Role of alpha smooth muscle actin in odontogenic differentiation of dental pulp stem cells.

Pulpotomy is an effective treatment for retaining vital pulp after pulp exposure caused by caries removal and/or trauma. The expression of alpha smooth muscle actin (α-SMA) is increased during the wound-healing process, and α-SMA-positive fibroblasts accelerate tissue repair. However, it remains largely unknown whether α-SMA-positive fibroblasts influence pulpal repair. In this study, we established an experimental rat pulpotomy model and found that the expression of α-SMA was increased in dental pulp after pulpotomy relative to that in normal dental pulp. In vitro results showed that the expression of α-SMA was increased during the induction of odontogenic differentiation in dental pulp stem cells (DPSCs) compared with untreated DPSCs. Moreover, α-SMA overexpression promoted the odontogenic differentiation of DPSCs via increasing mitochondrial function. Mechanistically, α-SMA overexpression activated the mammalian target of rapamycin (mTOR) signaling pathway. Inhibition of the mTOR signaling pathway by rapamycin decreased the mitochondrial function in α-SMA-overexpressing DPSCs and suppressed the odontogenic differentiation of DPSCs. Furthermore, we found that α-SMA overexpression increased the secretion of transforming growth factor beta-1 (TGF-ß1). In sum, our present study demonstrates a novel mechanism by which α-SMA promotes odontogenic differentiation of DPSCs by increasing mitochondrial respiratory activity via the mTOR signaling pathway.

An in vitro anti-inflammatory effect of Thai propolis in human dental pulp cells.

OBJECTIVE: To explore the potential for development of Thai propolis extract as a pulp capping agent to suppress pulpal inflammation from dental pulp infections. This study aimed to examine the anti-inflammatory effect of the propolis extract on the arachidonic acid pathway, activated by interleukin (IL)-1ß, in cultured human dental pulp cells. METHODOLOGY: Dental pulp cells, isolated from three freshly extracted third molars, were first characterized for their mesenchymal origin and treated with 10 ng/ml of IL-1ß in the presence or absence of non-toxic concentrations of the extract from 0.08 to 1.25 mg/ml, as determined by the PrestoBlue cytotoxic assay. Total RNA was harvested and analyzed for mRNA expressions of 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2). Western blot hybridization was performed to investigate COX-2 protein expression. Culture supernatants were assayed for released prostaglandin E2 levels. Immunofluorescence was conducted to determine involvement of nuclear factor-kappaB (NF-kB) in the inhibitory effect of the extract. RESULTS: Stimulation of the pulp cells with IL-1ß resulted in the activation of arachidonic acid metabolism via COX-2, but not 5-LOX. Incubation with various non-toxic concentrations of the propolis extract significantly inhibited upregulated COX-2 mRNA and protein expressions upon treatment with IL-1ß (p<0.05), resulting in a significant decrease in elevated PGE2 levels (p<0.05). Nuclear translocation of the p50 and the p65 subunits of NF-kB upon treatment with IL-1ß was also blocked by incubation with the extract. CONCLUSIONS: Upregulated COX-2 expression and enhanced PGE2 synthesis upon treatment with IL-1ß in human dental pulp cells were suppressed by incubation with non-toxic doses of Thai propolis extract via involvement of the NF-kB activation. This extract could be therapeutically used as a pulp capping material due to its anti-inflammatory properties.

Dental pulp stem cell-derived exosomes revitalize salivary gland epithelial cell function in NOD mice via the GPER-mediated cAMP/PKA/CREB signaling pathway.

