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Alginate/gelatin (Alg-Gel) hydrogels have been used experimentally, associated with mesenchymal stromal/stem cells (MSCs), to guide bone tissue formation. One of the main challenges for clinical application is optimizing Alg-Gel stiffness to guide osteogenesis. In this study, we investigated how Alg-Gel stiffness could modulate the dental pulp stem cell (DPSC) attachment, morphology, proliferation, and osteogenic differentiation, identifying the optimal conditions to uncouple osteogenesis from the other cell behaviors. An array of Alg-Gel hydrogels was prepared by casting different percentages of alginate and gelatin cross-linked with 2% CaCl2. We have selected two hydrogels: one with a stiffness of 11 ± 1 kPa, referred to as “low-stiffness hydrogel”, formed by 2% alginate and 8% gelatin, and the other with a stiffness of 55 ± 3 kPa, referred to as “high-stiffness hydrogel”, formed by 8% alginate and 12% gelatin. Hydrogel analyses showed that the average swelling rates were 20 ± 3% for the low-stiffness hydrogels and 35 ± 2% for the high-stiffness hydrogels. The degradation percentage was 47 ± 5% and 18 ± 2% for the low- and high-stiffness hydrogels, respectively. Both hydrogel types showed homogeneous surface shape and protein (Alg-Gel) interaction with CaCl2 as assessed by physicochemical characterization. Cell culture showed good adhesion of the DPSCs to the hydrogels and proliferation. Furthermore, better osteogenic activity, determined by ALP activity and ARS staining, was obtained with high-stiffness hydrogels (8% alginate and 12% gelatin). In summary, this study confirms the possibility of characterizing and optimizing the stiffness of Alg-Gel gel to guide osteogenesis in vitro without altering the other cellular properties of DPSCs.

Background: This study investigated the contact between adult dental pulp stem cells (DPSCs) and carbamide peroxide (CP), a bleaching agent that is a popular choice for at-home whitening products, using an in vitro model. Objectives: The aim of this study was to evaluate the impact of exposure to different concentrations and timings of a commonly used peroxide-based home tooth-whitening product on DPSCs. Materials and methods: Human DPSCs obtained from impacted third molars were cultured and exposed to various concentrations of carbamide peroxide (0.1%, 0.5%, and 1%). The effects of CP on DPSC proliferation and apoptosis were investigated by MTT assay and flow cytometry. Migration was investigated by micrographs of wound healing. An enzyme-linked immunosorbent assay (IL-6 and IL-8) was used to investigate the CP-stimulated cytokine production of DPSCs. Each experiment was performed three times with independent batches of DPSCs. Statistical analysis of the collected data was performed using one-way and two-way ANOVAs with the significance threshold set at p < 0.05. Tukey’s post hoc multiple comparison test was used to identify differences between groups. Results: Cell viability and adherence were lower in the CP-exposed cells compared to the non-stimulated cells, probably due to increased cell death (** p ≤ 0.01, **** p ≤ 0.0001). CP-stimulated DPSCs exhibited a dose-dependent release of IL-6 and IL-8 (**** p ≤ 0.0001). CP did not affect wound healing at any concentration tested. Conclusions: Human DPSCs were able to sense CP. Consequently, CP contributed significantly to cell apoptosis and local inflammatory responses through cytokine release.

Background : This study investigates the impact of environmental pollution on the quality and viability of dental stem cells (DSCs) from impacted third molars. By comparing DSCs from patients in industrial areas with high air pollution and those from non-industrial regions, the research assesses the adverse effects of heavy metals on stem cell proliferation. Methods : Impacted lower third molars were collected from 28 patients-10 from industrial and 18 from non-industrial areas. Patients were divided into two age groups: 18-27 years and 28-38 years old. Dental pulp was extracted under sterile conditions, and DSCs were isolated and cultured. Heavy metal concentrations in dental tissues were measured using atomic absorption/emission spectrometry. Results : The study found significantly higher concentrations of copper and lead in the dental tissues of patients in industrial areas. Cell viability was lower in samples from these areas, with a statistically significant difference in average doubling time and the number of cells obtained after the first passage. There was no significant impact of gender on heavy metal content, except for higher iron levels in men. Conclusions : Exposure to industrial pollutants negatively affects the viability and proliferation of DSCs, but there are no differences in differentiation in the osteogenic medium regarding cell mineralization. These studies highlight the importance of environmental factors for oral health, suggesting that residents of polluted areas may face greater difficulties in dental and regenerative treatments. Further research is needed to develop strategies to mitigate the effects and improve clinical outcomes for affected populations.

