Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs
Autor(a) principal: | |
---|---|
Data de Publicação: | 2012 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFPel - Guaiaca |
Texto Completo: | http://guaiaca.ufpel.edu.br/handle/123456789/2239 |
Resumo: | Physicochemical properties and biological applicability of materials to be used in Tissue Engineering (TE) have great interest in the development of innovations in biotechnology. In dentistry research incomes every day and clarify the possibility to implement therapies for regeneration of dental pulp in clinical practice in a short time period. Such translation will require the ability to build a pulp tissue that completely fills the root canal dentin and produce appropriate vascularization to perform the metabolic exchanges needed for human tissues. To do it, we need achieve some advances; standardization of techniques and materials, which produce completely safe results, is essential to do the translation from the lab assays to RCT in humans. Based on that, the aim of this study was to perform a systematic review of the literature to analyze the knowledge regarding the importance of the interface between stem cells and scaffolds. Thereafter, we identify some gaps of knowledge in this field, as well as the techniques that have been employed today with potential to establish the transition from laboratory research to clinical. Among the obtained results, we have detected that the scaffold s physical properties, although imperative in determining cellular behavior were, little exploited since the advent of pulp stem cells. So, we carried out a study to evaluate the influence of the pore size on the proliferation and differentiation of Dental Pulp Stem Cells (DPSCs) in vitro. In order to obtain two different pore sizes (150-250μm and 251-450μm), sodium chloride was sieved and used as the porogen-inducer. Tooth slices (1-mm thickness) were obtained from recently extracted third molars and after pulp tissue removal, scaffolds with both porogen inducer sizes were prepared using PLLA (Poly-L-lactic acid) inside the pulp chamber. DPSCs (1 x 105 cells) were seeded in the scaffolds with different porosities, in 24-well plates with specific medium. The cell proliferation was evaluated using the WST1 assay at 3, 7, 14 and 21 days intervals. Also, after 21 days of culture, the RNA of seeded cells was extracted using Trizol and RT-PCR technique was used to assess the differentiation of the DPSCs in odontoblasts, using putative odontoblast markers (DSPP, DMP1 and MEPE). RNA from fresh odontoblasts was used as a control. Cell proliferation rate was similar in both scaffolds except for the 14 days period, when the cells seeded in the scaffolds with larger porosities showed higher proliferation (p<0.05). After 21 days DPSCs seeded into the dentin slices expressed the differentiation odontoblastic markers, independently of the pore sizes. The two different pore sizes tested allowed the DPSCs proliferation and differentiation |
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http://lattes.cnpq.br/0691366323307735Sandra Beatriz Chaves TarquinoDemarco, Flávio Fernandohttp://lattes.cnpq.br/5439768560638141Tarquino, Sandra Beatriz ChavesConde, Marcus Cristian Muniz2014-08-20T14:30:09Z2014-01-272014-08-20T14:30:09Z2012-09-25CONDE, Marcus Cristian Muniz. Tissue Engineering applied to Pulp Regeneration: Assessment of Scaffold pore size influence on proliferation and differentiation of Dental Pulp stem Cells.. 2012. 115 f. Tese (Doutorado em Odontologia) - Universidade Federal de Pelotas, Pelotas, 2012.http://guaiaca.ufpel.edu.br/handle/123456789/2239Physicochemical properties and biological applicability of materials to be used in Tissue Engineering (TE) have great interest in the development of innovations in biotechnology. In dentistry research incomes every day and clarify the possibility to implement therapies for regeneration of dental pulp in clinical practice in a short time period. Such translation will require the ability to build a pulp tissue that completely fills the root canal dentin and produce appropriate vascularization to perform the metabolic exchanges needed for human tissues. To do it, we need achieve some advances; standardization of