BACKGROUND: Restoration of salivary gland function in Sjogren's syndrome (SS) is still a challenge. Dental pulp stem cells (DPSCs) derived exosomes had shown anti-inflammatory, anti-oxidative, immunomodulatory, and tissue function restorative abilities. However, the salivary gland function restoration potential of DPSCs-derived exosomes (DPSC-Exos) during SS has not been investigated yet. METHODS: DPSC-Exos was isolated by ultracentrifugation methods and characterized. Salivary gland epithelial cells (SGEC) were treated with interferon-gamma (IFN-γ) to mimic SS in vitro and cultured with or without DPSC-Exos. SGEC survival and aquaporin 5 (AQP5) expression were analyzed. mRNA sequencing and bioinformatics analysis were performed in IFN-γ vs. DPSC-Exos+ IFN-γ treated SGEC. Non-obese diabetic (NOD)/ltj female mice (SS model), were intravenously administered with DPSC-Exos, and salivary gland functions and SS pathogenicity were analyzed. Furthermore, the mRNA sequencing and bioinformatics predicted mechanism of the therapeutic effect of DPSC-Exos was further investigated both in vitro and in vivo using RT-qPCR, Western blot, immunohistochemistry, immunofluorescence, flowcytometry analysis. RESULTS: DPSC-Exos partially rescued IFN-γ triggered SGEC death. IFN-γ inhibited AQP5 expression in SGEC and DPSC-Exos reversed this effect. Transcriptome analysis showed GPER was the upregulated DEG in DPSC-Exos-treated SGEC with a positive correlation with salivary secretion-related DEGs. Pathway enrichment analysis revealed that DEGs were mainly attributed to estrogen 16 alpha-hydroxylase activity, extracellular exosome function, cAMP signaling, salivary secretion, and estrogen signaling. Intravenous injection of DPSC-Exos in NOD/ltj mice alleviated the SS syndrome as indicated by the increased salivary flow rate, attenuated glandular inflammation, and increased AQP5 expression. GPER was also upregulated in the salivary gland of DPSC-Exos-treated NOD/ltj mice compared with the PBS-treated NOD/ltj mice. IFN-γ+DPSC-Exos-treated SGEC showed higher expression of AQP5, p-PKA, cAMP, and intracellular Ca2+ levels compared with IFN-γ-treated SGEC. These effects were reversed by the inhibition of GPER. CONCLUSIONS: Our results showed that DPSC-Exos revitalize salivary gland epithelial cell function during SS via the GPER-mediated cAMP/PKA/CREB pathway suggesting the possible therapeutic potential of DPSC-Exos in SS-treatment.

Enhancement of mesenchymal stem cells' chondrogenic potential by type II collagen-based bioscaffolds.

BACKGROUND: Osteoarthritis (OA) is a common degenerative chronic disease accounting for physical pain, tissue stiffness and mobility restriction. Current therapeutic approaches fail to prevent the progression of the disease considering the limited knowledge on OA pathobiology. During OA progression, the extracellular matrix (ECM) of the cartilage is aberrantly remodeled by chondrocytes. Chondrocytes, being the main cell population of the cartilage, participate in cartilage regeneration process. To this end, modern tissue engineering strategies involve the recruitment of mesenchymal stem cells (MSCs) due to their regenerative capacity as to promote chondrocyte self-regeneration. METHODS AND RESULTS: In the present study, we evaluated the role of type II collagen, as the main matrix macromolecule in the cartilage matrix, to promote chondrogenic differentiation in two MSC in vitro culture systems. The chondrogenic differentiation of human Wharton's jelly- and dental pulp-derived MSCs was investigated over a 24-day culture period on type II collagen coating to improve the binding affinity of MSCs. Functional assays, demonstrated that type II collagen promoted chondrogenic differentiation in both MSCs tested, which was confirmed through gene and protein analysis of major chondrogenic markers. CONCLUSIONS: Our data support that type II collagen contributes as a natural bioscaffold enhancing chondrogenesis in both MSC models, thus enhancing the commitment of MSC-based therapeutic approaches in regenerative medicine to target OA and bring therapy closer to the clinical use.

The Inhibition of the Inducible Nitric Oxide Synthase Enhances the DPSC Mineralization under LPS-Induced Inflammation.

