Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo

Detalhes bibliográficos
Autor(a) principal: Andrade, Ana Laura Martins de
Data de Publicação: 2019
Tipo de documento: Tese
Idioma: por
Título da fonte: Repositório Institucional da UFSCAR
Texto Completo: https://repositorio.ufscar.br/handle/ufscar/12164
Resumo: Burning, a traumatic lesion of thermal origin affects organ tissues and may be classified according to the layers of affected tissue. The complexity of the tissue repair process has always made burning treatment a challenge, not only due to its severity but also due to its multiple complications. Photobiomodulation (PBM) therapy for burning, especially in the initial phases of the process, promotes better healing and shorter repair time. The use of stem cells is an area of cutaneous lesion treatment that has been greatly explored and has shown progress in the field of tissue engineering, especially with mesenchymal stem cells. The association of both therapies is believed to accelerate the burned tissue repair process, stimulating cell proliferation and aiding the growth of a tissue of quality. Thus, this study aimed to evaluate the laser PBM therapy (660nm) action on the mesenchymal stem cells (MSC) proliferation; identify the biological mechanisms involved in the laser PBM action; establish a efficient experimental model for full thickness burn; and investigate the association of the techniques (PBM and MSCs) on the treatment of full thickness burns. Thus, we performed fours studies, two in vitro and two in vivo. The in vitro studies were performed with two (2D) and tri (3D) dimensional cultures, using mesenchymal stem cells derived from adipose tissue (hASCs) and lase PBM with three different energies (1J, 2J and 6J), with four groups for 2D cultures and four groups for 3D cultures. The 3D culture used a gellan gum spongy-like hydrogel. In both cultures we evaluated the viability, cell proliferation, reactive oxygen species (ROSs) and the inflammatory mediator interleukin-8 (IL-8). The in vivo studies concluded that laser PBM stimulates hASCs proliferation, in addition to maintaining their viability, depending on the energy used in both cultures; in addition to contributing directly to the stimulation of the ROSs and acting in the IL-8 modulation in the 3D culture. The in vivo studies utilized 114 Wistar mice, 18 for the first and 96 for the seconds study. The first study was performed with the soldering iron burn method, with temperature of 150 ºC. The animals were randomized in three groups, G5: burned with 5 seconds of contact with the soldering iron; G10: burned with 10 seconds of contact with the soldering iron; and G20: burning with 20 seconds of contact with the soldering iron. Histopathologial analysis concluded that all groups resulted in full thickness burn, regardless of the time of application. For the second in vivo study, the animals were randomly distributed in four groups: CG: control group, LG: animals treated with lase PBM (660 nm) and 2 J of energy, TCG: animals treated with MSCs and GLCT: animals treated with laser PBM (660 nm and 2J of energy) plus MSCs). The groups treated with laser PBM received irradiations three times a week, in alternated days, for 21 days. The groups treated with MSCs received ten applications of cells in a total volume of 600 μL right after the burning. We performed photographic monitoring in the periods of 3, 7, 14 and 21 days after the beginning of the experiment to evaluate the lesion contraction. Animals were euthanized in four distinct periods (3, 7 , 14 and 21 days after the experimental procedure) and we collected blood and tissue for further histological, immunohistochemistry, immunoenzyme and birefringence analyses. We conclude that the association of MSCs and laser PBM was an efficient method to accelerate the repair process and formation of cutaneous tissue of quality.