The odontoblastic differentiation of dental pulp stem cells (DPSCs) associated with caries injury happens in an inflammatory context. We recently demonstrated that there is a link between inflammation and dental tissue regeneration, identified via enhanced DPSC-mediated dentinogenesis in vitro. Brain-derived neurotrophic factor (BDNF) is a nerve growth factor-related gene family molecule which functions through tropomyosin receptor kinase B (TrkB). While the roles of BDNF in neural tissue repair and other regeneration processes are well identified, its role in dentinogenesis has not been explored. Furthermore, the role of BDNF receptor-TrkB in inflammation-induced dentinogenesis remains unknown. The role of BDNF/TrkB was examined during a 17-day odontogenic differentiation of DPSCs. Human DPSCs were subjected to odontogenic differentiation in dentinogenic media treated with inflammation inducers (LTA or TNFα), BDNF, and a TrkB agonist (LM22A-4) and/or antagonist (CTX-B). Our data show that BDNF and TrkB receptors affect the early and late stages of the odontogenic differentiation of DPSCs. Immunofluorescent data confirmed the expression of BDNF and TrkB in DPSCs. Our ELISA and qPCR data demonstrate that TrkB agonist treatment increased the expression of dentin matrix protein-1 (DMP-1) during early DPSC odontoblastic differentiation. Coherently, the expression levels of runt-related transcription factor 2 (RUNX-2) and osteocalcin (OCN) were increased. TNFα, which is responsible for a diverse range of inflammation signaling, increased the levels of expression of dentin sialophosphoprotein (DSPP) and DMP1. Furthermore, BDNF significantly potentiated its effect. The application of CTX-B reversed this effect, suggesting TrkB`s critical role in TNFα-mediated dentinogenesis. Our studies provide novel findings on the role of BDNF-TrkB in the inflammation-induced odontoblastic differentiation of DPSCs. This finding will address a novel regulatory pathway and a therapeutic approach in dentin tissue engineering using DPSCs.

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults’ molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.

Alginate/gelatin (Alg-Gel) hydrogels have been used experimentally, associated with mesenchymal stromal/stem cells (MSCs), to guide bone tissue formation. One of the main challenges for clinical application is optimizing Alg-Gel stiffness to guide osteogenesis. In this study, we investigated how Alg-Gel stiffness could modulate the dental pulp stem cell (DPSC) attachment, morphology, proliferation, and osteogenic differentiation, identifying the optimal conditions to uncouple osteogenesis from the other cell behaviors. An array of Alg-Gel hydrogels was prepared by casting different percentages of alginate and gelatin cross-linked with 2% CaCl 2 . We have selected two hydrogels: one with a stiffness of 11 ± 1 kPa, referred to as "low-stiffness hydrogel", formed by 2% alginate and 8% gelatin, and the other with a stiffness of 55 ± 3 kPa, referred to as "high-stiffness hydrogel", formed by 8% alginate and 12% gelatin. Hydrogel analyses showed that the average swelling rates were 20 ± 3% for the low-stiffness hydrogels and 35 ± 2% for the high-stiffness hydrogels. The degradation percentage was 47 ± 5% and 18 ± 2% for the low- and high-stiffness hydrogels, respectively. Both hydrogel types showed homogeneous surface shape and protein (Alg-Gel) interaction with CaCl 2 as assessed by physicochemical characterization. Cell culture showed good adhesion of the DPSCs to the hydrogels and proliferation. Furthermore, better osteogenic activity, determined by ALP activity and ARS staining, was obtained with high-stiffness hydrogels (8% alginate and 12% gelatin). In summary, this study confirms the possibility of characterizing and optimizing the stiffness of Alg-Gel gel to guide osteogenesis in vitro without altering the other cellular properties of DPSCs.