Nitric oxide (NO) is a key messenger in physiological and pathological processes in mammals. An excessive NO production is associated with pathological conditions underlying the inflammation response as a trigger. Among others, dental pulp inflammation results from the invasion of dentin by pathogenic bacteria. Vital functions of pulp mesenchymal stem cells (DPSCs, dental pulp stem cells), such as mineralization, might be affected by the inducible NOS (iNOS) upregulation. In this context, the iNOS selective inhibition can be considered an innovative therapeutic strategy to counteract inflammation and to promote the regeneration of the dentin-pulp complex. The present work aims at evaluating two acetamidines structurally related to the selective iNOS inhibitor 1400W, namely CM544 and FAB1020, in a model of LPS-stimulated primary DPSCs. Our data reveal that CM544 and even more FAB1020 are promising anti-inflammatory compounds, decreasing IL-6 secretion by enhancing CD73 expression-levels, a protein involved in innate immunity processes and thus confirming an immunomodulatory role of DPSCs. In parallel, cell mineralization potential is retained in the presence of compounds as well as VEGF secretion, and thus their angiogenetic potential. Data presented lay the ground for further investigation on the anti-inflammatory potential of acetamidines selectively targeting iNOS in a clinical context.

Effects of Human Deciduous Dental Pulp-Derived Mesenchymal Stem Cell-Derived Conditioned Medium on the Metabolism of HUVECs, Osteoblasts, and BMSCs.

In this study, we assessed the effects of human deciduous dental pulp-derived mesenchymal stem cell-derived conditioned medium (SHED-CM) on the properties of various cell types. The effects of vascular endothelial growth factor (VEGF) in SHED-CM on the luminal architecture, proliferative ability, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) were determined. We also investigated the effects of SHED-CM on the proliferation of human-bone-marrow mesenchymal stem cells (hBMSCs) and mouse calvarial osteoblastic cells (MC3T3-E1) as well as the expression of ALP, OCN, and RUNX2. The protein levels of ALP were examined using Western blot analysis. VEGF blockade in SHED-CM suppressed the proliferative ability and angiogenic potential of HUVECs, indicating that VEGF in SHED-CM contributes to angiogenesis. The culturing of hBMSCs and MC3T3-E1 cells with SHED-CM accelerated cell growth and enhanced mRNA expression of bone differentiation markers. The addition of SHED-CM enhanced ALP protein expression in hBMSCs and MT3T3-E1 cells compared with that of the 0% FBS group. Furthermore, SHED-CM promoted the metabolism of HUVECs, MC3T3-E1 cells, and hBMSCs. These findings indicate the potential benefits of SHED-CM in bone tissue regeneration.

Potentiality and Inflammatory Marker Expression Are Maintained in Dental Pulp Cell Cultures from Carious Teeth.

OBJECTIVES: This investigation aimed to isolate and culture human dental pulp cells from carious teeth (cHDPCs) and compare their growth characteristics, colony-forming efficiency, mineralization potential and gene expression of Toll-like receptors (TLR)-2, TLR-4, TLR-9, tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-8, IL-17A, 1L-17R, IL-23A, nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK1), dentin matrix protein (DMP)-1, dentin sialophospho protein (DSPP), sex determining region Y-box 2 (SOX2) and marker of proliferation Ki-67 (MKi67) with cells isolated from healthy or non-carious teeth (ncHDPCs). METHODS: Pulp tissues were obtained from both healthy and carious teeth (n = 5, each) to generate primary cell lines using the explant culture technique. Cell cultures studies were undertaken by generating growth curves, a colony forming unit and a mineralization assay analysis. The expression of vimentin was assessed using immunocytochemistry (ICC), and the gene expression of above-mentioned genes was determined using quantitative real-time reverse-transcription polymerase chain reaction. RESULTS: ncHDPCs and cHDPCs were successfully isolated and cultured from healthy and inflamed human dental pulp tissue. At passage 4, both HDPC types demonstrated a typical spindle morphology with positive vimentin expression. No statistical difference was observed between ncHDPCs and cHDPCs in their growth characteristics or ability to differentiate into a mineralizing phenotype. ncHDPCs showed a statistically significant higher colony forming efficiency than cHDPCs. The gene expression levels of TLR-2, TLR-4, TLR-9, TNF-α, IL-6, IL-8, IL-17R, IL-23A, NF-κB, MAPK1, DMP1, DSPP and SOX2 were significantly higher in cHDPCs compared with ncHDPC cultures. CONCLUSION: cHDPCs retain their differentiation potential and inflammatory phenotype in vitro. The inflamed tooth pulp contains viable stem/progenitor cell populations which have the potential for expansion, proliferation and differentiation into a mineralizing lineage, similar to cells obtained from healthy pulp tissue. These findings have positive implications for regenerative endodontic procedures.

