DAYANE PIFFER LUCO
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Resumo IPEN-doc 23118 Laminin-based skin substitutes in a burn animal model2016 - STEFFENS, D.; MATHOR, M.B.; SOSTER, P.; VERGANI, G.; LUCO, D.P.; PRANKE, P.Available treatments for skin regeneration are insufficient for promoting healing. The current study has aimed to produce a cutaneous substitute uniting mesenchymal stem cells, keratinocytes, and a PDLLA biomaterial constructed by electrospinning to use in nude mice. Six groups were tested: (1) only PDLLA; (2) only PDLLA/Lam, a hydrolyzed scaffold with the binding of laminin; (3) PDLLA with cells; (4) PDLLA/Lam with cells (n = 6/group) and (5) animals injured without scaffolds (lesion control group) and (6) healthy control group (n = 4/group). All the animals had 1 cm2 defect performed on their backs, removing all the skin. The biomaterials( or scaffolds) were implanted in the mice for up to 9 days. Part of the defect was taken for histology and another for gene expression. Group 2 presented the best appearance with the softest wound. Gene expression analysis showed a considerable increase of TGFb1 expression, increased VEGF and balance of the BAX/ Bcl-2 ratio for the biomaterial groups when compared to the lesion group. Histological analysis showed well-formed tissue in the groups where the biomaterials and biomaterials plus cells were used. In some animals, in which biomaterials and cells were used, the epidermis was formed throughout the length of the wound. In conclusion, these biomaterials are capable of providing support for the growth of cells, indicating that they can be suitable biomaterials for use in tissue engineering.Resumo IPEN-doc 23899 Evaluation of electrospun matrices for the co-cultivation of mesenchymal stem cells and skin keratinocytes2014 - PRANKE, PATRICIA; STEFFENS, DANIELA; SANTI, BRUNA; LUCO, DAYANE P.; MATHOR, MONICA B.The regeneration of skin is an important field for tissue engineering. Currently available treatments are insufficient to prevent scar formation and promote healing of the patient, especially in large burns and chronic wounds. Due to the great need for skin substitutes with the ability of regenerating large amounts of skin, as well as the lack of an ideal replacement, the current study has aimed to produce a cutaneous substitute with a PDLLA polymer as a biomaterial. These, in turn, must be able to serve as a suitable support for cellular growth for the period of time required for tissue regeneration. For this purpose, scaffolds were constructed by the electrospinning technique and divided into 3 groups: 1) PDLLA matrices, 2) PDLLA/NaOH, which were PDLLA scaffolds hydrolyzed with a solution of NaOH 0.75M and 3) PDLLA/Lam, also hydrolyzed with NaOH and in which the protein laminin was linked by covalent binding. They were all constructed with 2 different fiber diameters, with the smallest at the top of the scaffold. These scaffolds were characterized by morphology and fiber diameter and their hydrophilicity or hydrophobicity features. Mesenchymal stem cells were then seeded onto the bottom of the scaffold and, after 24 hours, skin keratinocytes were seeded on the other side. This procedure was performed in all the groups. The groups were evaluated for cell adhesion on the day of the seeding and on days 7, 14 and 21 for viability with WST-8 assay. From day 7, the scaffolds were submitted to an air/liquid system of culture. As a result, the scaffolds presented well formed fibers which were randomly distributed. The treatment of the matrices with NaOH for 15 minutes did not substantially affect the structure of the fibers, but it was enough to hydrophilize the surface of the biomaterials, which is necessary for laminin linkage. The fiber diameter for all the groups was 4.58 μm for the largest fibers and 574 nm for the smallest. The pore size of the scaffolds obtained were approximately 27.5 μm and 3.44 μm, respectively, for the largest and smallest fibers. The linkage of the laminin was confirmed by immunofluorescence assay. For the biological analysis, cell adhesion was greater in the PDLLA/Lam scaffolds with absorbance of 2.268± 0.494, in comparison with 1.264±0.473 for the control (PDLLA scaffold) and 1.159±0.120 for the PDLLA/NaOH scaffold. On day 7 of the viability analysis, the absorbance for the PDLLA scaffold was 1.148±0.411, the PDLLA/NaOH group was 1.380±0.501 and the PDLLA/Lam was 1.990±0.255. On day 14, the absorbance for groups 1, 2 and 3 were 1.032±0.169, 0.755±0.016, and 1.636±0.313, respectively. On day 21, the results were 2.204±0.317, 1.437±0.024, 2.811±0.477 respectively for groups 1, 2, and 3. In general, in terms of the biological analysis, the PDLLA/Lam group showed the best results for cell adhesion and viability tests. Histological analysis is being processed for greater understanding of the behavior of the cells interacted within the scaffolds. In conclusion, the PDLLA scaffolds, mainly the PDLLA/Lam groups, showed good results for the cocultivation of the cells, with good cell adhesion and the presence of viable cells. These biomaterials were capable of providing support for the growth of the cells, which was observed by the increase in the absorbance over time. Therefore, although