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Cortical organoids derived from human induced pluripotent stem cells (hiPSCs) represent a powerful in vitro experimental system to investigate human brain development and disease, often inaccessible to direct experimentation. However, despite steady progress in organoid technology, several limitations remain, including high cost and variability, use of hiPSCs derived from tissues harvested invasively, unexplored three-dimensional (3D) structural features and neuronal connectivity. Here, using a cost-effective and reproducible protocol as well as conventional two-dimensional (2D) immunostaining, we show that cortical organoids generated from hiPSCs obtained by reprogramming stem cells from human exfoliated deciduous teeth (SHED) recapitulate key aspects of human corticogenesis, such as polarized organization of neural progenitor zones with the presence of outer radial glial stem cells, and differentiation of superficial- and deep-layer cortical neurons and glial cells. We also show that 3D bioprinting and magnetic resonance imaging of intact cortical organoids are alternative and complementary approaches to unravel critical features of the 3D architecture of organoids. Finally, extracellular electrical recordings in whole organoids showed functional neuronal networks. Together, our findings suggest that SHED-derived cortical organoids constitute an attractive model of human neurodevelopment, and support the notion that a combination of 2D and 3D techniques to analyze organoid structure and function may help improve this promising technology., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Ltd.)

Objective: The aim of this study was to explore the effect and mechanism of programmed cell death ligand 1 (PD-L1) in promoting the proliferation and osteo/odontogenic-differentiation of human dental pulp stem cells (hDPSCs) by mediating CCCTC-binding factor (CTCF) expression., Design: The interaction between PD-L1 and CTCF was verified through co-immunoprecipitation. hDPSCs transfected with PD-L1 overexpression and CTCF knockdown vectors were treated with lipopolysaccharide or an osteogenic-inducing medium. Inflammatory cytokines and osteo/odontogenic-differentiation related genes were measured. Osteo/odontogenic-differentiation of hDPSCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining., Results: Overexpression of PD-L1 inhibited LPS-induced pro-inflammatory cytokine upregulation, cell proliferation, ALP activity, and calcium deposition in hDPSCs and elevated the expression of osteo/odontogenic-differentiation related genes; however, such expression patterns could be reversed by CTCF knockdown. Co-immunoprecipitation results confirmed the binding of PD-L1 to CTCF, indicating that PD-L1 overexpression in hDPSCs increases CTCF expression, thus inhibiting the inflammatory response and increasing osteo/odontogenic-differentiation of hDPSCs., Conclusion: PD-L1 overexpression in hDPSCs enhances the proliferation and osteo/odontogenic-differentiation of hDPSCs and inhibit the inflammatory response by upregulating CTCF expression., Competing Interests: Declaration of Competing Interest The authors report no relationships that could be construed as a conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: In this study, we aimed to explore the mechanism underlying microRNA-20a (miR-20a) in viability, migration and osteogenic/odontoblastic differentiation of dental pulp stem cells (DPSCs)., Design: The results of Targetscan unveiled that interleukin (IL)- 8 might bind to miR-20a, which was confirmed by dual-luciferase reporter assay. MiR-20a expression was up-regulated or down-regulated in DPSCs, followed by measurement of cell viability and migration via MTT and wound healing assays. Then, IL-8 was over-expressed in miR-20a mimic-transfected DPSCs with or without nuclear factor (NF)-κB inhibitor. The effect of miR-20a on osteogenic/odontoblastic differentiation potential of DPSCs was determined by alkaline phosphatase and alizarin red S staining. The expression levels of IL-8, osteogenic differentiation indicators and NF-κB/p65 signaling pathway in cells were detected through quantitative real-time polymerase chain reaction (qRT-PCR) or western blot RESULTS: Contrary to the down-regulated miR-20a, up-regulated miR-20a not only reinforced the viability and migration of DPSCs, but also promoted cell osteogenic/odontoblastic differentiation potential and related gene expressions (alkaline phosphatase, osteocalcin, and dentin sialophosphoprotein (DSPP)). Furthermore, IL-8 was verified to be the target of miR-20a. IL-8 over-expression effectively suppressed the osteogenic/odontoblastic differentiation potential as well as inhibited the activation of NF-κB pathway in DPSCs induced by miR-20a mimic, but these effects could be counteracted by NF-κB inhibitor., Conclusion: MiR-20a up-regulation accelerated the viability, migration and osteogenic/odontoblastic differentiation potential of DPSCs by regulating NF-κB/p65 signaling pathway via targeting IL-8, which might be a potential treatment target for dental diseases., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Dental pulp stem cells (DPSCs) have a high capacity to differentiate into the neuronal cell lineage. Meanwhile, both Hippo signaling and melatonin are key regulators in neuronal differentiation of neuronal progenitor cells. Recently emerging evidences suggest the possible interaction between melatonin and Hippo signaling in different cell lines. But underlying mechanisms involved in the initiation or progression of neurogenic differentiation in DPSCs through this connection need to be explored. Therefore, the scope of this study is to investigate the effect of melatonin on Hippo signaling pathway through the expression of its downstream effector (YAP/p-YAP Y357 ) after the neuronal differentiation of DPSCs. In regard with this, DPSCs were incubated with growth and dopaminergic neuronal differentiation medium with or without melatonin (10 μM) for 21 days. The morphological changes were followed by phase contrast microscopy and differentiation of DPSCs was evaluated by immunofluorescence labelling with NeuN, GFAP, and tyrosine hydroxylase. Furthermore, we evaluated the presence of neural progenitor cells by nestin immunoreactivity. Hippo signaling pathway was investigated by evaluating the immunoreactivity of YAP and p-YAP Y357 . Our results were also supported by western-blot analysis and SOX2, PCNA and caspase-3 were also evaluated. The positive immunoreactivity for NeuN, tyrosine hydroxylase and negative immunoreactivity for GFAP showed the successful differentiation of DPSCs to neurons, not glial cells. Melatonin addition to dopaminergic media induced tyrosine hydroxylase and decreased significantly nestin expression. The expressions of PCNA and caspase-3 were also decreased significantly with melatonin addition into growth media. Melatonin treatment induced phosphorylation of YAP Y357 and reduced YAP expression. In conclusion, melatonin has potential to induce neuronal differentiation and reduce the proliferation of DPSCs by increasing phosphorylation of YAP Y357 and eliminating the activity of YAP, which indicates the active state of Hippo signaling pathway., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The natural polysaccharides are promising compounds for applications in regenerative medicine. Gellan gum (GG) is the bacteria-derived polysaccharide widely used in food industry. Simple modifications of its chemical properties make GG superior for the development of biocompatible hydrogels. Beside reversible cationic integration of GG chains, more efficient binding is accomplished with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). However, the side-products of polymer cross-linking might affect viability and differentiation of stem cells introduced into the hydrogels. We found that O-acylisourea (EDU) stimulates autophagy-based vacuolation in both periodontal ligament and dental pulp stem cells. 24-h treatment of cells with GG extracts cross-linked with 15 mM EDC developed large cytoplasmic vacuoles. Freshly prepared EDU (2-6 mM) but not 15 mM EDC solutions initiated vacuole development with concomitant reduction of cell viability/metabolism. Most of the vacuoles stained with acridine orange displayed highly acidic environment further confirmed by flow cytometric analysis. Western blot of the LC3 autophagy marker followed by a transmission electron microscopy indicated the process is autophagy-dependent. We propose that the high reactivity of EDU with intracellular components initiates autophagy, although the targets of EDU remain unknown. Nevertheless, a burst release of EDU from GG hydrogels might modulate negatively cellular processes and final effectiveness of tissue regeneration., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to investigate the effect of epigallocatechin gallate (EGCG) on the proliferation, mineralization, inflammation and hypoxia responses of human dental pulp stem cells (hDPSCs) in vitro and its effect on inflammatory pulp tissue in rats in vivo., Design: The optimum concentration of EGCG was selected by creating a dose response curve. Expression of odontogenic/osteogenic-related genes and inflammatory cytokines after stimulation with Lipopolysaccharide (LPS) was detected by real-time PCR. Under hypoxic conditions, cell proliferation and expression of reactive oxygen species (ROS) and superoxide dismutase (SOD) were detected.In vivo, the maxillary first molars of SD rats were pulpotomized and stimulated with 5 mg/mL LPS for 30 min. Normal saline and EGCG were used to flush the pulp chamber. After 2 months, samples were removed for micro-CT scanning and HE staining., Results: CCK-8 assay revealed that 10 μg/mL EGCG had no significant effect on the proliferation of hDPSCs. EGCG inhibited expression of IL-1β, IL-6, and TNF-α. Furthermore, EGCG rescued cell proliferation ability, increased SOD activity and reduced ROS expression under hypoxia.In vivo, reduced inflammatory cell accumulation was observed in the coronal pulp in the EGCG group, while in the control group, diffuse inflammatory cells were observed in the radicular pulp., Conclusion: EGCG had no obvious effects on calcified nodule formation but significantly