Microscopía electrónica con electrones secundarios y retrodispersos en el esmalte del diente fluorótico

Introducción: El objetivo del presente trabajo consistió en definir los patrones morfológicos del esmalte en dientes fluoróticos mediante la utilización de la Microscopía Electrónica de Barrido con electrones secundarios y retrodispersos. Material y Métodos: Se estudiaron 20 piezas dentarias permanentes de pacientes con fluorosis dental y 5 dientes controles. Todas las muestras fueron procesadas para su estudio morfológico. Resultados: Se distinguen tres zonas en dientes fluoróticos: zona externa, zona subsuperficial o lesión fluorótica y zona interna. La zona subsuperficial presenta un triple patrón morfológico característico. Discusión: Este estudio permite sistematizar patrones estructurales que en un futuro pueden constituir una base para el desarrollo de futuras estrategias reparativas en dientes fluoróticos.Introduction: The aim of the present study was to establish histological patterns of fluorotic teeth by scanning electron microscopy (SEM) using secondary and retrodispersive electrons. Material and Methods: We studied 20 permanent teeth belonging to pacients whith dental fluorosis and 5 control teeth. All samples were processed for morphological study and scanning electron microscopy (SEM). Results: We distinguished three areas in fluorotic teeth: external area, subsuperficial area (or fluorotic lesion) and internal area. Subsuperficial area showed three different types of morphological patterns, which were characteristic of fluorotic lesions. Discussion: This study provides systematic structural patterns which in the future can provide a basis for developing future strategies reparative in fluorotic teeth

Mineralization of human premolar occlusal fissures: a quantitative histochemical microanalysis

The mechanisms of cariogenesis in occlusal fissures remain elusive because of limited information about fissure structure and wall mineralization. The purpose of the present study was to determine the correlation between morphological patterns in occlusal fissures in human premolars and quantitative histochemical patterns of mineralization in the walls of these formations. We used scanning electron microscopy and quantitative X-ray microanalysis with the peak-tolocal background ratio method and microcrystalline calcium salts as standards. We distinguished three morphological patterns of fissures in scanning electron microscopic images. The wall of the fissures was less mineralized than the control enamel in all three types of fissures. Because the fissure walls are hypomineralized, we suggest that practicing dentists should take into account the degree of mineralization when they are preparing the fissures for the application of sealant.This work was partially supported by the Ministerio de Educación y Cultura (PB97-0840) and the Agencia Española de Cooperación Internacional (AECI)

Biofabrication of a Tubular Model of Human Urothelial Mucosa Using Human Wharton Jelly Mesenchymal Stromal Cells

Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin–agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results showed that the biofabrication methods used here succeeded in generating a tubular structure consisting of a stromal substitute with a stratified epithelial-like layer on top, especially using a medium containing epithelial growth and differentiation factors (EM), although differentiation was not complete. At the functional level, UM substitutes were able to synthesize collagen fibers, proteoglycans and glycosaminoglycans, although the levels of control UM were not reached ex vivo. Epithelial differentiation was partially achieved, especially with EM after 14 days of development, with expression of keratins 7, 8, and 13 and pancytokeratin, desmoplakin, tightjunction protein-1, and uroplakin 2, although at lower levels than controls. These results confirm the partial urothelial differentiative potential of HWJSC and suggest that the biofabrication methods explored here were able to generate a potential substitute of the human UM for future clinical use.CTS-115 Tissue Engineering Group and by the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministry of Science and Innovation, Instituto de Salud Carlos III, grant FIS PI21/0981 (cofinanced by FEDER funds, European Union)

Evaluation of cell viability and apoptotic patterns in stem cells isolated from human dental pulp

