous media for 

possible clinical applications. An interesting strategy applied to increase the aqueous 

solubility of some PS involves the formation of hydrophilic supramolecular species. A 

supramolecule is defined as a chemical species made up of two or more molecules 

linked by intermolecular interactions. In this sense, supramolecular chemistry uses an 

approach centered on molecule association, aiming at obtaining a certain property or 

functionality14. 

 Meglumine, also known as N-methylglucamine (Figure 1C), is an 

aminocarbohydrate derived from glucose capable of forming hydrophilic binary 

supramolecular species with compounds containing acidic hydrogen atoms in their 

structure14. The formation of meglumine compounds involves an acid-base reaction, in 

which the acidic hydrogen is transferred to the amine group of the aminocarbohydrate. 

To enable the formation of supramolecular species between meglumine and 

phthalocyanines, the latter are primarily functionalized with carboxylic groups, such as 

tetracarboxylated zinc phthalocyanine (FtZnTc, Figure 1D)14. 

Subsequently to the insertion of the acid groups, a reaction with the 

aminocarbohydrate can be conducted, producing species that are more soluble in 

water. This procedure allows for the insertion of up to four meglumine molecules, as 

observed in Figure 1E for zinc phthalocyanine tetracarboxy-N-methylglucamine 

(FtZnTcG). The presence of several hydroxyl groups in the supramolecule provides the 

sites where the hydrogen bonds are established by increasing the hydrophilicity of the 

phthalocyanines14. The absorption spectrum of phthalocyanines in solution consists of 

two main bands centered at 350nm and 670nm. Figure 2 illustrates the molecular 

absorption spectrum of the tetracarboxylated zinc phthalocyanine. 

Phthalocyanines have been used in aPDT in cancer treatments3, 4, 54, 71, 77, 

cutaneous e subcutaneous lesions6, in eradication of neoplasitic processes57, 67 and 



 

*Para metodologia completa, ver Apêndice 1. #Aprovação do Comitê de Ética, ver  Anexo A. 

47 

encapsulated virus7, due to its high selectivity and low phototoxicity characteristics and 

promotes an appreciable amount of ROS, capable of remaining in the triplet excited 

state for a longer period of time58. In this sense, the aim of this study was to evaluate in 

vivo the responses and tissue changes of aPDT application and its components as 

monotherapies, using phthalocyanine-tetracarboxyglucamine PS in PD-induced in rats.  

 

MATERIAL AND METHODS*  

Ethics comittee 

This project was approved by the Ethics Committee on Animal Experimentation 

(No. 07/2012)#. 

 

Samples 

Forty-two rats (Rattus norvegicus) of the albinus variation, Holtzman, adults, 

weighing between 300-330g were used. The animals were kept in plastic boxes, 3/4 

animals per box, and treated with water and food ad libitum before and during the 

whole experimental period. The animals were maintained in an environment with 

controlled light, humidity and temperature. 

 

Periodontal disease induction 

 The animals were anesthetized with a combination of ketamine (ketamine 

hydrochloride - Francotar 3% - Virbac do Brasil Ind. e Com. Ltda.) and xylazine 

(xylazine hydrochloride - Virbaxyl 2% - Virbac do Brasil Ind. e Com. Ltda.) at 

0,08mL/100g and 0,04mL/100g body weight, respectively. The ligatures were placed 

around the second maxillary molars on both sides. The hemimaxilae that received the 

therapies was chosen randomly (right or left). The PD was induced for 7 day before the 

beginning the therapies application of each group/period and 0-day period was 

considered the baseline. The ligatures remained in position throughout the experiment 

(Figure 3). 

 

Photodynamic Therapy 

Photosensitizer preparation 

Phthalocyanine-tetracarboxyglucamine was prepared from a stock solution of 

zinc-tetracarboxy-phthalocyanine at 1,1mg/mL in DMSO and subsequently diluted in 

phosphate buffered saline (pH=7.2) to a final concentration of 10µg/mL, in 

ointment consistency. Soon after the preparation, the PS was stored in light-protected 

poliproylene tubes maintained in a refrigerated environment until use. 

 



 

 

48 

Photosensitizer Application 

At the time of application, the animals’ mouth and the PS were light-protected.  

A blunt tip syringe containing 0,2mL of the PS was inserted into the gingival sulcus. 

