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Auditory neuropathy is the particular form of deafness in humans which cannot be treated by replacement therapy. Human dental pulp stem cells (hDPSCs) are derived from an ectomesenchymal neural crest cell population. Therefore, they possess a promising capacity for neuronal differentiation and repair. miR-124, a key regulator of neuronal development in the inner ear, is expressed at high levels in auditory and vestibular neurons. Here, we evaluated the possible effect of miR-124 in alteration of neural protein markers expression. Using quantitative reverse transcription-PCR (qRT-PCR) analyses and immunofluorescence staining, we studied the expression patterns of neural progenitor markers (Nestin, NOTCH1, and SOX2) and neural markers (β-tubulin III, GATA-3, and peripherin) upon transfection of hDPSCs with miR-124. The qRT-PCR results showed that Nestin was upregulated 6 h post-transfection. In contrast, Nestin expression exhibited a decreasing trend 24 h and 48 h post-transfection. Higher levels of β-tubulin III, 6 h and 16 h post transfection in RNA level as compared with control cells, were determined in transfected DPSCs. However, β-tubulin-III expression decreased 48 h post-transfection. The immunoflourescence results indicated that transfection of hDPSCs with miR-124, only affected Nestin among the studied neural progenitor and neural marker expression in protein level. Keywords: Sensorineural hearing loss, miR-124, Spiral ganglion neurons, Nestin, DPSCs

Background Dental pulp stem cells (DPSCs) are low immunogenic and hold immunomodulatory properties that, along with their well-established multi-potency, might enhance their potential application in autoimmune and inflammatory diseases. The present study focused on the ability of DPSCs to modulate the inflammatory microenvironment through PD1/PD-L1 pathway. Methods Inflammatory microenvironment was created in vitro by the activation of T cells isolated from healthy donors and rheumatoid arthritis (RA) patients with anti-CD3 and anti-CD28 antibodies. Direct and indirect co-cultures between DPSCs and PBMCs were carried out to evaluate the activation of immunomodulatory checkpoints in DPSCs and the inflammatory pattern in PBMCs. Results Our data suggest that the inflammatory stimuli trigger DPSCs immunoregulatory functions that can be exerted by both direct and indirect contact. As demonstrated by using a selective PD-L1 inhibitor, DPSCs were able to activate compensatory pathways targeting to orchestrate the inflammatory process by modulating pro-inflammatory cytokines in pre-activated T lymphocytes. The involvement of PD-L1 mechanism was also observed in autologous inflammatory status (pulpitis) and after direct exposure to pre-activated T cells from RA patients suggesting that immunomodulatory/anti-inflammatory properties are strictly related to their stemness status. Conclusions Our findings point out that the communication with the inflammatory microenvironment is essential in licensing their immunomodulatory properties. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-021-02664-4.

Background Alveolar bone loss is a frequent occurrence. Dental pulp stem cells (DPSCs) which have invasive accessibility and high osteogenic potential is a promising source for cell-based bone regeneration. EphrinB2 is involved in bone homeostasis and osteogenesis. The aim of this study was to investigate the effect and mechanism of ephrinB2 overexpression on osteogenic differentiation of DPSCs and bone defect repair. Methods EphrinB2 expression was analyzed during osteogenic induction of human DPSCs (hDPSCs). Endogenous ephrinB2 expression in hDPSCs was then upregulated using EfnB2 lentiviral vectors. The effect of ephrinB2 overexpression on osteogenic differentiation capacity of hDPSCs was investigated in vitro, and activation of ephrinB2-EphB4 bidirectional signaling in ephrinB2-overexpressing hDPSCs was detected. In vivo, a canine alveolar bone defect model was established and canine DPSCs (cDPSCs) were cultured, characterized, EfnB2-tranfected, and combined with a PuraMatrix scaffold. Micro-CT analysis was performed to evaluate the therapeutic effect of ephrinB2-overexpressing cDPSCs on bone defect repair. Results EphrinB2 was upregulated after osteogenic induction of hDPSCs. EphrinB2 overexpression enhanced osteogenic differentiation capacity of hDPSCs in vitro. Moreover, p-ephrinB2 instead of p-EphB4 was upregulated by ephrinB2 overexpression, and activation of ephrinB2-mediated reverse signaling promoted osteogenic differentiation of hDPSCs. In a canine bone defect model, ephrinB2 overexpression in cDPSCs significantly improved trabecular bone volume per tissue volume (BV/TV) and trabecular thickness, as demonstrated by radiographic analysis. Conclusions EphrinB2 overexpression enhanced osteogenic potential of DPSCs partially via upregulation of ephrinB2-mediated reverse signaling and effectively promoted alveolar bone defect repair.