techniques and materials, which produce completely safe results, is essential to do the translation from the lab assays to RCT in humans. Based on that, the aim of this study was to perform a systematic review of the literature to analyze the knowledge regarding the importance of the interface between stem cells and scaffolds. Thereafter, we identify some gaps of knowledge in this field, as well as the techniques that have been employed today with potential to establish the transition from laboratory research to clinical. Among the obtained results, we have detected that the scaffold s physical properties, although imperative in determining cellular behavior were, little exploited since the advent of pulp stem cells. So, we carried out a study to evaluate the influence of the pore size on the proliferation and differentiation of Dental Pulp Stem Cells (DPSCs) in vitro. In order to obtain two different pore sizes (150-250μm and 251-450μm), sodium chloride was sieved and used as the porogen-inducer. Tooth slices (1-mm thickness) were obtained from recently extracted third molars and after pulp tissue removal, scaffolds with both porogen inducer sizes were prepared using PLLA (Poly-L-lactic acid) inside the pulp chamber. DPSCs (1 x 105 cells) were seeded in the scaffolds with different porosities, in 24-well plates with specific medium. The cell proliferation was evaluated using the WST1 assay at 3, 7, 14 and 21 days intervals. Also, after 21 days of culture, the RNA of seeded cells was extracted using Trizol and RT-PCR technique was used to assess the differentiation of the DPSCs in odontoblasts, using putative odontoblast markers (DSPP, DMP1 and MEPE). RNA from fresh odontoblasts was used as a control. Cell proliferation rate was similar in both scaffolds except for the 14 days period, when the cells seeded in the scaffolds with larger porosities showed higher proliferation (p<0.05). After 21 days DPSCs seeded into the dentin slices expressed the differentiation odontoblastic markers, independently of the pore sizes. The two different pore sizes tested allowed the DPSCs proliferation and differentiationPropriedades físico-químicas e aplicabilidade biológica de materiais para aplicação em Engenharia Tecidual (ET) são de grande interesse e crescente importância no desenvolvimento de inovações na área de biotecnologia. Na odontologia as pesquisas avançam a cada dia e demonstram que no futuro será possível aplicar terapias para regeneração da polpa dental na prática clínica. Essa transição demandará a capacidade de construção de um tecido pulpar que preencha completamente o canal radicular, produza dentina e tenha uma vascularização suficiente para realização das trocas metabólicas teciduais. Para isso avanços ainda precisam acontecer; a padronização de técnicas e materiais que produzam resultados seguros é indispensável para que as terapias baseadas nos princípios da ET possam ser utilizadas em ensaios clínicos em humanos. O objetivo desse estudo foi realizar uma revisão sistemática da literatura para analisar as técnicas baseadas em Stem Cell-Based Therapy com potencial de estabelecer a transição das pesquisas laboratoriais para avalições clínicas em um futuro próximo. Após a revisão, algumas lacunas no conhecimento foram claras.. Assim, foi realizado um estudo para avaliar a influência do tamanho de poros de scaffolds, a base de PLLA, na proliferação e diferenciação de DPSCs. Para a obtenção de dois diferentes tamanhos de poros (150-250μm e 251-450μm), utilizamos cloreto de sódio como porógeno. Tooth Slices (1 mm de espessura) foram obtidos de terceiros molares humanos recém extraídos. O espaço correspondente à câmara pulpar foi preenchido com o sal, e então coberto com uma solução PLLA. Após a remoção a polimerização do PLLA e remoção do sal com água destilada foram semeadas DPSC (1 x 105 cells) nos scaffolds com diferentes porosidades. A proliferação celular foi avaliada após períodos específicos (3, 7, 14 e 21 dias) utilizando o método WST1. Após 21 dias em cultivo, realizamos o isolamento do RNA, do tecido produzido nos TS, utilizando Trizol®. Avaliamos a diferenciação odontoblástica através de RT-PCR através da expressão de marcadores odontoblásticos (DSPP, DMP1, MEPE), normalizados contra