The solution was applied around the entire second maxillary molars and, after 10 

minutes of incubation time in the dark, the light irradiation was performed. This 

application was done only once. 

 

Light source 

 The light source used to activate the phthalocyanine-tetracarboxyglucamine PS 

corresponded to a wavelength of 655 nm, 0,45W power density and 34J/cm2 dose (red 

LED, 11mm diameter, DMC Equipamentos Ltda, São Carlos, Brazil), coinciding with 

the maximum absorption band of phthalocyanine-tetracarboxyglucamine. The LED light 

was placed on the occlusal surface of the second maxillary molars and irradiation was 

maintained for 72 seconds. 

 

Experimental groups 

After 7 days of PD-induced, the animals were randomly divided the therapies 

application were performed according to their group (7animals/group/period): 

• Photosensitizer Group (PS): PS was applied around the entire second maxillary 

molars, into the sulcus (10 minutes of incubation time in the dark) and after, the 

PS  was removed with cotton swabs. 

• Red LED Light Group (LED): The LED light (655nm; 34J/cm2) was placed on 

the occlusal surface of the second maxillary molars and irradiation was 

maintained for 72 seconds. 

• Photodynamic Therapy Group (aPDT): Application of the PS followed by LED 

application. 

• PD Group (PD): Disease induction only, without treatment. 

 

The ligatures remained in position and they were removed at 7- and 15-days 

periods. Soon after, the animals were euthanized with an anesthetic overdose. The 

hemimaxillae were removed and fixed in 4% paraformol for 48h. Subsequently, the 

samples were washed in running water for 24h and placed in 70% alcohol, where they 

remained until the computerized microtomograph scanning. 

 

Histological processing 

After scanning, the samples were placed in a 7% EDTA solution, pH 7,2 (Synth, 

São Paulo, Brazil), buffered with sodium phosphate for decalcification. Following 



 

 

49 

laboratory procedures, the samples were then included in paraffin. Semi-serial sections 

were made along the axis of the tooth, at 4µm thickness. For each hemimaxillae, 

approximately 30 sections were obtained, divided into slides containing 3 sections 

each. Two slides from each hemimaxillae were stained by hematoxilin-eosin technique 

(HE) and used for, histometric, stereometric and histologic analysis. 

 

Three-dimensional Radiographic Analysis (µCT)  

The samples were scanned by means of X-ray beam scanning in a 

computerized microtomography system (Skyscan 1176, Aatselaar, Belgium, 2003). 

The parameters of the equipment were set as follows: Al 0.5mm filter; Voxel size: 

17.48µm; Voltage 50KV and electric current 500µA. After scanning, the 3D images for 

each sample were obtained through the equipment software (NRecon 1.6.1.5 - 

SkyScan N. V., Belgium, 2003). The images were rotated and repositioned in a 

standard orientation with the two-dimensional Dataviewer software (SkyScan 1176, 

Aartselaar, Belgium, 2003). for better viewing. At each 28 scans, a 18x18x18µm matrix 

was reconstructed and 3D images were generated for each sample. Subsequently, a 

volumetric analysis was performed by a trained examiner with no knowledge of the 

experimental groups. 

For this analysis, a specific software (ITK-SNAP 3.6.0 - Pennsylvania, USA) 

was used, where the examiner delimited a three-dimensional region of interest (ROI), 

of 217x193x269, driven by the morphometry of the samples. To maximize bone 

quantification and minimize teeth and root inclusion, the ROI was delimited from the 

distal first molar root to the mesial third molar root, and from the furcation  and 

interproximals region of the second molar to the end of the cortical bone, thus making 

sure that all the bone area in the furcation and interproximal region of the second molar 

were encompassed by the ROI, as displayed in Figure 4. After the ROI delineation, a 

binary image was generated and a single threshold was established for each sample, 

analyzing tissue similarity. Subsequently, the roots were digitally removed so as not to 

influence the results. The analyses and the resulting values are given in cubic 

millimeters (mm3). 

