universidade federal do rio grande do norte … · proliferação celular, para os dois tipos de...

UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE

CENTRO DE CIÊNCIAS DA SAÚDE

PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

ADESÃO, PROLIFERAÇÃO E GENOTOXICIDADE CELULAR DE

CELULOSE BACTERIANA MODIFICADA POR PLASMA

NAISANDRA BEZERRA DA SILVA

Natal-RN

2010

ii



PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

ADESÃO, PROLIFERAÇÃO E GENOTOXIDADE CELULAR DE CELULOSE

BACTERIANA MODIFICADA POR PLASMA

Naisandra Bezerra da Silva

Orientador: Prof. Dr. Clodomiro Alves Júnior

Co-orientadora: Profa Dra Silvia Regina Batistuzzo de Medeiros

Natal RN

2010

Tese apresentada ao programa de Pós-

Graduação em Ciências da Saúde da

Universidade Federal do Rio Grande do

Norte, como requisito para obtenção do

título de Doutora em Ciências da Saúde.

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PROGRAMAS DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE

Coordenadora do Programa de Pós-Graduação em Ciências da Saúde:

Profª. Drª. Técia Maria de Oliveira Maranhão

NATAL RN

2010

CATALOGAÇÃO NA FONTE

S586a

Silva, Naisandra Bezerra da.

Adesão, proliferação e genotoxicidade celular de celulose

bacteriana modificada por plasma / Naisandra Bezerra da Silva. –

Natal, 2011.

79f.

Orientador: Profº Drº Clodomiro Alves Júnior.

Coorientadora: Profª Drª Silvia Regina Batistuzzo de

Medeiros.

Tese (Doutorado) – Programa de Pós-Graduação em Ciências

da Saúde. Centro de Ciências da Saúde. Universidade Federal do

Rio Grande do Norte.

1. Enxerto de pele – tese. 2. Teste de biocompatibilidade –

tese. 3. Acetobacter Xylinus – tese. 4. Gluconacetobacter Xylinus

– tese. I. Alves Júnior, Clodomiro. II. Medeiros, Silvia Regina

Batistuzzo de. III. Título.

RN-UF/BS-CCS CDU: 611.018.18(043.2)

iv

NAISANDRA BEZERRA DA SILVA

ADESÃO, PROLIFERAÇÃO E GENOTOXIDADE CELULAR DE CELULOSE

BACTERIANA MODIFICADA POR PLASMA

Presidente da Banca: Prof. Dr. Clodomiro Alves Junior (UFRN)

BANCA EXAMINADORA:

Profa. Dra. Christina Alves Peixoto (UFPE)

Prof. Dr. Gilberto Weissmuller (UFRJ)

Prof. Dr. Carlos Augusto Galvão Barboza (UFRN)

Prof. Dr. Custódio Leopoldino de Brito Guerra Neto (UFRN)

NATAL

2010

v

“Escuta e serás sábio. O começo da sabedoria é o silêncio.”

Pitágoras

http://www.pensador.info/autor/Pitagoras/

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DEDICATÓRIA

A Deus, pela presença constante em minha vida, me guiando pelos caminhos

certos que me trouxeram paz e conquistas.

Aos meus pais: Vicente e Norma pela compreensão da minha ausência em

casa e pela confiança sempre em mim depositada, além dos ensinamentos que

moldaram o meu carácter.

vii

AGRADECIMENTOS

A Universidade Federal do Rio Grande do Norte (UFRN), ao Conselho

Nacional de Desenvolvimento Científico e Tecnológico (CNPq), e ao

Programa de Pós Graduação em Ciências da Saúde.(PPGCSa).

Ao Prof. Dr. Clodomiro Alves Junior pela oportunidade e confiança em mim

depositada. Homem sábio de grande inteligência, simples, humilde e amigo

que nas horas difíceis utilizou sua paciência e compreensão ao conduzir

minha orientação.

A Profa Dra Silvia Regina Batistuzzo de Medeiros, exemplo de pesquisadora,

pela efetiva colaboração na orientação desta pesquisa e pelos importantes

ensinamentos transmitidos.

Aos meus amigos Prof. Dr. Carlos Eduardo Bezerra de Moura e Prof. Dr.

Hugo de Oliveira Rocha por toda amizade, ajuda e ensinamentos passados

durante essa jornada.

Ao Prof. Dr. Francisco Miguel Portela Gama e Dra Susana Moreira, cujas

contribuições foram indispensáveis à realização desse trabalho.

Aos Professores Dr. Edivaldo da Silva Trindade e Dra. Célia Regina

Cavichiolo Franco por todas as contribuições com as análises de

microscopia.

A minha irmã Selma e ao meu noivo Marcello por toda torcida e incentivo.

Aos meus amigos Kaline, Cleysivam, Karina, que desde a graduação fazem

parte da minha vida como verdadeiros irmãos.

A Maria de Lourdes Freitas por sua ajuda e grande amizade.

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Ao pessoal do BIOPOL, LabPlasma e LBMG por tantos momentos

compartilhados. Especialmente à Jailma, Jeffersom, Joana, Juliana, Juliane,

Raniere, Manuella, Michelle, Roseane, Danilo, Igor e Aroldo por todas as

contribuições nos experimentos.

Aos amigos da Pós-Graduação Bianca, Joel, Nedinaldo, Ewerton e Antenor

pelos momentos de descontração entre as aulas e a amizade consolidada

durante o curso.

Ao Chefe do Departamento de Morfologia Prof. Celcimar Alves Câmara e

aos colegas da disciplina de Histologia Prof. Dr. Gustavo da Cunha Lima e

Profa Dra Christina da Silva Camillo por todo apoio.

A todos que contribuíram, direta ou indiretamente, para realização deste

trabalho.

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Sumário

Dedicatória ................................................................................................. vi

Agradecimentos.......................................................................................... vii

Lista de abreviaturas................................................................................... x

Resumo....................................................................................................... xi

1 INTRODUÇÃO......................................................................................... 01

1.1 Objetivos............................................................................................. 03

1.1.1 Objetivo geral.................................................................................. 03

1.1.2 Objetivos específicos...................................................................... 03

2 REVISÃO DA LITERATURA................................................................... 04

2.1 A celulose bacteriana........................................................................ 04

2.2 Modificação de superfície.................................................................. 05

2.3 Plasma................................................................................................ 06

2.4 Genotoxidade.................................................................................... 07

3 ANEXAÇÃO DOS ARTIGOS................................................................... 10

3.1 BC nanofibres: In vitro study of genotoxicity and cell proliferation . 10

3.2 Adhesion, cell proliferation and genotoxicity of bacterial cellulose

surfaces modified by plasma ……………………………………………..

18

3.3 Influence of argon-ion bombardment of titanium surfaces on the cell

behavior …………………………………………………………………..…

50

3.4 Effect of titanium surface modified by plasma energy source on

genotoxic response in vitro …………….…………………………………..

