Efeitos do Hidrogel/AnxA12-26 na periodontite em camundongos selvagens e geneticamente deficientes para Anexina A1 (AnxA1-/-)
Data
2024-06
Autores
Silva, Bruna Carolina 
Orientadores
Oliani, Sonia Maria
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A periodontite é uma inflamação crônica do periodonto, em que os patógenos bacterianos iniciam uma resposta inflamatória local, que estimula o sistema imune inato e pode progredir por meio dos tecidos periodontais pela ação de citocinas e mediadores biológicos. Diversos estudos clínicos e pré-clínicos têm investigado a eficácia de agentes modulatórios da resposta imune no tratamento adjunto da periodontite, e os resultados têm se mostrado promissores. Considerando as diversas atividades biológicas previamente descritas, a proteína Anexina A1 (AnxA1) é uma importante moduladora endógena do processo inflamatório. Várias pesquisas têm mostrado o efeito protetor do peptídeo mimético Ac2-26 nos processos inflamatórios agudos e crônicos em resposta à infecção. No entanto, as ações da AnxA1 têm sido pouco estudadas na periodontite. O objetivo deste estudo foi avaliar o efeito da aplicação local do Hidrogel/AnxA12-26 sobre a degradação dos tecidos periodontais mineralizados e não mineralizados, associado a progressão da periodontite em camundongos selvagens (WT) e geneticamente deficientes para AnxA1 (AnxA1-/-). A periodontite foi induzida com ligadura no 1° molar superior de camundongos machos C57BL/6, WT e AnxA1-/-(n = 10/grupo), seguida de aplicações locais de Hidrogel/AnxA12-26 e/ou água destilada (H2Od), por 7 e 14 dias. Os seguintes aspectos foram analisados: análises histopatológicas (Hematoxilina-Eosina, Azul de Toluidina e Picrosirius Red), imuno-histoquímicas para as expressões de ANXA1 e FPR2, ainda TNF-α, MMP-13, Runx2 por RT-qPCR, reabsorção óssea por Microtomografia computadorizada (µCT) e para análises de colágeno a Microscopia por Geração de Segundo-Harmônico (SHG). Após lesão, os animais tratados com Hidrogel/AnxA12-26 não apresentaram reações adversas e as avaliações histopatológicas, após 7 e 14 dias, mostraram redução das células inflamatórias no tecido gengival desse grupo experimental. Enquanto, nos camundongos WT e AnxA1-/-tratados com H2Od observamos exacerbado infiltrado inflamatório e aumento dos mastócitos de mucosa (MMCs). A aplicação local do hidrogel na mucosa diminuiu a expressão do mediador pró inflamatório TNF-α, enquanto nos AnxA1-/-a expressão foi significativamente aumentada. O tratamento Hidrogel/AnxA12-26, pelas análises de Picrosirius e SHG, mostra eficiência para impedir degradação do colágeno na matriz extracelular dos tecidos gengivais e redução na expressão de MMP-13. Diferentemente, os grupos tratados com água destilada apresentam maior perda do colágeno, com fibras desorganizadas e irregulares, e aumento na degradação de MMP-13. A maturação óssea foi avaliada pela expressão de Runx2 nos tecidos periodontais monstrando que os animais AnxA1-/- tiveram redução significativa da expressão de Runx2. Além disso, observamos imunorreatividade para ANXA1-FPR2 nas células ósseas (osteócitos, osteoblastos e osteoclastos) em todos os grupos com periodontite. Na análise de µCT, os camundongos AnxA1-/- demonstram maior perda óssea alveolar em 7 dias, enquanto os tratados com Hidrogel/AnxA12-26 não mostram efeitos significativos na periodontite experimental. No entanto, este tratamento reduz o número de osteoclastos nas lacunas de Howship no período de 7 dias. Em conjunto, nossos resultados mostram que o Hidrogel/AnxA12-26 é um biomaterial compatível com os tecidos gengivais, eficiente na regulação do processo inflamatório e remodelamento tecidual, contribuindo assim para aumentar o arsenal terapêutico na doença periodontal.
