Evaluación in Vitro de la Biocompatibilidad de Cuatro Cementos Selladores con Osteoblastos Humanos / In vitro Evaluation of Four Sealer Cements Biocompatibility with Human Osteoblasts

RESUMEN: El correcto sellado apical es un paso importante durante el tratamiento de conductos, para esto, se utilizan puntas de gutapercha y cemento sellador, de este último existen diversas formulaciones químicas en el mercado, por lo cual es importante tomar en cuenta los efectos que estas pueden tener en el proceso de cicatrización periapical. El propósito de este estudio fue evaluar la biocompatibilidad de cuatro cementos selladores con diferente composición química con osteoblastos humanos. Se prepararon extractos de cementos selladores a con dos concentraciones (10 mg/mL y 40 mg/mL) y dos tiempos de exposición (10 min y 8 h), estos fueron colocados en contacto con osteoblastos humanos para evaluar la proliferación y citotoxicidad a 24, 72 y 96 h con sus respectivos controles y blancos. Se realizó un análisis estadístico con ANOVA de un factor y la prueba de comparaciones múltiple de Bonferroni. Los resultados obtenidos, tanto en el ensayo de citotoxicidad como en el de proliferación, indicaron que el cemento a base de resina no es biocompatible con osteoblastos. El cemento a base de poli-dimetilxilosano fue el único que no mostró citotoxicidad a ningún de tiempo de exposición y concentración examinadas en este estudio.

ABSTRACT: Correct apical sealing is an important step during root canal treatment, hence, gutta-percha points and sealant are used. There are several chemical compositions on the market, so it is important to evaluate the effects of these in the periapical healing process. The aim of this study was to evaluate the biocompatibility of four sealer cements with different chemical composition placed in contact with human osteoblast. Different extracts were prepared at two concentrations (10 mg/mL and 40 mg/mL) and two exposure times (10 min and 8 h) these were placed in contact with human osteoblast to evaluate cytotoxicity and proliferation at 24, 48 and 72 h with their respective controls and blanks. A statistical analysis was performed with ANOVA of one factor and Bonferroni post hoc. Results obtained in cytotoxicity and proliferation assays, indicated that the resinbased cement is not biocompatible with osteoblast. The poly-dimethylxilosanbased cement was the only that did not show cytotoxicity at any time of exposure and concentration examined in this study.

Parasitaemia levels in Plasmodium chabaudi infected-mice modify IFN-gamma and IL-10 expression after a homologous or heterologous challenge.

CB6F1 mice infected with the nonlethal Plasmodium chabaudi chabaudi AS suffer parasitaemia levels up to 40% (full parasitaemia, FP) and develop both homologous and heterologous (against the lethal Plasmodium yoelii 17XL) protective immunity. However, if mice are treated with anti-malarial drug when parasitaemia is below 10% (low parasitaemia, LP), they only develop homologous immunity. For the better understanding of this interesting dissociation related to the degree of parasitaemia, in this work, we studied the genetic expression of some cytokines. We found that during primary parasitaemia both FP and LP mice showed at first a TNF-alpha, IL-2 and IFN-gamma response which is followed by an IL-4 and IL-10 response. When FP and LP mice were challenged with either the homologous (FP + AS and LP + AS mice) or the heterologous parasite (FP + 17XL and LP + 17XL mice), we observed that LP + 17XL mice, which failed to develop heterologous immunity and succumbed to the challenge, showed a stronger IFN-gamma and a weaker IL-10 expression than FP + 17XL mice, which developed heterologous immunity and survived the challenge. The importance and the possible implications of these findings are discussed.

Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I.

Assessment of cytokine expression has become crucial to understand host responses to infections as well as autoimmunity. Several approaches including Northern blot, RNase protection assay and enzyme-linked immunosorbent assay have been used for this purpose, but they are time consuming, labour intense, and relatively large quantity of the samples is usually required. Recently, a technique termed real-time reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to determine genetic expression with great sensitivity and specificity; however, specialized instrumentation and costly reagents are usually needed. We aimed at using low-cost reagents for real-time PCR. This was achieved by adapting a conventional RT-PCR protocol to the quantitative real-time format, by the addition of the SYBR Green I reagent. We validated the approach by assessing the cytokine gene expression of murine splenocytes upon stimulation with phorbol 12-myristate 12-acetate (PMA)-ionomycin. The results using this technique were compared with those obtained with the well-established gene array method. We conclude that the use of the SYBR Green I reagent during real-time RT-PCR provides a highly specific and sensitive method to quantify cytokine expression with accuracy and no post-PCR manipulation.

Mouse splenic CD4+ and CD8+ T cells undergo extensive apoptosis during a Plasmodium chabaudi chabaudi AS infection.

The presence and phenotype of apoptotic lymphocytes was studied in spleen cell suspensions taken from CB6F1 mice infected with Plasmodium chabaudi chabaudi AS. High levels of apoptotic cells were found, associated with high parasitaemias and splenomegaly. This was also accompanied by expansion and disarray of spleen white pulp. Apoptosis levels lowered when parasitaemia was cleared, but were still higher than in normal mice. At this time, the spleen was diminishing in size and the white pulp was contracting and rearranging. When parasitaemia was patent, the cells most affected by apoptosis were CD4+ T cells followed by CD8+ T cells, and to a lesser extent B220+ B cells. When parasitaemia was cleared, CD8+ T cells and B220+ B cells returned to basal levels of apoptosis, while CD4+ T cells still had higher apoptosis levels than normal mice. A similar pattern of lymphocyte subpopulation apoptosis was found in infected BALB/c mice, despite the fact that, for this mouse model, it has been reported that B cells are the cells that are most affected by apoptosis. We consider that the high levels of apoptosis in CD4+ T cells when parasitaemias are still high are not easily explained by a normal mechanism of down regulation of the immune response.