A novel hypoxic lncRNA, HRL-SC, promotes the proliferation and migration of human dental pulp stem cells through the PI3K/AKT signaling pathway.

BACKGROUND: Human dental pulp stem cells (hDPSCs) are critical for pulp generation. hDPSCs proliferate faster under hypoxia, but the mechanism by which long noncoding RNA (lncRNA) regulates this process is not fully understood. METHODS: Novel lncRNAs were obtained by reanalysis of transcriptome datasets from RNA-Seq under hypoxia compared with normoxia, and a differential expression analysis of target genes was performed. Bioinformatics analyses, including gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis and gene set enrichment analysis, were used to understand the function of key novel lncRNAs. hDPSCs were isolated from dental pulp tissue. EdU and scratch wound healing assays were used to detect the proliferation and migration of hDPSCs. qRT-PCR was used to detect changes in the RNA expression of selected genes. RNA fluorescence in situ hybridization, small interfering RNA, qRT-PCR and Western blot analysis were used to explore the function of key novel lncRNAs. RESULTS: We identified 496 novel lncRNAs in hDPSCs under hypoxia, including 45 differentially expressed novel lncRNAs. Of these, we focused on a key novel lncRNA, which we designated HRL-SC (hypoxia-responsive lncRNA in stem cells). Functional annotation revealed that HRL-SC was associated with hypoxic conditions and the PI3K/AKT signaling pathway. HRL-SC was mainly located in the cytoplasm of hDPSCs and had stable high expression under hypoxia. Knockdown of HRL-SC inhibited the proliferation and migration of hDPSCs and the expression levels of PI3K/AKT-related marker proteins. Furthermore, the AKT activator SC79 partially offset the inhibitory effect caused by the knockdown, indicating that HRL-SC promoted hDPSCs through the PI3K/AKT signaling pathway. CONCLUSIONS: Hypoxia-responsive lncRNA HRL-SC promotes the proliferation and migration of hDPSCs through the PI3K/AKT signaling pathway, and this understanding may facilitate the regenerative application of hDPSCs.

A role for the calcium-sensing receptor in the expression of inflammatory mediators in LPS-treated human dental pulp cells.

The aim of this study is to investigate the role of calcium-sensing receptor (CaSR) in the expression of inflammatory mediators of lipopolysaccharide (LPS)-treated human dental pulp cells (hDPCs). The expression profile of CaSR in LPS-simulated hDPCs was detected using immunofluorescence, real time quantitative PCR (RT-qPCR), and Western blot analyses. Then, its regulatory effects on the expression of specific inflammatory mediators such as interleukin (IL)-1ß, IL-6, cyclooxygenase 2 (COX2)-derived prostaglandin E2 (PGE2), tumor necrosis factor (TNF)-α, and IL-10 were determined by RT-qPCR and enzyme-linked immunosorbent assay (ELISA). LPS significantly downregulated the gene expression of CaSR, but upregulated its protein expression level in hDPCs. Treatments by CaSR agonist R568 or its antagonist Calhex231, and their combinations with protein kinase B (AKT) inhibitor LY294002 showed obvious effects on the expression of selected inflammatory mediators in a time-dependent manner. Meanwhile, an opposite direction was found between the action of R568 and Calhex231, as well as the expression of the pro- (IL-1ß, IL-6, COX2-derived PGE2, and TNF-α) and anti-inflammatory (IL-10) mediators. The results provide the first evidence that CaSR-phosphatidylinositol-3 kinase (PI3K)-AKT-signaling pathway is involved in the release of inflammatory mediators in LPS-treated hDPCs, suggesting that the activation or blockade of CaSR may provide a novel therapeutic strategy for the treatment of pulp inflammatory diseases.

