Bioactive Materials for Next-Generation Dentistry

Teeth were some of the first organs whose function was effectively restored by inert refilling materials that have become widely known to the general public; amalgams, polymeric resin composites, and gutta-percha are some such examples [...]This research was funded by a Basque Government Grant to Consolidated University Research Groups/Ikerketa Taldeak (IT1751-22)

El ultimatum de María Jesús

Duración (en horas): Más de 50 horas. Destinatario: Estudiante y DocenteEl planteamiento del presente proyecto está fundamentado en los principios del Aprendizaje Basado en Problemas, de manera que se presenta una situación problema (escenario) a partir del cual los estudiantes agrupados en pequeños grupos, deben poner en evidencia sus conocimientos previos en relación a los contenidos conceptuales del problema, plantear hipótesis, identificar sus necesidades de aprendizaje para el abordaje del problema, organizar su estudio de forma autodirigida, cooperativa y dinámica, aplicar el nuevo conocimiento obtenido al problema y reflexionar sobre el aprendizaje adquirido, tanto en su contenido, como en la forma y proceso en la cual lo han adquirido. Junto con la presentación del problema inicial, se van presentando de forma dinámica nuevas contextualizaciones del mismo y actividades para la aplicación de los nuevos conocimientos adquiridos. La propuesta se puso en marcha en el curso 2012/2013, en un grupo único de 12 estudiantes de primer curso de Grado en Odontología, dentro de la asignatura Giza Histologia/Histología Humana, de 6 créditos ECTS, impartida en el 2º cuatrimestre. La implementación de dicha metodología de enseñanza-aprendizaje, para los temas seleccionados, se realizó desde la primera semana de curso, y sin haber impartido contenidos previos. Las metodologías de enseñanza ABP (40% del temario) y tradicional (60% del temario) convivieron en el desarrollo de la asignatura, desde el principio hasta el final del curso. Tras el análisis de su implementación, podemos afirmar que este es un modelo que permite el desarrollo de todas las competencias sin excepción, tanto transversales como específicas, establecidas en el programa de la asignatura Giza Histología

Stem and Cancer Stem Cell Identities, Cellular Markers, Niche Environment and Response to Treatments to Unravel New Therapeutic Targets

Adult stem cells are a partially quiescent cell population responsible for natural cell renewal and are found in many different regions of the body, including the brain, teeth, bones, muscles, skin, and diverse epithelia, such as the epidermal or intestinal epithelium, among others [...

Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current state of knowledge on IPS cells advises that research on their regenerative properties is carried out in appropriate tissue and organ systems that permit a safe assessment of the long-term behavior of these reprogrammed cells. In the present paper, we discuss the mechanisms of cell reprogramming, current technical limitations of IPS cells for their use in human tissue engineering, and possibilities to overcome them in the particular case of dental regeneration

Identification of sugar moieties in chief cells of the rat fundic gastric glands

Many studies have been conducted to determine the composition of the glyconjugates of the mucus-secreting cells of the fundic glands of the stomach. However, the chief cells of these glands have been largely ignored because they secrete mainly zymogens with a lower glycosylation. The aim of this work was to analyze the glycoconjugates of the gastric chief cells by a battery of 17 different lectins, recognizing Fucose, N-AcetylGalactosamine, Galactose, N-Acetylneuramiinic acid, N-AcetylGlucosamine and Mannose containing oligosaccharides. Histochemical techniques were performed with several lectins and also combined with two pre-treatments; β-elimination, which removes O-linked oligosaccharides, and incubation with Peptide-N-Gycosidase F, which removes N-linked oligosaccharides. In addition, acid hydrolysis was performed before WGA histochemistry, and incubation with glucose oxidase before Con A labeling. Many lectins did not stain the chief cells. In addition, the presence of O-glycans in the apical cell membrane was demonstrated with the lectins AAL, HPA, MPA/MPL, PNA, RCA-I, and WGA. Some of these O-glycans were resistant to short-term β-elimination pre-treatments. Mannose-binding lectins stained the basal cytoplasm of the chief cells. The level of glycosylation of the chief cells was lower than that of the mucous cells. The presence of O-glycans in the apical cell membrane is consistent with the presence of mucins such as MUC1 in the apical membrane of chief cells. Moreover, Mannose-binding lectins revealed N-glycosylation in the basal cytoplasm. The knowledge of gastric chief cell glycoconjugates is relevant because of their potential involvement not only in in physiological but also in pathological processes, such as cancer.This work was supported by the University of the Basque Country UPV/EHU (Grant numbers: EHUA13/15, EHUA15/26); and Fundación Séneca, Comunidad Autónoma de la Región de Murcia (Grant number 04542/GERM/06)

Notch/Wnt Cross-Signalling Regulates Stemness Of Dental Pulp Stem Cells Through Expression Of Neural Crest And Core Pluripotency Factors