inhibited the inflammatory response of hDPSCs and inhibited apoptosis of hDPSCs caused by hypoxia injury. In vivo, EGCG exerts inhibitory effects on pulp tissue inflammation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objective: Although the osteogenic differentiation potential of mesenchymal stem cells of dental origin is well established, the roles of different marker proteins in this process remain to be clarified. Our aim was to compare the cellular and molecular changes, focusing in particular on mesenchymal stem cell markers, during in vitro osteogenesis in three dental stem cell types: dental follicle stem cells (DFSCs), periodontal ligament stem cells (PDLSCs) and dental pulp stem cells (DPSCs)., Design: Human DFSCs, PDLSCs and DPSCs were isolated, cultured and their osteogenic differentiation was induced for 3 weeks. Mineralization was assessed by von Kossa staining and calcium concentration measurements. The expression of mesenchymal and osteogenic markers was studied by immunocytochemistry and qPCR techniques. Alkaline phosphatase (ALP) activity and the frequency of STRO-1 positive cells were also quantified., Results: The three cultures all showed abundant mineralization, with high calcium content by day 21. The expression of vimentin and nestin was sustained after osteogenic induction. The osteogenic medium induced a considerable elevation of STRO-1 positive cells. By day 7, the ALP mRNA level had increased more than 100-fold in DFSCs, PDLSCs, and DPSCs. Quantitative PCR results indicated dissimilarities of osteoblastic marker levels in the three dental stem cell cultures., Conclusions: DFSCs, PDLSCs and DPSCs have similar functional osteogenic differentiation capacities although their expressional profiles of key osteogenic markers show considerable variations. The STRO-1 positive cell fraction expands during osteogenic differentiation while vimentin and nestin expression remain high. For identification of stemness, functional studies rather than marker expressions are needed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: This review aims to describe the basic characteristics of exosomes, and summarize their possible source and potential biological effects in pulp regeneration, providing new insights into the therapeutic role of exosomes for regenerative endodontics in the future., Design: A comprehensive review of scientific literature related to exosomes potentially used for pulp regeneration was conducted., Results: Dental mesenchymal stem cells (MSCs) play an important role in dental pulp regeneration. MSC-derived exosomes, as important biotransmitters in intercellular communication, have been shown to replicate the therapeutic effects of their parental cells. These exosomes have better stability, lower immunogenicity, higher safety and clinical efficiency, making it possible to apply them in pulp regeneration. Existing research suggests that exosomes could trigger the regeneration of dentin/pulp-like tissue in vivo, which may attribute to their role in promoting pulp angiogenesis, regulating dental cell proliferation, migration and differentiation, and providing neuroprotection., Conclusions: The applications of exosomes in the treatment of pulp regeneration have great potential, and exosomes may become ideal therapeutic biomaterial in regenerative endodontics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objectives: This study investigates the post-extraction storage period of human dental pulp stem cells (hDPSCs) for stem cell banking by investigating the viability, function, mineralization, and gene expression of hDPSCs isolated from extracted teeth after 1 h, 6 h and 24 h post-tooth extraction., Design: hDPSCs were extracted from the pulp of impacted third molar teeth after 1 h, 6 h, and 24 h after extraction. The mesenchymal stem cell (MSCs) properties of three groups of cells were analyzed using flow cytometry. Cell morphology and proliferation were analyzed using a light microscope and an MTT assay. The viability, function, mineralization, and gene expression of hDPSCs of 1 h, 6 h, and 24 h groups were also assessed., Results: The delayed harvesting of hDPSCs for 1, 6 or 24 h caused a 31 % reduction in mineral nodule formation and a reduction in the gene expression of osteocalcin (OCN) and vascular endothelial growth factor-alpha (VEGFA). However, the 1, 6 or 24 h, time delay had little effect on MTT cell proliferation, cell viability or morphology. The delayed of harvesting of hDPSCs for 1, 6 or 24 h also had little effect on the expression of MSCs positive (CD44, CD106, CD90) or negative surface markers (CD45 and CD11b)., Conclusions: Our results suggest that a 24 h delay in harvesting hDPSCs from extracted teeth can reduce their mineralization and gene activity but does not markedly reduce survival. Quicker hDPSCs harvesting is likely to yield more useful hDPSCs for experimentation and clinical treatment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: To compare proteomics and biological function of human dentin matrix molecules (hDMMs) and bovine dentin matrix molecules (bDMMs)., Design: Dentin powder from human or bovine teeth (n = 4) was demineralized in 10% (v/v) ethylenediaminetetraacetic acid for 7 days. The extracts were dialyzed, lyophilized and proteins were characterized using liquid chromatography-tandem mass spectrometry and shotgun proteomic analysis. To study biological function, mouse-derived undifferentiated dental pulp cells (OD21) were treated with 0.01, 0.1 or 1 μg/mL of hDMMs or bDMMs and proliferation was measured after 24 hours and 48 hours using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration was assessed after 24 hours using a Boyden chamber. Alizarin Red S staining was used to evaluate mineral formation., Results: There were 307 proteins identified, of which 93 proteins were common to both species. Gene Ontology functional analysis demonstrated similar pattern of biological process in both species which consisted mainly of tissue development and biomineralization. hDMMs and bDMMs both enhanced cell proliferation. After 24 hours, all concentrations of bDMMs promoted cell proliferation (p ≤ 0.05), while hDMMs did not affect proliferation. After 48 hours, groups with 1μg/mL of bDMMs and 0.01μg/mL of hDMMs had increased cell proliferation compared to control (p ≤ 0.0001). All concentrations of hDMMs and bDMMs enhanced cell migration and mineralization (p ≤ 0.0001)., Conclusion: bDMMs has similar biological functions as hDMMs. Moreover, bDMMs stimulated cell proliferation, migration and differentiation similar to hDMMs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: The aim of the present study was to investigate the biological roles and underlying mechanism of the long non-coding RNA maternally expressed gene 3 (MEG3) on osteogenic differentiation of human dental pulp stem cells (hDPSCs)., Methods: The expression levels of MEG3, microRNA-543 (miR-543), osterix, osteopontin, osteocalcin and runt-related transcription factor 2 (RUNX2) were measured by quantitative real-time PCR (qRT-PCR). Alkaline phosphatase (ALP) activity assay and alizarin red S staining (ARS) were used to measure the impacts exerted by MEG3, miR-543 on osteogenic differentiation. Cell proliferation was measured by MTT assay. In addition, the targeted relationships between miR-543, MEG3, and Smad ubiquitin regulatory factor 1 (SMURF1) were assessed through dual luciferase reporter assay., Results: During osteogenic induction, the expression of MEG3 was gradually reduced, whereas the expression of miR-543, osterix, osteopontin, osteocalcin and RUNX2 were gradually increased. Functional analysis implied that MEG3 overexpression or miR-543 inhibition reduced the cell proliferation, ALP activity, ARS levels, and decreased the expression of osteoblast-related proteins. Moreover, MEG3 promoted SMURF1 expression by directly targeting miR-543 as a competing endogenous RNA. Furthermore, overexpression of miR-543 or silencing SMURF1 could reverse the inhibitory effects of MEG3 on the osteogenic differentiation of hDPSCs., Conclusions: In conclusion, our study revealed that overexpression of MEG3 inhibited hDPSCs osteogenic differentiation via miR-543/SMURF1/RUNX2 regulatory network, which may contribute to the functional regulation and clinical applications of hDPSCs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study is to investigate the changes of human dental pulp stem cell (hDPSC) viability, proliferation and osteogenic differentiation in high glucose condition., Design: After 21 days of culture in low (5.5 mM) and high (20 mM) glucose medium, hDPSC viability and proliferation were assessed with respectively the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst assays. To investigate the influence of glucose on osteogenic differentiation hDPSCs were cultured for 28 days in low or high glucose medium with osteoinductive cocktail. Mineralization was examined by alizarin red staining/quantification and the expression of osteogenic-related genes [Runt-related transcription factor 2 (RUNX2), Osteocalcin (OCN), Collagen 1A1 (COL1A1)] analyzed by RT-qPCR., Results: We observed no significant difference (p >  0.05) on hDPSC proliferation or cell viability between low or high glucose groups. We did not highlight a significant difference after alizarin red staining and quantification between hDPSCs cultured with high or low glucose concentration in the culture medium. In the same manner, high glucose concentration did not appear to modify osteogenic gene expression: there was no significant difference in osteogenic-related gene expression between high or low glucose groups., Conclusion: Proliferation, viability, and osteogenic differentiation of hDPSCs were not changed by high glucose environment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to investigate the effects of two radiopaque agents, barium sulfate (BaSO 4 ) or zirconium oxide (ZrO 2 ) in double antibiotic paste (DAP), on the proliferation and mineral deposition of human dental pulp stem cells (DPSC)., Materials and Methods: Radiopaque antimicrobial medicaments composed of methylcellulose (MC) thickening polymer with BaSO 4 or ZrO 2 and either 1 or 5 mg/mL DAP (equal portions of metronidazole and ciprofloxacin) were used to investigate