57

4. COMENTÁRIOS, CRÍTICAS E SUGESTÕES........................................ 66

4.1 Expectativas futuras......................................................................... 66

4.2 Publicação/divulgação....................................................................... 68

4.3 Dificuldades....................................................................................... 70

5. REFERÊNCIAS....................................................................................... 71

Abstract....................................................................................................... 79

x

LISTA DE ABREVIATURAS

3T3 - Fibroblastos

CAs - Aberrações cromossômicas

CB - Celulose bacteriana

CBP - Celulose bacteriana modificada por plasma

CHO K1 - Chinese hamster ovary K1

CSAs - Aberrações tipo cromossômicas

CTAs - Aberrações do tipo cromatídica

DNA - ácido desoxirribonucleico

MEV - Microscopia de electronica de varredura

MFA - Microscopia de força atômica

MTT - 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT)

NFs - Nanofibras

XPS - X-ray photoelectron spectra

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RESUMO

A celulose bacteriana (CB) é utilizada, geralmente como uma membrana

temporária, na substituição de pele lesionada, em feridas, queimaduras,

ulcerações ou como enxerto. Modificações em sua superfície podem

determinar respostas no funcionamento celular dos tecidos adjacentes,

influenciando sua biocompatibilidade. Este estudo apresenta a primeira

avaliação da influência de nanopartículas de celulose bacteriana e de

membranas de celulose bacteriana com superfície modificada por plasma

(CBP) no comportamento e genotoxicidade celular. Inicialmente, a proliferação

celular foi avaliada com o teste MTT e danos ao DNA foram avaliados

utilizando-se os testes Cometa e Kado, sob a influencia das concentrações de

0,1; 0,5 e 1,0 mg/ml de nanofibras de CB em contato com fibroblastos 3T3 e

células CHO-K1. Os resultados obtidos nessas análises revelaram que a

proliferação celular, para os dois tipos de células, foi cerca de 15-20% menor

na presença de NFs, após 72h de cultivo celular, independentemente da

concentração utilizada, estas também não promoveram dano significativo ao

DNA. Em um segundo trabalho, membranas de celulose bacteriana foram

submetidas ao plasma em atmosfera contendo 70%N2 e 30% de O2.

Posteriormente foram caracterizadas por MEV e AFM e submetidas aos

ensaios cometa, micronúcleo, de adesão e proliferação celular. Os resultados

revelaram que o plasma modificou a superfície da CBP produzindo uma

rugosidade de aproximadamente 70± 5,1 nm. Na CBP, as células tornaram-se

mais alongadas com proliferação maior, provavelmente, influenciadas pelo

aumento da rugosidade da superfície. A nova superfície gerada também não foi

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genotóxica. Face ao exposto, este estudo gerou um novo biomaterial que pode

ser testado in vivo com futuro potencial para implante artificial.

Palavras chave: Modificação de superfície, Curativo biológico, teste de

biocompatibilidade, biomaterial, Acetobacter xylinus.

Adesão, proliferação e genotoxicidade celular de celulose bacteriana modificada por plasma

1. INTRODUÇÃO

A bioengenharia desenvolve uma série de dispositivos de complexidade

variada para aplicação em ortopedia, cardiologia, odontologia, terapia celular,

farmacologia, entre outros. Atualmente, nesta área, observa-se um crescente

interesse no desenvolvimento de novos biomateriais utilizados no reparo ou

substituição de tecidos e órgãos lesionados.

O sucesso clínico de dispositivos moldados com qualquer biomaterial

depende da interação entre as células do organismo implantado e a superfície

do material utilizado (1). Assim, para o desenvolvimento de um material

clinicamente aplicável, a modificação de superfície é uma ferramenta eficaz

que pode aumentar sua biofuncionalidade (2).

As características da superfície de um biomaterial como

rugosidade/topografia (3), energia (4) e carga (5) determinam sua interação com

fluidos corporais que, por sua vez desencadeiam uma cascata de reações

governando a resposta celular (adesão, proliferação e diferenciação), e

definem a biocompatibilidade do material (6). Diversas são as técnicas (métodos

mecânicos, químicos e físicos) utilizadas com o objetivo de modificar a

superfície e, portanto garantir uma resposta biológica funcionalmente

adequada. Dentre os métodos físicos destaca - se o tratamento à plasma (7-9),

sua utilização permite modificar as propriedades da superfície através da

criação de novos grupos funcionais, mudanças topográficas, adição/subtração

química à superfície. Neste contexto, materiais como titânio, hidroxiapatita e

polímeros (poliéster, TNT, algodão e quitosana), vêm sendo tratados a plasma

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no Laboratório de processamento de materiais por plasma (LabPlasma) da

UFRN.

Superfícies de titânio, submetidas ao plasma, revelaram que variações

na microtopografia ou na molhabilidade podem afetar a proliferação de células

tronco pré-osteoblasticas ou ainda, proporcionar menor efeito genotóxico em

células CHO-K1 (ovário de hamster chinês). Desse modo, os trabalhos:

“Influence of Argon-ion bombardment of titanium surfaces on the cell behavior”

e “Effect of titanium surface modified by plasma energy source on genotoxic

response in vitro”, anexados a este estudo, corroboram com a idéia de que as

propriedades da superfície podem alterar a resposta funcional da célula frente

a um biomaterial. Diante disso, surgiu a possibilidade de ampliar a aplicação da

celulose bacteriana através da modificação de sua superfície.

A celulose bacteriana (CB), sintetizada por Gluconacetobacter xylinus

tem se revelado um biomaterial extremamente versátil e promissor (10,11).

Apresenta diversas vantagens estruturais que a levam a ser considerada um

curativo biológico natural. É utilizada como substituto temporário da pele, no

tratamento de feridas, queimaduras, úlceras, corrupções, e abrasões dérmicas

(12-15); como prótese vascular, na liberação de medicamentos e na regeneração

tecidual guiada (10,16).

Diante das possíveis aplicações da celulose bacteriana na área de terapia

celular; que a interação material-tecido parece estar intimamente relacionada

com os tratamentos de superfície aplicados aos biomateriais; e que poucos

estudos biológicos têm se preocupado em avaliar o efeito do tratamento da

superfície sobre a biocompatibilidade da celulose bacteriana; este estudo

fornece dados pioneiros que podem gerar implicações na elaboração de novas

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estratégias para obtenção de uma celulose bacteriana com melhor

biocompatibilidade.

1.1 OBJETIVOS

1.1.1 OBJETIVO GERAL

Avaliar a adesão e proliferação celular sobre nanofibras de celulose

bacteriana, e superfícies de celulose bacteriana tratadas por plasma

verificando ainda suas genotoxicidades.

1.1.2 OBJETIVOS ESPECÍFICOS

Caracterizar nanofribas de celulose bacteriana através de microscopia

de transmissão;

Avaliar a proliferação de fibroblastos 3T3 frente a diferentes

concentrações de nanofibras de CB pelo método MTT.

Analizar a genotoxicidade de NFs de CB a partir dos testes cometa e

Ames;

Submeter membranas de celulose bacteriana ao tratamento a plasma,

modificando suas superfícies, e caracteriza-las através de XPS, MFA e

MEV;

Avaliar a proliferação e adesão de células CHO-K1, em membranas de

CB tratadas e sem tratamento a plasma, através dos métodos MTT e

cristal violeta;

Avaliar a genotoxicidade de membranas de CB tratadas e sem

tratamento a plasma pelos ensaios do cometa e micronúcleo.

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2. REVISÃO DA LITERARURA

O desenvolvimento de polímeros biodegradáveis com superfícies de

porosidades adequadas para o apoio mecânico e orientação tecidual, desperta

o interesse da engenharia de tecidos. Ainda, em alguns casos, superfícies

portadoras de fatores de crescimento para acelerar o processo de cura quando

colocadas in vivo (2). Nesse sentido, membranas de celulose bacteriana atraem

a atenção de diversos pesquisadores sendo utilizadas principalmente como

implante de pele ou de mucosas graças a sua semelhança, morfológica, à

associação de fibras colágenas da derme ou da lâmina própria.