 

Histometric analyses 

 For this analysis, a blind and calibrated examiner (Pearson’s correlation, r=0.93) 

selected 2 slides from each hemimaxillae. The furcation area was delimited according 

to the methodology reported by da Silva et al.,12 (2008). Measurements were taken 

using the Fiji ImageJ Launcher imaging software (National Institutes of Health, USA), 

evaluating the following areas: 



 

 

50 

• Furcation area: the area was defined, a 1000µm-zone under the furcation 

limited by the roots. The furcation and bone area were measured, thus 

obtaining the percentage of bone present in the furca region of each histological 

section.  

• Interproximal region (mesial and distal): a linear measurement of the cement-

enamel junction up to the top of the bone crest was performed, thus obtaining 

alveolar bone loss values. 

The furcation region measurements were obtained in percentage and 

interproximals region in micrometers. 

 

Stereometric Analysis 

This analysis was performed using a Leica DMLS light microscope (Leica - 

Reichert Diastar Products & Jung, Wetzlar, Germany) to select 2 sections per tooth, 

with intervals ranging from 40 to 50µm between them, and a Leica DFC 300 digital 

camera FX (Leica - Reichert Diastar Products & Jung, Wetzlar, Germany) to capture 

the furcation and interproximal region images at a 200x magnification. In a two-

dimensional plane, a 75x75 pixel area grid with 252 intersection points was placed in 

the regions of interest (supra-alveolar connective tissue at the furcation ceiling and 

papillae, in both mesial and distal sides) using the software Fiji ImageJ (National 

Institutes of Health, USA). Subsequently, the point counting technique was performed 

to analyze the proportion of tissue constituents (bone tissue, extracellular matrix, blood 

vessels, fibroblasts, inflammatory cells) at the angles of the grid. For the graphical 

representation of the stereometry, a percentage measurement of each tissue 

constituent corresponding to the total number of points was performed, based on Liu et 

al.33
 

(2006) and Odze et al.45
 

(1996). 

 

Histological analyses  

 Using a DIASTAR (Leica Reichert & Jung products, Germany) optical 

microscope with 4,0-10,0-fold objective and 10 ocular magnifications, the images were 

captured and sent to a microcomputer with the aid of a DXC-1107A/107AP video 

camera (Sony Electronics Inc, Japan). The inflammatory reactions of the connective 

tissue, bone resorption processes and tissue neoformation in each experimental group 

were evaluated by an experienced, blind and calibrated examiner for the experimental 

groups. 

 

 

 



 

 

51 

Statistical analyses 

The experimental data were tabulated using the Microsoft Excel for Mac 2011 

software (Apple Inc, USA) and analyzed statistically with the aid of the GraphPad Prism 

6.0 software (GraphPad Inc, USA). 

The data were evaluated applying the central point theorem, to verify if their 

arrangement respected a normal distribution, using the Shapiro-Wilk test. As all data 

were normally distributed, the parametric ANOVA (One Way) test was applied to verify 

the existence of statistical differences between the groups. Tukey’s post-test was 

subsequently applied, in order to detect differences among groups. For comparisons 

between the treatment periods, the ANOVA (Two Way) parametric test was applied. All 

tests were applied with a 95% confidence interval. 

 

RESULTS  

Three-dimensional Radiographic Analysis (µCT) 

No statistically significant difference was detected for bone volume in the region 

of the second maxillary molars of the treated hemimaxils. These data are displayed in 

Figure 5. 

 

Histometric Analysis 

The results of the furcation and interproximal analyses indicate that all the data 

were statistically the same for the bone area and bone loss, respectively, for region of 

the second maxillary molars molars of the treated hemimaxils of the experimental 

groups, as displayed in Figure 6. 

 

Stereometric Analysis 

Significant differences were observed in the percentage of bone tissue at 7 

days between the PS and LED light groups (*p<0.05). The percentage of blood vessels 

was also significantly different in the PD group between the 7 and 15 day groups 

(#p<0.05). The percentages of extracellular matrix and inflammatory cells were not 

significantly different. Regarding fibroblasts, a statistically significant difference was 

detected in the PD group comparing the 7 and 15 day periods (ap<0.05), the 

photosensitized group was statistically different compared to the PD, LED and aPDT 

groups (bcdp <0.05). These data are displayed in Figure 7. 

 

Histological Analysis 

In this evaluation, the morphological aspects of the gingival mucosa and bone 

tissue of the second maxillary molars of the rats were analyzed in the different groups. 