DPSC proliferation after 3 days, and alkaline phosphatase (ALP) activity and mineral deposition after 7 and 14 days. Radiopaque agents without DAP and Ca(OH) 2 were used as controls., Results: MC-BaSO 4 DAP and MC-ZrO 2 DAP at 1 or 5 mg/mL had no adverse effect on DPSC proliferation, compared to the media and MC controls. MC-ZrO 2 (DAP-free) greatly increased ALP activity after 7 days. DPSC mineral deposition was modestly reduced at 7 days by MC-BaSO 4 DAP and MC-ZrO 2 DAP, but not by DAP-free radiopaque agents, and was most reduced by 5 mg/mL DAP in the 14-day cultures., Conclusions: MC-BaSO 4 or MC-ZrO 2 medicaments containing up to 5 mg/mL of DAP supported the proliferation and early osteogenic differentiation of DPSC. Low DAP concentrations and short culture times led to more favorable effects on ALP activity and mineral deposition by DPSC. The findings suggest that radiopaque agents added for the purpose of detecting whether medicaments occupy the full extent of the root canal may have clinical applications. Radiopaque antibiotic medicaments containing low DAP concentrations may be an alternative to Ca(OH) 2 for regenerative endodontic procedures., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: The objective of this study was to evaluate the ability of three distinct agents on the induction of senescence in human dental pulp stem cells (DPSCs)., Design: DPSCs from three separate donors were treated with H 2 O 2 , doxorubicin and ultraviolet (UV) irradiation. The response of the cells to the three agents was assayed by specific staining for SA-βGal, RT-qPCR and flow cytometry., Results: The results showed that incubation with 100 μM H 2 O 2 and 20 nM Doxorubicin for seven days led to senescence in all donors' cells equally. Interestingly, UV irradiation for just one minute was sufficient to induce senescence in the cells. The SA-βGal positive senescent cells were arrested in G 1 phase and their S phase was significantly reduced as analyzed by flow cytometry. Significant increment in p21 and BTG1 expression and decrement in CCND1 expression also confirmed the cells have been arrested and get senescent via p53-p21 pathway., Conclusion: All three agents successfully triggered senescence in the cells. There was no significant difference in the capacity of the three donor's cells for senescence. To avoid premature senescence in stem cell in vitro, it is recommended to avoid unnecessary exposure of the cell to fluorescent and UV light. Moreover, to prevent ROS production, we recommend using a separate incubator with low oxygen content for cell culture, if possible., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: Angiogenesis is important in pulp-dentin formation. Among the regulatory factors, long noncoding RNA (LncRNA) is a class of functional RNA molecules that are not translated into protein and involved in regulating multiple physiological processes. The different expression of LncRNA and its target gene in dental pulp stem cells (DPSCs) were explored and may provide a theoretical basis for future regulation of dental pulp angiogenesis., Methods: In this study, we cultured DPSCs from healthy dental pulp tissues and divided them into two groups: the normal DPSCs and the DPSCs cultured in vascular induction medium. In total, 40,173 LncRNA probes and 20,730 protein coding mRNAs were detected through microarray, which were then verified by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method., Results: The result of differential expressions measured in LncRNA through microarray showed that 376 LncRNAs increased significantly and 426 were downregulated among the two groups of cells. Moreover, the mRNA microarray in normal cultured DPSCs showed that 629 LncRNAs were significantly upregulated, while 529 of them were downregulated compared with the DPSCs that were cultured in vascular induction medium. Gene ontology (GO) analysis inferred the molecular function of mRNAs. Pathway analysis showed that 52 signaling pathways were involved in the differentiation process of DPSCs. qRT-PCR analysis, conducted for validation, showed results consistent with the microarray analysis., Conclusions: We found that a number of different regulators are involved in inducing vascular differentiation of DPSCs, which provides a foundation for subsequent experiments., Competing Interests: Declaration of Competing Interest The authors deny any conflicts of interest related to this study., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to evaluate the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) and human umbilical cord mesenchymal stem cells (hUCMSCs) in three-dimensional co-culture system which was established with the help of bone morphogenetic protein-2 (BMP-2) and hydrogel., Methods: hDPCs and hUCMSCs were cultured in different concentrations of hydrogel to explore the more suitable concentrations for subsequent experiments. hUCMSCs and hDPCs induced by BMP-2 were co-cultured in the hydrogel. MTT assay was used to measure the cell viability. The differentiation into odontoblast-like cells were measured by the mRNA expression of dentin salivary phosphoprotein (DSPP), dentin matrix protein-1 (DMP-1), alkaline phosphatase and osteocalcin. Alizarin red staining was performed for the formation of mineralized nodules., Results: hUCMSCs and hDPCs could grow and proliferate in hydrogel scaffold. The growth rate of cells in lower concentrations hydrogels were higher than that of high concentrations hydrogels (P < 0.05). The study showed that 0.25% hydrogel scaffold was more suitable for subsequent experiments than other groups. Compared with hUCMSCs-monoculture and hDPCs-monoculture, the co-culture groups exhibited more proliferative potential, alkaline phosphatase activity and mineralization nodule formation (P < 0.05). The mRNA expression in co-culture groups were higher than that of hUCMSCs-monoculture, closed to or even higher than that of hDPCs-monoculture., Conclusion: 0.25% hydrogel was the suitable concentration in co-culture system for subsequent experiments. The co-culture groups had stronger abilities of odontoblastic differentiation and mineralization than cells-monoculture groups, indicated that the co-culture conditions could regulate cell proliferation and differentiation within a certain range., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: To compare three different xeno-free protocols for neural differentiation of human dental pulp stem cells (DPSC)., Methods: DPSC were treated with three different media to induce neural differentiation namely N1 (DMEM for 5 days), N2 (PSC neural induction media for 7 days) and N3 (neural media with B27 supplement, 40 ng/ml bFGF and 20 ng/ml EGF for 21 days). Cell proliferation (MTS assay), morphology, gene (qPCR for NESTIN, VIMENTIN, TUB-3, ENO2, NF-M and NF-H) and protein expression (flow cytometry) of neurogenic markers were assessed at different time points and compared to untreated cells (DMEM supplemented with 10% FBS). Statistical analysis was performed with global significance level of 5%., Results: N1 and N2 formulations increased the genetic expression of two out of six genes TUB-3, NF-M and TUB-3, NF-H, respectively, whereas N3 elevated the expression of all genes by the late stage. N3 also stimulated protein expression for NESTIN, TUB-3 and NF-H. Cells treated with both N2 and N3 presented neuron-like morphology, decreased proliferation and expression of stemness genes at protocol end point., Conclusion: N3 was the most effective formulation in promoting a neurogenic shift in gene and protein expression. Cells provided with the N3 formulation exhibited neuron-like morphology, elaborating axonal-like projections concomitant with cell cycle withdrawal and reduced expression of stemness genes indicating greater commitment to a neurogenic lineage., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: The objectives of this study were (a) to determine the differentially expressed microRNAs that can target heat shock protein B8 (HspB8) during in vitro expansion of dental pulp stem cells (DPSCs); (b) to identify microRNAs involved in posttranscriptional regulation of HspB8 expression; and (c) to determine if HspB8-targeting microRNAs play roles on osteogenic differentiation of DPSCs., Design: DPSCs were established from rat first molars and expanded in vitro until the passage that cells lost osteogenic potential. TargetScan was used to predict the microRNAs that target HspB8 mRNA. Stem-loop quantitative RT-PCR was conducted to identify the HspB8-targeting microRNAs that were upregulated in late passages. The microRNAs mimics were transfected into DPSCs to assess their effects on HspB8 expression and on osteogenic differentiation., Results: let-7b-5p, miR-98-5p, miR-215, miR-219a-1-3p and miR-295-5p were found to consistently increase expression in DPSCs after expansion. HspB8 mRNA and/or protein were significantly decreased in the DPSCs after transfection of miR-215 and miR-219a-1-3p mimics; whereas no significant reduction was seen after transfecting let-7b-5p, miR-98-5p and miR-295-5p mimics. When subjecting the transfected DPSCs to osteogenic induction, reduction of calcium deposition or osteogenic marker expression were observed with miR-215, miR-219a-1-3p and miR-295-5p transfection., Conclusions: Increased expression of miR-215 and miR-219a-1-3p downregulates HspB8 expression, which contributes to the reduction of osteogenic capability of DPSCs. Increased expression of miR295-5p also causes a reduction of osteogenic differentiation, but not involved in HspB8., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: The purpose of this study was to compare the effects of two fast-setting pulp-capping materials, Biodentine (BD) and iRoot Fast Set (FS) root repair material, on the attachment, viability, migration, and differentiation of human dental pulp stem cells (hDPSCs)., Methods: A comparative study was conducted between BD and FS material disks. Scanning electron microscope (SEM) images were used to observe the attachment of hDPSCs on the disks. A live/dead assay was used to assess the cell viability. Transwell assay was performed to study cell migration. Cell differentiation was determined by quantitative real-time polymerase chain reaction (qRT-PCR) for the analysis of osteogenic differentiation