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 &lt, 0.005), osteopontin (OPN) (p &lt, 0.0005) and alkaline phosphatase (ALP) (p &lt, 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 &lt, 0.05) signaling pathway, the activation of the extracellular matrix-related gene (ECMG) (p &lt, 0.05) and the Ca2+ signaling pathway (p &lt, 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.

Circular RNAs (circRNAs) have been found to be a crucial role in stem cell-associated bone regeneration. However, the functions and underlying mechanisms of circRNAs in the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain largely unclear. We found that overexpression of circRNA124534 unexpectedly promoted DPSCs osteogenesis in vitro and in vivo. Our results confirmed circRNA124534, acting as a miRNA sponge, directly interacts with miR-496 and consequently regulates β-catenin, which in turn exerts osteogenesis of DPSCs. Enforced expression of miR-496 reversed the osteogenesis of circRNA124534, and suppression of miR-496 enhanced the osteogenic differentiation of DPSCs by promoting β-catenin. In conclusion, our findings demonstrate functions of circRNA124534 in modulating osteogenic differentiation through the miR-496/β-catenin pathway; thus, providing a novel potential target for therapy.

Background: Dental pulp stem cells (DPSCs) are a unique source for future clinical application in dentistry such as periodontology or endodontics. However, DPSCs are prone to apoptosis under abnormal conditions. Taxifolin is a natural flavonoid and possesses many pharmacological activities including anti-hypoxic and anti-inflammatory. We aimed to elucidate the mechanisms of taxifolin protects DPSC under hypoxia and inflammatory conditions. Methods: DPSCs from human dental pulp tissue was purchased from Lonza (cat. no. PT-5025. Basel, Switzerland)) and identified by DPSC’s biomarkers. DPSC differentiation in vitro following the manufacturers’ instructions. ARS staining and Oil red staining verify the efficiency of differentiation in vitro after 2 weeks. The changes of various genes and proteins were identified by Q-PCR and western-blot, respectively. Cell viability was determined by the CCK-8 method, while apoptosis was determined by Annexin V/PI staining. Results: DPSC differentiation in vitro shows that hypoxia and TNF-α synergistically inhibit the survival and osteogenesis of DPSCs. A final concentration of 10 μM Taxifolin can significantly reduce the apoptosis of DPSCs under inflammation and hypoxia conditions. Taxifolin substantially increases carbonic anhydrase IX (CA9) expression but not HIF1a, and inhibitions of CA9 expression nullify the protective role of taxifolin under hypoxia and inflammatory condition. Conclusion: Taxifolin significantly increased the expression of CA9 when it inhibits DPSC apoptosis and taxifolin synergistically to protect DPSCs against apoptosis with CA9 under hypoxia and inflammatory conditions. Taxifolin can be used as a potential drug for clinical treatment of DPSC-related diseases.

