Small Molecules Promote the Rapid Generation of Dental Epithelial Cells from Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.

From Pluripotent Stem Cells to Organoids and Bioprinting: Recent Advances in Dental Epithelium and Ameloblast Models to Study Tooth Biology and Regeneration.

Ameloblasts are the specialized dental epithelial cell type responsible for enamel formation. Following completion of enamel development in humans, ameloblasts are lost and biological repair or regeneration of enamel is not possible. In the past, in vitro models to study dental epithelium and ameloblast biology were limited to freshly isolated primary cells or immortalized cell lines, both with limited translational potential. In recent years, large strides have been made with the development of induced pluripotent stem cell and organoid models of this essential dental lineage - both enabling modeling of human dental epithelium. Upon induction with several different signaling factors (such as transforming growth factor and bone morphogenetic proteins) these models display elevated expression of ameloblast markers and enamel matrix proteins. The advent of 3D bioprinting, and its potential combination with these advanced cellular tools, is poised to revolutionize the field - and its potential for tissue engineering, regenerative and personalized medicine. As the advancements in these technologies are rapidly evolving, we evaluate the current state-of-the-art regarding in vitro cell culture models of dental epithelium and ameloblast lineage with a particular focus toward their applicability for translational tissue engineering and regenerative/personalized medicine.

Células madre en odontología: nuevas perspectivas / Stem cells in dentistry: new perspectives

En el campo de la odontología, prevalecen actualmente alternativas terapéuticas con una filosofía conservadora. Sin embargo, con el advenimiento de los tratamientos con células madre (CM), se amplían las posibilidades terapéuticas, que buscan la combinación y el equilibrio entre la intervención tradicional y las posibilidades de reposición de estructuras anatómicas dañadas, a través de la regeneración de tejidos utilizando células madre o sus derivados (AU)

In the dentistry field, therapeutic alternatives with a conservative philosophy currently prevail. However, with the advent of stem cell (SC) treatments, therapeutic possibilities are expanding, seeking a combination and balance between traditional intervention and the pos- sibility of replacing damaged anatomical structures through tissue regeneration, using stem cells or their derivatives (AU)

Does cryopreservation affect the biological properties of stem cells from dental tissues? a systematic review

Abstract This systematic review evaluated if different cryopreservation protocols could affect biological properties (Cell survival rate (CSR), proliferation, differentiation, maintenance of stem cell markers) of stem cells obtained from dental tissues (DSC) post-thaw. An electronic search was carried out within PubMed and ISI Web Science by using specific keyword. Two independent reviewers read the titles and abstracts of all reports respecting predetermined inclusion/exclusion criteria. Data were extracted considering the biological properties of previously cryopreserved DSCs and previously cryopreserved dental tissues. DSCs cryopreserved as soon as possible after their isolation presents a CSR quite similar to the non-cryopreserved DSC. Dimethyl sulfoxide (DMSO) [10%] showed good results related to cell recovery post-thaw to cryopreserve cells and tissues for periods of up to 2 years. The cryopreservation of DSC in a mechanical freezer (-80°C) allows the recovery of stem cells post-thaw. The facilities producing magnetic field (MF), demand a lower concentration of cryoprotectant, but their use is not dispensable. It is possible to isolate and cryopreserve dental pulp stem cell (DPSC) from healthy and diseased vital teeth. Cryopreservation of dental tissues for late DSC isolation, combined with MF dispensability, could be valuable to reduce costs and improve the logistics to develop teeth banks.

Resumo Essa revisão sistemática avaliou se diferentes protocolos de criopreservação podem afetar as propriedades biológicas (taxa de sobrevivência celular, proliferação, diferenciação, manutenção dos marcadores de superfície) de células-tronco isoladas de tecidos dentais (DSC) após o descongelamento. Uma busca eletrônica foi realizada no PubMed e no ISI Web of Science utilizando palavras-chave específicas. Dois revisores independentes avaliaram os títulos e resumos de todos os estudos respeitando critérios de inclusão e exclusão previamente determinados. Os dados foram extraídos considerando as propriedades biológicas de DSC, e DSC isoladas de tecidos previamente criopreservados. DSC criopreservadas logo após seu isolamento apresentaram propriedades biológicas muito semelhantes às observadas em DSC não criopreservadas. Dimetil sulfóxido (DMSO) [10%] demonstrou bons resultados relacionados com a recuperação celular após descongelamento de células e tecidos, por períodos de até 2 anos. A criopreservação de DSC em freezer mecânico (-80 °C) permite a recuperação de células-tronco pós-descongelação. A utilização de freezer com campo magnético (MF), proporciona a utilização de uma menor concentração de crioprotector, mas a sua utilização não é dispensável. É possível isolar e criopreservar e criopreservar células-tronco da polpa dental (DPSC) de dentes vitais saudáveis e doentes. Criopreservação de tecidos dentais após o isolamento de DSC, combinados com MF, podem ser valiosas estratégias para reduzir custos e melhorar a logística no desenvolvimento de bancos de dentes.