gene expression: alkaline phosphatase (ALP), collagen type I (COL1) and osteocalcin (OCN)., Results: SEM images indicated that hDPSCs showed a well-spreading morphology on both BD and FS disks. FS significantly increased the proliferation and migration of hDPSCs on day 7 (P＜0.05). Neither BD nor FS promoted the expression of osteogenic genes during the observation period., Conclusions: BD and FS both were beneficial to hDPSC attachment, and they had similar effects on cell osteogenic differentiation, whereas FS performed better than BD on hDPSCs proliferation and migration., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: This short review summarizes our current knowledge about dental stem cell aging and about possible targets for the regulation of cellular senescence., Design: A literature search was performed using a combination of keywords, e.g., stem cells, replicative senescence, differentiation potential, dental pulp, dental follicle and periodontal ligament., Results: Previous studies have shown that cellular senescence occurs while the proliferation of dental stem cells. Moreover, the differentiation potential was significantly decreased in senescent stem cells and senescent cells secrete also factors that are harmful to the adjacent tissue cells. Moreover, many targets for the regulation of cellular senescence are considered; for example pathways related to the nutrient sensing such as the 5' adenosine monophosphate-activated protein kinase (AMPK) pathway., Conclusions: The regulation of cellular senescence will play a crucial role in the clinical use of stem cells. However, there is no cell culture protocol available that prevents dental stem cell senescence. Therefore, more knowledge about molecular processes in stem cells is needed before and after the induction of senescence., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to investigate the anti-inflammatory effect of betamethasone on LPS-stimulated human dental pulp stem cells (DPSCs) and its associated mechanism. The osteo-/odontogenic differentiation and osteoclast effect of betamethasone on DPSCs and stem cells from human exfoliated deciduous teeth (SHED) were evaluated., Design: The proliferative effect of betamethasone on DPSCs was analyzed using a cholecystokinin octapeptide assay. The anti-inflammatory effect of betamethasone was investigated using quantitative polymerase chain reaction (qPCR) and ELISA. The anti-inflammatory mechanism was explored using qPCR, Western blot, and immunofluorescence staining. The osteo-/odontogenic differentiation and osteoclast effect of betamethasone on DPSCs and SHED were detected by qPCR., Results: 1 μg L -1 betamethasone was found to have the strongest effect on DPSCs proliferation. The expression of pro-inflammatory cytokines and mediators, as well as prostaglandin E 2 (PGE 2 ) were significantly decreased following treatment with betamethasone in LPS- stimulated DPSCs. They were also decreased in response to an NF-κB inhibitor, Bay 11-7082. Betamethasone and Bay 11-7082 significantly inhibited the expression of p-p65 and promoted the nuclear exclusion of p65. Gene expression associated with osteo-/odontogenic differentiation was significantly up-regulated in betamethasone and osteogenic media (OM) treated groups. The ratio of the receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) at the mRNA level was suppressed in DPSCs and elevated in SHED., Conclusions: Betamethasone has an anti-inflammatory effect on LPS- stimulated DPSCs through a blockade of NF-κB activation and exhibits an osteo-/odonto-inductive effect on DPSCs and SHED. Although betamethasone displays an osteoclast effect on SHED., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: To study the effects of polyphenol resveratrol on TNFα-induced inflammatory signaling as well as the underlying mechanism in human dental pulp stem cells (DPSCs)., Materials and Methods: Human DPSCs were cultured and treated by TNFα in the presence or absence of resveratrol. NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways were analyzed by Western blotting and immunofluorescence staining. Interleukin 6 (IL6) and interleukin 8 (IL8) mRNA levels were analyzed by reverse transcription polymerase chain reaction. For the mechanistic study, autophagy was examined and further manipulated by gene silencing of Atg5 using siRNAs. Statistical analysis was performed by Student's t- test, and values of p < 0.05 were considered significant., Results: Upon TNFα treatments, neither degradation of IκBα nor the phosphorylation and nuclear translocation of p65 NF-κB were inhibited by resveratrol at different concentrations. In contrast, resveratrol dramatically inhibited TNFα-induced phosphorylation of c-Jun N-terminal kinase (JNK) MAPK. Furthermore, resveratrol activated autophagy, as evidenced by the accumulated autophagic puncta formed by lipid bound LC3B in resveratrol-treated cells. Intriguingly, both resveratrol and JNK inhibitor SP600125 suppressed TNFα-induced IL6 and IL8 mRNA expression (P < 0.05). Silencing autophagy gene Atg5 led to the hyper-activation of JNK and augmented TNFα-induced IL6 and IL8 mRNA expression (P < 0.05)., Conclusions: The results suggest that resveratrol suppresses TNFα-induced inflammatory cytokines expressed by DPSCs through regulating the inhibitory autophagy-JNK signaling cascade. Resveratrol might be beneficial to ameliorate pulpal damage during the acute phase of inflammation in vital pulp therapy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Magnesium, the second most abundant cation in cellular fluid, is critical for mineralization of hard tissues. Among the molecules involved in cellular Mg 2+ homeostasis, functional impairment of Mg 2+ permeable ion channel TRPM7 or Mg 2+ transporter CNNM4 have been found to result in severe hypomineralization of the enamel and dentin. However, molecular expressions of TRPM7, CNNM4 and their respective homologues have not been fully investigated in adult odontoblasts., Design: Expressions of TRPM6, TRPM7, CNNM1, CNNM2, CNNM3, CNNM4 were screened in acutely dissociated rat odontoblasts by single cell RT-PCR. Among these candidates, expression levels of TRPM7 and CNNM4 were compared along the odontoblast layer by immunohistochemical analysis. Finally, the coexpression pattern of TRPM7 and CNNM4 in subcellular regions was examined by immunocytochemical analysis., Results: ScRT-PCR revealed high expression rate of TRPM7 and CNNM4 in odontoblasts, with CNNM4 detected almost exclusively in TRPM7-positive odontoblasts. However, CNNM2 and CNNM3 were detected in only a small population of odontoblasts, and TRPM6 and CNNM1 were not detected even in the pulp tissue. Immunohistochemical analysis revealed higher CNNM4 expression in the apical odontoblast layer than the coronal area, in contrast to the ubiquitous expression of TRPM7. Lastly, immunocytochemical analysis revealed colocalization of CNNM4 with TRPM7 in the odontoblastic process., Conclusions: CNNM4 and TRPM7 may serve as main Mg 2+ regulators in odontoblasts, possibly with selective involvement of CNNM4 in apical dentin formation or mineralization. Colocalization of TRPM7 and CNNM4 in the odontoblastic process suggest functional coupling of these two molecules to maintain Mg 2+ homeostasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: Despite the increased interest in stem cells isolated from remnant pulp of deciduous teeth, no specific marker has been yet established for them. The present study aimed to investigate whether SSEA-4 (stage-specific embryonic antigen) would be a suitable marker to isolate stem cells from Human Exfoliated Deciduous teeth (SHEDs) in order to increase its differentiation potential toward osseous tissue., Design: The SHEDs were isolated and the expression patterns of mesenchymal, hematopoietic and embryonic stem cell markers were assessed. The cells were then divided into two groups of SSEA-4(+) and unsorted SHEDs and the cell proliferation rate and population-doubling-time (PDT) were calculated. Subsequently, the differentiation potentials were examined through alizarin-red staining and Quantitative real time-PCR (qRT-PCR)., Results: Isolated cells were spindle-shaped with a high expression of mesenchymal stem cell markers and weak expression of hematopoietic markers. The mean expression of Oct-4 was 68.77%±1.28. Despite similar proliferation rates between SSEA-4(+) and unsorted SHEDs, because of differences in the shape of the growth curves, PDT was lower in unsorted SHEDs (P = 0.2 × 10 -4 ). Alizarin-red staining showed similar calcium deposition in both groups. Upon differentiation, the expression of osteocalcin was higher in unsorted SHEDs (P = 0.043), while, the expression of alkaline phosphatase was lower (P<0.001). The parathyroid hormone receptor (PTHR) expression was not significantly different (P = 0.0625)., Conclusions: The results of the present study revealed that SHEDs have high differentiation potentials even in the unsorted cells. Although the SSEA-4-positive SHEDs showed slightly better osteogenic potential, the differences were not abundant to link SSEA-4 expression with superior differentiation potency., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To evaluate to the effect of metformin on attachment of human dental pulp stem cells (hDPSCs) and their proliferation and osteogenic differentiation on biphasic hydroxyapatite/beta-tricalcium phosphate granules of macro-porous biphasic calcium phosphate (MBCP)., Materials and Methods: This in vitro study included four groups: A:hDPSCs + MBCP + Metfromin, B:hDPSCs + MBCP, C:hDPSCs + Metformin and D:hDPSCs (control). Attachment of hDPSCs to bone granules in groups A and B was observed by scanning electron microscopy on days 1 and 7 of cultivation. Cell viability was assessed by MTT assay on days 1, 3, and 7 after cell seeding. Differentiation of the hDPSCs was assessed by measurement of alkaline phosphatase activity on days 3, 7, 14 and 21 after cell culturing in standard and osteogenic media. The data was analyzed by two-way ANOVA at a significance level of p = 0.05., Results: The hDPSCs had firmly attached to the surface of MBCP granules, especially in group A. The MTT values increased in all groups from day 1 to day 7 (p < 0.001). The highest MTT values were observed