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Item Amniotic membrane applied to burns healing: Pre-clinical study(CDRR Editors) Amorim, Fernanda Cláudia Miranda; Arisawa, Emilia Ângela Loschiavo; Sant’Anna, Luciana Barros; Fonseca, Khetyma Moreira; Costa, Davidson Ribeiro; Rodrigues, Ana Beatriz Mendes; Carvalho, Jancineide Oliveira deThis preclinical study aimed to evaluate the tissue repair process of burns treated with human amniotic membrane (hAM) patches in rats. Twenty-four rats were subjected to superficial burns of partial thickness, and randomly allocated into two groups: Control and Treated Group, subdivided into two experimental periods of 7th and 14th days. The lesions were evaluated by digitalized images (macroscopy) and by the analysis of histological sections stained in H&E to quantify the number of inflammatory cells and fibroblasts present in the different experimental times (histomorphometry). The histomorphometric analyses were performed blindly. Statistical analysis employed Kolmogorov-Smirnov and Mann Whitney tests, with 95% confidence interval at 5% significance level (p <0.05). Macroscopically, the lesions of Treated group presented a crust formation before Control Group, and there were no signs of infection in both groups. Microscopically, the qualitative analysis showed a faster evolution in the healing process of the Treated groups compared to the Control, with reduction of the inflammatory infiltrate, intense fibroblasts proliferation and better organization of the collagen fibers. The quantitative analysis showed statistically significant results regarding the reduction of inflammatory cells (p<0.0001) at 7th and 14 th day and increased proliferation of fibroblasts at 14th day (p<0.0001) in lesions treated with hAM compared to Control group. The results of this preclinical study demonstrated that the application of hAM patches reduces the inflammatory process and accelerates the onset of the proliferative phase in burn injuries.Item Antimicrobial Effect of the Amniotic Membrane Isolated and Associated with Photodynamic Therapy(MDPI) Santos, Amanda Cerquearo Rodrigues dos; Teodoro, Guilherme Rodrigues; Ferreira-Strixino, Juliana; Sant’Anna, Luciana BarrosMicrobial control through alternative therapies, such as the amniotic membrane (AM) and antimicrobial photodynamic therapy (aPDT), has been gaining prominence with the advancement of bacterial resistance to conventional treatments. This study aimed to evaluate the antimicrobial effect of AM isolated and associated with aPDT using the PHTALOX® as a photosensitizer (PS) against Staphylococcus aureus and Pseudomonas aeruginosa biofilms. The groups studied were: C+; L; AM; AM+L; AM+PHTX; and AM+aPDT. The irradiation parameters were 660 nm, 50 J.cm−2, and 30 mW.cm−2. Two independent microbiological experiments were carried out in triplicate, and the results were analyzed by CFU/mL counting and a metabolic activity test, both statistically analyzed (p < 0.05). The integrity of the AM was verified after the treatments by a scanning electron microscope (SEM). The groups AM, AM+PHTX, and, mainly, AM+aPDT showed a statistical difference When compared to C+ regarding the decrease in CFU/mL and metabolic activity. SEM analysis showed significant morphological alterations in the AM+PHTX and AM+aPDT groups. The treatments with AM isolated or associated with PHTALOX® were adequate. The association had potentiated the biofilm effect, and the morphological differences presented by AM after treatment did not hinder its antimicrobial effect, encouraging its use in biofilm formation locals.Item Effects of Preservation Methods in the Composition of the Placental and Reflected Regions of the Human Amniotic Membrane(Karger) Moraes, Jéssica Tereza Guedes de Oliveira; Costa, Maíra Maftoum; Alves, Paula Cristina Santos; Sant’Anna, Luciana BarrosThe human amniotic membrane (AM) is emerging as an in- teresting biomaterial for regenerative medicine due to its biological and mechanical proprieties. The beneficial effects of the AM are probably related to its bioactive factors pro- duced by local cells and stored in the stromal matrix. How- ever, the search for a preservation method capable of pre- serving AM properties remains a challenge. The aim of this study was to evaluate important features of 2 anatomical re- gions of the human AM (reflected and placental amnion) af- ter different preservation methods. For this purpose, human placentas were harvested and processed for AM isolation and storage