histological analysis is still in progress, these scaffolds promise to be suitable biomaterials for use in tissue engineering.Artigo IPEN-doc 21376 Criopass laser efficiency as an enhancer of caffeine and caffeisilane C permeation: 3D human skin equivalent as a model barrier2015 - LOPES, PATRICIA S.; LUCO, DAYANE P.; OLIVEIRA, PEDRO G.; RUFINO, IASMIN M.; HERBST, ESTELA B.; GRECCO, GIULIA; MATHOR, MONICA B.; SILVA, HERSON D.T.; ANDREO, MARCIO A.; LEITE SILVA, VANIA R.Artigo IPEN-doc 21373 Determinação da citotoxicidade da cafeina e cafeina com alginato siloxanetriol em adipocitos humanos obtidos de células mesenquimais de lipoaspirado2015 - LOPES, PATRICIA S.; LUCO, DAYANE P.; OLIVEIRA, PEDRO G.; MATHOR, MONICA B.; LEITE SILVA, VANIA R.Artigo IPEN-doc 21245 Development of a biomaterial associated with mesenchymal stem cells and keratinocytes for use as a skin substitute2015 - STEFFENS, DANIELA; MATHOR, MONICA B.; SANTI, BRUNA T.S.; LUCO, DAYANE P.; PRANKE, PATRICIAResumo IPEN-doc 20671 Cytotoxicity, phototoxicity and genotoxicity evaluation of sucupira oil: a potential new cosmetic ingredient2014 - UEMURA, M.T.; OSTROKY, E.A.; MATHOR, M.B.; ANDREO FILHO, N.; KANEKO, T.M.; LUCO, D.P.; BARA, M.T.F.; PAULA, J.R.; LOPES, P.S.Resumo IPEN-doc 20670 Techniques for efficient isolation of human Langerhans cells: assessment of potential allergenic compounds2014 - LUCO, D.P.; STEFFENS, D.; LOPES, P.S.; UEMURA, M.T.; MATHOR, M.B.Resumo IPEN-doc 20669 Three-dimensional human skin model to evaluate the phototoxicity of UVA and UVB photoprotection histological analysis2014 - LUCO, DAYANE P.; LOPES, PATRICIA S.; LEITE-SILVA, VANIA R.; UEMURA, MARCELLY T.; MATHOR, MONICA B.Resumo IPEN-doc 20668 Phototoxicity evaluation of two photoprotectors active cosmetic ingredients at a three-dimensional human skin model2013 - LUCO, DAYANE P.; LOPES, PATRICIA S.; MATHOR, MONICA B.Dissertação IPEN-doc 20224 Padronização de técnicas de isolamento de células de Langerhans imaturas e desenvolvimento de um modelo tridimensional de pele humana para testes de sensibilidade in vitro2014 - LUCO, DAYANE P.A pele é o maior órgão do corpo humano e constitui a principal defesa do organismo contra agentes físicos e químicos, sendo também fundamental para evitar a perda de água por dessecação. Formada por três camadas distintas, mas complementares, sendo as duas principais denominadas derme e epiderme, contendo diferentes tipos celulares, como fibroblastos, queratinócitos, melanócitos, células de Merkel e células de Langerhans, sendo que estas últimas desempenham um papel fundamental na hipersensibilidade de contato. Devido à importância da manutenção da pele saudável para a vida humana, existe uma crescente necessidade da elaboração de substitutos de tecidos para o tratamento de feridos e doentes, assim como, há grande demanda de pele para testes químicos das áreas farmacêutica e cosmética. Outro fator de fundamental importância para o desenvolvimento de métodos alternativos in vitro, é a pressão mundial para que estes testes substituam os modelos animais. Esta abordagem vai de encontro aos novos conceitos de substituição, redução e refinamento na utilização de animais em estudos científicos, ditando o futuro da cultura celular e bioengenharia de tecidos. Graças ao grande desenvolvimento do cultivo celular e descoberta de que as células cultivadas podem ser reagrupadas de acordo com o delineamento experimental, se torna possível à criação de equivalentes dermoepidérmicos para estudos in vitro, como por exemplo, testes de cito e fototoxicidade ou avaliação da fase inicial da reação alérgica e processos de sensibilização da pele. Neste caso, se faz necessária a obtenção de grande quantidade de células de Langerhans imaturas. As células de Langerhans (CLs) são células dendríticas imaturas localizadas na epiderme e epitélio superficial que desempenham um papel central na imunidade da pele, agindo como verdadeiras sentinelas capazes de captar antígenos de contato. Desta forma, foram testados quatro diferentes protocolos para extração e criopreservação destas células, sendo ainda analisadas as suas características morfológicas e fenotípicas. Obtivemos resultados não expressivos quanto ao isolamento, pureza e marcação positiva para CD1a no Protocolo 2 (Expansor de Pele). Os Protocolo 1A (Coleta de Sobrenadante) e 3 (Epiderme + Gradiente de Ficoll Paque) ofereceram altos níveis de células marcadas positivamente para CD1a, apresentando a mesma qualidade de marcação. No entanto, o Protocolo 3 forneceu um maior número de células viáveis, e uma maior pureza da amostra, uma vez que só utiliza a epiderme para a obtenção da suspensão de células, o que o coloca como modelo a ser seguido em posteriores experimentos. Os métodos aqui apontados como mais promissores, podem ser reproduzidos em laboratórios de cultura celular convencionais, contribuindo para aumentar a reprodutibilidade e confiabilidade de resultados experimentais relativos às CLs. Da mesma forma, avaliamos a utilização dos equivalentes de pele humana para a realização de testes in vitro de cito e fototoxicidade, os quais podem de fato reduzir a utilização de modelos animais para identificação do perfil tóxico de uma substância ou de formulações mais complexas.