in group C followed by the control group and groups A and B (p < 0.001). Alkaline phosphatase activity also increased in all groups between days 3 to 21 (p < 0.001) except between days 7 and 14 in standard media (p = 0.094). In standard media, groups with MBCP granules (A and B) showed higher activity (p < 0.05). In osteogenic media, the groups with metformin (A and C) showed higher alkaline phosphatase activity (p < 0.05)., Conclusion: This in vitro study showed that 100 Mol/L metformin increased attachment and proliferation of hDPSCs on biphasic granules. Osteogenic differentiation of hDPSCs also increased in the presence of metformin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to compare the behavior of dental pulp stem cells (DPSCs) after isolation using solutions containing either collagenase/dispase or collagenase alone., Design: DPSCs were isolated by two digestion methods (collagenase/dispase or collagenase alone) from human third molars. Immunophenotypic features were confirmed by flow cytometry for cell markers STRO-1, cluster of differentiation (CD) 146, CD45, and collagen type-I. The proliferation potential of cells was evaluated by 5-bromo-2'-deoxyuridine (brdU) incorporation assay, and finally they were assessed for multi-lineage differentiation potential. Data were analyzed using one-way analysis of variance and independent t-tests., Results: DPSCs isolated by either method showed similar levels of STRO-1, CD45, and collagen type-I and similar incorporation of brdU (P > 0.05). However, DPSCs obtained by collagenase I/dispase treatment had significantly higher numbers of CD146 + cells and osteogenic and chondrogenic capacities compared to those obtained by treatment with collagenase I alone (P < 0.05). On the other hand, more STRO-1+/CD164-DPSCs were found in the collagenase alone group with higher adipogenic potential., Conclusions: Different enzyme solutions gave rise to different populations of DPSCs. Dispase enhanced isolation of CD146 + DPSCs probably by disrupting the basement membranes of blood vessels and releasing DPCSs embedded in the perivascular niche. Furthermore, the differentiation potential of DPSCs was influenced by the change in enzyme solution., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Progression of pulpitis is facilitated by the immune system's response to bacteria, enhancing the production of inflammatory regulators. Bacterial lipopolysaccharide (LPS) is the major structural component of the outer wall of all Gram-negative bacteria and a potent activator of the immune system. Apoptosis is believed to play an important role in the inflammatory process of pulpitis. SIRT6 is a member of class III of histone deacetylases (HDACs), also called sirtuins (SIRTs). The role of SIRT6 in apoptosis in pulpitis is unknown. In this study, we found that the expression of SIRT6 in human dental pulp cells (hDPCs) was down-regulated by treatment with LPS. MTT and LDH assays revealed that overexpression of SIRT6 in hDPCs attenuated cell death induced by LPS. Consistently, our results demonstrated that SIRT6 was able to protect hDPCs from apoptosis. We found that SIRT6 could interact with Ku70, an important apoptosis regulator, by the immunoprecipitation (IP) experiment. SIRT6 physically binds to Ku70. Overexpression of SIRT6 reduced acetylation of Ku70 and promoted interaction of Ku70 with the proapoptotic protein Bax. These studies underscore an essential role of SIRT6 in the survival of hDPCs in stress situations., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Three-dimensional (3D) cell culture methods are of high importance to studies of biological processes. This is particularly the case with spheroid cultures, which create 3D cell aggregates without the use of exogenous materials. Compared to conventional monolayer cultures, cellular spheroid cultures have been demonstrated to improve multilineage potential and extracellular matrix production. To address this issue in depth, we present a more comprehensive analysis of 3D human dental pulp cell (hDPC) spheroids., Design: hDPC spheroids were fabricated by the pellet culture method and were cultured without adding any reagent to induce differentiation. The gene-expression profiles of the 3D and two-dimensional (2D) cultured hDPCs were compared by complementary DNA microarray analysis. Odontoblastic and osteoblastic differentiation marker gene expression was evaluated by quantitative real-time PCR (RT-qPCR). Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to examine the morphology of hDPC spheroids and extracellular matrix components., Results: Compared with 2D monolayer culture, microarray analysis identified 405 genes and 279 genes with twofold or greater differential expression after 3 days and 28 days of 3D culture, respectively. In 3D hDPC spheroids, gene ontology analysis revealed upregulation of extracellular matrix-related genes and downregulation of cell growth-related genes. RT-qPCR analysis showed higher expression levels of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM revealed the morphological characteristics of the fibrillar collagen-rich matrix and cell-cell interactions., Conclusions: The present findings provide clues to understanding the mechanisms of pellet-cultured hDPCs and contribute to future research in the comparative studies of different 3D culture methods., (Copyright © 2018. Published by Elsevier Ltd.)

Objective: This study aimed to compare the proteomic profiling of human dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) under in vitro osteogenic induction, which imitates the microenvironment during osteo-/odontogenesis of DPSCs and PDLSCs., Design: The proteomic profiles of osteoinduced DPSCs and PDLSCs from a single donor were compared using the isobaric tag for relative and absolute quantitation (iTRAQ) technique and subsequent bioinformatics analysis., Results: A total of 159 differentially expressed proteins in PDLSCs and DPSCs were identified, 82 of which had a higher expression level in PDLSCs, while 77 were more highly expressed in DPSCs. Among these enriched proteins, certain members from the collagen, heat shock protein and protein S100 families may distinguish osteoinduced PDLSCs and DPSCs. Gene ontology (GO) classification revealed that a large number of the enriched terms distinguishing PDLSCs and DPSCs are involved in catalytic activity, protein binding, regulation of protein metabolic processes and response to stimulus. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated several involved pathways, including the fatty acid biosynthesis pathway, pantothenate and CoA biosynthesis pathway, arachidonic acid metabolism pathway and PPAR signaling pathway. Further verification showed that the mineralization and migration capacities of PDLSCs were greater than those of DPSCs, in which heat shock protein beta-1, Protein S100-A10 and S100-A11 may play a part., Conclusions: Less than 5% of the differentially expressed proteins make up the comparative proteomic profile between osteoinduced PDLSCs and DPSCs. This study helps to characterize the differences between osteoinduced PDLSCs and DPSCs in vitro., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To investigate the role of Schisandrin C in odontoblastic differentiation, and its relations between autophagy and mitochondrial biogenesis in human dental pulp cells (HPDCs)., Design: Fresh third molars were used, and cultured for HDPCs. Western blotting technique, Alizarin red S staining, alkaline phosphatase (ALP) activity, and confocal microscopy were used to detect autophagy, mitochondrial biogenesis, and odontoblastic differentiation. To understand the mechanism of Schisandrin C, the HDPCs were treated with lipopolysaccharide (LPS), autophagy and heme oxygenase-1 (HO-1) inhibitors: 3-Methyladenine (3-MA) and Zinc protoporphyrin IX (ZnPP), respectively., Results: LPS decreased the expression of autophagy molecules [autophagy protein 5 (ATG-5), beclin-1, and microtubule-associated protein 1A/1B light chain 3 (LC3-I/II)] and mitochondrial biogenesis molecules [heme oxygenase-1 (HO-1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)], and disrupted odontoblastic differentiation. The down-regulation of autophagy and mitochondrial biogenesis with 3-MA and ZnPP inhibited odontoblastic differentiation. However, Schisandrin C restored the expression of all the above molecules, even with LPS and inhibitor treatment. This result demonstrates that autophagy and mitochondrial biogenesis plays an essential role in odontoblastic differentiation, and Schisandrin C activates these systems to promote odontoblastic differentiation of HDPCs., Conclusion: Schisandrin C has potential characters to regulate odontoblastic differentiation, and may be recommended for use as a compound for pulp homeostasis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Stem cells from pulp tissue are a promising cell-based therapy for neurodegenerative patients based on their origin in the neural crest. The aim of this study was to differentiate and evaluate the ability of human dental pulp stem cells from permanent teeth (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) to differentiate into spiral ganglion neurons., Design: After isolation and characterization of mesenchymal stem cell properties, DPSC and SHED were treated with the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell-derived neurotrophic factor (GDNF). The differentiation was identified by immunostaining and qRT-PCR analysis of neuronal markers and measuring intracellular calcium activity., Results: After 2 weeks of induction, morphological changes were observed in both DPSC and SHED. The differentiated cells expressed neuron-specific class III beta-tubulin, GATA binding protein 3 (GATA3) and tropomyosin receptor kinase B, protein markers of spiral ganglion neurons. These cells also showed upregulation of the genes encoding these proteins, namely GATA3 and neurotrophic receptor tyrosine kinase 2. Intracellular calcium dynamics that reflect neurotransmitter release were observed in differentiated DPSC and SHED., Conclusion: These results demonstrate that dental pulp stem cells from permanent and deciduous teeth can differentiate into spiral ganglion neuron-like cells., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Dental