2.1 A celulose bacteriana (CB)

A celulose bacteriana é um exopolissacarídeo liberado na superfície

bacteriana através de poros na membrana plasmática sob a forma de longas

fibrilas, que podem se agregar originando uma película (Figura 01). Sua

biogênese geralmente dependente da fosforilação da glicose por glucoquinases

(17) e ocorre na interface entre o meio de cultura e a superfície gasosa (18,19).

Apresenta diversas características que a diferem da celulose vegetal, é obtida

livre de lignina e hemicelulose (10). Possui maior cristalinidade, resistência à

tração, elasticidade e durabilidade (20). Sua estrutura forma uma barreira física

contra infecções e promove alta capacidade de absorver e reter água.

Morfologicamente é composta por feixes densos de microfibrilas muito finas de

tamanho nanométrico. Embora muito porosa, seus poros são relativamente

pequenos (21,22), e podem influenciar a sua capacidade de liberação controlada

de drogas e infiltração celular (23-26). A modificação da superfície de membranas

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de CB, resultando em poros maiores ou maior rugosidade pode ser uma forma

de expandir o seu uso.

Figura 01: Membrana de celulose bacteriana

A celulose bacteriana mostrou ser biocompatível quando associada ao

tecido ósseo, à pele, vasos sangüíneos, fibroblastos e hepatócitos de ratos e

coelhos (27-32). Apesar de ser biodegradável no ambiente, permanece por

longos períodos em contato com os tecidos quando implantada. Segundo Islam

e Tanaka (33) uma exposição contínua a determinados compostos pode gerar

efeitos bioacumulativos que alteram diversas funções celulares ou ainda

proporcionam efeitos mutagênicos, carcinogênicos e/ou teratogênicos. Apesar

dos avanços recentes, a celulose bacteriana ainda não é amplamente utilizada

ou investigada principalmente quanto à resposta celular, genotoxicidade e seus

riscos mutagênicos.

2.2 Modificação de superfície

As propriedades de superfície de um biomaterial são determinantes para

respostas das células na interface célula-biomaterial, consequentemente

afetam o crescimento e a qualidade do tecido neoformado (34). Dentre estas

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propriedades a microtopografia/rugosidade tem apresentado alto poder de

alterar a adesão, migração, morfologia, apoptose, ativação

gênica/diferenciação e ativação de macrófagos (35-38).

Na busca por uma superfície ideal, de acordo com suas possibilidades

de aplicação, várias pesquisas têm sido desenvolvidas com o intuito de

modificar as superfícies por diversos tipos de processos(8). Estes envolvem

métodos mecânicos, químicos e físicos de tratamento de superfície, gerando

assim, vários graus de texturas (39,40). Entretanto, dentre os diferentes métodos

existentes, aqueles que utilizam o plasma como fonte energética são os que

mais avançam nos últimos anos, devido principalmente, à sua versatilidade (41).

O tratamento à plasma merece destaque pois permite modificações na

superfície do material sem comprometer suas propriedades intrínsecas como o

módulo de elasticidade e resistência (42) . Ainda, por necessitar de pequeno

tempo de tratamento, possuir baixo custo e não ser prejudicial ao meio

ambiente (42).

2.3 Plasma

O plasma é o quarto estado da matéria e consiste numa atmosfera

contendo elétrons, íons, partículas neutras e partículas energéticas (43). Em

nível laboratorial, pode ser obtido através de uma descarga elétrica em um gás,

pela aplicação de uma diferença de potencial entre dois elétrodos inseridos

numa câmara, com pressões inferiores a 100 Pa. A diferença de potencial

resulta em uma aceleração de elétrons que colidirão com átomos e moléculas

neutras do gás, produzindo íons e novos elétrons. Os íons produzidos são

acelerados para o eletrodo polarizado catodicamente, participando de uma

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série de efeitos. Dentre os efeitos pode-se citar o aquecimento do cátodo pela

transferência de energia dos íons; a criação de defeitos pelo arrancamento dos

átomos da superfície (sputtering), colisões elásticas e a reação superficial

acelerada devido a presença de espécies mais ativas que em processos

convencionais (44).

No processo de erosão por plasma (plasma etching) as condições da

descarga são modificadas de modo a diminuir a taxa de sputtering. Isto

significa dizer que apenas íons do gás da descarga estão presentes no plasma.

Se a atmosfera é composta por elementos muito reativos como o oxigênio, por

exemplo, íons desse elemento reagem com átomos da superfície que se

pretende modificar e, por volatilização dos produtos da reação, serão criados

poros na superfície (45).

Esta técnica permite produzir uma textura uniforme superficialmente,

devido a facilidade do controle do processo para gerar a rugosidade, quer seja

através do aumento da energia de bombardeamento e /ou número de íons

bombardeantes(41,42).

2.4 Genotoxicidade

Testes de citotoxicidade in vitro desempenham um papel importante na

avaliação da biocompatibilidade de novos materiais utilizados para a confecção

de implantes. O padrão internacional ISO-10993-5 (Avaliação biológica de

dispositivos médicos, testes de citotoxicidade in vitro), define os métodos de

ensaio a serem utilizados para a determinação de citotoxicidade in vitro.

Células são semeadas em contato com o material testado, ou com um extrato,

8


sendo as avaliações feitas considerando-se diferentes parâmetros, tais como

potencial genotóxico, aderência celular, proliferação, alterações morfológicas e

alterações do metabolismo (47).

Um composto/material é considerado genotóxico quando é capaz de

promover lesões na molécula de DNA, contudo estas lesões podem ser

reparadas ou não pelos sistemas de reparo celular. Caso o reparo não ocorra e

haja permanência da lesão, uma mutação pode ser instalada. Deste modo, um

agente é dito mutagênico quando é capaz de modificar a seqüência

nucleotídica original da molécula de DNA.

A quantificação do risco genotóxico e mutagênico de biomateriais, pode

ser realizada através de diversos bioensaios entre estes merecem destaque o

teste Cometa e micronúcleo que têm sido utilizados internacionamelmente

como um sistema de triagem para detectar a predisposição de risco genotóxico

e/ou mutagênico de novos químicos e drogas.

O teste cometa (single cell gel eletrpphoresis – SCGE) detecta danos no

DNA que não foram corrigidos, através de imagens semelhantes a um cometa

geradas por migração eletroforética dos fragmentos de DNA em relação ao

núcleo principal da célula (48,49).

Micronúcleos são pequenos corpos extra nucleares, expressos em

células que estão se dividindo, podendo ser fragmentos de cromossomos ou

cromossomos inteiros mal segregados durante a mitose. Assim, os

micronúcleos não são incluídos no núcleo da célula filha durante a telófase

(50,51). A indução de micronúcleos pode ser resultado de compostos

clastogênicos (agentes que induzem a quebra cromossomal através de

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interações com o DNA) ou aneugênicos (agentes que induzem ganho ou perda

de cromossomos interferindo na maquinaria de formação do fuso mitótico)

(52,50).

O teste de Ames, desenvolvido por Ames et al. (1973) utiliza diferentes

linhagens de Salmonella typhimurium, derivadas da linhagem parental LT2,

auxotrófica para a histidina (his-), geradas em laboratório para detectar

mutações do tipo deslocamento de quadro de leitura, substituição de pares de

bases ou danos oxidativos ao DNA. Essas linhagens não se proliferam em

meio de cultura mínimo, sem histidina, a menos que ocorram mutações que

restaurem a síntese desse aminoácido. A frequência de mutação reversa pode

ser medida pela contagem do número de colônias que crescem em meio

mínimo após a exposição de uma população bacteriana frente a um agente

mutagênico (54).