at 2 different conditions: room temperature for 18 h in DMEM (fresh AM) and −80°C in DMEM/glycerol solu- tion for 30 days (cryopreserved AM). After the storage peri- od, the structural integrity of the membrane was assessed by histological and Picrosirius polarization analysis, cellular vi- ability analysis was performed using the MTT assay, and the soluble proteins were quantified with the Qubit Protein Assay Kit. Both preservation protocols reduced the cell viabili- ty, mainly in the placental amnion region of the AM, but pre- served the morphology of epithelial and stromal layers, as well as the organization and distribution of collagen fibers. There was a reduction in soluble proteins only in fresh AM. Importantly, the cryopreserved AM group presented the same concentration as the control group. In conclusion, the cryopreservation using DMEM/glycerol was ideal for pre- serving the structural integrity and soluble protein content, indicating the feasibility of this method in preserving AM for its use in regenerative medicine.Item Feridas em pés diabéticos tratadas com membrana amniótica humana e terapia a laser de baixa intensidade: um estudo clínico piloto(HMP) Santos, Joelita de Alencar Fonseca; Nicolau, Renata Amadei; Sant’Anna, Luciana Barros; Paterno, Josne Carla; Cristovam, Priscila Cardoso; Gomes,, Jose Álvaro Pereira; Santos, José Diego Marques; Arisawa, Emilia Angela Lo SchiavoCONTEXTO: A terapia a laser de baixa intensidade (LLLT) e a aplicação de membrana amniótica humana (HAM) demonstraram ser opções viáveis para uso na cicatrização de feridas. OBJETIVO: Este estudo buscou comparar LLLT e HAM a um tratamento controle (hidrogel, solução salina e gaze) em pessoas com diabetes mellitus (DM) e úlceras nos pés. MÉTODOS: Usando um delineamento de estudo clínico piloto prospectivo, foram recrutados pacientes que receberam atendimento em um centro de saúde especializado no tratamento de feridas nos pés diabéticos entre novembro de 2016 e agosto de 2017. Os pacientes elegíveis deveriam ter entre 30 e 59 anos de idade; diagnóstico de DM tipo 2 (níveis de glicemia capilar pós-prandial entre 140 e 350 mg/dL); e ter úlceras nos pés em estágio 2 ou 3, não infectadas e em granulação, medindo menos de 7 cm por 3 cm. Pacientes imunossuprimidos e desnutridos ou aqueles com neoplasias ou em estado crítico não eram elegíveis para participar. Os pacientes receberam o tratamento controle (2 mg de hidrogel, solução salina e gaze), HAM (adesivos de HAM descongelado, aplicados com bordas sobrepostas) ou LLLT (sessão de fototerapia, 2 mg de hidrogel, solução salina e gaze) por 28 dias. Variáveis, medidas da área da ferida, pontuações da Pressure Ulcer Scale for Healing (PUSH) e pontuações da Visual Analog Scale (VAS) foram usadas para avaliar o progresso da melhora da ferida e a dor nos dias 7, 14, 21 e 28. Estatísticas descritivas foram usadas para analisar os perfis antropométricos e clínicos dos participantes. O teste de Kolmogorov-Smirnov foi usado para analisar a distribuição da amostra. O teste de Kruskal-Wallis com pós-teste de Dunn foi usado para avaliar as diferenças nas pontuações PUSH e VAS e no tamanho da ferida para análise intergrupo, e o teste U de Mann-Whitney foi usado para os mesmos resultados na análise intragrupo. O nível de significância foi de 5% ( P < 0,05). RESULTADOS: Vinte e sete (27) pacientes participaram (idade média, 51,4 anos; índice de massa corporal médio, 26,5 kg/m 2 ), com 9 pacientes em cada grupo de tratamento. Não foram observadas diferenças estatisticamente significativas nas variáveis clínicas ou antropométricas entre os grupos, mas as áreas médias basais das feridas foram diferentes (2,6 cm2 para o controle, 1,9 cm2 para o LLLT e 5,5 cm2 para os grupos HAM). As comparações intragrupo mostraram uma redução significativa na pontuação PUSH no grupo LLT entre os dias 0 e 21 (8,2 vs 4,9; P < 0,01) e dias 21 a 28 (4,9 vs 3,2; P < 0,001). Em todos os grupos de tratamento, a redução percentual foi significativamente diferente entre os dias 7 e 28. Nenhum resultado foi significativamente diferente entre os grupos. CONCLUSÃO: A área da ferida da úlcera do pé diabético, bem como os escores PUSH e VAS, apresentaram maior melhora em pacientes com DM que receberam LLLT ou HAM do que no grupo controle, mas as diferenças não foram significativas. Estudos maiores são necessários para comparar essas modalidades de tratamento.Item Evaluation of the antimicrobial action of plasma activated water on amniotic membrane(Springer) Almeida, Felipe