Periodontitis is a chronic inflammation of the periodontium, in which bacterial pathogens initiate a local inflammatory response, which stimulates the innate immune system and can progress through the periodontal tissues through the action of cytokines and biological mediators. Several clinical and preclinical studies have investigated the effectiveness of immune response modulatory agents in the adjunctive treatment of periodontitis, and the results have been promising. Considering the various biological activities previously described, the Annexin A1 (AnxA1) protein is an important endogenous modulator of the inflammatory process. Several studies have shown the protective effect of the Ac2-26 mimetic peptide on acute and chronic inflammatory processes in response to infection. However, the actions of AnxA1 have been poorly studied in periodontitis. The objective of this study was to evaluate the effect of local application of Hydrogel/AnxA12-26 on the degradation of mineralized and non mineralized periodontal tissues, associated with the progression of periodontitis in wild-type (WT) and genetically deficient mice for AnxA1 (AnxA1-/-). Periodontitis was induced by ligature the 1st upper molar of male C57BL/6, WT and AnxA1-/- mice (n = 10/group), followed by local applications of Hydrogel/AnxA12-26 and/or distilled water (H2Od), for 7 and 14 days. The following aspects were analyzed: histopathological analyzes (Hematoxylin-Eosin, Toluidine Blue and Picrosirius Red), immunohistochemistry for the expressions of ANXA1 and FPR2, as well as TNF-α, MMP-13, Runx2 by RT-qPCR, bone resorption by Microcomputed Tomography (µCT) and for collagen analysis, Second Harmonic Generation Microscopy (SHG After injury, the animals treated with Hydrogel/AnxA12-26 showed no adverse reactions and histopathological evaluations after 7 and 14 days showed a reduction in inflammatory cells in the gingival tissue of this experimental group. However, in the WT and AnxA1-/- mice treated with H2Od we observed an exacerbated inflammatory infiltrate and an increase in mucosal mast cells (MMCs). Local application of the hydrogel to the mucosa decreased the expression of the pro-inflammatory mediator TNF-α, while in AnxA1-/-the expression was significantly increased. The Picrosirius and SHG analyses of the hydrogel/AnxA12-26 treatment showed that it was effective in preventing collagen degradation in the extracellular matrix of gingival tissues and reducing the expression of MMP-13. In contrast, the groups treated with distilled water showed greater loss of collagen, with disorganized and irregular fibres, and increased degradation of MMP-13. Bone maturation was assessed by the expression of Runx2 in periodontal tissues, showing that AnxA1-/-animals had a significant reduction in Runx2 expression. Furthermore, we observed immunoreactivity for ANXA1-FPR2 in bone cells (osteocytes, osteoblasts and osteoclasts) in all groups with periodontitis. In the µCT analysis, AnxA1-/- mice show higher alveolar bone loss at 7 days, while those treated with Hydrogel/AnxA12-26 show no significant effects on experimental periodontitis. However, this treatment reduces the number of osteoclasts in Howship's gaps at 7 days. Taken together, our results show that Hydrogel/AnxA12-26 is a biomaterial compatible with gingival tissues, efficient in regulating the inflammatory process and tissue remodeling, thus contributing to increasing the therapeutic arsenal in periodontal disease.
Periodontitis is a chronic inflammation of the periodontium, in which bacterial pathogens initiate a local inflammatory response, which stimulates the innate immune system and can progress through the periodontal tissues through the action of cytokines and biological mediators. Several clinical and preclinical studies have investigated the effectiveness of immune response modulatory agents in the adjunctive treatment of periodontitis, and the results have been promising. Considering the various biological activities previously described, the Annexin A1 (AnxA1) protein is an important endogenous modulator of the inflammatory process. Several studies have shown the protective effect of the Ac2-26 mimetic peptide on acute and chronic inflammatory processes in response to infection. However, the actions of AnxA1 have been poorly studied in periodontitis. The objective of this study was to evaluate the effect of local application of Hydrogel/AnxA12-26 on the degradation of mineralized and non mineralized periodontal tissues, associated with the progression of periodontitis in wild-type (WT) and genetically deficient mice for AnxA1 (AnxA1-/-). Periodontitis was induced by ligature the 1st upper molar of male C57BL/6, WT and AnxA1-/- mice (n = 10/group), followed by local applications of Hydrogel/AnxA12-26 and/or distilled water (H2Od), for 7 and 14 days. The following aspects were analyzed: histopathological analyzes (Hematoxylin-Eosin, Toluidine Blue and Picrosirius Red), immunohistochemistry for the expressions of ANXA1 and FPR2, as well as TNF-α, MMP-13, Runx2 by RT-qPCR, bone resorption by Microcomputed Tomography (µCT) and for collagen analysis, Second Harmonic Generation Microscopy (SHG After injury, the animals treated with Hydrogel/AnxA12-26 showed no adverse reactions and histopathological evaluations after 7 and 14 days showed a reduction in inflammatory cells in the gingival tissue of this experimental group. However, in the WT and AnxA1-/- mice treated with H2Od we observed an exacerbated inflammatory infiltrate and an increase in mucosal mast cells (MMCs). Local application of the hydrogel to the mucosa decreased the expression of the pro-inflammatory mediator TNF-α, while in AnxA1-/-the expression was significantly increased. The Picrosirius and SHG analyses of the hydrogel/AnxA12-26 treatment showed that it was effective in preventing collagen degradation in the extracellular matrix of gingival tissues and reducing the expression of MMP-13. In contrast, the groups treated with distilled water showed greater loss of collagen, with disorganized and irregular fibres, and increased degradation of MMP-13. Bone maturation was assessed by the expression of Runx2 in periodontal tissues, showing that AnxA1-/-animals had a significant reduction in Runx2 expression. Furthermore, we observed immunoreactivity for ANXA1-FPR2 in bone cells (osteocytes, osteoblasts and osteoclasts) in all groups with periodontitis. In the µCT analysis, AnxA1-/- mice show higher alveolar bone loss at 7 days, while those treated with Hydrogel/AnxA12-26 show no significant effects on experimental periodontitis. However, this treatment reduces the number of osteoclasts in Howship's gaps at 7 days. Taken together, our results show that Hydrogel/AnxA12-26 is a biomaterial compatible with gingival tissues, efficient in regulating the inflammatory process and tissue remodeling, thus contributing to increasing the therapeutic arsenal in periodontal disease.
Descrição
Citação
SILVA, Bruna Carolina. Efeitos do Hidrogel/AnxA12-26 na periodontite em camundongos selvagens e geneticamente deficientes para Anexina A1 (AnxA1-/-). 2024. 74 f. Dissertação (Mestrado em Biologia Estrutural e Funcional) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2024.