Introducción: El desarrollo de sustitutos biológicos mediante Ingeniería Tisular requiere de la utilización de una fuente de células madre que, además de ser capaz de autorenovarse y diferenciarse, mantengan una funcionalidad y viabilidad óptima justo en el momento de su uso. El estudio de la viabilidad celular constituye un importante control de calidad, especialmente en aquellas poblaciones celulares con un alto potencial para su utilización en Ingeniería Tisular, como son las células madre de la pulpa dental (DPSC). El objetivo de este trabajo es la evaluación de la viabilidad durante los tres primeros subcultivos de células madre de la pulpa dental humana (hDPSC). Material y métodos: Se obtuvieron 3 subcultivos consecutivos de hDPSC de terceros molares humanos sanos (N = 3) mediante un proceso de digestión enzimática. La concentración intracelular de los iones sodio (Na), potasio (K), y cloro (Cl) fue determinada mediante microscopía analítica por energía dispersiva de rayos X (EPXMA). El porcentaje de células en apoptosis (fragmentación de DNA) fue determinado mediante el ensayo de fluorescencia TUNEL en el primer y tercer subcultivo. Resultados: En el segundo subcultivo se detectó un descenso significativo del potasio (p = 0,011) indicando un descenso en la viabilidad celular. En el tercer subcultivo, los niveles de cloro y sodio aumentaron de forma significativa (p = 0,010 y p = 0,002), generando un perfil iónico compatible con células en estado apoptótico. La fluorescencia a partir del ensayo TUNEL reveló 2,76 ± 1,80 % de células apoptóticas en el tercer subcultivo, mientras que en el primer subcultivo, dicha proporción fue de 0,55 ± 0,27%. Discusión: Las hDPSC se encuentran de forma nativa en la pulpa dental y estas condiciones nativas se pueden alterar cuando pasan a las condiciones de un medio de cultivo in vitro. Estas alteraciones pueden ser la respuesta a un proceso de adaptación. Dicha adaptación, junto con el estrés adicional generado por el tratamiento enzimático realizado para digerir la matriz extracelular, tiene como consecuencia una pérdida transitoria y leve de la viabilidad producida por un mecanismo de apoptosis. En resumen, los 3 primeros subcultivos de hDPSC deberán ser descartados para su uso en ingeniería tisular, por ser células en un estado de apoptosis activa.Introduction: The development of biological substitutes by Tissue Engineering needs the use of a stem cell source able not only to self-replicate and differentiate, but also to maintain optimal functionality and cell viability when used. The evaluation of cell viability is considered an important quality control, especially in cell lineages with high capabilities for being used in tissue engineering, as dental pulp stem cells (DPSC). The aim of this research is to evaluate the cell viability through three human dental pulp stem cells (hDPSC) subcultures. Materials and methodology:Three consecutive hDPSC subcultures were obtained from human sound third molars (N = 3) by enzymatic digestion. Intracellular ionic concentration of sodium (Na), potassium (K) and chlorine (Cl) was determined by electron probe X-ray microanalysis (EPXMA). Apoptotic cells percentage (DNA fragmentation) was determined by TUNEL fluorescence assay at first and third subculture Results: A significant decrease of potassium was detected at second subculture (p = 0,011), suggesting a loss of cell viability. At third subculture, chlorine and sodium statistically increased (p = 0,010 y p = 0,002), inducing an apoptotic-like ionic profile. Fluorescence from TUNEL assay revealed 2,76 ± 1,80 % of apoptotic cells at third subculture, while that ratio was 0,55 ± 0,27% at first subculture. Discussion: hDPSC are natively stored in the dental pulp and these native conditions may be altered by the in vitro cell culture conditions. In this regard, these alterations could be in response to an adaptative process. This adaptative process, besides cell stress as a consequence of an enzymatic treatment for the extracellular matrix digestion, produces a mild and temporary loss of cell viability by apoptosis. In summary, the first, second and third subculture of hDPSC should be discarded for the use in tissue engineering as they are apoptotic

Type I and type VII agarose characterization for use in tissue engineering

Objetivos: El objetivo del presente trabajo es el desarrollo de un nuevo protocolo para la inclusión de geles de agarosa en parafina que permita su estudio en histología, y la posterior caracterización de dos tipos distintos de agarosa, Tipo I y Tipo VII, para su uso en Ingeniería Tisular. Métodos: Se evaluaron distintos protocolos que permitiesen un correcto procesamiento de los geles de agarosa permitiendo obtener cortes de dichos geles incluidos en parafina. Una vez optimizado el protocolo, se realizó una caracterización de las agarosas tipo I y VII para su utilización en Ingeniería Tisular. Para ello se obtuvieron geles de agarosa de ambos tipos a concentraciones del 2% y 2.5%, y en cuyo interior había condrocitos. Se evaluó la permeabilidad de la membrana celular mediante la medida por espectrofotometría del ADN liberado al medio, el tamaño celular de los condrocitos cultivados y el estado celular mediante inmunohistoquímica frente a PCNA y Vimentina. También se realizó un estudio sobre la resistencia a la compresión que presentan los distintos geles con el paso del tiempo. Resultados: Tres de los protocolos propuestos hacían posible el procesamiento de los geles y permitieron la caracterización de los distintos tipos de agarosa. En este caso, el estudio de las agarosas tipo I y VII ha demostrado que los condrocitos crecen correctamente en ambos tipos de agarosas, pero que la tipo VII favorece el desarrollo de los mismos frente a la Tipo I. Además, la agarosa pierde elasticidad con el paso de los días soportando a la vez menos fuerza de compresión. Conclusiones: El Protocolo propuesto para el procesamiento y posterior inclusión de geles de agarosa en parafina en este trabajo permite realizar histología en los mismos, y ha permitido confirmar que la agarosa tipo VII favorece el desarrollo y supervivencia celular.Purpose: The objective of the present work is the development of a new protocol for the inclusion of paraffinembedded agarose gels to allow their study in histology and the subsequent characterization of two different types of agarose, Type I and Type VII, for their use in Tissue Engineering. Method: Different protocols were evaluated for a correct processing of the agarose gels allowing to obtain cuts of paraffin-embedded agarose gels. Once the protocol was optimized, a characterization of type I and VII agarose was performed for their use in tissue engineering. For this purpose, agarose gels of both types were obtained at concentrations of 2% and 2.5%, and in which chondrocytes were present. The permeability of the nuclear membrane was measured by spectrophotometry of the DNA released, the cell size of the cultured chondrocytes and the cellular state by immunohistochemistry against PCNA and Vimentin. A study on the compressive strength of different gels over time was also performed. Results: Three of the proposed protocols allowed the processing of the gels and allowed the characterization of the different types of agarose. In this case, the study of the type I and VII agarose has shown that chondrocytes grow correctly in both types of agarose, but type VII favors the development of the same against Type I. In addition, the agarose loses elasticity with the day pass while supporting less compressive force. Conclusions: The proposed protocol for the processing and subsequent inclusion of paraffin-agarose gels in this work allows the histology to be carried out in the same ones, and has allowed to confirm that type VII agarose favors the development and cellular survival