pulp stem cells (DPSCs) are multipotent and play an important role in repairing damaged and/or defective dentinogenesis/osteogenesis. Recent studies have documented the implication of miR-143 in osteogenic differentiation of DPSCs. Nevertheless, the detailed mechanisms of miR-143 involved in the osteogenic differentiation of DPSCs remain to be further elaborated., Methods: Isolated DPSCs were incubated with osteogenic differentiation medium to induce osteogenic differentiation. qRT-PCR and western blot were performed to determine the expressions of miR-143 and tumor necrosis factor α (TNF-α). Luciferase reporter assay was used to confirm whether TNF-α was a target of miR-143. Osteogenic differentiation of DPSCs was evaluated by alkaline phosphatase (ALP) activity assay, ALP staining, and western blot analyses of osteogenic-markers including bone morphogenetic protein 2 (BMP2), ALP, runt-related transcription factor 2 (RUNX2) and collagen type I (COLI)., Results: miR-143 was downregulated and TNF-α was upregulated during osteogenic differentiation of DPSCs. miR-143 posttranscriptionally regulated TNF-α expression in DPSCs by binding to its 3'UTR. miR-143 overexpression suppressed osteogenic differentiation of DPSCs, as demonstrated by the decrease of ALP activity, ALP positive cell ratio, as well as BMP2, ALP, RUNX2, and COLI expressions. Moreover, miR-143 reversed TNF-α-induced osteogenic differentiation of DPSCs. Finally, the osteogenic differentiation of DPSCs induced by miR-143 inhibitor was attenuated following inactivation of nuclear factor kappa B (NF-κB) signaling pathway., Conclusion: miR-143 suppressed the osteogenic differentiation of DPSCs by blockade of NF-κB signaling pathway via targeting TNF-α., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study is to investigate the role of the WNT7B protein in the migration and differentiation of human dental pulp cells (HDPCs)., Design: The effect of recombinant human WNT7B (rhWNT7B) on the proliferation and migration of HDPCs was evaluated by 5-ethynyl-2'-deoxyuridine (EdU), immunofluorescence staining of Ki67, flow cytometry and scratch assay; the differentiation of HDPCs was measured by alkaline phosphatase (ALP) staining, alizarin red staining, ALP activity, qPCR and western blot. The activation of the WNT/beta-catenin (WNT/β-catenin) and c-Jun N-terminal kinase (JNK) pathways was analysed by western blot, immunocytochemistry and dual luciferase assays. XAV939 and SP600125，the inhibitors of the WNT/β-catenin and JNK pathways, were further applied to verify the mechanism., Results: rhWNT7B repressed the proliferation but did not affect the apoptosis of HDPCs. In the presence of rhWNT7B, ALP and alizarin red staining were increased substantially in the HDPCs with osteogenic induction; the gene expression of Runx2 and Col1 in HDPCs was quite elevated compared with that induced in osteogenic medium without WNT7B measured by qPCR; The ALP activity was also increased with rhWNT7B stimulation in HDPCs after 7-day odontogenic culture; Western blot revealed that the expression of dentin sialophosphoprotein (DSPP) of HDPCs was up-regulated significantly with the addition of WNT7B as well. Further study showed that rhWNT7B activated the WNT/β-catenin and JNK signalling pathways in the differentiation of HDPCs. XAV939 and SP600125 can partly offset the effect of the WNT7B-induced differentiation of HDPCs., Conclusion: WNT7B promoted the differentiation of HDPCs partly through the WNT/β-catenin and JNK signalling pathways., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Biphasic Calcium Phosphate (BCP) with a ratio of 20/80 Hydroxyapatite (HA)/Beta-tricalcium phosphate (β-TCP) promotes the differentiation of human dental pulp cells (HDPCs). In the current study, the genotoxicity of locally produced BCP of modified porosity (65%) with a mean pore size of 300micrometer (μm) was assessed using Comet and Ames assays. HDPCs were treated with BCP extract at three different inhibitory concentrations which were obtained based on cytotoxicity test conducted with concurrent negative and positive controls. The tail moment of HDPCs treated with BCP extract at all three concentrations showed no significant difference compared to negative control (p>0.05), indicating that BCP did not induce DNA damage to HDPCs. The BCP was evaluated using five tester strains of Salmonella typhimurium TA98, TA100, TA102, TA1537 and TA1538. Each strain was incubated with BCP extract with five different concentrations in the presence and absence of metabolic activation system (S9) mix. Concurrently, negative and positive controls were included. The average number of revertant colonies per plate treated with the BCP extract was less than double as compared to the number of revertant colonies in negative control plate and no dose-related increase was observed. Results from both assays suggested that the BCP of modified porosity did not exhibit any genotoxic effect under the present test conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To investigate the role of the EphrinB2 signaling pathway in the osteogenesis/odontogenesis of human dental pulp stem cells (DPSCs)., Design: The endogenous expression levels of EphrinB2 and its cognate receptors EphB2 and EphB4 in DPSCs were analyzed by qRT-PCR and Western blotting after 7, 14 and 21 days of osteogenic/odontogenic induction culture. Additionally, the phosphorylation of EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction, were also investigated by Western blots. Subsequently, we investigated whether supplementation of recombinant EphrinB2-Fc within the induction milieu can enhance the osteogenic/odontogenic differentiation of DPSCs., Results: Endogenous gene and protein expression levels of EphrinB2, EphB2 and EphB4 were upregulated in induced versus non-induced DPSCs, over 21 days of osteogenic/odontogenic induction. Western blots showed increase in phosphorylated EphrinB2, EphB4 and ERK1/2 proteins at early time-points following osteogenic induction. Preliminary investigation of a concentration range (0, 0.5, 1 and 2 μg/ml) of recombinant EphrinB2-Fc within osteogenic induction media, showed that 0.5 μg/ml was optimal for enhancing the osteogenic/odontogenic differentiation of DPSCs over a culture duration of 14 days. Subsequently, more comprehensive qRT-PCR analysis with 0.5 μg/ml EphrinB2-Fc revealed significant upregulation of several key osteogenic marker genes in treated versus untreated DPSCs after 21 days of osteogenic/odontogenic induction. By 7 days of osteogenic induction, DPSCs treated with 0.5 μg/ml EphrinB2-Fc exhibited significantly more calcium mineralization (Alizarin red S staining) and alkaline phosphatase activity than the untreated control., Conclusions: EphrinB2 signaling plays a key role in the osteogenic/odontogenic differentiation of DPSCs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Pulpitis is a multi-factorial disease that could be caused by complex interactions between genetics, epigenetics and environmental factors. We aimed to evaluate the role of Enhancer of Zeste Homolog 2 (EZH2) in the inflammatory response of human dental pulp cells (HDPCs) and dental pulp tissues., Methods: The expressions of inflammatory cytokines in HDPCs treated by EZH2 complex or EZH2 siRNA with or without rhTNF-α were examined by quantitative real-time polymerase chain reaction (q-PCR). The levels of secreted inflammatory cytokines including IL-6, IL-8, IL-15, CCL2 and CXCL12 in culture supernatants were measured by Luminex assay. In rat pulpitis model, the effects of EZH2 on dental pulp tissues were verified by histology. We invested the mechanisms of the effect of EZH2 on the inflammatory factors by ChIP assay., Results: EZH2 down-regulation inhibited the expression of inflammatory factors, including IL-6, IL-8, IL-15, CCL2 and CXCL12 in HDPCs. EZH2 complex promoted the expression and secretion of these inflammatory factors in HDPCs, while EZH2 silencing could attenuate the promotion of inflammatory factors that were induced by rhTNF-α. In pulpitis models of rats, EZH2 down-regulation inhibited the inflammatory process of dental pulp while EZH2 complex showed no significant facilitation of pulpal inflammation. In addition, EZH2 could bind on the promoters of IL-6, IL-8 and CCL2, but not IL-15 and CXCL12, to affect the transcription of these proinflammatory cytokines., Conclusions: In HDPCs, EZH2 could induce inflammation, while EZH2 down-regulation could attenuate the inflammatory responses. EZH2 plays an important role in this inflammatory process of dental pulp., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Angiopoietin-like 4 (Angptl4) is an angiogenesis modulating signaling factor and as such involved in blood vessel formation but also in hard tissue resorption. Here we hypothesized that the hypoxia mimetic agent L-mimosine (L-MIM) and hypoxia stimulate the production of Angptl4 in the dental pulp., Material and Methods: Monolayer and spheroid cultures of primary human dental pulp-derived cells (DPC) were treated with L-MIM or hypoxia. Furthermore, tooth slice cultures were performed. The production of Angptl4 was assessed at mRNA and protein levels using reverse transcription qPCR and immunoassays, respectively. To assess the involvement of hypoxia inducible factor (HIF)-1α (HIF-1signaling, inhibitor studies with echinomycin and Western Blot analysis for HIF-1α were performed in DPC monolayer cultures.(HIF-1 RESULTS: L-MIM and hypoxia increased production of Angptl4 at mRNA and protein levels in monolayer cultures of DPC. The increase of Angptl4 was paralleled by an increase of HIF-1α and inhibited by echinomycin. Angptl4 protein levels were also elevated in spheroid cultures. In tooth slice cultures, the pulp tissue expressed and released Angptl4 under normoxic and hypoxic conditions and in the presence of L-MIM. There was a trend for an increase in Angptl4 mRNA levels and a trend for a decrease in the protein levels of the supernatants., Conclusions: Our results suggest that the hypoxia mimetic agent L-MIM and hypoxia can increase Angptl4 production in DPC involving HIF-1α. However, the increase in the cell culture supernatants does not translate in an increased release in tooth slice organ cultures., (Copyright © 2017. Published by Elsevier Ltd.)

