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Doctoral Thesis
DOI
https://doi.org/10.11606/T.23.2019.tde-07012020-192457
Document
Author
Full name
Hector Valentin Caballero Flores
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Machado, Manoel Eduardo de Lima (President)
Beltrán, Enrique Hair Salas
Marques, Marcia Martins
Nabeshima, Cleber Keiti
Title in Portuguese
Desenvolvimento e caracterização de matrizes tridimensionais porosas a base de quitosana, gelatina e dentina em pó para aplicação em endodontia regenerativa
Keywords in Portuguese
Dentina em pó
Gelatina
Genipina
Matrizes sólidas
Quitosana
Abstract in Portuguese
A composição e arquitetura das matrizes desempenham um papel importante na regeneração tecidual, e a interação entre seus componentes pode influenciar no desenvolvimento celular. Assim, o objetivo do presente estudo foi desenvolver e analisar as características morfológicas, físico-químicas, biológicas e antimicrobianas de matrizes reticuladas ou não a base de quitosana, gelatina e dentina em pó. Matrizes sólidas de quitosana pura; quitosana e gelatina reticuladas ou não com genipina; quitosana, gelatina e dentina em pó reticuladas ou não, foram elaboradas através do método de congelamento e liofilização. As características morfológicas das matrizes foram analisadas por microscopia eletrônica de varredura. As características físico-químicas foram analisadas ensaio de degradação, embebição e liberação de proteínas. A citotoxicidade do meio condicionado com as matrizes nas células-tronco da papila dental foi avaliada pelo ensaio de MTT. A citotoxicidade das matrizes foi avaliada pelo ensaio de contato direto. A morfologia e adesão celular nas matrizes foram avaliadas por microscopia eletrônica de varredura e a proliferação celular pelo ensaio de MTT. A diferenciação das células nas matrizes foi avaliada pelo ensaio de vermelho de alizarina. O efeito antimicrobiano das matrizes foi avaliado pelo método de cultura bactéria, e a adesão das bactérias nas matrizes foi observada por microscopia eletrônica de varredura. Os dados obtidos foram comparados com nível de significância de 5%. Assim, todas as matrizes apresentaram estruturas porosas. A reticulação da matriz reduziu a taxa de degradação e embebição. Com exceção da quitosana pura, todas as matrizes liberaram proteínas. A adição de dentina em pó nas matrizes reticuladas compostas por quitosana e gelatina promoveu maior liberação de proteínas, adesão, proliferação e diferenciação celular. Adicionalmente, conferiu-lhe efeito bacteriostático sobre E. faecalis, limitando sua adesão à superfície da matriz. Assim, conclui-se que a matriz reticulada composta por quitosana e gelatina com adição de dentina em pó apresenta características morfológicas, físico-químicas, biológicas e antimicrobianas adequadas para seu potencial emprego na regeneração das estruturas do complexo dentinho-pulpar. Sendo a adição de dentina em pó determinante na melhoria dessas características.
Title in English
Development and characterization of three-dimensional porous chitosan, gelatin and dentin powder scaffolds for regenerative endodontics
Keywords in English
Chitosan
Dentin powder
Gelatin
Genipin
Solid matrix
Abstract in English
The composition and architecture of the scaffolds play an important role in the tissue regeneration, and the interaction between its components can influence the cellular development. Thus, the aim of this study was to develop and analyze the morphological, physicochemical, biological and antimicrobial properties of chitosan-based scaffold associated to gelatin and dentin powder. Solid scaffolds of chitosan-based only; chitosan-based scaffold associated to gelatin cross-linked or not by genipina; chitosan-based associated to gelatin and dentin powder cross-linked or not by genipin, were prepared by the freeze-drying method. The morphology of the scaffolds was analyzed by scanning electron microscopy. The physico-chemical properties were analyzed for degradation, imbibition and release of proteins. The cytotoxicity of the conditioned medium by scaffolds on the stem cells of the dental papilla was evaluated by the MTT assay. The cytotoxicity of the scaffolds was evaluated by direct contact assay. The cell morphology and adhesion on the scaffolds were evaluated by scanning electron microscopy, and cell proliferation by the MTT assay. Cell differentiation on the scaffolds was assessed by the alizarin red assay. The antimicrobial effect of the scaffolds was evaluated by the bacterial culture method, and the adhesion of the bacteria to the scaffolds was observed by scanning electron microscopy. The data obtained were statistically compared at significance level of 5%. All the scaffolds had porous structures. The crosslinking of the scaffolds reduced the degradation and swelling ratio. With the exception of scaffold chitosan-based only, all scaffolds released proteins. The addition of dentin powder in the crosslinked scaffolds of chitosan-based associated to gelatin promoted greater protein release, adhesion, proliferation and cell differentiation. In addition, it resulted to bacteriostatic effect on E. faecalis, limiting its adhesion to the scaffold surface. Thus, it is concluded that the chitosan-based scaffold associated to gelatin and dentin powder cross-linked by genipina has morphological, physico-chemical, biological and antimicrobial properties suitable for its potential use in the endodontic regeneration, and the dentin powder is important for the improvement of these properties.
 
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Publishing Date
2020-09-15
 
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