Interações biológicas de cimentos ósseos a base de silicato de cálcio com diferentes soluções ativadoras: estudo in vitro e in vivo
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Data
2020-02-19
Autores
Santos, Hanna Flavia Santana dos
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Universidade Estadual Paulista (Unesp)
Resumo
Os cimentos de silicatos de cálcio (CaSiO3) podem ser utilizados em tratamentos de reparo ósseo, tanto para aplicações médicas quanto odontológicas. A fim de atender às necessidades da engenharia de tecidos e aprimorar o leque de opções foram produzidos três cimentos de silicatos de cálcio utilizando α-wollastonita como precursora, juntamente com soluções ativadoras com potencial hidrogeniônico neutro, com três diferentes cátions, Na+, K+ e NH4+. A topografia superficial dos cimentos foi analisada por microscopia eletrônica de varredura com canhão de emissão por campo (MEV-FEG). Estudos in vitro, in vivo, relacionados a capacidade de interação com microrganismos e testes biomecânicos foram realizados para avaliar a influência de diferentes soluções ativadoras de fosfato e carbonato nos cimentos produzidos. Nos testes in vitro foram utilizadas células mesenquimais provenientes de fêmures de ratos. Após períodos pré-determinados foram realizados os testes de viabilidade celular, conteúdo de proteína total (PT), atividade de fosfatase alcalina (ALP) e formação de nódulos de mineralização. Para análise da interação microbiana a formação dos biofilmes monotípicos de S. aureus, P. aeruginosa e C. albicans foi mensurada. Posteriormente, os cimentos obtidos, a base de silicato de cálcio, foram submetidos ao estudo in vivo utilizando 20 ratos Wistars que passaram por procedimento cirúrgico para confecção de um defeito crítico de 3,0 mm nas tíbias direita e esquerda. Após a eutanásia, as peças foram submetidas a análise histológica, histomorfométrica e ao teste biomecânico de flexão de três pontos. Os dados obtidos foram estatisticamente analisados pelo teste Anova um e dois fatores, com nível de significância de 5%. Foi possível analisar que nenhum cimento se mostrou citotóxico, e todos permitiram a adesão e proliferação celular. As células em contato com os cimentos e meios de cultura se mostraram metabolicamente ativas pela expressão de proteínas total, porém, o grupo G-NH4 obteve destaque devido a expressão superior de fosfatase alcalina, demonstrando maior indução de diferenciação celular. Na análise histológica observou-se proliferação óssea em todos os grupos, sem diferença estatística entre os grupos. Na mensuração de força máxima e força de ruptura observou-se diferença estatisticamente significante entre o grupo G-NH4 e os demais grupos. Sendo assim, o silicato de cálcio produzido com solução ativadora de fosfato de amônio bifásico, na fase α-wollastonita, se mostrou promissor para engenharia de materiais para regeneração tecidual óssea.
Calcium silicate cements (CaSiO3) can be used in bone repair treatments, both for medical and dental applications. In order to meet the needs of tissue engineering and improve the range of options, three calcium silicate cements were produced using αwollastonite as a precursor, with an activating solution of neutral potential hydrogen ionic, made of three different cations Na+, K+ and NH4 +. A superficial topography of the cements was analyzed by Field Emission Gun Scanning Electron Microscopy (SEMFEG). In vitro, in vivo studies related to the ability to interact with microorganisms and biomechanical tests were carried out to evaluate the influence of different phosphate and carbonate activating solutions in the cements produced. In in vitro tests, mesenchymal cells from rat femurs were used. After predetermined periods, cell viability tests, total protein content (PT), alkaline phosphatase activity (ALP) and formation of mineralization nodules were performed. For the analysis of microbial interaction, the formation of monotypic biofilms from S. aureus, P. aeruginosa and C. albicans was measured. Subsequently, the obtained cements, based on calcium silicate, were subjected to an in vivo study using 20 Wistars rats that underwent a surgical procedure to make a 3.0 mm critical defect in the right and left tibiae. After euthanasia, the pieces were submitted to histological, histomorphometric analysis and biomechanical flexion test of three points. The data obtained were statistically analyzed by the Anova test, one and two factors, with a significance level of 5%. It was possible to analyze that no cement was shown to be cytotoxic, and all allowed cell adhesion and proliferation. The cells in contact with the cements and culture media were shown to be metabolically active by the expression of total proteins, however, the G-NH4 group was highlighted due to the superior expression of alkaline phosphatase, demonstrating greater induction of cell differentiation. In the histological analysis, bone proliferation was observed in all groups, with no statistical difference between groups. In measuring the maximum strength and tensile strength it showed a statistically significant difference between the G-NH4 group and the other groups. Thus, the calcium silicate produced with a biphasic ammonium phosphate activating solution, in the α-wollastonite phase, proved to be promising for engineering materials for bone tissue regeneration.
Calcium silicate cements (CaSiO3) can be used in bone repair treatments, both for medical and dental applications. In order to meet the needs of tissue engineering and improve the range of options, three calcium silicate cements were produced using αwollastonite as a precursor, with an activating solution of neutral potential hydrogen ionic, made of three different cations Na+, K+ and NH4 +. A superficial topography of the cements was analyzed by Field Emission Gun Scanning Electron Microscopy (SEMFEG). In vitro, in vivo studies related to the ability to interact with microorganisms and biomechanical tests were carried out to evaluate the influence of different phosphate and carbonate activating solutions in the cements produced. In in vitro tests, mesenchymal cells from rat femurs were used. After predetermined periods, cell viability tests, total protein content (PT), alkaline phosphatase activity (ALP) and formation of mineralization nodules were performed. For the analysis of microbial interaction, the formation of monotypic biofilms from S. aureus, P. aeruginosa and C. albicans was measured. Subsequently, the obtained cements, based on calcium silicate, were subjected to an in vivo study using 20 Wistars rats that underwent a surgical procedure to make a 3.0 mm critical defect in the right and left tibiae. After euthanasia, the pieces were submitted to histological, histomorphometric analysis and biomechanical flexion test of three points. The data obtained were statistically analyzed by the Anova test, one and two factors, with a significance level of 5%. It was possible to analyze that no cement was shown to be cytotoxic, and all allowed cell adhesion and proliferation. The cells in contact with the cements and culture media were shown to be metabolically active by the expression of total proteins, however, the G-NH4 group was highlighted due to the superior expression of alkaline phosphatase, demonstrating greater induction of cell differentiation. In the histological analysis, bone proliferation was observed in all groups, with no statistical difference between groups. In measuring the maximum strength and tensile strength it showed a statistically significant difference between the G-NH4 group and the other groups. Thus, the calcium silicate produced with a biphasic ammonium phosphate activating solution, in the α-wollastonite phase, proved to be promising for engineering materials for bone tissue regeneration.
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Biocerâmica, Reparo ósseo, Diferenciação osteoblástica, Biofilme, Bioceramics, Bone repair, Osteoblastic differentiation, Biofilm