Biomaterial a base de celulose bacteriana com aplicação na regeneração de tecido cutâneo
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Data
2019-03-08
Autores
Martín Bedoya, José Gregorio
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A celulose bacteriana (CB) é sintetizada por vários tipos de bactérias, com maior eficiência pela Gluconacetobacter xylinus. A CB é um biomaterial que têm uma estrutura de rede de nano fibras tridimensional com propriedades como biocompatibilidade, não toxicidade, estabilidade mecânica e alto teor de umidade, que a torna promissora para diversas aplicações, sendo que na engenharia de tecidos sua multifuncionalidade abrange a substituição temporária de tecido epitelial em casos de queimaduras, úlceras, lesões, carreador de fármacos, dentre outros propósitos. Apesar da CB apresentar potencial para ser utilizado como curativo, faz-se necessário a realização de modificações de sua superfície, com a incorporação de moléculas de adesão celular, para ajustar as deficiências da CB nativa, a fim de melhorar a adesão, migração, proliferação e diferenciação celular, tornando-se promissora para aplicações biomédicas. Deste modo, este trabalho objetivou desenvolver um curativo à base de CB contendo peptídeo promotor de adesão celular RGD (Arginina-Glicina-Ácido aspártico), sequência encontrada em várias proteínas da matriz extracelular (MEC), e avaliar in vitro sua viabilidade e proliferação de fibroblastos, com a finalidade de aplicação em regeneração tecidual de pele. O peptídeo RGD utilizado (JGB2) foi escolhido por meio de estudos computacionais, sendo sintetizado pelo método da fase sólida (estratégia Fmoc), purificado por CLAE e caracterizado por espectrometria de massas. Membranas de CB foram produzidas por G. xylinuns, purificadas e funcionalizadas pela esterificação de dois resíduos de lisina, para incorporação do peptídeo JGB2. Diferentes amostras da CB foram caracterizadas a partir de análises de liberação prolongada do peptídeo JGB2, medidas de ângulo de contato, resistência à tração, análises de microscopia eletrônica de varredura (MEV-FEG), difração de raios-X (DRX) e espectroscopia de absorção na região de infravermelho com Transformada de Fourier (FT-IR). Os resultados obtidos mostraram que a funcionalização da membrana com lisina não resultou em alterações significativas na estrutura e morfologia nas membranas. Comparativamente à CB pura, a funcionalização da CB, aumentou a quantidade de peptídeo adsorvido e quadruplicou o tempo de liberação do peptídeo da membrana, características desejáveis para aplicação em processos de regeneração tecidual. Também foi possível constatar que a membrana funcionalizada com o aminoácido lisina contendo o peptídeo JGB2, apresentou desempenho superior nos ensaios in vitro de citotoxicidade, e, por conseguinte, possui grande potencial para ser estudada mais detalhadamente e eventualmente ser aplicada como biomaterial na reparação tecidual de pele.
Bacterial celulose (BC) is synthesizedby some bacteria, with greater efficiency by Gluconacetobacter xylinus.BC is a biomaterial that has a network 3-D structure of nanofibers with properties like biocompatibility, non-toxicity, mechanical stability, and high moisture content, whichmakes it promising fora wide range of applications, being that in the engineering of tissue its multifunctionality includes the temporary substitution of epithelial tissue in cases of burns, ulcers, injuries, drug carriers, among other purposes. Although CB present potential to be used as a dressing, it is necessary to perform modifications of its surface with the incorporation of cell adhesion moleculesto adjust the deficiencies of the native CB, in order to improve cell adhesion, migration, proliferation and differentiation, making it a promising material for biomedical applications.In this way, this work aimed to develop a dressing based on BC containing peptide promoter of cell adhesion RGD (Arginine-Glycine-Aspartic acid), sequence found in several extracellular matrix proteins (ECM) and to evaluate in vitroits viability and proliferation of fibroblasts, with the purpose of application in skin tissue regeneration. The RGD peptide used (JGB2) was chosen by means of computational studies, synthesized by the solid phase method (Fmoc strategy), purified by HPLC and characterized by mass spectrometry. CB membranes were produced by G. xylinuns, purified and functionalized by the esterification of two lysine residues, for incorporation of the JGB2 peptide.Different CB samples were characterized from analyzes of prolonged release of theJGB2 peptide, measurements of contact angle, tensile strength, scanning electron microscopy (SEM-FEG), X-ray diffraction (XRD) and spectroscopy absorption in the infrared region with Fourier Transform (FT-IR). The results showed that membrane functionalization with lysine did not result in significant changes in membrane structure and morphology. Compared to pure CB, CB functionalization increased the amount ofadsorbed peptide and quadrupled membrane peptide release time, desirable characteristics for application in tissue regeneration processes. It was also possible to verify that the functionalized membrane containing the JGB2 peptide presented superior performance in the in vitrocytotoxicity assays, and therefore has great potential to be studied in more detail and eventually be applied as biomaterial in skin tissue repair.
Bacterial celulose (BC) is synthesizedby some bacteria, with greater efficiency by Gluconacetobacter xylinus.BC is a biomaterial that has a network 3-D structure of nanofibers with properties like biocompatibility, non-toxicity, mechanical stability, and high moisture content, whichmakes it promising fora wide range of applications, being that in the engineering of tissue its multifunctionality includes the temporary substitution of epithelial tissue in cases of burns, ulcers, injuries, drug carriers, among other purposes. Although CB present potential to be used as a dressing, it is necessary to perform modifications of its surface with the incorporation of cell adhesion moleculesto adjust the deficiencies of the native CB, in order to improve cell adhesion, migration, proliferation and differentiation, making it a promising material for biomedical applications.In this way, this work aimed to develop a dressing based on BC containing peptide promoter of cell adhesion RGD (Arginine-Glycine-Aspartic acid), sequence found in several extracellular matrix proteins (ECM) and to evaluate in vitroits viability and proliferation of fibroblasts, with the purpose of application in skin tissue regeneration. The RGD peptide used (JGB2) was chosen by means of computational studies, synthesized by the solid phase method (Fmoc strategy), purified by HPLC and characterized by mass spectrometry. CB membranes were produced by G. xylinuns, purified and functionalized by the esterification of two lysine residues, for incorporation of the JGB2 peptide.Different CB samples were characterized from analyzes of prolonged release of theJGB2 peptide, measurements of contact angle, tensile strength, scanning electron microscopy (SEM-FEG), X-ray diffraction (XRD) and spectroscopy absorption in the infrared region with Fourier Transform (FT-IR). The results showed that membrane functionalization with lysine did not result in significant changes in membrane structure and morphology. Compared to pure CB, CB functionalization increased the amount ofadsorbed peptide and quadrupled membrane peptide release time, desirable characteristics for application in tissue regeneration processes. It was also possible to verify that the functionalized membrane containing the JGB2 peptide presented superior performance in the in vitrocytotoxicity assays, and therefore has great potential to be studied in more detail and eventually be applied as biomaterial in skin tissue repair.
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Palavras-chave
Materiais biomédicos, Celulose, Peptídeos, Cicatrização de ferimentos, Citotoxicidade