Publication: Análisis de la biocompatibilidad/citotoxicidad de nanopartículas de plata en pulpa dental
Authors
Manzanera Mondéjar, Ángeles
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Escuela Internacional de Doctorado
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Guerrero Gironés, Julia ; López García, Sergio ; Rivas Mundiña, Berta
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Abstract
Objetivo: Analizar la biocompatibilidad y citotoxicidad de dos tipos de NPsAg, MG-89 y MG-85, con tamaños diferentes y morfologías similares, para comprobar qué dosis son compatibles con células pulpares mesenquimales y si existe diferencias entre ambos materiales.
Material y métodos: Para este estudio de biocompatibilidad se utilizaron dos muestras de NPsAg, MG 89, de morfología esférica y de tamaño 3,67±1,06 nm; y MG 85, de morfología quasi-esférica y de tamaño 18,83±8,86 nm. Los ensayos llevados a cabo fueron: análisis de la concentración de inhibición media (IC50), análisis de la actividad metabólica (MTT) (24, 48, 72 horas), ensayo de la migración celular (24, 48, 72 horas) (cierre de herida), ensayo de inmunofluorescencia (72 horas), medición de las especies reactivas de oxígeno (ROS), ensayo de apoptosis/necrosis (Anexina V/PI) y ensayo de liberación de citoquinas (ELISA para la producción de IL-6 e IL-8). El análisis estadístico se realizó por vía ANOVA unidireccional y la prueba post-hoc de Tukey (p<0,05).
Resultados: Los resultados mostraron un IC50 de 29,9 μg/ml para MG-89, mientras que para MG-85 fue de 21,01 μg/ml por su mayor citotoxicidad. La actividad metabólica se vio afectada en ambos materiales en concentraciones de 50 μg/ml y 25 μg/ml, aunque la viabilidad celular era similar al control en concentraciones inferiores a 5 μg/ml. La migración celular y morfología mostraron resultados equiparables al control para MG-89 cuando las concentraciones eran inferiores a 10 μg/ml. La producción de ROS aumentó para todas las muestras de manera estadísticamente muy significativa desde las dosis más bajas. El ensayo de apoptosis/necrosis muestra una tendencia a una apoptosis programada celular en concentraciones altas, con desplazamiento gradual a necrosis cuando se reduce la concentración. Los resultados de ELISA para la producción de IL-6 e IL-8, indican una respuesta similar al control en todas las concentraciones a excepción de 50 μg/ml y 25 μg/ml, donde la producción de citocinas aumenta para MG-85 y disminuye para MG-89.
Conclusiones: La muestra de NPsAg MG-89 presentó mejores resultados en biocompatibilidad y menos efectos inflamatorios cuando se comparó con MG-85 en celulares mesenquimales de pulpa dental. Los resultados mostraron que únicamente dosis bajas de ambos materiales presentan resultados similares a los del control, siendo esta equivalencia mayor para aquellas NPsAg de menor tamaño y morfología regular (MG-89). La posible respuesta antiinflamatoria de la MG-89 en la concentración de 25 μg/ml, siembra el interés por futuras investigaciones in vitro que permitan dilucidar posibles usos e inclusión en materiales dentales.
Objective: To analyse the biocompatibility and cytotoxicity of two types of AgNPs, MG-89 and MG-85, presenting different sizes but similar morphologies; in order to determine the doses compatibles with dental pulp stem cells and whether differences exist between both materials. Material and methods: Two samples of AgNPs were used for this biocompatibility study: MG-89, spherical morphology and size 3,67± 1,06 nm; and MG-85 size 18,83±8,86 nm and quasi-spherical morphology. The assays performed included: half-maximal inhibitory concentration (IC50), metabolic activity (MTT) (24, 48, 72 hours), cell migration (24, 48, 72 hours) (wound healing), measurement of reactive oxygen species (ROS), apoptosis/necrosis assay (Annexin V/PI), immunofluorescence and inflammatory cytokine release (ELISA for IL-6 and IL-8 production). Statistical analysis was carried out via on-way ANOVA and Tukey’s post-hoc test (p<0,05). Results: The results for the IC50 test were 29,9 μg/ml for MG-89, whereas for MG-85 showed 21,01 μg/ml, indicating higher citotoxicity for the last one. Metabolic activity was reduced in both materials at concentrations of 50 μg/ml and 25 μg/ml, although cell viability was comparable to the control results at concentrations below 5 μg/ml. Cell migration and morphology showed results similar to the control for MG-89 at concentrations below 10 μg/ml. ROS production increased significantly in all samples, even at the lowest doses. The apoptosis/necrosis assay revealed a predominance of programmed apoptosis at higher concentrations, with a gradual shift towards necrosis when concentrations were lowered. ELISA results for cytokines production indicated a similar response to control at all concentrations, except for 50 μg/ml and 25 μg/ml, where IL-6 and IL-8 production increased for MG-85 and decreased for MG-89. Conclusions: The sample MG-89 demonstrated higher biocompatibility and fewer inflammatory effects when compared to MG-85 in dental pulp stem cells. These findings show that only low doses produce results similar to the control, with this equivalence being more evident for the smaller and more regularly shaped AgNPs (MG-89). The posible anti-inflammatory response in MG-89 at 25 μg/ml raises the interest for further in vitro studies to explore potential applications and incroporation into dental materials.
Objective: To analyse the biocompatibility and cytotoxicity of two types of AgNPs, MG-89 and MG-85, presenting different sizes but similar morphologies; in order to determine the doses compatibles with dental pulp stem cells and whether differences exist between both materials. Material and methods: Two samples of AgNPs were used for this biocompatibility study: MG-89, spherical morphology and size 3,67± 1,06 nm; and MG-85 size 18,83±8,86 nm and quasi-spherical morphology. The assays performed included: half-maximal inhibitory concentration (IC50), metabolic activity (MTT) (24, 48, 72 hours), cell migration (24, 48, 72 hours) (wound healing), measurement of reactive oxygen species (ROS), apoptosis/necrosis assay (Annexin V/PI), immunofluorescence and inflammatory cytokine release (ELISA for IL-6 and IL-8 production). Statistical analysis was carried out via on-way ANOVA and Tukey’s post-hoc test (p<0,05). Results: The results for the IC50 test were 29,9 μg/ml for MG-89, whereas for MG-85 showed 21,01 μg/ml, indicating higher citotoxicity for the last one. Metabolic activity was reduced in both materials at concentrations of 50 μg/ml and 25 μg/ml, although cell viability was comparable to the control results at concentrations below 5 μg/ml. Cell migration and morphology showed results similar to the control for MG-89 at concentrations below 10 μg/ml. ROS production increased significantly in all samples, even at the lowest doses. The apoptosis/necrosis assay revealed a predominance of programmed apoptosis at higher concentrations, with a gradual shift towards necrosis when concentrations were lowered. ELISA results for cytokines production indicated a similar response to control at all concentrations, except for 50 μg/ml and 25 μg/ml, where IL-6 and IL-8 production increased for MG-85 and decreased for MG-89. Conclusions: The sample MG-89 demonstrated higher biocompatibility and fewer inflammatory effects when compared to MG-85 in dental pulp stem cells. These findings show that only low doses produce results similar to the control, with this equivalence being more evident for the smaller and more regularly shaped AgNPs (MG-89). The posible anti-inflammatory response in MG-89 at 25 μg/ml raises the interest for further in vitro studies to explore potential applications and incroporation into dental materials.
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