Objective: Stem cell-based tissue repair and regeneration require the regulation of cell migration and adhesion. As a regulator of cell polarization, Cdc42 (cell division control protein 42) plays a basic role at the initial stage of cell migration and adhesion. This study explores the effect of Cdc42 on the polarized migration and adhesion of hDPSCs (human dental pulp stem cells)., Design: HDPSCs were isolated from extracted third molars and transfected with siRNA targeted against Cdc42. Scratch wound assays and transwell assays were performed to detect the migration of human dental pulp stem cells. Polarization assays were applied to explore the polarized movement of Golgi bodies and nuclei. Western blot was used to examine the expression of related proteins., Results: The expression of Cdc42 was knocked down by siRNA transfection, which inhibited the migration of hDPSCs in both the scratch wound assays and transwell assays. Meanwhile, the proportion of polarized hDPSCs during migration was also decreased, and the adhesion ability of hDPSCs was downregulated. Western blot demonstrated that these effects were dependent on FAK (focal adhesion kinase), β-catenin and GSK3β (Glycogen synthase kinase-3β)., Conclusion: Our study demonstrates that Cdc42 plays an essential role during the polarized movement and adhesion of hDPSCs., (Copyright © 2017. Published by Elsevier Ltd.)

Objectives: This study aimed to simplify the collection, isolation and cryopreservation procedure of human dental pulp stem cells (DPSCs) to ease the establishment of dental stem cell banking., Design: Extracted third molars were collected and stored either in growth medium or in gentamicin-saline (480μg/ml) for 6, 9 or 12h. DPSCs were isolated and subjected to cryopreservation by a controlled-rate or rapid freezing method in 5 or 10% DMSO. Flow cytometry and growth pattern of DPSCs before and after cryopreservation were conducted., Results: Rate of contamination by which the extracted teeth were stored in control and gentamicin-saline were 9.1% (N=33) and 2.3% (N=43), respectively. Successful cell isolation rate of teeth preserved in gentamicin-saline at 6h (92.9%) was comparable to those of growth media group (90.3%). At 9 and 12h, the rates dropped significantly to 75% and 54%, respectively. Cryopreservation by controlled-rate freezing either in 5 or 10% DMSO resulted in a significantly higher percentage of viable cells than by rapid freezing. Cells conserved by controlled-rate freezing in 5% DMSO showed a pattern of growth similar to control unfrozen cells; 10% DMSO significantly deteriorated the growth pattern of the cells. After thawing, DPSCs conserved by controlled-rate freezing still expressed stemness characteristics, although hematopoietic stem cell markers were slightly increased., Conclusion: Gentamicin-saline was effective in preserving human teeth for DPSC isolation. Controlled-rate freezing in 5% DMSO gave the highest rate of cell viability. This study simplifies the storage conditions and proposes a simple method for cryopreservation of DPSCs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Effects of melatonin on the proliferation and differentiation of human dental pulp cells (hDPCs) remain unclear. The purpose of this study was to investigate the effect of melatonin on the proliferation and differentiation of the hDPCs., Design: Primary hDPCs were obtained from the third molar of volunteer aged from 18 to 25. CCK8 assay evaluated the effect of melatonin upon cell proliferation at day 1, 2, 3, 4, 5. After 7days' osteogenic induction with melatonin or vehicle, alkaline phosphatase (ALP) activity was measured with a commercial kit. Then levels of dentin sialophosphoprotein (DSPP) were determined by immunocytochemical staining and western blot analysis, followed by quantitative real-time reverse transcription-Polymerase chain reaction (qRT-PCR) to analyse mRNA levels of ALP and DSPP. Finally hDPCs exposed to osteogenic medium containing melatonin or vehicle for 14days were stained with alizarin red to detect mineralization nodules formation., Results: Melatonin significantly inhibited the proliferative ability of the hDPCs in a concentration- and time-dependent manner. The hDPCs cultured in osteogenic induction medium with melatonin presented an increase of ALP activity, expression of DSPP, mRNA levels of ALP and DSPP, and mineralization nodules formation., Conclusions: These findings indicate that melatonin at physiological concentrations can inhibit proliferation and promote the differentiation of hDPCs, which might give some new insights into the mechanism of regulating DPCs to achieve dentine regeneration., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: This study provides an in vivo evaluation of the inflammatory response, levels of cell proliferation and apoptosis, and the presence of necrosis after dental bleaching with two concentrations of hydrogen peroxide (H 2 O 2 )., Design: Wistar rats were divided into Control (placebo gel), BLUE (20% H 2 O 2 , 1×50min), and MAXX (35% H 2 O 2 , 3×15min) groups. At 2 and 30days, the rats were killed (n=10). The jaws were processed for histology analysis and PCNA and Caspase-3-cleaved immunohistochemistry, and data were submitted to the Mann-Whitney or ANOVA test (P<0.05)., Results: At 2days, the MAXX group showed necrosis and the BLUE group revealed moderate inflammation on the occlusal third of the crown (P<0.05). At 30days, tertiary dentin had formed and there was an absence of inflammation. The level of cell proliferation was higher in the middle third of the BLUE group (P<0.05), and cervical of MAXX at 2days (P<0.05), decreasing at 30days. The apoptosis was present at 2days, particularly in the cervical third of the crown in the bleached groups (P<0.05), with a decrease only at 30days in the BLUE group (P<0.05)., Conclusions: The concentration of H 2 O 2 influences effects on the pulp tissue, where a higher concentration of H 2 O 2 can cause necrosis in the pulp and a prolonged effect within the apoptotic process; lower concentrations of H 2 O 2 provide moderate inflammation, cell proliferation and apoptosis with a reduction of these processes over time., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/β-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter., Design: A piggyBac transposon plasmid (pTA-LEN) was introduced into HDDPCs, using the Neon ® transfection system. After G418 selection, the emerging colonies were assessed for EGFP-derived fluorescence by fluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using RNA isolated from these colonies to examine stem cell-specific transcript expression. Osteoblastic or neuronal differentiation was induced by cultivating the LEF1-positive cells with differentiation-inducing medium., Results: RT-PCR analysis confirmed the expression of several stem cell markers, including OCT3/4, SOX2, REX1, and NANOG, in LEF1-positive HDDPCs, which could be differentiated into osteoblasts and neuronal cells., Conclusions: The isolated LEF1-positive HDDPCs exhibited the properties of stem cells, suggesting that LEF1 might serve as a marker for SHED., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: The aim of this study was to investigate correlations between dental pulp cell count of odontoblasts, subodontoblasts and fibroblasts and age, within different age groups. Formulation of regression equations using the dental pulp cell count for predicting age was attempted., Design: Eighty-one extracted teeth were grouped into two age groups (6-25 years, 26-80 years). The teeth were demineralized and histological sections were prepared for cell count. Regression equations were generated from regression analysis of cell count and tested for age estimation., Results: The number of dental pulp cells were found to increase until around the third decade of life and following this, the odontoblasts and subodontoblasts cell numbers began to decline while the fibroblasts seemed to remain almost stationary. The Pearson correlation test revealed a significant positive correlation between the cell number for all type of cells and age in the 6-25 years group (r=+0.791 for odontoblasts, r=+0.600 for subodontoblasts and r=+0.680 for fibroblasts). In the 26-80 years age group, a significant negative correlation of the odontoblasts (r=-0.777) and subodontoblasts (r=-0.715) with age was observed but for fibroblasts, the correlation value was negligible (r=-0.165). Regression equations generated using odontoblasts and subodontoblasts cell number were applicable for age estimation. The standard error of estimates (SEEs) were around±5years for 6-25 years and±8years for 26-80 years age groups. The mean values of the estimated and chronological ages were not significantly different., Conclusions: A significant correlation between the cell count of odontoblasts and subodontoblasts with age was demonstrated. Regression equations using odontoblasts and subodontoblasts cell number can be used to predict age with some limitations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to investigate transforming growth factor-β-induced gene product-h3 (βig-h3) expression in dental pulp tissue and its effects on odontoblastic differentiation of dental pulp cells (DPCs)., Design: A rat direct pulp capping model was prepared using perforated rat upper first molars capped with mineral trioxide aggregate cement. Human DPCs (HDPCs) were isolated from extracted teeth. βig-h3 expression in rat dental pulp tissue and HDPCs was assessed by immunostaining. Mineralization of HDPCs was assessed by Alizarin red-S staining. Odontoblast-related gene expression in HDPCs was analyzed by quantitative RT-PCR., Results: Expression of βig-h3 was detected in rat dental pulp tissue, and attenuated by direct pulp capping, while expression of interleukin-1β and tumor necrosis factor-α was increased in exposed pulp tissue. βig-h3 expression was also detected in HDPCs, with reduced expression during odontoblastic differentiation. The above cytokines reduced βig-h3 expression in HDPCs, and promoted their mineralization. Recombinant βig-h3 inhibited the expression of odontoblast-related genes and mineralization of HDPCs, while knockdown of βig-h3 gene expression promoted the expression of odontoblast-related genes in HDPCs., Conclusions: The present findings