o GAPDH. O RNA de odontoblastos humanos foi o controle positivo. As taxas de proliferação celular foram similares nos dois grupos experimentais. Entretanto, após 14 dias de cultivo, as células cultivadas nos scaffolds com maiores porosidades apresentaram uma taxa de proliferação significativamente maior (p<0,05). Após 21 dias de cultivo nos TS, as DPSCs expressaram os três MO, independente do tamanho dos poros. Os tamanhos de poros aplicados por nós foram capazes de sustentar a proliferação e diferenciação das DPSC semeadas nos TSapplication/pdfporUniversidade Federal de PelotasPrograma de Pós-Graduação em OdontologiaUFPelBROdontologiaPolpa dentáriaEngenharia tecidualScaffoldCélulas troncoDental pulpTissue engineeringScaffoldStem cellsCNPQ::CIENCIAS DA SAUDE::ODONTOLOGIAEngenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCsTissue Engineering applied to Pulp Regeneration: Assessment of Scaffold pore size influence on proliferation and differentiation of Dental Pulp stem Cells.info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFPel - Guaiacainstname:Universidade Federal de Pelotas (UFPEL)instacron:UFPELORIGINALTese_marcus_cristian_muniz_conde.pdfapplication/pdf3350332http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2239/1/Tese_marcus_cristian_muniz_conde.pdfa91e9eb1b05eba094cafdfee07a22a72MD51open accessTEXTTese_marcus_cristian_muniz_conde.pdf.txtTese_marcus_cristian_muniz_conde.pdf.txtExtracted Texttext/plain208082http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2239/2/Tese_marcus_cristian_muniz_conde.pdf.txt36e7f83dc677782c2282b48bcc174d8bMD52open accessTHUMBNAILTese_marcus_cristian_muniz_conde.pdf.jpgTese_marcus_cristian_muniz_conde.pdf.jpgGenerated Thumbnailimage/jpeg1746http://guaiaca.ufpel.edu.br/xmlui/bitstream/123456789/2239/3/Tese_marcus_cristian_muniz_conde.pdf.jpge1220843d6188504ff33f21d197f1daaMD53open access123456789/22392023-06-19 18:47:17.114open accessoai:guaiaca.ufpel.edu.br:123456789/2239Repositório InstitucionalPUBhttp://repositorio.ufpel.edu.br/oai/requestrippel@ufpel.edu.br || repositorio@ufpel.edu.br || aline.batista@ufpel.edu.bropendoar:2023-06-19T21:47:17Repositório Institucional da UFPel - Guaiaca - Universidade Federal de Pelotas (UFPEL)false |
dc.title.por.fl_str_mv |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs |
dc.title.alternative.eng.fl_str_mv |
Tissue Engineering applied to Pulp Regeneration: Assessment of Scaffold pore size influence on proliferation and differentiation of Dental Pulp stem Cells. |
title |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs |
spellingShingle |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs Conde, Marcus Cristian Muniz Polpa dentária Engenharia tecidual Scaffold Células tronco Dental pulp Tissue engineering Scaffold Stem cells CNPQ::CIENCIAS DA SAUDE::ODONTOLOGIA |
title_short |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs |
title_full |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs |
title_fullStr |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs |
title_full_unstemmed |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs |
title_sort |
Engenharia Tecidual aplicada à regeneração pulpar: Análise da influência das porosidades de um scaffold sobre a proliferação e diferenciação odontoblástica de DPSCs |
author |
Conde, Marcus Cristian Muniz |
author_facet |
Conde, Marcus Cristian Muniz |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/0691366323307735 |
dc.contributor.advisorLattes.por.fl_str_mv |
Sandra Beatriz Chaves Tarquino |
dc.contributor.advisor-co1.fl_str_mv |
Demarco, Flávio Fernando |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://lattes.cnpq.br/5439768560638141 |
dc.contributor.advisor1.fl_str_mv |
Tarquino, Sandra Beatriz Chaves |
dc.contributor.author.fl_str_mv |
Conde, Marcus Cristian Muniz |
contributor_str_mv |
Demarco, Flávio Fernando Tarquino, Sandra Beatriz Chaves |
dc.subject.por.fl_str_mv |
Polpa dentária Engenharia tecidual Scaffold Células tronco |
topic |
Polpa dentária Engenharia tecidual Scaffold Células tronco Dental pulp Tissue engineering Scaffold Stem cells CNPQ::CIENCIAS DA SAUDE::ODONTOLOGIA |
dc.subject.eng.fl_str_mv |
Dental pulp Tissue engineering Scaffold Stem cells |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS DA SAUDE::ODONTOLOGIA |