Restoration of BDNF, DARPP32, and D2R Expression Following Intravenous Infusion of Human Immature Dental Pulp Stem Cells in Huntington's Disease 3-NP Rat Model.

Huntington's disease (HD) is a neurodegenerative inherited genetic disorder, which leads to the onset of motor, neuropsychiatric and cognitive disturbances. HD is characterized by the loss of gamma-aminobutyric acid (GABA)ergic medium spiny neurons (MSNs). To date, there is no treatment for HD. Mesenchymal stem cells (MSCs) provide a substantial therapeutic opportunity for the HD treatment. Herein, we investigated the therapeutic potential of human immature dental pulp stem cells (hIDPSC), a special type of MSC originated from the neural crest, for HD treatment. Two different doses of hIDPSC were intravenously administrated in a subacute 3-nitropropionic acid (3NP)-induced rat model. We demonstrated hIDPSC homing in the striatum, cortex and subventricular zone using specific markers for human cells. Thirty days after hIDPSC administration, the cells found in the brain are still express hallmarks of undifferentiated MSC. Immunohistochemistry quantities analysis revealed a significant increase in the number of BDNF, DARPP32 and D2R positive stained cells in the striatum and cortex in the groups that received hIDPSC. The differences were more expressive in animals that received only one administration of hIDPSC. Altogether, these data suggest that the intravenous administration of hIDPSCs can restore the BDNF, DARPP32 and D2R expression, promoting neuroprotection and neurogenesis.

Rho kinase inhibitor induced human dental pulp stem cells to differentiate into neurons.

AIMS: Neurological diseases due to neuron loss have become major public health problems. Current treatment reduces symptoms; however, there is no cure for neurological diseases. Therefore, stem cells may be an alternative therapy. Human dental pulp stem cells (hDPSCs) are an attractive source for cell-based approaches due to their high regenerative potential. The Rho kinase (ROCK) inhibitor Y-27632 promoted the neuronal differentiation of several stem cell types. However, its neuronal-inductive effect on hDPSCs has not been reported. Thus, the aim of our study was to investigate whether Y-27632 can induce the neuronal differentiation of hDPSCs. MAIN METHODS: hDPSCs were isolated from human third molars using an enzymatic method and were subsequently characterized. Cytotoxicity was evaluated using an MTT assay. The optimal concentration to induce neural differentiation was assessed using 1-50 µM Y-27632 as evaluated by Cresyl violet and immunofluorescence staining of neurofilaments and ßIII-tubulin, respectively. Ten µM Y-27632 was used for neuronal induction for 72 h, and differentiation was confirmed based on the expression of neurogenic markers (MAP2, Brn3a, and ChAT) and intracellular calcium activity. KEY FINDINGS: Our findings indicate that Y-27632 was not cytotoxic to hDPSCs and 10 µM Y-27632 was the lowest concentration that induced the morphological changes of hDPSCs into neuronal cells with Cresyl violet-positive staining and significantly enhanced the fluorescence intensity of neurofilament and ßIII-tubulin. The neuronal genes' expression and intracellular calcium activity were upregulated after induction with Y-27632. SIGNIFICANCE: At the optimal concentration and time, Rho kinase inhibitor induces hDPSC differentiation into neuronal cells.