O ensaio de aberrações cromossômicas é uma técnica citogenética

clássica onde danos no DNA, em nível cromossômico são acessados durante a

metáfase celular. Aberrações cromossômicas (CAs) são mudanças na estrutura

ou no número normal dos cromossomos, que podem ocorrem

espontaneamente ou resultar de um tratamento químico ou físico. Levando-se

em conta a morfologia, as CAs podem ser divididas em duas classes:

aberrações tipo cromossômicas (CSAs), as quais consistem em quebras de

dupla fita de DNA envolvendo um ou múltiplos cromossomos; e as aberrações

do tipo cromatídica (CTAs), as quais envolvem somente uma das duas

cromátides de um cromossomo ou de diversos cromossomos (55,56).

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3. ANEXAÇÃO DOS ARTIGOS

3.1 BC nanofibres: In vitro study of genotoxicity and cell proliferation

Revista: Toxicology letters

Fator de Impacto: 3.24 - A2

11


12


13


14


15


16


17


18


3.2 Adhesion, cell proliferation and genotoxicity of bacterial cellulose

surfaces modified by plasma

Revista: Submetido ao Journal Biomedical Materials Research Part A

Fator de Impacto: 2.7 - A2

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Adhesion, cell proliferation and genotoxicity of bacterial cellulose

surfaces modified by plasma

Naisandra B. Silva1*, Carlos E. B. Moura1, Hugo A. O. Rocha2, Silvia R.

Batistuzzo3, Fabia K. Andrade4 , Renata A. N. Pértile4 , Francisco M. Gama4,

Clodomiro Alves Jr5.

1Department of Morphology, Bioscience Center (BC), Federal University of Rio Grande do Norte (UFRN), Natal-RN, ZC 59072-970, Brazil.

2Department of Biochemistry, (BC), Federal University of Rio Grande do Norte.

3Department of Cell Biology and Genetics, (BC), Federal University of Rio Grande do Norte.

4IBB, Institute for Biotechnology and Bioengineering, University of Minho, Campus de Gualtar, ZC 4710-057 Braga, Portugal.

5Department of Mechanical Engineering - Center of Technology, Federal University of Rio Grande do Norte.

* Corresponding Author:

Federal University of Rio Grande do Norte

Department of Morphology, Bioscience Center

Natal-RN, ZC 59072-970, Brazil.

Email: [email protected]; Tel.: +55-84-32153431.

20


Abstract: Bacterial cellulose (BC) has a wide range of potential applications,

namely as temporary substitute skin in the treatment of skin wounds, such as

burns, ulcers and grafts. Surface properties determine the functional response

of cells, an important factor for the successful development of biomaterials. This

work evaluates the influence of bacterial cellulose surface treatment by plasma

(BCP) on the cellular behavior and its genotoxicity potential. The modified

surface was produced by plasma discharge in N2 and O2 atmosphere, and the

roughness produced by ion bombardment characterized by scanning electron

microscopy (SEM) and atomic force microscopy (AFM). Cell adhesion, viability

and proliferation on BCP were analysed using crystal violet staining and the 3-

[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium (MTT) method. Genotoxicity

was evaluated using the comet and cytokinesis block micronucleus assay. The

results show that the plasma treatment changed surface roughness, producing

an ideal cell attachment, evidenced by more elongated cell morphology and

improved proliferation. The excellent biocompatibility of BCP was confirmed by

genotoxicity tests, which showed no significant DNA damage. The BCP has

therefore great potential as a new artificial implant.

Key words: bacterial cellulose, biocompatibility, biomaterial, substitute skin,

roughness.

21


INTRODUCTION

In recent years rapid progresses have been made in the development of

new biomaterials from natural or synthetic polymers for biomedical

applications. In this context, the bacterial cellulose (BC) produced by

Gluconacetobacter xylinum has proved to be a remarkably versatile and

promising biomaterial. It can be used in a wide variety of technological

applications, such as paper products, electronics, acoustics, and biomedical

devices [1][2].

Bacterial cellulose has been demonstrated to be a valuable temporary

skin substitute for the treatment of skin wounds, such as burns, ulcers, grafts,

as an adjuvant in dermal abrasions [3-5], as a vascular graft, in the release of

drugs and in tissue guided regeneration [1,6].

Bacterial cellulose has several characteristics that differ from plant

cellulose, such as higher crystallinity, tensile strength, elasticity and durability

[7]. Its nanostructure is a physical barrier against infection and increases water

absorption and retention. It has been shown to be biocompatible when

associated with bone tissue, skin tissue, blood vessels, fibroblasts and

hepatocytes of rats and rabbits [9-13] and the nanofibres did not exhibit

genotoxicity [14]. Despite recent advances, with the creation of new

biomaterials using bacterial cellulose, few studies have investigated cellular

responses or genotoxic and mutagenic risks.

Despite its excellent properties, BC is composed of dense microfibrils

with relatively small pores [15][6] that can influence its ability to perform as a

drug release system and to control cell infiltration [16-19]. Modification of the

22


membrane surface, resulting in larger pores or increased roughness, may be a

way to expand its use.

The use of plasma as an energetic source for surface modification seems

to be a good option for the surface modification of BC, not compromising its

mechanical properties. Plasma can also be beneficial in changing surface

topography, increasing surface roughness, in addition to cleaning / sterilizing

and increasing surface wettability [20][21].

Plasma is the fourth state of matter. It is produced by an electric

discharge in gas, by applying a voltage between two electrodes inserted in a

chamber, at low pressures. Because of this voltage, electrons are accelerated

and collide with plasma particles, forming neutral particles, ions, excited

particles and electrons. The ions produced are accelerated to the cathodically

polarized electrode, taking part in a series of effects [22]. Among these effects

is the breaking of molecule chains, the formation of new functional groups

and/or morphological alterations, such as microporosity [23].

To improve BC-cell interaction, the former was treated by plasma and

characterized according to both changes generated on the surface and cellular

response. In the present work we used a plasma atmosphere containing a

mixture of 70% N2 and 30% O2 because of the results found in our earlier study,

which elsewhere was shown to significantly increase polyester surface

roughness[24][25], resulting in altered surface wettability.

Therefore, the objective of this study was to evaluate the effect of

bacterial surface modification, resulting from plasma treatment, on in vitro cell

response and to evaluate the genotoxicity of this new biomaterial. The results of

23


this research may have strong implications for the creation of strategies to

improve the clinical application of this material.

MATERIAL AND METHODS

Preparation of BC substrates

The bacterial cellulose membranes were produced according to Moreira

et al. [14] and subsequently treated in a plasma reactor, using an atmosphere

with a gas mixture containing N2 (70%) and O2 (30%). The plasma reactor used

for the treatment (Fig. 1)) consists mainly of a reaction chamber, a vacuum

system, and a power system. In addition to two electrodes and an adjusting

ring, the plasma chamber also included a glass cylinder (400 mm in length and

320 mm in diameter), for a total volume of 0.32m3. The ends of the tube were

sealed by two stainless steel flanges. The connection of the bottom flange held

vacuum, pressure sensors and thermocouples. The power supply had a

continuously adjustable output up to 1500V DC and 2A current. The samples,

measuring 2.5 cm × 5.0 cm, were fixed on the inner chamber, using an

adjustment ring, and placed between two electrodes, 4 cm from the cathode, as

suggested by Costa et al. [24]. This distance was necessary to avoid thermal

alterations on the surface during processing, given that the cathode reached

temperatures above 150oC during previous experiments.

All treatments were performed under the same conditions (Table 1).

