Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration

Detalhes bibliográficos
Autor(a) principal: Silva, Isabela Sanches Pompeo da
Data de Publicação: 2023
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
Texto Completo: https://www.teses.usp.br/teses/disponiveis/25/25148/tde-17012024-155854/
Resumo: The search for minimally invasive regenerative strategies that induce reparative dentin formation by stimulating resident pulp cells has been studied in the field of Regenerative Dentistry. In this study, the incorporation of bovine bone matrices to gelatin methacrylate (GelMA) was proposed, intending to develop a photopolymerizable biomimetic hydrogel for dentin engineering. Initially, the manufacturing parameters of GelMA were selected in dose-response tests, based on the study of the degradability and viability of pulp cells, with the hydrogels being manufactured with 15% of GelMA and 0.075% of photoinitiator (LAP; Lithium Acylphosphinate), and photoactivated for 30s. Next, microparticles were produced from bovine bone tissue submitted to the decellularization processes (BMdc), where the cellular content was removed, and deproteinization (BMdp), where all organic content was eliminated. From an initial screening, the concentration of 1% (w/v) of bone matrix was selected, as it promotes better cell interaction. Thus, the following groups were established: GelMA-BMdc GelMA incorporated with 1% BMdc; GelMA-BMdp - GelMA incorporated with 1% BMdp; GelMA-nHA - GelMA incorporated with 1% nano-hydroxyapatite; GelMA hydrogel without incorporation (negative control). The hydrogels were prepared, injected, and light-cured in 96-compartment plates, with human pulp cells seeded in their structure. Biological assays of cell viability and proliferation (Live/Dead and Alamar Blue), and odontoblastic differentiation (ALP activity and Alizarin Red) were performed. The hydrogels were evaluated by scanning electron microscopy (SEM), to observe the architecture and evaluate the porosity (ImageJ), and by Fourier transform infrared spectroscopy (FTIR), to evaluate the chemical structure. In addition, a degradability test with enzymatic challenge was performed. Data were analyzed using ANOVA and Tukey tests (=5%). Viable and proliferative cells were observed in all groups without significant differences. The incorporation of bone matrices resulted in a significant increase in ALP activity and mineralized matrix deposition compared to GelMA, and the GelMA-BMdc group showed significantly higher values than all other experimental groups. The FTIR-ATR spectra confirmed the composition of bone matrices and their complexation to gelatin, as well as the chemical binding of BMdc to the structure of GelMA. Incorporating bone matrices did not change the pattern of GelMA degradability; however, BMdp and nHA altered the porosity pattern, generating smaller pore diameter values than pure GelMA. This effect was not observed for GelMA-BMdc. Thus, we conclude that the incorporation of bovine bone matrix submitted to decellularization to GelMA resulted in a stable and porous hydrogel with high bioactivity on human pulp cells, presenting itself as an alternative biomaterial that can be injected and photopolymerized in situ in pulp exposures for the regeneration of dentin.