Dose-responses of Stem Cells from Human Exfoliated Teeth to Infrared LED Irradiation

Despite several reports regarding tissue regeneration, including pulp repair induced by different light sources, only limited data have been reported concerning the effects of light-emitting diodes (LED) on stem cells from human exfoliated deciduous teeth (SHEDs). The aim of this study was to evaluate the effects of different energy densities of infrared LED on the cell viability, number of cells and mineralized tissue production by SHEDs. SHEDs were obtained from near-exfoliation primary teeth (n=3), seeded in plain DMEM (104 cells/cm2), and irradiated by a LED prototype (LEDTable 850 nm, 40 mW/cm2) delivering 0 (control), 2, 4, 8, 15 or 30 J/cm2 (n=9). Cell viability (MTT assay), cell proliferation (trypan blue assay), and mineralized nodule (MN) formation (alizarin red stain) were assessed 12 and 72 h post-irradiation. Data were subjected to Kruskal-Wallis and Mann-Whitney tests (α=0.05). Cells irradiated with 2 or 4 J/cm2 exhibited higher metabolism at 72 h, and all energy densities provided increase in cell proliferation after 12 h. Regarding MN formation, the best results were observed at 72 h after SHED irradiation with 8 and 15 J/cm2. It was concluded that the cell viability, cell number and MN formation by pulp cells are enhanced after exposure to infrared LED irradiation. Overall, the greatest SHED biostimulation was obtained with 4 and 8 J/cm2.

Apesar de diversos estudos envolvendo regeneração tecidual, incluindo o reparo pulpar induzido por diferentes fontes de luz, dados limitados têm sido reportados a respeito dos efeitos da irradiação com diodos emissores de luz (LED) sobre células-tronco de dentes decíduos esfoliados (SHEDs). O objetivo do presente estudo foi avaliar os efeitos de diferentes doses de energia (DE) do LED infravermelho sobre a viabilidade celular, número de células viáveis e produção de nódulos mineralizados (NM) por SHEDs. As células foram obtidas a partir de dentes decíduos próximos ao período de esfoliação (n=3), semeadas em DMEM completo (10⁴ células/cm2) e irradiadas utilizando um protótipo de LED (LEDTable 850 nm, 40 mW/cm²⁾ com as doses de 0 (controle), 2, 4, 8, 15 ou 30 J/cm² (n=9). A viabilidade celular (MTT), o número de células viáveis (trypan blue assay) e a formação de NM (alizarin red stain) foram realizados 12 e 72 h após a irradiação. Os dados foram avaliados utilizando os testes Kruskal-Wallis e Mann-Whitney (α=0,05). As células irradiadas com 2 ou 4 J/cm² exibiram uma maior viabilidade em 72 h, e todas as DE aumentaram o número de células viáveis após 12 h. Para a formação de NM, os melhores resultados foram observados 72 h após a irradição das SHEDs, com as doses de 8 e 15 J/cm². Concluiu-se que a viabilidade celular, o número de células e a formação de NM por células pulpares são aumentados após exposição ao LED infravermelho. De um modo geral, a melhor bioestimulação celular (SHEDs) foi obtida com 4 e 8 J/cm².

Micrometric measurement of the density of stained odontoblast processes

The embryological, structural and functional unit of the dentine-pulp complex shares the odontoblast, located in the border of the dentine pulp, with basal nuclei and organelles. The odontoblast process emerges from its apical pole. It is formed by microtubules, microfilaments and vesicles covered by membranes penetrating the dentinal tubules, isolated from the inter-tubular matrix, along the extent of the dentine. The objective of this study was to evaluate the efficacy of three staining techniques: hematoxylin-eosin, periodic acid-Schiff and Schmorl, by staining the process, from beginning to end, and compare the results with the erosion technique. Thirty human teeth were employed in the trial; after their extraction the pulp was fixated, the pieces demineralized in nitric acid at 8%, the collagen filaments eliminated with Type II Collage-nase, the tissue was stained, and the measurements were made. The portions with no pulp were prepared with the erosion technique. Results: Comparing the best results obtained by staining with the values obtained with the erosion technique, the former showed lower values. Conclusion: Staining techniques show lower density of the staining processes compared with the dentinal tubules in the erosion technique.

Histochemical detection of sugar residues in lizard teeth (Liolaemus gravenhorsti): a lectin-binding study

The structural diversity of the many oligosaccharide chains of surface glycoconjugates renders them likely candidates for modulators of cell-interactions, cellular movements, differentiation, and cellular recognition. A selection of different lectins was used to investigate the appearance of cellular distribution and changes in sugar residues during tooth development in the polyphyodont lizard, Liolaemus gravenhorsti. Lectins from three groups were used: (1) N-acetylgalactosamine specificity: BS-1, PNA, RCA-120; (2) N-acetylglucosamine specificity: ECA; and (3) fucose specificity: UEA 1 and LTA.. Digital images were processed using Scion Image. Grayscale graphics in each image were obtained. The lectins used showed a strong, wide distribution of the L-fucose and N-acetylgalactosamine at the cell surface and in the cytoplasm of multinucleate odontoclast cell, while mononuclear odontoclast cells showed no binding, suggesting some roles that the residues sugar might play in the resorption of dentine or with multinucleation of odontoclast after the attachment to the dentine surface in this polyphyodont species. Further studies must be planned to determine the specific identities of these glycoconjugates,and to elucidate the roles played by these sugar residues in the complex processes related to odontogenesis in polyphyodont species.