Dental pulp stem cells (DPSCs) from adult teeth express neural crest (NC) markers together with core transcriptional factors associated with stem cell pluripotency, such as Oct4a, Sox2, c-Myc, Rex1, Stella/Dppa3, Ssea1/Fut4, Lin28 and Nanog. The possibility to boost the natural stemness features of DPSCs by mild methods, that do not involve gene and/or chromatin modification or gene transfection, is highly desirable for cell therapy. Canonical Wnt and Notch are two highly conserved developmental signalling pathways that are involved in NC emergence and stem cell self-renewal. We determined that both pathways coordinate to regulate the expression of core pluripotency and NC factors in DPSCs. Pharmacological inhibition of the Notch pathway for 48 h, by the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), abolished the expression of NC and core factors. In addition, it induced a silencing of the canonical Wnt signalling and a clear reduction in the stemness potential of DPSCs, as shown by a reduced ability to generate mature, fully differentiated osteoblasts and adipocytes. Conversely, pharmacological activation of the Wnt pathway for 48 h, by either the glycogen synthase kinase 3 beta (GSK3-beta) inhibitor 6-bromoindirubin-3'-oxime (BIO) or the human recombinant protein Wnt-3a, not only largely increased the expression of NC and core factors, but also increased the efficiency of DPSCs to differentiate into mature osteoblasts and adipocytes. These results showed that a short preconditioning activation of Wnt/Notch signalling by small molecules and/or recombinant proteins enhanced the stemness and potency of DPSCs in culture, which could be useful for optimising the therapeutic use of these and other tissue-specific stem cells.Technical and human support provided by the analytical microscopy service of SGIKER (UPV/EHU, MINECO, GV/EJ, ERDF and ESF) is gratefully acknowledged. This work was funded by the UPV/EHU (GIU16/66, UFI 11/44) and the Basque Government (GV/EJ; IT831-13). V.U. received a fellowship from The Global Training Grant (GV/EJ) to fund a research stage at The Institute of Cancer Research (London, UK)

Vasculogenesis from Human Dental Pulp Stem Cells Grown in Matrigel with Fully Defined Serum-Free Culture Media

The generation of vasculature is one of the most important challenges in tissue engineering and regeneration. Human dental pulp stem cells (hDPSCs) are some of the most promising stem cell types to induce vasculogenesis and angiogenesis as they not only secrete vascular endothelial growth factor (VEGF) but can also differentiate in vitro into both endotheliocytes and pericytes in serum-free culture media. Moreover, hDPSCs can generate complete blood vessels containing both endothelial and mural layers in vivo, upon transplantation into the adult brain. However, many of the serum free media employed for the growth of hDPSCs contain supplements of an undisclosed composition. This generates uncertainty as to which of its precise components are necessary and which are dispensable for the vascular differentiation of hDPSCs, and also hinders the transfer of basic research findings to clinical cell therapy. In this work, we designed and tested new endothelial differentiation media with a fully defined composition using standard basal culture media supplemented with a mixture of B27, heparin and growth factors, including VEGF-A165 at different concentrations. We also optimized an in vitro Matrigel assay to characterize both the ability of hDPSCs to differentiate to vascular cells and their capacity to generate vascular tubules in 3D cultures. The description of a fully defined serum-free culture medium for the induction of vasculogenesis using human adult stem cells highlights its potential as a relevant innovation for tissue engineering applications. In conclusion, we achieved efficient vasculogenesis starting from hDPSCs using serum-free culture media with a fully defined composition, which is applicable for human cell therapy purposes.This work was financed by the «Ramón y Cajal» program RYC-2013-13450 (JRP); UPV/EHU (GIU16/66, UFI 11/44 and COLAB19/03; FU); MINECO Retos I + D + I (SAF2015-70866-R; JRP, PID2019-104766RB-C21; JRP); Basque Government (GV/EJ; IT831-13; GI) TERSAFURNA-2020333039 and ELKARTEK KK-2019-00093. J.L. has a grant from UPV/EHU (DOKBERRI 2019(DOCREC19/49)). I.I. has a grant from the Basque Government (PRE_2019_2_0300 or GIU16/66)

Cell Reprogramming, IPS Limitations, and Overcoming Strategies in Dental Bioengineering

The procurement of induced pluripotent stem cells, or IPS cells, from adult differentiated animal cells has the potential to revolutionize future medicine, where reprogrammed IPS cells may be used to repair disease-affected tissues on demand. The potential of IPS cell technology is tremendous, but it will be essential to improve the methodologies for IPS cell generation and to precisely evaluate each clone and subclone of IPS cells for their safety and efficacy. Additionally, the current state of knowledge on IPS cells advises that research on their regenerative properties is carried out in appropriate tissue and organ systems that permit a safe assessment of the long-term behavior of these reprogrammed cells. In the present paper, we discuss the mechanisms of cell reprogramming, current technical limitations of IPS cells for their use in human tissue engineering, and possibilities to overcome them in the particular case of dental regeneration

Human DenPulp Stem Cells Grown in Neurogenic Media Differentiate Into Endothelial Cells and Promote Neovasculogenesis in the Mouse Brain

Dental pulp stem cells (DPSCs) have the capacity to give rise to cells with neuronal-like phenotypes, suggesting their use in brain cell therapies. In the present work, we wanted to address the phenotypic fate of adult genetically unmodified human DPSCs cultured in Neurocult (TM) (Stem Cell Technologies), a cell culture medium without serum which can be alternatively supplemented for the expansion and/or differentiation of adult neural stem cells (NSCs). Our results show that non-genetically modified human adult DPSCs cultured with Neurocult NS-A proliferation supplement generated neurosphere-like dentospheres expressing the NSC markers Nestin and glial fibrillary acidic protein (GFAP), but also the vascular endothelial cell marker CD31. Remarkably, 1 month after intracranial graft into athymic nude mice, human CD31+/CD146+ and Nestin+ DPSC-derived cells were found tightly associated with both the endothelial and pericyte layers of brain vasculature, forming full blood vessels of human origin which showed an increased laminin staining. These results are the first demonstration that DPSC-derived cells contributed to the generation of neovasculature within brain tissue, and that Neurocult and other related serum-free cell culture media may constitute a fast and efficient way to obtain endothelial cells from human DPSCs.This work was funded by "Ramon y Cajal" program RYC-2013-13450 (JRP) and RYC 2012-11137 (JME); Spanish Ministry of Economy and Competitiveness SAF2015-70866-R; UPV/EHU (GIU16/66, UFI 11/44); and Basque Government (GV/EJ; IT831-13). JL and OP-A obtained a Ph.D. fellowship from the University of the Basque Country (UPV/EHU)