Publicação
Macrophage Activation by the Skin2 Dermal Matrix
| Resumo: | Autograft skin transplantation remains the gold standard to treat severe skin loss, but due to its limitations, regenerative therapies have been developed. Commercially available skin substitutes have not yet achieved effective anatomical, functional, and aesthetic results. In this context, this dissertation is involved in the development of the dermal layer of a biosynthetic skin substitute: Skin2. It intends to study the matrix-macrophages interactions to preview the body response to the Skin2 substitute, where macrophages are crucially involved, and to understand the role of macrophages and their secreted mediators in the dermal matrix degradation in the context of a regenerating wound. Additionally, it investigates alternative fabrication processes to those applied in previous studies. Freeze-dried 3D porous scaffolds and films made of chitosan, gelatin, polycaprolactone and combinations of these polymers were fabricated with an endotoxin level respecting the established medical limit. Sodium hydroxide, hydrogen peroxide and dry heat proved to be effective depyrogenation methods. Crosslinking by dehydrothermal and vapor phase glutaraldehyde achieved efficient results, but further research should be conducted regarding films crosslinking by the latter method. Macrophages studies were carried in vitro using THP-1 cells as model. Macrophage behaviour was first studied without the presence of the dermal matrix. Differentiation occurred as expected; macrophages were viable 15 days after their differentiation and polarization towards an M1 phenotype proved effective. The expression of TNF-α gene by cells cultured in contact with chitosan and gelatin blended films treated at 180 ◦C suggested a polarization towards an M1 phenotype. Moreover, when in the presence of macrophages, degradation of films was very low or even absent, while chitosan and crosslinked chitosan/gelatin scaffolds were degraded. Further research should be conducted in several domains, especially in the optimization of immunostaining techniques and polarization studies of macrophages in contact with the dermal matrix. |
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| Autores principais: | Gomes, Madalena Lopes Natário Pinto |
| Assunto: | Chitosan gelatin polycaprolactone freeze-drying film depyrogenation |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Autograft skin transplantation remains the gold standard to treat severe skin loss, but due to its limitations, regenerative therapies have been developed. Commercially available skin substitutes have not yet achieved effective anatomical, functional, and aesthetic results. In this context, this dissertation is involved in the development of the dermal layer of a biosynthetic skin substitute: Skin2. It intends to study the matrix-macrophages interactions to preview the body response to the Skin2 substitute, where macrophages are crucially involved, and to understand the role of macrophages and their secreted mediators in the dermal matrix degradation in the context of a regenerating wound. Additionally, it investigates alternative fabrication processes to those applied in previous studies. Freeze-dried 3D porous scaffolds and films made of chitosan, gelatin, polycaprolactone and combinations of these polymers were fabricated with an endotoxin level respecting the established medical limit. Sodium hydroxide, hydrogen peroxide and dry heat proved to be effective depyrogenation methods. Crosslinking by dehydrothermal and vapor phase glutaraldehyde achieved efficient results, but further research should be conducted regarding films crosslinking by the latter method. Macrophages studies were carried in vitro using THP-1 cells as model. Macrophage behaviour was first studied without the presence of the dermal matrix. Differentiation occurred as expected; macrophages were viable 15 days after their differentiation and polarization towards an M1 phenotype proved effective. The expression of TNF-α gene by cells cultured in contact with chitosan and gelatin blended films treated at 180 ◦C suggested a polarization towards an M1 phenotype. Moreover, when in the presence of macrophages, degradation of films was very low or even absent, while chitosan and crosslinked chitosan/gelatin scaffolds were degraded. Further research should be conducted in several domains, especially in the optimization of immunostaining techniques and polarization studies of macrophages in contact with the dermal matrix. |
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