Voltage, current, pressure and cathode temperature were measured and

controlled on the control panel. After stabilization, the plasma was initiated to

quantify the time process. At the end of processing, BC nanostructure was

24


analyzed using a Nova NanoSEM 200 scanning electron microscope

(Netherlands), with accelerating voltage of 5kV.

Surface roughness was assessed by measuring the roughness

parameter, Ra (roughness average), Rp (mean peak) and Rv (valleys) using

atomic force microscopy (AFM - SPM9500J3 Shimadzu). Each sample was

scanned at six different randomly selected locations; the total scan area was

approximately 15 x 15 µm2. The values were calculated from the 5 x 5 µm2 area

in the middle of the scanned area.

Determination of contact angles – wettability

Water contact angles were measured using a face contact angle meter

(OCA 20, Dataphysics, Germany). The contact angle of the untreated and

treated bacterial cellulose surfaces was measured by the sessile drop method

[26], in which a 2µL droplet of ultra pure water (Milli Q) was placed on a

horizontal BC surface and observed with a face contact angle meter. The

images were captured and the angle formed by the tangent of the droplet with

the surface was measured.

Analysis of X-ray photoelectron spectra (XPS)

XPS analysis was performed using an ESCALAB 200A, VG

Scientific (UK) with PISCES software for data acquisition and analysis.

For analysis, an achromatic Al (Kα) X-ray source operating at 15kV (300

W) was used, and the spectrometer, calibrated to the Ag 3d5/2 (368.27 eV)

reference, was operated in CAE mode with 20 eV pass energy. Data acquisition

was performed at a pressure below 1.E-6 Pa. Survey scan spectra were

obtained at pass energy of 50 eV, while for C1s, O1s and N1s individual high-

25


resolution spectra were taken at pass energy of 20 eV and a 0.1 eV energy

step. Spectra analysis was performed using peak fitting with Gaussian-

Lorentzian peak shape and Shirley type background subtraction (or linear,

taking into account the data). The binding energy (eV) scales were referenced

to the hydrocarbon component (C-C) in the C 1s spectra at 285 eV.

Cell culture

Chinese hamster ovary (CHO-K1) cells were cultivated in Dulbecco’s

Modified Eagle’s Medium (DMEM – Invitogen), supplemented with 10% fetal

bovine serum (FBS -Gibco, Germany) 100 UI/mL of penicillin and 100 μg/mL

of streptomycin. The culture was incubated at 37oC in a humidified atmosphere

of approximately 5% CO2. Confluent cells, grown to confluence in 75 mL TPP

culture flasks were then detached with 0.15 % trypsin (Cultlab, Brasil) for 5 min,

5 mL of complete medium was added, and the cells were centrifuged at 1800

rpm for 5 min. After the pellet was suspended in 1mL of fresh medium, CHO K1

was counted with a Neubauer chamber and used in the assays. The cells were

seeded on Discs of BC (1cm in diameter) with treated (BCP) and untreated

plasma (BC). In all the negative controls the cells were cultivated on the plastic

tissue culture plate. For comet assay and micronuclei respectively the positive

controls were treated with 100 mM/mL of hydrogen peroxide and 0.1µg/mL of

mitomycin C. Each treatment was performed in triplicate to ensure

reproducibility.

26


Cell adhesion

CHO-K1 cells were detached from the subculture with 0.15% trypsin

(Cultlab, Brazil) for 5 min, resuspended, and plated on BC membranes placed

on 24-well plate at a cell population density of 5 X 105 cells/mL in DMEM

supplemented with 10% FBS and antibiotics. The media were removed from

three samples out of nine in each group (BC, BCP) at 1, 2 and 3 hours of

incubation, respectively, and the unattached cells were washed away twice with

PBS at 37oC. The material was subsequently fixed in methanol PA ice for 5 min

and washed again with PBS. Attached cells were stained with a solution of

0.2% crystal violet in 2% methanol for 5 min at room temperature. Stained cells

were washed with PBS and the bound dye was solubilized with 100 mL of

solubilization buffer (v/v mixture of 0.1M Na3C6H5O7 and 50% ethanol) to be

transferred to 96-well plates. Absorbance (optical density, OD) was measured

using ELISA analyzer (Spectra µQuant – Biotec – MKX 200) at 570 nm. Cell

morphology was also observed by SEM at 24h and 48h to inspect cell

accommodation in the membranes. Cell-seeded samples were fixed with 1%

glutaraldehyde buffer (pH 7.4) for 1h, dehydrated using a gradation series of

ethanol/distilled water mixtures (30%, 50%, 70%, 90%, 100%). The critical

drying point of specimens was determined with liquid CO2 and specimens were

sputter-coated with gold and examined by SEM (JEOL JSM-6360 LV).

27


Proliferation assays

For cell proliferation assays, cells were seeded on biomaterial disks in

24-well plates (TPP) at 5.0 x 104 cells/disk for BCP and BC in triplicate. After

incubation for 2, 4 and 6 days, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-

tetrazolium bromide - Invitrogen) solution, prepared using DMEM, was added to

each well and incubated for 4h. The MTT was subsequently removed from the

wells and 1mL of absolute ethanol added and mixed. Aliquots (200µL) of the

culture medium were transferred to 96-well plates and quantified

spectrophotometrically (µQuant – Biotec – MKX 200) for absorbance with a

microplate reader (TPP) at 570 nm.

Cytokinesis block micronucleus (CBMN)

Exponentially growing cells were cultured on BC or BCP membranes or

plastic well plates for controls until reaching 80% confluence. CHO-K1 cells

were then washed twice with phosphate-buffered saline (PBS) prior to a 72h

incubation in fresh medium (DMEM) containing 10% FBS and 5.0µg/mL

cytochalasin-B (cyt-B, Sigma) at 37oC, as described by Fenech [27], with some

alterations [28]. At the end of cyt B treatment, the cells were washed twice with

PBS, trypsinized and harvested by centrifugation at 1500 rpm for 5 min. The

pellet was resuspended in 5% KCL at ambient temperature for 5 min. They

were then re-centrifuged, fixed in 20:1 (v/v) methanol/acetic acid, placed on a

clean microscope slide and stained with 5% (v/v) Giemsa for 10 min at 60ºC.

For each bacterial cellulose membrane sample, 3000 binucleated cells (i.e.

1000 from each of the two slides prepared from the triplicate cultures), were

scored for MN presence, which was assessed using bright-field optical

28


microscopy at a magnification of 200-1000x. All sides were coded for blind

analysis.

Comet assay

The alkaline comet assay was performed, as described by Singh et al.

[29], with some adaptations. The cells that were on the BC, BCP and controls

(1.0 x 106 - 25µL) were embedded in 75µL of 0.75% low melting point agarose.

After the agarose solidified the slides were placed in lysis buffer (2.5 M NaCl,

100 mM EDTA and 10 mM Tris; pH 10.0) containing freshly added 1% (v/v)

Triton X-100 and 10% (v/v) dimethyl sulphoxide for 12h at 4oC. After treatment

with lysis buffer, the slides were washed in PBS and incubated in freshly

prepared alkaline buffer solution (300 mM NaOH and 1 mM EDTA; pH ≥ 13) for

20 min, and DNA was submitted to electrophoresis for 30 min at 25 V

(0.90V/cm) and 300 mA, after which the buffer solution was neutralized with 0.4

M Tris-HCl (pH 7.5) and the DNA stained with 50µL ethidium bromide

(20µg/µL). All the above steps were conducted under dim light to prevent

additional DNA damage. Each electrophoresis run was considered valid only if

the negative and positive controls yielded the expected results. Observations

were made at 400x magnification using a florescence microscope (Nikon

Eclipse E200). At least 100 randomly selected images were analyzed from each

sample and the DNA damage was analyzed with the DEMO version of Comet

Assay IV software and CASP software package. Tail moment comet parameter

measures were used as DNA damage indicators, since they are considered the

most informative and reliable.