MicroRNA-20a elevates osteogenic/odontoblastic differentiation potential of dental pulp stem cells by nuclear factor-κB/p65 signaling pathway via targeting interleukin-8.

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.

The GLUT1-mTORC1 axis affects odontogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) are considered valuable for regenerative therapy. Although glucose transporter 1 (GLUT1) is known to play a critical role in cell differentiation, its mechanism of the odontogenic differentiation of hDPSCs remains unclear. This study was conducted to investigate the effect and underlying mechanisms of GLUT1 on odontogenic differentiation of hDPSCs. hDPSCs was treated with phloretin (Phl), a GLUT1 inhibitor. The impact of GLUT1 on the odontogenic differentiation of hDPSCs was analysed using quantitative real-time polymerase chain reaction, alizarin-red staining, and western blotting. Glucose uptake by hDPSCs was significantly inhibited by Phl treatment. Overall, inhibition of GLUT1 upregulated the expression of DSPP, DMP1, RUNX2, and OCN and increased the formation of mineralised nodules on odontogenic induction of hDPSCs. The levels of phosphorylated mTOR and ribosomal protein S6 kinase 1 (p70S6K) were increased after GLUT1 inhibition and decreased by an mTOR inhibitor (rapamycin, Rapa) during the odontogenic induction of hDPSCs. Moreover, mTOR suppression decreased the expression of the genes described above and formation of mineralised nodules. These results suggest that inhibition of GLUT1 promoted the odontogenic differentiation of hDPSCs via the mTORC1-p70S6K axis, providing a foundation for further application of hDPSCs in regenerative therapy.

Complement C5aR/LPS-induced BDNF and NGF modulation in human dental pulp stem cells.

Stem cells with the ability to differentiate into a variety of cells and secrete nerve regeneration factors have become an emerging option in nerve regeneration. Dental pulp stem cells (DPSCs) appear to be a good candidate for nerve regeneration given their accessibility, neural crest origin, and neural repair qualities. We have recently demonstrated that the complement C5a system, which is an important mediator of inflammation and tissue regeneration, is activated by lipoteichoic acid-treated pulp fibroblasts, and governs the production of brain-derived nerve growth factor (BDNF). This BDNF secretion promotes neurite outgrowth towards the injury site. Here, we extend our observation to DPSCs and compare their neurogenic ability to bone marrow-derived mesenchymal stem cells (BM-MSCs) under inflammatory stimulation. Our ELISA and immunostaining data demonstrate that blocking the C5a receptor (C5aR) reduced BDNF production in DPSCs, while treatment with C5aR agonist increased the BDNF expression, which suggests that C5aR has a positive regulatory role in the BDNF modulation of DPSCs. Inflammation induced by lipopolysaccharide (LPS) treatment potentiated this effect and is C5aR dependent. Most important, DPSCs produced significantly higher levels of C5aR-mediated BDNF compared to BM-MSCs. Taken together, our data reveal novel roles for C5aR and inflammation in modulation of BDNF and NGF in DPSCs.

General Control Nonrepressed Protein 5 Modulates Odontogenic Differentiation Through NF-κB Pathway in Tumor Necrosis Factor-α-Mediated Impaired Human Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) from pulpitis patients showed defective osteogenic differentiation. However, as the most well-studied histone acetyltransferase, the impaired general control nonrepressed protein 5 (GCN5) plays essential roles in various developmental processes. The aim of this study was to investigate the effect of GCN5 on DPSCs odontogenic differentiation. The healthy dental pulp tissues were obtained from the extracted impacted third molar of patients with the informed consent. DPSCs were treated with a high concentration of tumor necrosis factor-alpha (TNF-α) (100 ng/mL) and odontogenic differentiation-related gene and GCN5 protein level by Western blot analysis. Proliferation of the DPSCs was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Immunofluorescence staining detected GCN5 and NF-κB signaling for p-p65. The mechanism of GCN5 regulating odontogenic differentiation of DPSCs was determined by small interfering RNA analysis. Our data suggested that TNF-α can significantly reduce mineralization and the expression of dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein at higher concentration (100 ng/mL). Meanwhile, it showed that the inflammation in microenvironment resulted in a downregulation of GCN5 expression and GCN5 knockdown caused decreased odontogenic differentiation of DPSCs was also found. In addition, the knockdown of GCN5 increased the expression of phosphorylation of p65, thus activating NF-κB pathway of DPSCs. Meanwhile, NF-κB pathway inhibitor pyrrolidinedithiocarbamic acid reversed the siGCN5 decreased odontogenic differentiation of DPSCs. Altogether, our findings indicated that in inflammatory microenvironments GCN5 plays a protective role in pulpitis impaired odontogenic differentiation of DPSCs by activating NF-κB pathway, which may provide a potential approach to dentin regeneration.

