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)

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

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

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)

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)

Wnt-3a Induces Epigenetic Remodeling in Human Dental Pulp Stem Cells

Dental pulp stem cells (DPSCs) from adult teeth show the expression of a very complete repertoire of stem pluripotency core factors and a high plasticity for cell reprogramming. Canonical Wnt and Notch signaling pathways regulate stemness and the expression of pluripotency core factors in DPSCs, and even very short-term (48 h) activations of the Wnt pathway induce a profound remodeling of DPSCs at the physiologic and metabolic levels. In this work, DPSC cultures were exposed to treatments modulating Notch and Wnt signaling, and also induced to differentiate to osteo/adipocytes. DNA methylation, histone acetylation, histone methylation, and core factor expression levels where assessed by mass spectroscopy, Western blot, and qPCR. A short-term activation of Wnt signaling by WNT-3A induced a genomic DNA demethylation, and increased histone acetylation and histone methylation in DPSCs. The efficiency of cell reprogramming methods relies on the ability to surpass the epigenetic barrier, which determines cell lineage specificity. This study brings important information about the regulation of the epigenetic barrier by Wnt signaling in DPSCs, which could contribute to the development of safer and less aggressive reprogramming methodologies with a view to cell therapy.This work was funded by the UPV/EHU (GIU16/66, UFI 11/44; to F.U.), the Basque Government (GV/EJ; Ikerketa Taldeak IT831-13; to G.I. and ELKARTEK KK-2019-00093; to F.U.) and ISCIII (DTS18/00142; to N.S.)