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spelling Andrade, Ana Laura Martins deParizotto, Nivaldo Antoniohttp://lattes.cnpq.br/5527703735628408Brassolatti, Patriciahttp://lattes.cnpq.br/8403640648196148http://lattes.cnpq.br/6270999048662693092bf715-ce7e-4390-92b9-18792975ec3d2020-01-24T13:03:27Z2020-01-24T13:03:27Z2019-12-03ANDRADE, Ana Laura Martins de. Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo. 2019. Tese (Doutorado em Fisioterapia) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12164.https://repositorio.ufscar.br/handle/ufscar/12164Burning, a traumatic lesion of thermal origin affects organ tissues and may be classified according to the layers of affected tissue. The complexity of the tissue repair process has always made burning treatment a challenge, not only due to its severity but also due to its multiple complications. Photobiomodulation (PBM) therapy for burning, especially in the initial phases of the process, promotes better healing and shorter repair time. The use of stem cells is an area of cutaneous lesion treatment that has been greatly explored and has shown progress in the field of tissue engineering, especially with mesenchymal stem cells. The association of both therapies is believed to accelerate the burned tissue repair process, stimulating cell proliferation and aiding the growth of a tissue of quality. Thus, this study aimed to evaluate the laser PBM therapy (660nm) action on the mesenchymal stem cells (MSC) proliferation; identify the biological mechanisms involved in the laser PBM action; establish a efficient experimental model for full thickness burn; and investigate the association of the techniques (PBM and MSCs) on the treatment of full thickness burns. Thus, we performed fours studies, two in vitro and two in vivo. The in vitro studies were performed with two (2D) and tri (3D) dimensional cultures, using mesenchymal stem cells derived from adipose tissue (hASCs) and lase PBM with three different energies (1J, 2J and 6J), with four groups for 2D cultures and four groups for 3D cultures. The 3D culture used a gellan gum spongy-like hydrogel. In both cultures we evaluated the viability, cell proliferation, reactive oxygen species (ROSs) and the inflammatory mediator interleukin-8 (IL-8). The in vivo studies concluded that laser PBM stimulates hASCs proliferation, in addition to maintaining their viability, depending on the energy used in both cultures; in addition to contributing directly to the stimulation of the ROSs and acting in the IL-8 modulation in the 3D culture. The in vivo studies utilized 114 Wistar mice, 18 for the first and 96 for the seconds study. The first study was performed with the soldering iron burn method, with temperature of 150 ºC. The animals were randomized in three groups, G5: burned with 5 seconds of contact with the soldering iron; G10: burned with 10 seconds of contact with the soldering iron; and G20: burning with 20 seconds of contact with the soldering iron. Histopathologial analysis concluded that all groups resulted in full thickness burn, regardless of the time of application. For the second in vivo study, the animals were randomly distributed in four groups: CG: control group, LG: animals treated with lase PBM (660 nm) and 2 J of energy, TCG: animals treated with MSCs and GLCT: animals treated with laser PBM (660 nm and 2J of energy) plus MSCs). The groups treated with laser PBM received irradiations three times a week, in alternated days, for 21 days. The groups treated with MSCs received ten applications of cells in a total volume of 600 μL right after the burning. We performed photographic monitoring in the periods of 3, 7, 14 and 21 days after the beginning of the experiment to evaluate the lesion contraction. Animals were euthanized in four distinct periods (3, 7 , 14 and 21 days after the experimental procedure) and we collected blood and tissue for further histological, immunohistochemistry, immunoenzyme and birefringence analyses. We conclude that the association of MSCs and laser PBM was an efficient method to accelerate the repair process and formation of cutaneous tissue of quality.A queimadura é definida como uma lesão traumática de origem térmica que acomete os tecidos orgânicos e pode ser classificada de acordo com a camada de pele atingida. Levando em consideração a