29


Statistical analysis

All results are expressed as mean ± SD. Paired t- test was used to

evaluate the statistical significance of surface differences. A 95% confidence

level was selected to define significance for all statistical tests, and differences

were considered significant when p < 0.05.

RESULTS AND DISCUSSION

Surface characterization and wettability

It is well known that not only surface material chemistry but also surface

properties such as roughness/topography have an effect on protein adsorption,

cytoskeleton alignment and cellular responses (e.g. cell attachment,

adhesion, spreading, proliferation and differentiation) of biomaterials [30-33]. In

this study, we evaluated the surface roughness generated in bacterial cellulose

membranes modified by plasma.

The hydrophilicity/hydrophobicity of a material is a relevant

biocompatibility property that can be estimated by through contact angle

measurementsusing liquids with well characterized properties In this work, the

water contact angles were measured. The results showed that the incorporation

of nitrogen using the plasma treatment did not modify the contact angles (BC:

26.68 ± 1.187; BCP: 28.50 ± 0.532); however, the nanofibrous BC structure was

altered, to a significant extent, as can be seen by SEM (Fig. 2). It is well known

that plasma modification leads to increased surface roughness. These changes

in surface topography are mostly caused by the physical erosion promoted by

plasma ions [34]. In the case of BC, the plasma treatment seems to disrupt the

30


fibres to some extent, leading to a more porous and rough material, also

detected by AFM. Figures 2b and 2d show the nanofibrous structure of BC and

BCP membranes, respectively. The BCP surface appeared rougher (Fig. 2d),

as compared to the untreated, smoother BC (Fig. 2b).

A detailed knowledge of each single surface feature through AFM image

analysis is crucial when studying the behaviour of a cell that comes into contact

with the surface. This is because the cell can extend filopodia, i.e., highly motile

nanostructures, to gather information from the surrounding environment by

interacting individually with every single topographical surface feature [35][36].

AFM surface topography and profiles of 2 samples with different surface

roughness are shown in figure 3. The results obtained in the analysis of

surfaces without cells, show higher peaks on the BCP surface (495.5 nm), while

on BC it was 308.7 nm (Fig. 3). Average roughness (Ra) was significantly

higher in the treated cellulose (Ra=70.312 ± 5.1 nm) while BC showed Ra of

42.752 ± 6.0 nm. Mean peak (Rp) and mean valley (Rv) were also higher in

BCP (541.397 ± 62.8 nm and 402.538 ± 33.7 nm, respectively), while for BC the

values were 159.872 ± 24.3 nm and 149.092 ± 18.1 nm, respectively. The

plasma treatment thus led to a rough surface texture. Increased porosity and

roughness may have a positive effect on nutrient permeability and cell

communication when using BC in tissue engineering applications.

X-ray photoelectron spectroscopy (XPS)

The O/C atomic ratio of the untreated and plasma- treated BC were

0.82 and 0.83, respectively (Table 2), comparable to the theoretical value of

pure cellulose (0.83). With respect to nitrogen content, the incorporation of

31


about 1.49% of nitrogen can be observed after plasma modification.

The chemical surface modifications introduced in the plasma were

analysed through deconvolution of the XPS spectra. The C 1s and N 1s peaks

in the BC and BCP are shown in figure 4 and table 3. The binding energy of C ls

and its spectra deconvolution are well documented. There is general

agreement on the assignment of components C1, C2, C3 and C4 of the C 1s

peak in wood-derived material. C1 corresponds to carbon linked only to

hydrogen or carbon (-C-H, -C-C); C2 is assigned to carbon linked to a single

oxygen atom (-C-O), whereas C3 binds two non-carbonyl oxygen atoms (O-C-

O), or a single carbonyl oxygen atom (-C=O) and C4 represents carbon atoms

linked to a carbonyl and a non-carbonyl oxygen atom (O–C=O). The

appearance of C1 and C4 may be due to either contamination or a chemical

change in cellulose structure, as reported in the literature [37-39]. In this work,

BC exhibited 4 carbon peaks, corresponding to C1, C2, C3 and C4 and BCP

showed an additional peak (C5), which corresponds to carbon linked to three

oxygen atoms (O-CO-O). According to Hua et al. [40], the formation of the C4

and C5 functionality is due to modification of O-C-O linkages. It is noteworthy

that the formation of C5 is possible only through the cleavage of the C1-C2

carbons of the pyranoside ring followed by ex situ post plasma oxidation [40].

The N 1s component was decomposed in only one peak at a binding

energy of 400.7 eV. In carbonaceous materials, this value (400.5 + 0.3 eV) is

generally assigned to pyrrole (or pyridone) nuclei N5 [41][42].

32


Cell adhesion and proliferation

The results from the adhesion assay showed that BCP substrate was

unable to enhance the adhesion of CHO-K1 after 1, 2 and 3h incubation after

plating (figure 5A). Indeed, the cell adhesion is similar on BC and BCP. This is

partly in line with the result of previous studies, which showed that fibroblast

attachment to microgrooved substrates is low at early stages of the culture,

drastically increasing at later phases [43][44]. A reasonable explanation for this

observation is that the formation of organized cell-substrate contact junctions is

hampered on textured surfaces. As a consequence, no stable cell adhesion can

be ensured, resulting initially in reduced attachment percentages. The cell is

likely in the process of exploring and probing the surface [43].

Surface roughness can affect cell adhesion due to increased or

decreased contact area, which is proportionate to the interfacial adhesive force.

Therefore, surface roughness may influence the contact area of the cell

membrane with the substrate [45]. According to Mansur and Mansur [46] and

Dunn and Zicha [47], cell spreading is usually divided into three main interaction

levels: (a) not spread: cells were still spherical in appearance, protrusions or

lamellipodia were not yet produced; (b) partially spread: at this stage, cells

begin to spread laterally at one or more sides, but the plasma membrane

extensions are not completely confluent; and (c) fully spread. The latter model

(c) yields the best result for material cell hosting, which was observed on the

BCP surface (Fig. 2C and D).

The cells on the membranes underwent morphological changes to

stabilize the cell–biomaterial interface. In BCP they spread and established

33


close contact with the material, adopting a flattened morphology and showing

filopodia anchoring the cells to the surface (Fig. 2C and D). In the round state

(Fig. 2A and B), the surface to volume ratio of the cell is reduced and cell

proliferation and nutrient supply also decreases. Contact adhesion is a

precondition for cell growth. Therefore, the observation of nonspreading cells

are indicative of undesirable surface properties [48]. Thus, greater proliferation

was indeed evident in BCP, with roughness of 70.312 ± 5.1 nm at 2, 4 and 6

days (Fig. 5B). Similar results were found by Khan et al. [45] and Fan et al. [49]

for neurons.

Genotoxicity using the Comet assay and Cytokinesis block micronucleus

(CBMN)

In vitro genotoxicity tests play an important role in evaluating the

biocompatibility of novel implant materials. The international standard ISO-

10993-3 (biological evaluation of medical devices: tests for in vitro genotoxicity)

defines the test methods to be used for the determination of in vitro

genotoxicity. There are two common types of genotoxicity tests: 1) the comet

assay is a versatile and sensitive method for measuring single and double-

strand breaks in DNA [50]; 2) the in vitro cytokinesis block micronucleus assay

is used to detect substances or materials that induce the formation of small

membrane- bound DNA fragments in the cytoplasm of interphase cells.