Generation of genipin cross-linked fibrin-agarose hydrogel tissue-like models for tissue engineering applications

Generation of biomimetic and biocompatible artificial tissues is the basic research objective for tissue engineering (TE). In this sense, the biofabrication of scaffolds that resemble the tissues’ extracellular matrix (ECM) is an essential aim in this field. Uncompressed and nanostructured fibrin-agarose hydrogels (FAH and NFAH respectively) emerged as promising scaffold in TE, but its structure and biomechanical properties must be improved in order to broad their TE applications. Here we generated and characterized novel membranelike models with increased structural and biomechanical properties based on the chemical cross-linking of FAH and NFAH with genipin (GP at 0.1, 0.25, 0.5 and 0.75%). Furthermore, scaffolds were subjected to rheological (G, G’, G” modulus), ultrastructural and ex vivo biocompatibility analyses. Results showed that all GP concentrations increased the stiffness (G) and especially the elasticity (G’) of FAH and NFAH. Ultrastructural analyses demonstrated that GP and nanostructuration of FAH allowed controlling the porosity of FAH. In addition, biological studies revealed that higher concentration of GP significantly decreased the cell viability. Finally, this study demonstrated the possibility to generate natural FAH and NFAH with improved structural and biomechanical properties by using GP. However, further in vivo studies are needed in order to demonstrate the biocompatibility, biodegradability and regeneration capability of these cross-linked scaffolds

Development of a diagnostic algorithm in periodontal disease and identification of genetic expression patterns: A preliminary report

AbstractBackground/purposeTo identify genetic expression patterns that can be used to define an appropriate diagnostic algorithm of clinical use in periodontal disease.Materials and methodsTotal RNA was extracted from 13 samples corresponding to normal human gingiva (NHG) and human gingiva affected by periodontal disease (PDHG). A comprehensive gene expression analysis was carried out by microarray analysis using Affymetrix Human Genome U133 plus 2.0 oligonucleotide arrays.ResultsSixty-six probe sets (genes and expressed sequence tags – EST) overexpressed in all samples of one of the comparison groups, were used for the diagnostic algorithm. All samples, including an independent test sample, were correctly classified as normal or periodontally affected using the diagnostic algorithm. In addition, 2596 genes/EST were upregulated and 1542 genes/EST were downregulated in PDHG, with numerous gene functions impaired in PDHG, especially those related to the immune response, cell-cell junctions, and extracellular matrix remodeling.ConclusionOur study reveals differential gene expression profiles in NHG and PDHG. The proposed diagnostic algorithm could have clinical usefulness for differential diagnosis in periodontal disease

Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa fibroblasts and keratinocytes from small biopsies of normal oral mucosa using enzymatic treatments. Then we determined the viability of the cultured cells by electron probe quantitative X-ray microanalysis, and we demonstrated that most of the cells in the primary cultures were alive and had high K/Na ratios. Once cell viability was determined, we used the cultured fibroblasts and keratinocytes to develop an artificial oral mucosa construct by using a fibrin-agarose extracellular matrix and a sequential culture technique using porous culture inserts. Histological analysis of the artificial tissues showed high similarities with normal oral mucosa controls. The epithelium of the oral substitutes had several layers, with desmosomes and apical microvilli and microplicae. Both the controls and the oral mucosa substitutes showed high suprabasal expression of cytokeratin 13 and low expression of cytokeratin 10. All these results suggest that our model of oral mucosa using fibrin-agarose scaffolds show several similarities with native human oral mucosa.This work was supported by the grants FIS 03/0141 and FIS 04/1306 from the Spanish National Ministry of Health (Instituto de Salud Carlos III) and by CM 2005/011 from Junta de Andalucía