complexidade do processo de reparo tecidual envolvido nesse tipo de lesão, o tratamento de queimaduras sempre foi um desafio tanto pela sua gravidade, como pela multiplicidade de complicações. De acordo com a literatura, a utilização da terapia por fotobiomodulação a laser (FBM) no tratamento de queimaduras, especialmente em fases iniciais do processo, promove uma melhor cicatrização e menor tempo de reparo. Outra grande área que vêm sendo investigada para o tratamento de lesões cutâneas e tem se destacado no ramo da engenharia tecidual é a utilização de células-tronco, em especial as mesenquimais. Acredita-se que a associação de ambas as terapias são capaz de acelerar o processo de reparação tecidual de queimaduras, de forma a estimular a proliferação celular e auxiliar a formação de um tecido de qualidade. Assim, o objetivo deste estudo foi avaliar a ação da FBM a laser (660nm) na proliferação de células-tronco mesenquimais (CTMs); identificar os mecanismos biológicos envolvidos na ação da FBM a laser; estabelecer um modelo experimental eficaz de queimadura de espessura total; e investigar a associação das técnicas (FBM e CTMs) no tratamento de queimadura de espessura total. Para isso foram realizados quatro estudos, sendo dois in vitro e dois estudos in vivo. Para a realização dos estudos in vitro foram realizados cultivos bidimensionais (2D) e tridimensionais (3D), sendo utilizado células tronco mesenquimais derivadas de tecido adiposo (hASCs) e a FBM a laser com três diferentes energias (1 J, 2 J e 6 J), sendo assim realizado quatro grupos para o cultivo 2D e quatro grupos para o cultivo 3D. Ressalta-se que para o cultivo 3D foi utilizado um hidrogel esponjoso de gellan gum. Foram avaliados para ambos os cultivos a viabilidade, proliferação celular, espécie reativas de oxigênio (EROs) e o mediador inflamatório interleicina-8 (IL-8). Os estudos in vitro concluíram que a FBM a laser é capaz de estimular a proliferação das hASCs, além de manter a viabilidade das mesmas, dependendo da energia empregada em ambos os cultivos, além de contribuir diretamente para a estimulação de EROs e atuar na modulação da IL-8 no cultivo 3D. Para os estudos in vivo foram utilizados 114 ratos, da linhagem Wister, sendo 18 para o primeiro estudo e 96 para o segundo. Para o primeiro estudo, foi realizado o modelo de queimadura por ferro de solda, com temperatura de 150ºC e os animais foram aleatorizados em três grupos, G5: realização da queimadura com 5 segundos de contado do ferro de solda; G10: realização da queimadura com 10 segundos de contado do ferro de solda; e G20: realização da queimadura com 20 segundos de contado do ferro de solda. Foram realizadas análises histopatológicas que concluíram que independente do tempo de aplicação, todos os grupos resultaram em uma queimadura de espessura total. Para o segundo estudo in vivo, os animais foram distribuídos ao acaso em quatro grupos: GC: grupo controle, GL: animais tratados com a FBM a laser (660nm) e 2 J de energia, GCT: animais tratados com CTMs e GLCT: animais tratados com a associação da FBM a laser (660nm e 2 J de energia) e CTMs. Os grupos tratados pela FBM a laser receberam as irradiações três vezes por semana, em dias alternados durante 21 dias. Os grupos tratados com CTMs receberam dez aplicações das células em volume total de 600μL logo após a indução da queimadura. Foi realizado o monitoramento fotográfico nos períodos de 3, 7, 14 e 21 dias após o início do experimento para avaliar o grau de contração da lesão. Os animais foram sacrificados em quatro períodos distintos (3º, 7º, 14º e 21º dia após o procedimento experimental) sendo realizada a coleta de sangue e dos tecidos para posterior análises histológica, imunohistoquimica, imunoenzimática e de birrefringência. O estudo concluiu que a associação das CTMs com a FBM a laser foi eficaz para acelerar o processo de reparo e formar um tecido cutâneo de qualidade.