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spelling Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regenerationAvaliação de hidrogeis de gelatina metacrilada contendo matriz óssea bovina descelularizada aplicados para regeneração dentináriaBone matrixDentinDentinaEngenharia tecidualGelatinGelatinaHidrogéisHydrogelsMatriz ósseaRegeneraçãoRegenerationTissue engineeringThe search for minimally invasive regenerative strategies that induce reparative dentin formation by stimulating resident pulp cells has been studied in the field of Regenerative Dentistry. In this study, the incorporation of bovine bone matrices to gelatin methacrylate (GelMA) was proposed, intending to develop a photopolymerizable biomimetic hydrogel for dentin engineering. Initially, the manufacturing parameters of GelMA were selected in dose-response tests, based on the study of the degradability and viability of pulp cells, with the hydrogels being manufactured with 15% of GelMA and 0.075% of photoinitiator (LAP; Lithium Acylphosphinate), and photoactivated for 30s. Next, microparticles were produced from bovine bone tissue submitted to the decellularization processes (BMdc), where the cellular content was removed, and deproteinization (BMdp), where all organic content was eliminated. From an initial screening, the concentration of 1% (w/v) of bone matrix was selected, as it promotes better cell interaction. Thus, the following groups were established: GelMA-BMdc GelMA incorporated with 1% BMdc; GelMA-BMdp - GelMA incorporated with 1% BMdp; GelMA-nHA - GelMA incorporated with 1% nano-hydroxyapatite; GelMA hydrogel without incorporation (negative control). The hydrogels were prepared, injected, and light-cured in 96-compartment plates, with human pulp cells seeded in their structure. Biological assays of cell viability and proliferation (Live/Dead and Alamar Blue), and odontoblastic differentiation (ALP activity and Alizarin Red) were performed. The hydrogels were evaluated by scanning electron microscopy (SEM), to observe the architecture and evaluate the porosity (ImageJ), and by Fourier transform infrared spectroscopy (FTIR), to evaluate the chemical structure. In addition, a degradability test with enzymatic challenge was performed. Data were analyzed using ANOVA and Tukey tests (=5%). Viable and proliferative cells were observed in all groups without significant differences. The incorporation of bone matrices resulted in a significant increase in ALP activity and mineralized matrix deposition compared to GelMA, and the GelMA-BMdc group showed significantly higher values than all other experimental groups. The FTIR-ATR spectra confirmed the composition of bone matrices and their complexation to gelatin, as well as the chemical binding of BMdc to the structure of GelMA. Incorporating bone matrices did not change the pattern of GelMA degradability; however, BMdp and nHA altered the porosity pattern, generating smaller pore diameter values than pure GelMA. This effect was not observed for GelMA-BMdc. Thus, we conclude that the incorporation of bovine bone matrix submitted to decellularization to GelMA resulted in a stable and porous hydrogel with high bioactivity on human pulp cells, presenting itself as an alternative biomaterial that can be injected and photopolymerized in situ in pulp exposures for the regeneration of dentin.A busca por estratégias regenerativas minimamente invasivas que estimulem a formação de dentina reparadora a partir do estímulo às células pulpares residentes, tem sido estudada no campo da Odontologia Regenerativa. Neste estudo, foi proposta a incorporação de matrizes ósseas bovinas à gelatina metacrilada (GelMA), com a proposta de desenvolver um hidrogel biomimético fotopolimerizável para engenharia da dentina. Inicialmente, os parâmetros de fabricação do GelMA foram selecionados em ensaios dose-resposta, baseados no estudo da degradabilidade e viabilidade de células pulpares, sendo os hidrogéis fabricados com 15% de GelMA e 0,075% de fotoiniciador (LAP; Lithium Acylphosphinate), e fotoativados por 30s. Na sequência, foram produzidas micropartículas de tecido ósseo bovino submetido aos processos de descelularização (BMdc), onde o conteúdo celular foi removido, e de desproteinização (BMdp), onde todo conteúdo orgânico foi eliminado. A partir de um screening inicial, a concentração de 1% (p/v) de matriz óssea