Application of dental pulp stem cells in oral maxillofacial tissue engineering.

In the maxillofacial area, soft and hard tissue abnormalities are caused by trauma, tumors, infection, and other causes that expose the maxillofacial region to the surface of the human body. Patients' normal physiological function and appearance are interfered with, and their mental health is adversely impacted, reducing their overall life quality. The pursuit of appropriate medical treatments to correct these abnormalities is thus vital. Autologous stem cell regeneration technology mainly focused on tissues has lately emerged as a significant problem in the medical community. Because of the capacity of dental pulp stem cells (DPSCs) to self-renew, the use of DPSCs from the human pulp tissues of deciduous teeth or permanent teeth has gained popularity among scientists as a stem cell-based therapy option. Aside from that, they are simple to extract and have minimal immunogenicity. As a result, bone tissue engineering may be a critical component in treating maxillofacial and periodontal bone abnormalities. DPSCs activity in maxillofacial and periodontal tissue-engineered bone tissue was investigated in this research.

Towards Induction of Angiogenesis in Dental Pulp Stem Cells Using Chitosan-Based Hydrogels Releasing Basic Fibroblast Growth Factor.

INTRODUCTION: Chitosan is a natural biopolymer that attracted enormous attention in biomedical fields. The main components of regenerative endodontic procedures (REPs), as well as tissue engineering, are scaffolds, stem cells, and growth factors. As one of the basic factors in the REPs is maintaining vascularization, this study was aimed at developing basic fibroblast growth factor- (bFGF-) loaded scaffolds and investigating their effects on the angiogenic induction in human dental pulp stem cells (hDPSCs). METHODS: Poly (ε-caprolactone) (PCL)/chitosan- (CS-) based highly porous scaffold (PCL/CS) was prepared and evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analyses. The adhesion and survival potency of seeded cells were assessed by SEM and MTT assays, respectively. The amount of angiogenic markers was investigated in gene and protein levels by real-time PCR and western blotting assays, respectively. RESULTS: Based on our findings, the SEM and FTIR tests confirmed the appropriate structure of synthesized scaffolds. Besides, the adhesion and survival rate of cells and the levels of VEGFR-2, Tie2, and Angiopoietin-1 genes were increased significantly in the PCL/CS/bFGF group. Also, the western blotting results showed the upregulation of these markers at protein levels, which were considerably higher at the PCL/CS/bFGF group (P < 0.05). CONCLUSIONS: On a more general note, this study demonstrates that the bFGF-loaded PCL/CS scaffolds have the potential to promote angiogenesis of hDPSCs, which could provide vitality of dentin-pulp complex as the initial required factor for regenerative endodontic procedures.

Enhancing osteogenic potential of hDPSCs by resveratrol through reducing oxidative stress via the Sirt1/Nrf2 pathway.