In this work, bacterial cellulose membranes, untreated or plasma treated,

did not induce DNA damage on exposed CHO-K1 cells, as measured by the

comet test. Indeed, the respective values of tail moment 6.61 ± 1.27 and 7.07 ±

1.36 are not significantly different. DNA migration was demonstrated with BC

34


and BCP (Fig. 6), but without a significant increase when compared to the

negative control (0.97 ± 0.3). A significant difference in tail moment was only

observed in the positive control (p <0.001). It has been generally estimated that

some 104 lesions are induced in a mammalian cell genome every day by

spontaneous chemical reactions and during cellular metabolism. The most

predominant among these are spontaneously generated or products of reactive

oxygen species (ROS reaction) [51]. ROS are the major source of endogenous

DNA damage in aerobic organisms and the hydroxyl radical is the most reactive

of these radicals. The interaction of hydroxyl radicals with the genomic DNA

results in a plethora of modified bases and sugars, 8, 5'- cyclopurine - 2'-

deoxynucleosides, DNA - protein cross-links, abasic sites and strand breaks

[52]. Bacterial cellulose may promote an increase in ROS, but since it does not

cause significant damage to DNA, no oxidative stress leading to cell death is

generated.

Cytokinesis block micronucleus (CBMN) frequency is shown in table 4.

Other nuclear alterations, such as nucleoplasmic bridges, buttons, and nuclear

bubbles were not found in this work. Cells grown on the BCP and BC surfaces

did not reveal a statistically significant number of micronuclei. Taken together,

the results of these tests show that neither treated nor untreated bacterial

cellulose surfaces exhibit genotoxicity. However, it is important to emphasize

that an in-depth and complete biocompatibility analysis, taking into account

genotoxic and mutagenic effects, has only been carried out in bacterial cellulose

in studies conducted by Schmitt et al. [53] and Moreira et al. [14] and others,

such as Miyamoto et al. [13], take into account only aspects related to the

appearance of inflammation or tissue rejection to confirm biocompatibility. Our

35


results provide the literature with pioneering data on the genotoxicity of bacterial

cellulose surfaces modified by plasma as the energy source.

CONCLUSIONS

This work presents the first evaluation of the influence of plasma

treatment on the modification of bacterial cellulose surfaces. The main following

conclusions may be highlighted: plasma, in a nitrogen and oxygen atmosphere,

changed the surface of bacterial cellulose; the treatment promoted an increase

in roughness of 42.752 ± 4.0 nm to 70.312 ± 5.1 nm in the treated cellulose; cell

adhesion of these surfaces, for up to three hours, is low; in BCP, cell

morphology became more elongated and cell proliferation was highest, likely

owing to the increase in surface roughness; the new surface generated was not

genotoxic. Given the above findings, this work generated a new biomaterial that

can be tested in vivo, with potential use as a new artificial implant.

Acknowledgements

Funding through the FCT (Fundação Ciência e Tecnologia)/CNPq

(National Council for Scientific and Technological Development) bilateral

program is gratefully acknowledged.

Conflict of Interest

No benefit of any kind will be received either directly or indirectly by the

author(s).

36


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Figure Legends:

Figure 1. Schematic representation of the plasma reactor

Figure 2. SEM micrographs of bacterial cellulose with adhered cells : (A) and

(B) show morphology of round CHO-K1 cells in BC at 24 and 48h; scale bars

=2 μm and 6500 X and 9000 X magnification; (C) and (D)show elongated

CHO-K1 cells in BCP at 24 and 48 hours, scale bars = 5 μm and 3000 X and

3500 X magnification. (b) and (d) show the nanofibrous structure BC and BCP

membranes, respectively; scale bars = 1 μm and 100000 X magnification.

Figure 3. Three-dimensional AFM images and surface profile of two lines of

analysis (A-B and C-D) in the top and bottom diameter of the samples. (A)

Untreated bacterial cellulose; (B) Treated bacterial cellulose

Figure 4. Deconvolution of the carbon peak for (a) BC and (b) BCP and (c)

nitrogen peak for BCP.

Figure 5. (A) Histogram of CHO K1 cell adhesion in treated and untreated

bacterial cellulose at 1, 2 and 3h. (B) Histogram of cell proliferation at 2, 4 and 6

days. Each bar represents an average of CHO-K1±standard deviation (n=3 in

each group containing three equal experiments, *p < 0.001).

Figure 6. Effect of a plasma-treated bacterial cellulose (BCP) and untreated

surface (BC) on DNA according to comet assay experiments. PC: positive

control (H2O2); NC: negative control culture plastic; Tail moment values

represented as the mean ± standard deviation obtained from three experiments

*p < 0.001.

44


Figure 01

Figure 02

45


Figure 03

46


Figure 04

47


Figure 05

Figure 06

48


Table 1 - Conditions in all the treatments carried out in

plasma reactor

Parameter Value

Time 30 min

N2 Flow 10 sccm

O2 Flow 4 sccm

Current 0,2 A

Pressure 4 mbar

Voltage 425 V

Table 2 - BC elemental composition analysed by XPS

Surface

Modification

Oxygen

(%)

Carbon

(%)

Nitrogen

(%)

O/C N/C

BC 45.20 54.79 0.01 0.82 0.0001

BCP 44.82 53.69 1.49 0.83 0.028

49


Table 3 - Concentration of different carbon groups on

bacterial cellulose surface treated with plasma.

Bacterial

Cellulose

Binding Energy

(eV)

Peaks

(%)

Untreated

C1 285.0 7.22

C2 286.67 64.56

C3 287.96 23.15

C4 289.04 5.05

Treated

C1 285.0 10.47

C2 286.69 59.15

C3 288.11 21.41

C4 289.15 6.17

C5 290.14 2.80

Table 4 - Mean frequency of binucleated cells with

micronuclei (BNMN) in 3000 cells for PC, NC,

BCP and BC groups, and their percentage

***p<0.001.

Treatments BNMN %

Positive control *** 85±17.09 0.28

Negative control 13± 3.51 0.04

BCP 15± 1.73 0.05

BC 14± 4.73 0.04

50


3.3 Influence of argon-ion bombardment of titanium surfaces on the cell

behavior

Revista: Surface & Coatings Technology

Fator de Impacto: 1.86 - B1

51


52


53


54


55


56


57


3.4 Effect of titanium surface modified by plasma energy source on

genotoxic response in vitro

Revista: Toxicology

Fator de Impacto: 2.83 A2

58


59


60


61


62


63


64


65


66


4. COMENTÁRIOS, CRÍTICAS E SUGESTÕES

A possibilidade de ingressar em uma Pós-graduação com caráter

multidisciplinar foi fundamental para o desenvolvimento deste trabalho.

Pesquisadores das áreas da saúde (biológos, farmacêuticos, veterinários,

dentistas) e engenharia atuaram de forma interdisciplinar para a realização do

projeto “Funcionalização de superfícies de nanofibras de celulose bacteriana

para aplicação em regeneração tecidual - CNPq nº 27/2006 - Projetos

Conjuntos de P&D&I /Portugal - CNPq/GRICES” do qual esta pesquisa fez

parte.

