Biocompatibilidade da resina acrílica de prótese ocular: análise in vitro e in vivo
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Data
2018-11-06
Autores
Silva, Emily Vivianne Freitas da [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O objetivo desse estudo foi avaliar a influência de diferentes ciclos e métodos de polimerização da resina acrílica (RA) branca de próteses oculares sobre a biocompatibilidade de células da conjuntiva humana e resposta inflamatória do tecido subcutâneo de ratos. Para isso, foram confeccionados corpos de prova em RA termopolimerizados em água aquecida (RNAA), por energia de microondas (RNTM) e quimicamente ativados (RNQA). Para a análise in vivo, a resposta inflamatória desses 3 grupos (n=20/grupo) foi avaliada no tecido subcutâneo de 20 ratos Wistar por 7, 15, 30 e 60 dias (d). Células inflamatórias foram contadas no tecido adjacente ao corpo de prova após coloração com hematoxilina e eosina. A análise imunohistoquímica foi realizada para a detecção de IL-1β, IL-6, TNFα, IL-17 e CCL20. Para a análise in vitro, diferentes ciclos de polimerização para cada método citado foram avaliados, totalizando 11 grupos (n=8/grupo). Foram realizadas análises de grau de conversão (GC), MTT, Alamar Blue, ELISA, RT-PCR em tempo real e dupla marcação de Anexina V e iodeto de propídio. Dados quantitativos foram submetidos à Análise de Variância e ao teste de Tukey com significância de 5%. Os resultados qualitativos foram comparados visualmente. Na análise in vivo, houve infiltrado inflamatório moderado para os grupos RNTM e RNQA e leve para o grupo RNAA após 7 d. O infiltrado inflamatório e a imunomarcação dos alvos testados diminuiu gradativamente ao longo dos 60 d. O grupo RNTM exibiu mais células inflamatórias, com exceção do grupo RNAA, que apresentou mais eosinófilos e linfócitos após 15 d, e do grupo RNQA, onde foi observado mais macrófagos em 15 d e neutrófilos em 60 d. Os grupos RNAA e RNQA apresentaram maior imunomarcação de IL-1β após 7 d. O grupo RNQA apresentou maior imunomarcação de IL-1β (15 e 30 d), IL-6 (30 e 60 d), IL-17 (15 e 30 d) e TNF-α (7 d). Os grupos RNAA e RNTM apresentaram maior imunomarcação de TNF-α nos períodos de 15 e 30 d, enquanto o grupo RNTM, aos 60 d. Na análise in vitro, todos os grupos apresentaram proliferação celular maior que 75%. O ciclo longo de polimerização em microondas apresentou menor GC e percentual de proliferação celular no MTT e resultou em grande liberação de IL-2. No ensaio de Alamar Blue, esse grupo apresentou baixo percentual de proliferação celular, assim como o grupo que recebeu ciclo longo de polimerização em água aquecida e grupos submetidos à ativação química. Maior liberação de IL-6 foi observada nos grupos submetidos à ativação química e de IL-23 para o ciclo curto de polimerização em microondas. Maior expressão gênica de TGF β1 ocorreu para o grupo que recebeu ciclo longo de polimerização em água aquecida seguido de 30 min de armazenamento em água. Maior expressão gênica de CASP9 ocorreu para o grupo ativado quimicamente sobre a bancada. Pode-se concluir que os métodos de polimerização por meio de energia de microondas (ciclos longo e curto) e por ativação química desencadearam uma resposta inflamatória mais intensa. Dentre os métodos de polimerização recomendados pelo fabricante, a polimerização em água aquecida apresentou resultados mais satisfatórios.