suggest that βig-h3 in DPCs may be involved in reparative dentin formation and that its expression is likely to negatively regulate this process., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: The purpose of this study was to investigate the role of a novel mutant DLX3 on the odontogenic differentiation of human dental pulp cells (hDPCs) in tricho-dento-osseous (TDO) syndrome., Design: hDPCs were obtained from the healthy premolars, stably-expressing wild-type DLX3 (WT), novel mutant DLX3 (Mu) and control vector (NC) cells were generated using recombinant lentiviruses. The proliferation rates of WT-hDPCs and Mu-hDPCs were measured by CCK8 assay. Odonto-differentiation of hDPCs was assessed by alkaline phosphatase (ALP) activity assay, and mineralization ability was assessed by Alizarin red staining. Odontogenic markers, including DMP-1, DSPP, Nes, ALP, and DLX5, were analyzed using real-time polymerase chain reaction (qPCR). DMP-1 and DSPP expressions were further confirmed by Western blotting., Results: CCK8 results showed that the novel mutant DLX3 decreased the proliferation rate of hDPCs compared with wild-type DLX3. qPCR showed that the novel mutant DLX3 weakened odontogenic differentiation by downregulating the expression of odontogenic genes. These results were further confirmed by Western blotting and ALP activity assay. Additionally, Alizarin red staining showed that the novel mutant DLX3 decreased the mineralization of hDPCs compared with wild-type DLX3., Conclusions: Novel de novo mutation of DLX3 significantly decreases the proliferation rate and inhibits the odontogenic differentiation and mineralization of hDPCs, suggesting that this novel mutation of DLX3 can influence the dentinogenesis in TDO syndrome., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Cold-sensitive ion channels, such as transient receptor potential melastatin (TRPM) 8 and transient receptor potential ankyrin (TRPA) 1, may play a crucial role in the nociceptive function of odontoblasts, whereas expression of these TRP channels in human native odontoblasts remains to be elucidated. This study aimed to analyze the expression of TRPM8 and TRPA1 in freshly isolated native human odontoblasts., Design: Odontoblasts were isolated from freshly extracted healthy human teeth (n=4); after removing the inner pulp tissues from the pulp chambers, odontoblasts remaining on the dentin surface were washed out with phosphate buffered saline and collected. Reverse transcription-polymerase chain reaction was employed to compare the expression levels of TRPM8, TRPA1, and dentin matrix acidic phosphoprotein 1 (DMP1) mRNAs between the isolated odontoblasts and the inner pulp tissues. The isolated cells were subjected to immunolocalization of TRPM8 and nestin. Paraformaldehyde-fixed, EDTA-demineralized frozen sections obtained from freshly extracted healthy human teeth (n=4) were also analyzed immunohistochemically using anti-nestin, TRPM8, and TRPA1 antibodies., Results: Expression levels of TRPM8 and DMP1 in the isolated odontoblasts were significantly higher than those in the inner pulp tissues (p<0.05). Expression of TRPM8 and nestin was observed in the odontoblastic layer of the dental pulp tissue and isolated odontoblasts, while expression of TRPA1 was not detected., Conclusions: TRPM8, but not TRPA1, was detected in freshly isolated native human odontoblasts at the protein and mRNA levels, suggesting that odontoblasts play an important role in detecting external cold stimulation via TRPM8 in healthy condition., (Copyright © 2016. Published by Elsevier Ltd.)

Objective: The dissemination of stem cells into tissues requiring inflammatory and reparative response is fundamentally dependent upon their chemotactic migration. Expression of TNF-α is up regulated in inflamed pulps. Dental pulp cells are also known to express integrin α6 subunit. Expression of integrin subunit α6 has been linked to the acquisition of migratory potential in a wide variety of cell types in both pathological and physiological capacities. Therefore, in this study we examined the effects of a pleiotropic cytokine TNF-α on the migration of hDPSCs and investigated its relationship with expression of integrin α6 in hDPSCs during chemotactic migration., Design: hDPSC cultures were established. Protein expression profile of α6 integrin subunit was determined. Effect of exogenous TNF-α (50ng/mL) on hDPSCs' migration potential was evaluated by transwell inserts and in vitro scratch assay. Upregulation/downregulation of TNF-α mediated migration was assayed in presence/absence of integrin α6 respectively. To suppress integrin α6 expression, cells were transfected with integrin α6 siRNA and then cell migration and cytoskeletal changes were evaluated., Results: Our results showed significant increase of hDPSCs' migration after stimulation with TNF-α. By knockdown of integrin α6, which is upregulated by TNF-α, we observed a decrease in the TNF-α directed chemotaxis of hDPSCs., Conclusion: In this study, we show that activation of integrin α6 brought about by TNF-α led to an increase in migratory activity in DPSCs in vitro thus describing a novel association between a cytokine TNF-α and α6 chain of an adhesion receptor integrin in regulating migration of hDPSCs., (Copyright © 2016. Published by Elsevier Ltd.)

Objective: The present study aims to investigate whether reparative dentinogenesis could be guided at central pulpal sites or at a distance from the amputated pulp of miniature pig teeth, by using set calcium silicate-based carriers containing human recombinant bioactive molecules., Design: Pulp exposures were performed in 72 permanent teeth of 4 healthy miniature swine. The teeth were capped with pre-manufactured implants of set calcium silicate-based material containing BMP-7, TGFβ1 or WnT-1, for 3 weeks. Conical-shaped intrapulpal implants were exposed in the central pulp core, while disc-shaped extrapulpal implants were placed at a distance from the amputated pulp. Implants without bioactive molecules were used as controls. Thickness and forms of new matrix mineralized deposition were assessed histologically at post-operative periods of 3 weeks by light microscopy., Results: Intrapulpal applications: Calcified structures composed of osteodentine were found in contact with the BMP-7 implants. An inhomogeneous calcified tissue matrix was found around the WnT-1 carriers. A two-zone calcified structure composed of osteodentine and a thicker tubular matrix zone was seen at the TGFβ1 carrier-pulp interface. Extrapulpal applications: The space between WnT-1 implants and pulp periphery had been invaded by soft tissue with traces of calcified foci. Thick calcified structures composed of osteodentine were found surrounding pulp exposure sites in response to application of BMP-7. Spindle-shaped cells associated with atubular calcified matrix or elongated polarized cells associated with tubular dentine-like matrix were found along the cut dentinal walls of the TGFβ1 group., Conclusion: The present experiments indicated that set calcium silicate could be used as carrier for biologically active molecules. TGFβ1 was shown to be an effective bioactive molecule in guiding tertiary dentine formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objectives: Insulin-like growth factor 1 (IGF-1) is a broad-spectrum growth-promoting factor that plays a key role in natural tooth development. Human dental pulp stem cells (hDPSCs) are multipotent and can influence the reparative regeneration of dental pulp and dentin. This study was designed to evaluate the effects of IGF-1 on the proliferation and differentiation of human dental pulp stem cells., Methods: HDPSCs were isolated and purified from human dental pulps. The proliferation and osteo/odontogenic differentiation of hDPSCs treated with 100ng/ml exogenous IGF-1 were subsequently investigated., Results: MTT assays revealed that IGF-1 enhanced the proliferation of hDPSCs. ALP activity in IGF-1-treated group was obviously enhanced compared to the control group from days 3 to 9. Alizarin red staining revealed that the IGF-1-treated cells contained a greater number of mineralization nodules and had higher calcium concentrations. Moreover, western blot and qRT-PCR analyses demonstrated that the expression levels of several osteogenic genes (e.g., RUNX2, OSX, and OCN) and an odontoblast-specific marker (DSPP) were significantly up-regulated in IGF-1-treated hDPSCs as compared with untreated cells (P<0.01). Interestingly, the expression of phospho-ERK and phospho-p38 were also up-regulated, indicating that the MAPK signaling pathway is activated during the differentiation of hDPSCs., Conclusions: IGF-1 can promote the proliferation and osteo/odontogenic differentiation of hDPSCs by activating MAPK pathways., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the cytotoxicity and mineralization effects of TEGDMA in human dental pulp cells (hDPCs) under hypoxic and normoxic culture conditions., Design: Cell viability was evaluated using XTT assay after incubation periods of 24, 48, or 72h. The expression of mineralization-related genes (osteonectin, osteopontin, dentin sialophosphoprotein, collagen type 1) and heme oxygenase 1 (HO-1) was assessed by quantitative real-time polymerase chain reaction at 24 and 72h., Results: In XTT assay, viability was higher in 0.3, 1, 2, 4, and 5mM groups in the presence of 21% O 2 after 24h (p<0.05). Additionally, while 0.3, 1, 2mM groups had higher cell viability in the presence of 21% O 2 after 48h (p<0.05), in 3mM groups cell viability was higher under 3% O 2 than 21% O 2 after both 24 and 48h (p<0.05). 1-3mM groups had higher cell viability under 3% O 2 after 72h (p<0.05). There was no difference between 4 and 5mM groups with regards to cell viability after 48 or 72h (p>0.05). In the gene expression study, TEGDMA-treated hDPCs showed lower mineralization potential in the presence of 3% than with 21% O 2 (p<0.05). hDPCs revealed higher HO 1 expression in 0.3 and 1mM groups under hypoxic than under normoxic conditions after a 72-h time period (p<0.001)., Conclusions: Hypoxic conditions increased cell survival in accordance with the culture period but inhibited the odontoblastic differentiation of hDPCs treated with TEGDMA., (Copyright © 2016 Elsevier Ltd. All rights reserved.)