As previously described by several authors, dental pulp stem cells (DPSCs), when adequately stimulated, may acquire a neuronal-like phenotype acting as a favorable source of stem cells in the generation of nerves. Besides, it is known that hypoxia conditioning is capable of stimulating cell differentiation as well as survival and self-renewal, and that multiple growth factors, including Epidermal Growth factor (EGF) and basic fibroblast growth factor (bFGF), are often involved in the induction of the neuronal differentiation of progenitor cells. In this work, we investigated the role of hypoxia in the commitment of DPSCs into a neuronal phenotype. These cells were conditioned with hypoxia (O2 1%) for 5 and 16 days; subsequently, we analyzed the proliferation rate and morphology, and tested the cells for neural and stem markers. Moreover, we verified the possible autocrine/paracrine role of DPSCs in the induction of neural differentiation by comparing the secretome profile of the hypoxic and normoxic conditioned media (CM). Our results showed that the hypoxia-mediated DPSC differentiation was time dependent. Moreover, conditioned media (CM derived from DPSCs stimulated by hypoxia were able, in turn, to induce the neural differentiation of SH-SY5Y neuroblastoma cells and undifferentiated DPSCs. In conclusion, under the herein-mentioned conditions, hypoxia seems to favor the differentiation of DPSCs into neuron-like cells. In this way, we confirm the potential clinical utility of differentiated neuronal DPSCs, and we also suggest the even greater potential of CM-derived-hypoxic DPSCs that could more readily be used in regenerative therapies.

The clinical use of bioactive material in the field of biomedical tissue engineering has become increasingly of interest in practice. This study investigates how BiodentineTM (BD), a tricalcium silicate cement, in culture media, affects the odonto/osteogenic differentiation potential of in vitro cultured human dental pulp stem cells (hDPSCs). hDPSCs were extracted and characterized for their expression profile by flow cytometry. Then, hDPSCs were cultured in media containing BD for 3 weeks to study the impact of BD on the odonto/osteogenesis pathway, compared to the positive control (osteogenic media) and negative control (cell culture media). Odonto/osteogenic differentiation of hDPSCs treated with BD was assessed by measuring the level of expression of odonto/osteogenic markers by flow cytometry, ELISA and Alizarin red stain. Additionally, the expression profile of the genes involved in the odonto/osteogenesis pathway was investigated, using PCR array. Our results indicate that hDPSCs treatment with BD results in an increased tendency for odonto/osteogenic differentiation. The BD treated group demonstrates a significant increase in the expression of odonto/osteogenic markers, osteocalcin (OCN) (p < 0.005), osteopontin (OPN) (p < 0.0005) and alkaline phosphatase (ALP) (p < 0.0005), and the presentation of calcium deposits by ARS, compared to the negative control by using t-test and ANOVA. Moreover, the BD-treated group is marked by the upregulation of genes related to the odonto/osteogenesis pathway, compared to the control groups, specifically the genes that are involved in the bone morphogenic protein (BMP) (p < 0.05) signaling pathway, the activation of the extracellular matrix-related gene (ECMG) (p < 0.05) and the Ca2+ signaling pathway (p < 0.05), compared to day 1 of treatment by using ANOVA. BD shows a stimulatory effect on the odonto/steogenic capacity of hDPSCs, suggesting BD as a good candidate and a very promising and useful means to be applied in regenerative medicine to regenerate dentine tissue in clinical settings.

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.

As previously described by several authors, dental pulp stem cells (DPSCs), when adequately stimulated, may acquire a neuronal-like phenotype acting as a favorable source of stem cells in the generation of nerves. Besides, it is known that hypoxia conditioning is capable of stimulating cell differentiation as well as survival and self-renewal, and that multiple growth factors, including Epidermal Growth factor (EGF) and basic fibroblast growth factor (bFGF), are often involved in the induction of the neuronal differentiation of progenitor cells. In this work, we investigated the role of hypoxia in the commitment of DPSCs into a neuronal phenotype. These cells were conditioned with hypoxia (O 2 1%) for 5 and 16 days; subsequently, we analyzed the proliferation rate and morphology, and tested the cells for neural and stem markers. Moreover, we verified the possible autocrine/paracrine role of DPSCs in the induction of neural differentiation by comparing the secretome profile of the hypoxic and normoxic conditioned media (CM). Our results showed that the hypoxia-mediated DPSC differentiation was time dependent. Moreover, conditioned media (CM derived from DPSCs stimulated by hypoxia were able, in turn, to induce the neural differentiation of SH-SY5Y neuroblastoma cells and undifferentiated DPSCs. In conclusion, under the herein-mentioned conditions, hypoxia seems to favor the differentiation of DPSCs into neuron-like cells. In this way, we confirm the potential clinical utility of differentiated neuronal DPSCs, and we also suggest the even greater potential of CM-derived-hypoxic DPSCs that could more readily be used in regenerative therapies.

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.