The aim of this study was to evaluate the influence of different cycles and methods of white color acrylic resin (AR) for ocular prosthesis on the biocompatibility of human conjunctival cells and on the inflammatory response of rat subcutaneous tissue. For this, AR specimens were prepared in water bath (NRWB), by microwave energy (NRME), and chemically activated (ANR). For in vivo analysis, the inflammatory response of these 3 groups (n=20/group) was assessed in subcutaneous tissue of 20 Wistar rats at 7, 15, 30 and 60 days (d). Inflammatory cells were counted in the tissue adjacent to specimen after staining with hematoxylin and eosin. The immunohistochemical analysis was performed for the detection of IL-1β, IL-6, TNFα, IL-17, and CCL20. For in vitro analysis, different cycles of polymerization for each method were evaluated, with a total of 11 groups (n=8/group). The degree of conversion (DC), MTT, ELISA, real-time RT-PCR and Annexin V and propidium iodide assays were performed. Quantitative data were submitted to Analysis of Variance and Tukey test with a 5% significance. Qualitative data were submitted to visual comparison. In in vivo analysis, there was a moderate inflammatory infiltrate for groups NRME and ANR, and a light infiltrate for the group NRWB after 7 d. The inflammatory infiltrate and the immunolabeling of tested targets decreased gradually during the 60 d. The group NRME exhibited the highest number of inflammatory cells, except for the group NRWB, which presented a higher number of eosinophils and lymphocytes after 15 d, and for the group ANR, where a higher number of macrophages and neutrophils were observed at 15 d and at 60 d, respectively. Groups NRWB and ANR showed higher IL-1β immunolabeling after 7 d. The group ANR had the highest immunolabeling of IL-1β (15 and 30 d), IL-6 (30 and 60 d), IL-17 (15 and 30 d), and TNF-α (7 d). Groups NRWB and NRME showed greater immunolabeling in the periods of 15 and 30 d, while the group NRME had also high results at 60 d. In in vitro analysis, all groups showed cell proliferation higher than 75%. The long cycle of polymerization using microwave energy resulted in lower DC and lower percentage of cell proliferation in the MTT assay and in large release of IL-2. In the Alamar Blue assay, this group had a low percentage of cell proliferation, as well as the group that received a long cycle of polymerization in water bath and groups submitted to chemical activation. A higher release of IL-6 was observed in groups submitted to chemical activation and of IL-23, for the short cycle of polymerization in microwave. Higher TGF β1 gene expression occurred for the group that received long cycle of polymerization in water bath followed by 30 min of storage in water. Higher CASP 9 gene expression occurred for the chemically activated group on bench. It can be concluded that the polymerization by microwave energy (long and short cycles) and by chemical activation resulted in higher inflammatory response. Among methods recommended by the manufacturer, the water bath polymerization showed more satisfactory results.
The aim of this study was to evaluate the influence of different cycles and methods of white color acrylic resin (AR) for ocular prosthesis on the biocompatibility of human conjunctival cells and on the inflammatory response of rat subcutaneous tissue. For this, AR specimens were prepared in water bath (NRWB), by microwave energy (NRME), and chemically activated (ANR). For in vivo analysis, the inflammatory response of these 3 groups (n=20/group) was assessed in subcutaneous tissue of 20 Wistar rats at 7, 15, 30 and 60 days (d). Inflammatory cells were counted in the tissue adjacent to specimen after staining with hematoxylin and eosin. The immunohistochemical analysis was performed for the detection of IL-1β, IL-6, TNFα, IL-17, and CCL20. For in vitro analysis, different cycles of polymerization for each method were evaluated, with a total of 11 groups (n=8/group). The degree of conversion (DC), MTT, ELISA, real-time RT-PCR and Annexin V and propidium iodide assays were performed. Quantitative data were submitted to Analysis of Variance and Tukey test with a 5% significance. Qualitative data were submitted to visual comparison. In in vivo analysis, there was a moderate inflammatory infiltrate for groups NRME and ANR, and a light infiltrate for the group NRWB after 7 d. The inflammatory infiltrate and the immunolabeling of tested targets decreased gradually during the 60 d. The group NRME exhibited the highest number of inflammatory cells, except for the group NRWB, which presented a higher number of eosinophils and lymphocytes after 15 d, and for the group ANR, where a higher number of macrophages and neutrophils were observed at 15 d and at 60 d, respectively. Groups NRWB and ANR showed higher IL-1β immunolabeling after 7 d. The group ANR had the highest immunolabeling of IL-1β (15 and 30 d), IL-6 (30 and 60 d), IL-17 (15 and 30 d), and TNF-α (7 d). Groups NRWB and NRME showed greater immunolabeling in the periods of 15 and 30 d, while the group NRME had also high results at 60 d. In in vitro analysis, all groups showed cell proliferation higher than 75%. The long cycle of polymerization using microwave energy resulted in lower DC and lower percentage of cell proliferation in the MTT assay and in large release of IL-2. In the Alamar Blue assay, this group had a low percentage of cell proliferation, as well as the group that received a long cycle of polymerization in water bath and groups submitted to chemical activation. A higher release of IL-6 was observed in groups submitted to chemical activation and of IL-23, for the short cycle of polymerization in microwave. Higher TGF β1 gene expression occurred for the group that received long cycle of polymerization in water bath followed by 30 min of storage in water. Higher CASP 9 gene expression occurred for the chemically activated group on bench. It can be concluded that the polymerization by microwave energy (long and short cycles) and by chemical activation resulted in higher inflammatory response. Among methods recommended by the manufacturer, the water bath polymerization showed more satisfactory results.
Descrição
Palavras-chave
Olho artificial, Resinas acrílicas, Citotoxicidade imunológica, Teste de materiais, Ocular prosthesis