Santos de; Doria, Anelise Cristina Osório Cesar; Sant’Anna, Luciana Barrosntroduction The potential use of the Amniotic Membrane (AM) in different applications has been widely studied and the need to guarantee the sterility of this material is essential for its use. Due to the increase in bacterial resistance, it is important to research new sterilization techniques. Material and methods The aim of this study was to analyze the antimicrobial action of plasma-activated reverse osmosis (RO) water on contaminated amniotic membrane using ATCC® strain of Escherichia coli (25922), Klebsiella pneumoniae (13883), Staphylococcus aureus (6538). The AM was contaminated, after which the PAW was placed in contact with the AM for 90 min. Plasma, gliding arc of argon and compressed air were used to activate the water. Results The results obtained were a cell viability of 11% with the refrigerated PAW and 15% with the ambient PAW for K. pneumoniae, 9% with the refrigerated PAW and 13% with the ambient PAW for S. aureus and 10% with the refrigerated PAW and 14% with the ambient PAW for E. coli, observing a better antimicrobial action of the refrigerated PAW. A greater reduction in CFU was observed when using refrigerated PAW + AM. Conclusion It is concluded that PAWs have a significant antimicrobial action, but not enough to perform AM sterilization.Item Amniotic membrane modulates MMP9 and MMP12 gene and protein expression in experimental model of the hepatic fibrosis(Academia Brasileira de Ciências) Alves, Ana Paula da Silva; Teixeira, Roberta Jenniffer Maciel; Silva, Raissa Monteiro da; Canevari, Renata de Azevedo; Sant’Anna, Luciana BarrosHepatic fibrosis is characterized by excessive deposition of collagen in the hepatic parenchyma, which disturbs the normal architecture and function. We have shown that human amniotic membrane (AM) can be used as a patch on the whole liver surface, resulting in an extremely significant reduction in collagen deposition. The aim of this study was to investigate the effects of AM on the matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 12 (MMP12) genes and proteins expression by real time quantitative PCR and immunohistochemistry, respectively, as well as image analysis on biliary fibrosis induced in rats by the bile duct ligation (BDL).Two weeks after the BDL, an AM fragment was applied onto the liver, and four weeks later, the liver samples were collected. MMP9 and MMP12 genes were significantly over expressed in group treated with AM. The immunoexpression of MMP9 and MMP12 was observed in all groups. However, the quantitative image analysis demonstrated an increase of the area occupied only by MMP12 in the livers of AM-treated rats with respect to BDL rats. These findings suggest that the AM exerts its beneficial effects on biliary fibrosis by increasing the MMP12, which in turn reduces the excessive collagen deposition on liver tissue.Item Photobiomodulation and amniotic membrane for treat tendon injury in rats(Academia Brasileira de Ciências) Nicodemo, Mariana de Castro; Arisawa, Emilia Angela Lo Schiavo; Sant’Anna, Luciana Barros; Martins, Rodrigo Álvaro Brandão LopesTendons, complex fibrous structures, are subjected to great tensions, which can give rise to the so-called tendinopathies. This study aimed to evaluate photobiomodulation and human Amniotic Membrane applied as single or combined therapies to treat induced Achilles tendon lesions. Seventy-five rats were divided into five groups (n=15): C- control Sham surgery; I- tendon injury; LA- tendon injury treated with photobiomodulation; AM- tendon injury treated with Amniotic Membrane; LAM- tendon injury + photobiomodulation and Amniotic Membrane, subdivided into three groups (n=5) with analysis at 3, 7, and 14 days. The tendon injuries were made with a 20 g weight released from a mini guillotine onto the ankle in dorsiflexion. AM and LAM groups received an Amniotic Membrane fragment while LA and LAM groups received transcutaneous photobiomodulation, using a 660 nm wavelength laser. The inflammatory cells showed statistical differences between groups C and I (p<0.05), I and AM (p<0.01), I and LA (p<0.05), and I and LAM (p<0.01). Both photobiomodulation and Amniotic Membrane were shown to enhance tendon repair, and the association of photobiomodulation plus Amniotic Membrane was the most effective treatment. We conclude that the association of photobiomodulation plus Amniotic Membrane was effective in accelerating and improving the tendon regeneration process.