description |
Physicochemical properties and biological applicability of materials to be used in Tissue Engineering (TE) have great interest in the development of innovations in biotechnology. In dentistry research incomes every day and clarify the possibility to implement therapies for regeneration of dental pulp in clinical practice in a short time period. Such translation will require the ability to build a pulp tissue that completely fills the root canal dentin and produce appropriate vascularization to perform the metabolic exchanges needed for human tissues. To do it, we need achieve some advances; standardization of techniques and materials, which produce completely safe results, is essential to do the translation from the lab assays to RCT in humans. Based on that, the aim of this study was to perform a systematic review of the literature to analyze the knowledge regarding the importance of the interface between stem cells and scaffolds. Thereafter, we identify some gaps of knowledge in this field, as well as the techniques that have been employed today with potential to establish the transition from laboratory research to clinical. Among the obtained results, we have detected that the scaffold s physical properties, although imperative in determining cellular behavior were, little exploited since the advent of pulp stem cells. So, we carried out a study to evaluate the influence of the pore size on the proliferation and differentiation of Dental Pulp Stem Cells (DPSCs) in vitro. In order to obtain two different pore sizes (150-250μm and 251-450μm), sodium chloride was sieved and used as the porogen-inducer. Tooth slices (1-mm thickness) were obtained from recently extracted third molars and after pulp tissue removal, scaffolds with both porogen inducer sizes were prepared using PLLA (Poly-L-lactic acid) inside the pulp chamber. DPSCs (1 x 105 cells) were seeded in the scaffolds with different porosities, in 24-well plates with specific medium. The cell proliferation was evaluated using the WST1 assay at 3, 7, 14 and 21 days intervals. Also, after 21 days of culture, the RNA of seeded cells was extracted using Trizol and RT-PCR technique was used to assess the differentiation of the DPSCs in odontoblasts, using putative odontoblast markers (DSPP, DMP1 and MEPE). RNA from fresh odontoblasts was used as a control. Cell proliferation rate was similar in both scaffolds except for the 14 days period, when the cells seeded in the scaffolds with larger porosities showed higher proliferation (p<0.05). After 21 days DPSCs seeded into the dentin slices expressed the differentiation odontoblastic markers, independently of the pore sizes. The two different pore sizes tested allowed the DPSCs proliferation and differentiation |
publishDate |
2012 |
dc.date.issued.fl_str_mv |
2012-09-25 |
dc.date.accessioned.fl_str_mv |
2014-08-20T14:30:09Z |
dc.date.available.fl_str_mv |
2014-01-27 2014-08-20T14:30:09Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
CONDE, Marcus Cristian Muniz. Tissue Engineering applied to Pulp Regeneration: Assessment of Scaffold pore size influence on proliferation and differentiation of Dental Pulp stem Cells.. 2012. 115 f. Tese (Doutorado em Odontologia) - Universidade Federal de Pelotas, Pelotas, 2012. |
dc.identifier.uri.fl_str_mv |
http://guaiaca.ufpel.edu.br/handle/123456789/2239 |
identifier_str_mv |
CONDE, Marcus Cristian Muniz. Tissue Engineering applied to Pulp Regeneration: Assessment of Scaffold pore size influence on proliferation and differentiation of Dental Pulp stem Cells.. 2012. 115 f. Tese (Doutorado em Odontologia) - Universidade Federal de Pelotas, Pelotas, 2012. |
url |
http://guaiaca.ufpel.edu.br/handle/123456789/2239 |
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Universidade Federal de Pelotas |
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Programa de Pós-Graduação em Odontologia |
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UFPel |
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BR |
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Odontologia |
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Universidade Federal de Pelotas |
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