foi selecionada, por promover melhor interação celular. Assim, os seguintes grupos foram estabelecidos: GelMA-BMdc GelMA incorporado com 1% de BMdc; GelMA-BMdp - GelMA incorporado com 1% de BMdp; GelMA-nHA - GelMA incorporado com 1% de nano-hidroxiapatita; GelMA hidrogel sem incorporação (controle negativo). Os hidrogéis foram preparados, injetados e fotopolimerizados em placas de 96 compartimentos, sendo células pulpares humanas semeadas em sua estrutura. Os ensaios biológicos de viabilidade e proliferação celular (Live/Dead e Alamar Blue), e de diferenciação odontoblástica (Atividade de ALP e Alizarin Red) foram realizados. Os hidrogéis foram avaliados por microscopia eletrônica de varredura (MEV), para observação da arquitetura e avaliação da porosidade (ImageJ), e por espectroscopia no infravermelho por transformada de Fourier (FTIR), para avaliar a estrutura química. Além disso, foi realizado ensaio de degradabilidade com desafio enzimático. Os dados foram submetidos à análise pelos testes de ANOVA e Tukey (=5%). Células viáveis e proliferativas foram observadas em todos os grupos sem diferenças significativas. A incorporação das matrizes ósseas resultou em aumento significante da atividade de ALP e deposição de matriz mineralizada em comparação ao GelMA, sendo que o grupo GelMA-BMdc apresentou valores significativamente superiores a todos os demais grupos experimentais. Os espectros FTIR-ATR confirmaram a composição das matrizes ósseas e sua complexação à gelatina, bem como ligação química da BMdc à estrutura do GelMA. A incorporação das matrizes ósseas não alterou o padrão de degradabilidade do GelMA; no entanto, a BMdp e nHA alteraram o padrão de porosidade, gerando menores valores de diâmetro de poro em comparação ao GelMA puro. Este efeito não foi observado para o GelMA-BMdc. Assim, concluímos que a incorporação de matriz óssea bovina submetida à descelularização ao GelMA resultou em um hidrogel estável e poroso, com elevada bioatividade sobre células pulpares humanas, apresentando-se como uma alternativa de biomaterial passível de ser injetado e fotopolimerizado in situ em exposições pulpares visando a regeneração da dentina.Biblioteca Digitais de Teses e Dissertações da USPPassos, Diana Gabriela Soares dosSilva, Isabela Sanches Pompeo da2023-09-28info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttps://www.teses.usp.br/teses/disponiveis/25/25148/tde-17012024-155854/reponame:Biblioteca Digital de Teses e Dissertações da USPinstname:Universidade de São Paulo (USP)instacron:USPReter o conteúdo por motivos de patente, publicação e/ou direitos autoriais.info:eu-repo/semantics/openAccesseng2024-02-05T18:04:02Zoai:teses.usp.br:tde-17012024-155854Biblioteca Digital de Teses e Dissertaçõeshttp://www.teses.usp.br/PUBhttp://www.teses.usp.br/cgi-bin/mtd2br.plvirginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.bropendoar:27212024-02-05T18:04:02Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)false
dc.title.none.fl_str_mv Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
Avaliação de hidrogeis de gelatina metacrilada contendo matriz óssea bovina descelularizada aplicados para regeneração dentinária
title Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
spellingShingle Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
Silva, Isabela Sanches Pompeo da
Bone matrix
Dentin
Dentina
Engenharia tecidual
Gelatin
Gelatina
Hidrogéis
Hydrogels
Matriz óssea
Regeneração
Regeneration
Tissue engineering
title_short Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
title_full Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
title_fullStr Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
title_full_unstemmed Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
title_sort Evaluation of gelatin methacryloyl hydrogels containing decellularized bovine bone matrix applied for dentin regeneration
author Silva, Isabela Sanches Pompeo da
author_facet Silva, Isabela Sanches Pompeo da
author_role author
dc.contributor.none.fl_str_mv Passos, Diana Gabriela Soares dos
dc.contributor.author.fl_str_mv Silva, Isabela Sanches Pompeo da
dc.subject.por.fl_str_mv Bone matrix
Dentin
Dentina
Engenharia tecidual
Gelatin
Gelatina
Hidrogéis
Hydrogels
Matriz óssea
Regeneração
Regeneration
Tissue engineering
topic Bone matrix
Dentin
Dentina
Engenharia tecidual
Gelatin
Gelatina
Hidrogéis
Hydrogels
Matriz óssea
Regeneração