4.1 Expectativas Futuras

Nossos resultados poderão servir de subsídios na escolha de uma

superfície de celulose bacteriana mais adequada ao uso terapêutico. Nesse

sentido, este estudo poderá resultar nos seguintes processos e produtos:

1. Uso do plasma a baixa pressão para modificação de superfícies de

celulose bacteriana;

2. Confecção de scaffolds de celulose bacteriana tratada por plasma

para testes in vivo;

3. Desenvolvimento de uma biomembrana de CBP que poderá ser

testada na liberação controlada de fármacos;

4. Realização de ensaios in vivo com a celulose bacteriana tratada por

plasma como curativo biológico;

67


5. Avaliação da resposta biológica da CB e CBP frente a células tronco,

na diferenciação endotelial e epitelial.

Ainda, levando em consideração que todo biomaterial deve ser testado

in vitro quanto a sua biocompatibilidade, genotoxicidade e mutagenicidade,

antes da sua aplicação in vivo e da liberação de seu uso para produção de

implantes, (52-54) existe a possibilidade de criação da linha de pesquisa:

Genotoxicidade e mutagenicidade de biomateriais.

Atualmente sou professora assistente da disciplina de Histologia da

Universidade Federal do Rio Grande do Norte, concluir o curso de doutorado é

indispensável à minha progressão e ascensão como pesquisadora. Atuo na

base de pesquisa: Plasticidade Morfofuncional dos Sistemas Orgânicos.

Microscopia Celular e Tecidual. Pretendo, a partir de agora, fortalecer nossa

linha de pesquisa: Biologia Celular, caracterização estrutural e ultraestrutural,

adesão, proliferação, diferenciação e morte celular. Orientar alunos na

graduação e pós-graduação e elaborar novos projetos que serão submetidos

às instituições de fomento. Ressalto ainda, que com os excelentes resultados

obtidos, durante o desenvolvimento deste trabalho, surgiu a oportunidade de

ingressar em um pós-doutorado com a aprovação do projeto “Produção,

caracterização e avaliação da atividade antitumoral de nanogeis de fucanas

extraídas de algas do litoral do Nordeste - EDITAL MCT/CNPQ Nº 62/2008”.

Cooperação internacional entre a Universidade Federal do Rio Grande do

Norte e Universidade do Minho – Braga/Portugal.

68


4.2 Publicação/divulgação

Para atingir nossos objetivos, foram desenvolvidas diversas parcerias

científicas as quais resultaram em artigos pioneiros, com resultados inéditos,

de impacto científico e de reconhecimento internacional. O ponto chave que

nos levou ao ineditismo foi a modificação das superfícies de celulose

bacteriana por plasma e a avaliação, em nível molecular, de sua genotoxidade.

O trabalho submetido “Adhesion, cell proliferation and genotoxicity of

bacterial cellulose surfaces modified by plasma” tem grandes possibilidades de

ampliação com estudos relacionados à avaliação da acomodação do

citoesqueleto celular frente a superfícies modificadas, ou a expressão de

integrinas correlacionadas à adesão celular. As metodologias aplicadas nesse

estudo poderão ser utilizadas para avaliações de resposta celular frente à

superfície de novos biomateriais, nanogéis ou nanopartículas.

Além dos artigos concluídos, outros trabalhos foram publicados em anais

de congresso versando sobre a temática: modificação de superfícies de

biomateriais. Sendo o penúltimo deles, listado abaixo, premiado com menção

honrosa no III International Symposium in Biochemistry of Macromolecules and

Biotechnology.

1. Silva NB, Moura CEB, Freitas ML, Batistuzzo SR, Gama FMP, Rocha HAO,

Alves Jr. C. Celulose Bacteriana: Genotoxidade e efeito na proliferação

celular. In: V COLAOB - Congresso Latino Americano de Órgãos Artificiais e

Biomateriais, 2008, p. 89.

http://lattes.cnpq.br/4717410137206021




69


2. Sá JC, Alves MBM, Rocha HAO, Silva NB, Moura CEB, Alves Jr. C.

Influencia da rugosidade e molhabilidade de superfícies de titânio

modificadas por Plasma na Proliferação de células-tronco. In: V COLAOB -

Congresso latino de órgãos Artificiais e Biomateriais, 2008, p.111.

3. Moura CEB, Rocha HAO, Silva NB, Alves MBM, Sá JC, Papa PC, Alves Jr.

C. Proliferação de pre-oesteoblastos sobre superficie de titãnio

bombardeada por íons de argônio. In: XXVIII Congresso Brasileiro de Vácuo

Aplicado na Indústria. Natal, 2007. p.1.

4. Tavares JCM, Barbosa JS, Cabral TM, Silva NB, Moura CEB, Sá JC.; Alves

MBM, Rocha HAO, Alves Jr. C, Medeiros SRB. DNA Damage induced for

titanium Plasma treated surface. In: III International Symposium in

Biochemistry of Macromolecules and Biotechnology - SBBq, 2006, Natal.

(MENÇÃO HONROSA)

5. Barbosa JS, Tavares JCM, Cabral TM, Silva NB, Moura CEB, Sá JC, Alves

MBM, Rocha HAO, Alves Jr. C, Medeiros SRB. Mutagenicity evaluation of

Plasma treated on titanium surface. In: III International Symposium in

Biochemistry of Macromolecules and Biotechnology - SBBq, 2006, Natal.






70


4.3 Dificuldades

Ao longo da concretização deste estudo, surgiram algumas dificuldades

principalmente relacionadas à obtenção de reagentes, e utilização

microscópica. A UFRN é carente de um núcleo de microscopia voltado a

análises biológicas. Microscópios do tipo Confocal ou de Varredura ambiental

são imprescindíveis ao avanço de pesquisas que utilizam a interface células-

biomaterial. Projetos como CT-infra, são alternativas para sanar essa

deficiência, nesse sentido o grupo interdisciplinar que trabalha na área de

biomateriais na UFRN vem tendo a preocupação em elaborar projetos que

contemplam esses equipamentos.

71


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http://www.sciencedirect.com/science/journal/03009084

http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=%23TOC%236236%232006%23999119988%23637667%23FLA%23&_cdi=6236&_pubType=J&view=c&_auth=y&_acct=C000037878&_version=1&_urlVersion=0&_userid=687335&md5=84883b799e2f60e6ff788e1b0450a8ea

79


ABSTRACT

Bacterial cellulose (BC) is generally used as a temporary membrane to replace

damaged skin, in wounds, burns ulcers or as a graft. Changes to its surface

may determine cell function response in adjacent tissues, influencing its

biocompatibility. This study presents the first evaluation of the influence of

bacterial cellulose nanoparticles and bacterial cellulose membranes with

plasma-modified surface (BCP) on behavior and cell genotoxicity. Cell

proliferation was evaluated using the MTT test and DNA damage by the comet

assay and Kado test, all at concentrations of 0.1, 0.5 and 1.0 mg/ml of BC

nanofibers in contact with 3T3 fibroblasts and CHO-K1 cells. Results reveal that

cell proliferation for both types of cells was around 15-20% lower in the

presence of NFs, after 72h of cell culture, regardless of concentration used; nor

did they cause any significant DNA damage. In a second study, bacterial

cellulose membranes were submitted to plasma in atmosphere containing

70%N2 and 30% O2. They were later characterized by SEM and AFM and

submitted to comet, micronucleus, adhesion and cell proliferation assays.

Results show that plasma altered the BCP surface, producing roughness of

approximately 70± 5.1 nm. In BCP, cells became more elongated with greater

proliferation, likely due to the increase in surface roughness. The new surface

generated was also not genotoxic. Thus, this study created a new biomaterial

with future potential for artificial implantation that can be tested in vivo.

Keywords: Surface modification, biological dressing, biocompatibility test,

biomaterial, Acetobacter xylinus.

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