 

 

52 

The animals periodontium in the PD group exhibited changes at 7 days:  An 

exacerbated inflammatory process in the lamina propria of the interdental papilla, was 

observed, frequently ulcerated. The junctional epithelium was located apically to the 

cement-enamel junction. Bone in the furcation region showed moderate resorption. At 

the 15-day period, a decrease in the inflammatory process and a slight bone resorption 

in the furcation were observed. The PS, light and aPDT groups presented microscopic 

characteristics similar to those described for the PD group, but less intense. Thus, 

histological descriptions were similar for the experimental groups. Figure 6 illustrates 

the periodontal tissues of the different periods. 

 
DISCUSSION 

 The aim of this study was to evaluate in vivo alterations and tissue responses 

after the application of aPDT and its components as monotherapies using the 

phthalocyanine-tetracarboxyglucamine PS in rats presenting induced PD.  

The inflammatory profiles were not statistically different and indicated a trend of 

lower amounts of inflammatory cells in the aPDT group at 7 days. The histological 

analysis indicated no significant differences between the groups within the analyzed 

periods, suggesting that the applied therapies did not cause tissue damage. These 

findings are in agreement with the study by Al Habashneh et al.,2 (2015), that 

mentioned that PS application without a light source does not cause damage to healthy 

tissues, and that the bacterial destruction process occurs only when PS is used in 

conjunction with a light emitter. In the study conducted by Melo et al.,14 (2014) several 

conditions were analyzed for the selective photoinactivation of S. aureus and E. coli, in 

plankton cultures and biofilms, by using the PSs hypericin and zinc phthalocyanine and 

new synthesized hydrosoluble derivatives, such as phthalocyanine 

tetracarboxyglucamine (FcZnTcG), at various concentrations, incubation times and 

light densities, among others. The authors verified that the employed photoinactivation 

parameters contributed to the inhibition of 100% of the bacterial cells without damaging 

the cells used as the host model (VERO epithelial cell line). This indicates that the PS 

concentrations and incubation time should be low in order to facilitate selective 

accumulation in microorganisms, faster in bacteria than in epithelial cells, so that low 

dose irradiation leads to damage only to microorganisms and not healthy tissue. In the 

present study, a PS concentration of 10 µg/mL and an incubation time of 10 minutes 

were used. These parameters were defined based on the results reported by Melo et 

al.,14 (2014) in both cell and microorganism cultures. The absence of differences 

between the experimental groups could be related to these parameters, which were not 

sufficient to achieve any effect on the microorganisms located in the subgingival 



 

 

53 

ligature. 

The results of the three-dimensional radiographic and histometric analyses 

indicate no differences between the groups and analyzed periods, indicating that the 

applied therapies did not interfere in PD progression, since the ligatures were placed 

around the second molars on both sides of the maxillae and remained in position 

throughout the experimental period, simulating a clinical situation of PD progression in 

patients. Contrarily to other studies that evaluated aPDT associated with for PD 

treatment in animals5, 15, 19, 50, the present study analyzed aPDT with the PS 

phthalocyanine-tetracarboxyglucamine under disease conditions without other 

treatments. With the applied methodology, the ligature placed to induce PD was not 

removed, thus maintaining a large amount of biofilm in the region. This condition may 

have interfered in the results, suggesting that aPDT may have acted only on the 

superficial layer of the biofilm, and was not able to completely eliminate the bacteria 

present in the area.  

Fontana et al.,18 (2009) investigated the effects of aPDT on bacteria derived 

from human dental biofilms under planktonic conditions and in in vitro biofilms. The 

objective was to compare the susceptibility of bacteria to aPDT after sensitization with 

methylene blue and exposure to red light at 665nm. The results indicate that aPDT 

eliminated about 63% of the bacteria in the planktonic phase, whereas the light effect 

resulted in much lower decrease in microorganism counts  (31% were killed) in biofilms 

derived from the same samples. Thus, biofilm bacteria presented resistance to the 

aPDT, with elimination not exceeding 32% in relation to the controls. The authors 

concluded that, although aPDT was less effective in treating bacteria in biofilms than in 

planktonic cultures, the difference was only two-fold, while another study60 

demonstrated that antibiotics are approximately 250 times less effective under these 

conditions. Müller et al.,39 (2007) reported less than 1 log10 bacterial destruction in oral 

biofilms comprising six species developed on bovine enamel disks a