Regeneration
Tissue engineering
description The search for minimally invasive regenerative strategies that induce reparative dentin formation by stimulating resident pulp cells has been studied in the field of Regenerative Dentistry. In this study, the incorporation of bovine bone matrices to gelatin methacrylate (GelMA) was proposed, intending to develop a photopolymerizable biomimetic hydrogel for dentin engineering. Initially, the manufacturing parameters of GelMA were selected in dose-response tests, based on the study of the degradability and viability of pulp cells, with the hydrogels being manufactured with 15% of GelMA and 0.075% of photoinitiator (LAP; Lithium Acylphosphinate), and photoactivated for 30s. Next, microparticles were produced from bovine bone tissue submitted to the decellularization processes (BMdc), where the cellular content was removed, and deproteinization (BMdp), where all organic content was eliminated. From an initial screening, the concentration of 1% (w/v) of bone matrix was selected, as it promotes better cell interaction. Thus, the following groups were established: GelMA-BMdc GelMA incorporated with 1% BMdc; GelMA-BMdp - GelMA incorporated with 1% BMdp; GelMA-nHA - GelMA incorporated with 1% nano-hydroxyapatite; GelMA hydrogel without incorporation (negative control). The hydrogels were prepared, injected, and light-cured in 96-compartment plates, with human pulp cells seeded in their structure. Biological assays of cell viability and proliferation (Live/Dead and Alamar Blue), and odontoblastic differentiation (ALP activity and Alizarin Red) were performed. The hydrogels were evaluated by scanning electron microscopy (SEM), to observe the architecture and evaluate the porosity (ImageJ), and by Fourier transform infrared spectroscopy (FTIR), to evaluate the chemical structure. In addition, a degradability test with enzymatic challenge was performed. Data were analyzed using ANOVA and Tukey tests (=5%). Viable and proliferative cells were observed in all groups without significant differences. The incorporation of bone matrices resulted in a significant increase in ALP activity and mineralized matrix deposition compared to GelMA, and the GelMA-BMdc group showed significantly higher values than all other experimental groups. The FTIR-ATR spectra confirmed the composition of bone matrices and their complexation to gelatin, as well as the chemical binding of BMdc to the structure of GelMA. Incorporating bone matrices did not change the pattern of GelMA degradability; however, BMdp and nHA altered the porosity pattern, generating smaller pore diameter values than pure GelMA. This effect was not observed for GelMA-BMdc. Thus, we conclude that the incorporation of bovine bone matrix submitted to decellularization to GelMA resulted in a stable and porous hydrogel with high bioactivity on human pulp cells, presenting itself as an alternative biomaterial that can be injected and photopolymerized in situ in pulp exposures for the regeneration of dentin.
publishDate 2023
dc.date.none.fl_str_mv 2023-09-28
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://www.teses.usp.br/teses/disponiveis/25/25148/tde-17012024-155854/
url https://www.teses.usp.br/teses/disponiveis/25/25148/tde-17012024-155854/
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv
dc.rights.driver.fl_str_mv Reter o conteúdo por motivos de patente, publicação e/ou direitos autoriais.
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Reter o conteúdo por motivos de patente, publicação e/ou direitos autoriais.
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.coverage.none.fl_str_mv
dc.publisher.none.fl_str_mv Biblioteca Digitais de Teses e Dissertações da USP
publisher.none.fl_str_mv Biblioteca Digitais de Teses e Dissertações da USP
dc.source.none.fl_str_mv
reponame:Biblioteca Digital de Teses e Dissertações da USP
instname:Universidade de São Paulo (USP)
instacron:USP
instname_str Universidade de São Paulo (USP)
instacron_str USP
institution USP
reponame_str Biblioteca Digital de Teses e Dissertações da USP
collection Biblioteca Digital de Teses e Dissertações da USP
repository.name.fl_str_mv Biblioteca Digital de Teses e Dissertações da USP - Universidade de São Paulo (USP)
repository.mail.fl_str_mv virginia@if.usp.br|| atendimento@aguia.usp.br||virginia@if.usp.br
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