Ismael Ángel Rodríguez

The effects of fibrin and fibrin-agarose on the extracellular matrix profile of bioengineered oral mucosa

Several studies have developed efficient oral mucosa constructs using different types of scaffold. However, the changes in the morphology and gene and protein expression profile that could occur in these artificial constructs remain unknown. This study compared the histology and expression of several extracellular matrix molecules in human artificial oral mucosa developed using two different types of scaffolds: fibrin and fibrin‐agarose. To that end, bioengineered oral mucosa stromas were constructed from biopsy samples of human oral mucosa and the substitute generated was analyzed at different periods of time in culture. Histological analysis was carried out by light and transmission electron microscopy and the expression of collagen types I, III, and VI, the proteoglycans decorin and biglycan, and the different chains of laminin, were assessed by immunoperoxidase technique. This study found that fibrin scaffolds accelerated fibroblast growth and remodeling of the scaffold, thus enhancing collagen fibrillogenesis. In the fibrin‐agarose scaffold, the morphology and organization of the fibroblasts did not change during the culture period. All extracellular matrix proteins analyzed were expressed in both scaffolds. However, in fibrin scaffolds, these proteins were widely distributed and replaced the scaffold during the follow‐up period. These results show that the substitutes generated showed histological and molecular similarities with native human oral mucosa stroma. In addition, it was observed that the nature of the biomaterial influenced the behaviour of the oral stromal fibroblasts, thereby modulating their growth, protein synthesis, and collagen fibrillogenesis. Copyright

An early and late cytotoxicity evaluation of lidocaine on human oral mucosa fibroblasts

Local anesthetic drugs are extensively used in dentistry. However, the cytotoxic effects of these pharmaceutical compounds remain unclear. In this work, we have evaluated the cell viability and cell function of human oral mucosa fibroblasts exposed to different concentrations of lidocaine for increasing incubation times, using a global screening methods including structural, metabolic and microanalytical analyses. Our results demonstrate that lidocaine is able to alter cell viability and function even at low concentrations and times, although the effect of lidocaine concentration was more important than the incubation time. First, the structural analysis methods revealed that ≥5% concentrations of lidocaine are able to significantly reduce cell viability. Then, the metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-1) assays suggest that concentrations starting from 1% were able to significantly hinder cell physiology. Finally, electron-probe X-ray microanalysis confirmed the deleterious effects of lidocaine and allowed us to demonstrate that these effects are associated to an apoptosis process of cell death. Therefore, care should be taken when lidocaine is clinically used, and the lowest efficient concentrations should always be used. Furthermore, these results suggest that the comprehensive evaluation method used in this work is accurate and efficient for screening of local anesthetics.

Análisis de biocompatibilidad de cementos ionómero de vidrio de alta viscosidad

espanolObjetivo:. El objetivo del presente trabajo ha sido analizar la biocompatibilidad de dos cementos ionomero de vidrio de alta viscosidad (CIVAV) en un modelo experimental in vitro de fibroblastos gingivales humanos utilizando criterios morfologicos, bioquimicos y metabolicos. Metodos: Fibroblastos gingivales humanos fueron cultivados en placa de 24 pocillos en una concentracion de 2x105 celulas/500 µl de medio cultivo DMEM. Las celulas fueron expuestas, durante 72 horas, a discos de 2 mm de diametro y 1 mm de espesor de dos CIVAV EQUIA Forte Fil (EFT) (GC Corporation, Japan) y EQUIA Fil (EFL) (GC Corporation, Japan). Para analizar las posibles alteraciones morfologicas, las celulas fueron examinadas mediante microscopia optica. Se utilizo para analizar proliferacion celular la tecnica de WTS-1 y para detectar alteraciones en la membrana nuclear se utilizo test de ADN libre. El control positivo fue fibroblastos cultivados en medio DMEM (CM) y el control negativo fue fibroblastos incubados en 2% triton X-100 (CT). Para determinar diferencias globales entre todos los grupos experimentales se empleo la prueba de Kruskal-Wallis y una p Resultados: La microscopia optica no mostro alteraciones morfologicas en las celulas expuestas a los ionomeros analizados. Asimismo, la identificacion de ADN mostro un comportamiento homogeneo entre EFT, EFL y el control positivo no existiendo diferencias estadisticamente significativas (p Conclusion: Los CIVAV muestran un comportamiento de biocompatibilidad aceptable en un modelo experimental in vitro de fibroblastos gingivales humanos. Estos biomateriales podrian ser materiales de eleccion para resolver de manera mas simple obturaciones que necesitan realizarse en cavidades en dentina profunda. EnglishObjetive: The objetive of this study was to analize the biocompatibility of two high viscosity glass ionomer cements (CIVAV) in in vitro experimental model of human gingival fibroblast using by metabolic, biochemical and morphology criteria.. Methods: Human gingival fibroblasts were plated on 24-well plate inserts at final concentration of 2x105 cells/500 ul of DMEM culture medium were used. Cells were exposed during 72 hours to discs of 2 mm diameter and 1 mm of thickness of a CIVAV EQUIA Forte Fil (EFT) (GC Corporation, Japan), and EQUIA Fil (EFL) (GC Corporation). To analyze morphological alterations, cells were examined by light microscopy. In addition, for cell proliferation analysis, WTS-1 technique was used and nuclear membrane alterations were determined by quantification of free DNA. The fibroblasts cultured in DMEM medium (CM) were positive control and fibroblasts incubated in 2% Triton X (CT) were negative control. To determine global differences between the experimental groups Kruskal-Wallis test was used (p Results: The results did not show significant morphological changes in cells exposed to biomaterials. Moreover, DNA test showed similar result in EFT, EFL and positive control without differences statistically significant (p Conclusion: CIVAV showed an acceptable behavior of biocompatibility in the in vitro experimental model of human gingival fibroblasts. These biomaterials could be selected in restauration of cavities with highly compromised dentine surface.