Collagen membranes are used in oral surgery for bone defects treatment acting as a barrier that does not allow the invasion of soft tissue into the growing bone. To improve biocompatibility collagen membranes were coated with graphene oxide (GO), a graphene derivative. The aim of this study was to investigate the biocompatibility of GO coated collagen membranes on human dental pulp stem cells (DPSCs) focusing on biomaterial cytotoxicity, ability to promote DPSCs differentiation process and to control inflammation event induction. DPSCs were cultured on uncoated membranes and on both 2 and 10 mu g mL(-1) GO coated membranes up to 28 days. Alamar blue and LDH cytotocicity assay, PGE2 ELISA assay, real time RT-PCR for RUNX2, BMP2, SP7, TNF alpha and COX2 genes expression were performed. Proliferation is higher on GO coated membranes at days 14 and 28. LDH assay evidences no cytotoxicity. BMP2 and RUNX2 expression is higher on coated membranes, BMP2 at early and RUNX2 and SP7 at late experimental times. PGE2 levels are lower on GO coated membranes at days 14 and 28, both TNF alpha and COX2 expression is significantly decreased when GO is applied. GO coated membranes are not toxic for DPSCs, induce a faster DPSCs differentiation into odontoblasts/osteoblasts and may represent good alternative to conventional membranes thus ensuring more efficient bone formation and improving the clinical performance.

OBJECTIVES: Stem cells have the ability to rescue and/or repair injured tissue. In humans, it is possible to isolate different types of stem cells from the body. Among these, dental pulp stem cells (DPSCs) are relatively easily obtainable and exhibit high plasticity and multipotential capabilities. In particular they represent a gold standard for neural-crest-derived bone reconstruction in humans and can be used for the repair of body defects in low-risk autologous therapeutic strategies. SOURCES: An electronic search was conducted on PubMed databases and supplemented with a manual study of relevant references. RESULTS: All research described in this review highlight that DPSCs are mesenchymal stem cells that could be used in clinical applications. Unfortunately, very few clinical trials have been reported. Major obstacles imposed on researchers are hindering the translation of potentially effective therapies to the clinic. Both researchers and regulatory institutions need to develop a new approach to this problem, drawing up a new policy for good manufacturing practice (GMP) procedures. We strongly suggest that only general rules be standardized rather than everything. Importantly, this would not have an effect on the safety of patients, but may very well affect the results, which cannot be identical for all patients, due to physiological diversity in the biology of each patient. Alternatively, it would be important to study the role of specific molecules that recruit endogenous stem cells for tissue regeneration. In this way, the clinical use of stem cells could be successfully developed. CONCLUSIONS: DPSCs are mesenchymal stem cells that differentiate into different tissues, maintain their characteristics after cryopreservation, differentiate into bone-like tissues when loaded on scaffolds in animal models, and regenerate bone in human grafts. In summary, all data reported up to now should encourage the development of clinical procedures using DPSCs. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved Objectives: Stem cells have the ability to rescue and/or repair injured tissue. In humans, it is possible to isolate different types of stem cells from the body. Among these, dental pulp stem cells (DPSCs) are relatively easily obtainable and exhibit high plasticity and multipotential capabilities. In particular they represent a gold standard for neural-crest-derived bone reconstruction in humans and can be used for the repair of body defects in low-risk autologous therapeutic strategies. Sources: An electronic search was conducted on PubMed databases and supplemented with a manual study of relevant references. Results: All research described in this review highlight that DPSCs are mesenchymal stem cells that could be used in clinical applications. Unfortunately, very few clinical trials have been reported. Major obstacles imposed on researchers are hindering the translation of potentially effective therapies to the clinic. Both researchers and regulatory institutions need to develop a new approach to this problem, drawing up a new policy for good manufacturing practice (GMP) procedures. We strongly suggest that only general rules be standardized rather than everything. Importantly, this would not have an effect on the safety of patients, but may very well affect the results, which cannot be identical for all patients, due to physiological diversity in the biology of each patient. Alternatively, it would be important to study the role of specific molecules that recruit endogenous stem cells for tissue regeneration. In this way, the clinical use of stem cells could be successfully developed. Conclusions: DPSCs are mesenchymal stem cells that differentiate into different tissues, maintain their characteristics after cryopreservation, differentiate into bone-like tissues when loaded on scaffolds in animal models, and regenerate bone in human grafts. In summary, all data reported up to now should encourage the development of clinical procedures using DPSCs.

Background Dental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntington's disease (HD), have not been fully investigated. Results To examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile. Conclusions Our results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.