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CAPES: Código do Financiamento 001FAPESP: 2015/ 17648-9porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Fisioterapia - PPGFtUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessQueimaduraFotobiomodulaçãoLaserterapiaCélulas-troncoBurnPhotobiomodulationLasertherapyStem cellsCIENCIAS DA SAUDE::FISIOTERAPIA E TERAPIA OCUPACIONALEfeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivoEffects of laser photobiomodulation (660nm) associated with mesenchemical stem cells in full thickness burns: in vitro and in vivo studyinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis60016342667-df05-4210-9304-197722e71222reponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALTESE- A.L.M.Andrade_BCO.pdfTESE- A.L.M.Andrade_BCO.pdfTese_Andrade_ALMapplication/pdf4008146https://repositorio.ufscar.br/bitstream/ufscar/12164/3/TESE-%20A.L.M.Andrade_BCO.pdf0f11f577e2be4f18d62c428cb7b7679dMD53Carta-comprovante-da-versão-final-de-teses-e-dissertações.pdfCarta-comprovante-da-versão-final-de-teses-e-dissertações.pdfCarta orientadorapplication/pdf141991https://repositorio.ufscar.br/bitstream/ufscar/12164/5/Carta-comprovante-da-vers%c3%a3o-final-de-teses-e-disserta%c3%a7%c3%b5es.pdff8ec8bb5e1085a0cc845851138bb0a47MD55CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufscar.br/bitstream/ufscar/12164/6/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD56TEXTTESE- A.L.M.Andrade_BCO.pdf.txtTESE- A.L.M.Andrade_BCO.pdf.txtExtracted texttext/plain225878https://repositorio.ufscar.br/bitstream/ufscar/12164/7/TESE-%20A.L.M.Andrade_BCO.pdf.txt29306564f7657cab35bbce2a5156456dMD57Carta-comprovante-da-versão-final-de-teses-e-dissertações.pdf.txtCarta-comprovante-da-versão-final-de-teses-e-dissertações.pdf.txtExtracted texttext/plain1332https://repositorio.ufscar.br/bitstream/ufscar/12164/9/Carta-comprovante-da-vers%c3%a3o-final-de-teses-e-disserta%c3%a7%c3%b5es.pdf.txt956524802caf0e8eb7efa5ca369ef791MD59THUMBNAILTESE- A.L.M.Andrade_BCO.pdf.jpgTESE- A.L.M.Andrade_BCO.pdf.jpgIM Thumbnailimage/jpeg6313https://repositorio.ufscar.br/bitstream/ufscar/12164/8/TESE-%20A.L.M.Andrade_BCO.pdf.jpgd561b6c6a97f51a97381b8d685238062MD58Carta-comprovante-da-versão-final-de-teses-e-dissertações.pdf.jpgCarta-comprovante-da-versão-final-de-teses-e-dissertações.pdf.jpgIM Thumbnailimage/jpeg13777https://repositorio.ufscar.br/bitstream/ufscar/12164/10/Carta-comprovante-da-vers%c3%a3o-final-de-teses-e-disserta%c3%a7%c3%b5es.pdf.jpg9a77e571e91af4276ce99f95c7b61ba6MD510ufscar/121642023-09-18 18:31:48.367oai:repositorio.ufscar.br:ufscar/12164Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:48Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false
dc.title.por.fl_str_mv Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
dc.title.alternative.eng.fl_str_mv Effects of laser photobiomodulation (660nm) associated with mesenchemical stem cells in full thickness burns: in vitro and in vivo study
title Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
spellingShingle Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
Andrade, Ana Laura Martins de
Queimadura
Fotobiomodulação
Laserterapia
Células-tronco
Burn
Photobiomodulation
Lasertherapy
Stem cells
CIENCIAS DA SAUDE::FISIOTERAPIA E TERAPIA OCUPACIONAL
title_short Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
title_full Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
title_fullStr Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
title_full_unstemmed Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
title_sort Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo
author Andrade, Ana Laura Martins de
author_facet Andrade, Ana Laura Martins de
author_role author
dc.contributor.authorlattes.por.fl_str_mv http://lattes.cnpq.br/6270999048662693
dc.contributor.author.fl_str_mv Andrade, Ana Laura Martins de
dc.contributor.advisor1.fl_str_mv Parizotto, Nivaldo Antonio
dc.contributor.advisor1Lattes.fl_str_mv http://lattes.cnpq.br/5527703735628408
dc.contributor.advisor-co1.fl_str_mv Brassolatti, Patricia
dc.contributor.advisor-co1Lattes.fl_str_mv http://lattes.cnpq.br/8403640648196148
dc.contributor.authorID.fl_str_mv 092bf715-ce7e-4390-92b9-18792975ec3d
contributor_str_mv Parizotto, Nivaldo Antonio
Brassolatti, Patricia
dc.subject.por.fl_str_mv Queimadura
Fotobiomodulação
Laserterapia
Células-tronco
topic Queimadura
Fotobiomodulação
Laserterapia
Células-tronco
Burn
Photobiomodulation
Lasertherapy
Stem cells
CIENCIAS DA SAUDE::FISIOTERAPIA E TERAPIA OCUPACIONAL
dc.subject.eng.fl_str_mv Burn
Photobiomodulation
Lasertherapy
Stem cells
dc.subject.cnpq.fl_str_mv CIENCIAS DA SAUDE::FISIOTERAPIA E TERAPIA OCUPACIONAL
description Burning, a traumatic lesion of thermal origin affects organ tissues and may be classified according to the layers of affected tissue. The complexity of the tissue repair process has always made burning treatment a challenge, not only due to its severity but also due to its multiple complications. Photobiomodulation (PBM) therapy for burning, especially in the initial phases of the process, promotes better healing and shorter repair time. The use of stem cells is an area of cutaneous lesion treatment that has been greatly explored and has shown progress in the field of tissue engineering, especially with mesenchymal stem cells. The association of both therapies is believed to accelerate the burned tissue repair process, stimulating cell proliferation and aiding the growth of a tissue of quality. Thus, this study aimed to evaluate the laser PBM therapy (660nm) action on the mesenchymal stem cells (MSC) proliferation; identify the biological mechanisms involved in the laser PBM action; establish a efficient experimental model for full thickness burn; and investigate the association of the techniques (PBM and MSCs) on the treatment of full thickness burns. Thus, we performed fours studies, two in vitro and two in vivo. The in vitro studies were performed with two (2D) and tri (3D) dimensional cultures, using mesenchymal stem cells derived from adipose tissue (hASCs) and lase PBM with three different energies (1J, 2J and 6J), with four groups for 2D cultures and four groups for 3D cultures. The 3D culture used a gellan gum spongy-like hydrogel. In both cultures we evaluated the viability, cell proliferation, reactive oxygen species (ROSs) and the inflammatory mediator interleukin-8 (IL-8). The in vivo studies concluded that laser PBM stimulates hASCs proliferation, in addition to maintaining their viability, depending on the energy used in both cultures; in addition to contributing directly to the stimulation of the ROSs and acting in the IL-8 modulation in the 3D culture. The in vivo studies utilized 114 Wistar mice, 18 for the first and 96 for the seconds study. The first study was performed with the soldering iron burn method, with temperature of 150 ºC. The animals were randomized in three groups, G5: burned with 5 seconds of contact with the soldering iron; G10: burned with 10 seconds of contact with the soldering iron; and G20: burning with 20 seconds of contact with the soldering iron. Histopathologial analysis concluded that all groups resulted in full thickness burn, regardless of the time of application. For the second in vivo study, the animals were randomly distributed in four groups: CG: control group, LG: animals treated with lase PBM (660 nm) and 2 J of energy, TCG: animals treated with MSCs and GLCT: animals treated with laser PBM (660 nm and 2J of energy) plus MSCs). The groups treated with laser PBM received irradiations three times a week, in alternated days, for 21 days. The groups treated with MSCs received ten applications of cells in a total volume of 600 μL right after the burning. We performed photographic monitoring in the periods of 3, 7, 14 and 21 days after the beginning of the experiment to evaluate the lesion contraction. Animals were euthanized in four distinct periods (3, 7 , 14 and 21 days after the experimental procedure) and we collected blood and tissue for further histological, immunohistochemistry, immunoenzyme and birefringence analyses. We conclude that the association of MSCs and laser PBM was an efficient method to accelerate the repair process and formation of cutaneous tissue of quality.
publishDate 2019
dc.date.issued.fl_str_mv 2019-12-03
dc.date.accessioned.fl_str_mv 2020-01-24T13:03:27Z
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dc.identifier.citation.fl_str_mv ANDRADE, Ana Laura Martins de. Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo. 2019. Tese (Doutorado em Fisioterapia) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12164.
dc.identifier.uri.fl_str_mv https://repositorio.ufscar.br/handle/ufscar/12164
identifier_str_mv ANDRADE, Ana Laura Martins de. Efeitos da fotobiomodulação a laser (660nm) associada às células tronco mesenquimais em queimaduras de espessura total: estudo in vitro e in vivo. 2019. Tese (Doutorado em Fisioterapia) – Universidade Federal de São Carlos, São Carlos, 2019. Disponível em: https://repositorio.ufscar.br/handle/ufscar/12164.
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