CONTEXT: The osteogenic potential of the human dental pulp stromal cells (hDPSCs) was reduced in the state of oxidative stress. Resveratrol (RSV) possesses numerous biological properties, including osteogenic potential, growth-promoting and antioxidant activities. OBJECTIVE: This study investigates the osteogenic potential of RSV by activating the Sirt1/Nrf2 pathway on oxidatively stressed hDPSCs and old mice. MATERIALS AND METHODS: The hDPSCs were subjected to reactive oxygen species (ROS) fluorescence staining, cell proliferation assay, ROS activity assay, superoxide dismutase (SOD) enzyme activity, the glutathione (GSH) concentration assay, alkaline phosphatase staining, real-time polymerase chain reaction (RT-PCR) and Sirt1 immunofluorescence labelling to assess the antioxidant stress and osteogenic ability of RSV. Forty female Kunming mice were divided into Old, Old-RSV, Young and Young-RSV groups to assess the repair of calvarial defects of 0.2 mL RSV of 20 mg/kg/d for seven days by injecting intraperitoneally at 4 weeks after surgery using micro-computed tomography, nonlinear optical microscope and immunohistochemical analysis. RESULTS: RSV abates oxidative stress by alleviating the proliferation, mitigating the ROS activity, increasing the SOD enzyme activity and ameliorating the GSH concentration (RSV IC50 in hDPSCs is 67.65 ± 9.86). The antioxidative stress and osteogenic capabilities of RSV were confirmed by the up-regulated gene expression of SOD1, xCT, RUNX2 and OCN, as well as Sirt1/Nrf2. The collagen, bone matrix formation and Sirt1 expression, are significantly increased after RSV treatment in mice. DISCUSSION AND CONCLUSIONS: For elderly or patients with oxidative stress physiological states such as hypertension, heart disease, diabetes, etc., RSV may potentially improve bone augmentation surgery in regenerative medicine.

In-vitro analysis on the potential use of dental pulp mesenchymal stem cells on arecoline-induced oral epithelial cells.

To assess the protective role of the secretome of dental pulp mesenchymal stem cells on arecoline-induced epithelial-mesenchymal transition and senescence on epithelial cells of the oral mucosa. Effect of varying concentrations of arecoline extract and dental pulp mesenchymal stem cell condition media (DPSC-CM) were noted on oral mucosal epithelial cells. MTT assay, Annexin V-FITC/PI assay, and the quantitative gene expressions of BCL2, PUMA, BAD, BAX, CASP3, CASP9, CASP12, TGFB1, CST3, COL1A2, COL3A1, TIMP1, TIMP2, CDH1, and CDH2 were assessed. Oral mucosal epithelial cells exposed only to the arecoline were the control. 50% and 100% DPSC-CM decreased apoptosis-related gene expression in the cells exposed with 25 µM arecoline compared to the control. 50% DPSC-CM attenuated the expression of all fibrotic genes and EMT-related genes. 20% and 100% DPSC-CM showed differential effects on fibrotic and EMT-related genes. DPSC-CM inhibited apoptosis, and attenuated expression of fibrotic and EMT-related genes on arecoline treated human oral epithelial cells.

Delivery of affordable and scalable encapsulated allogenic/autologous mesenchymal stem cells in coagulated platelet poor plasma for dental pulp regeneration.

The main goal of regenerative endodontics procedures (REPs) is to revitalize teeth by the regeneration of healthy dental pulp. In this study, we evaluated the potential of combining a natural and accessible biomaterial based on Platelet Poor Plasma (PPP) as a support for dental pulp stem cells (DPSC) and umbilical cord mesenchymal stem cells (UC-MSC). A comparison study between the two cell sources revealed compatibility with the PPP based scaffold with differences noted in the proliferation and angiogenic properties in vitro. Additionally, the release of growth factors including VEGF, HGF and DMP-1, was detected in the media of cultured PPP and was enhanced by the presence of the encapsulated MSCs. Dentin-Discs from human molars were filled with PPP alone or with MSCs and implanted subcutaneously for 4 weeks in mice. Histological analysis of the MSC-PPP implants revealed a newly formed dentin-like structure evidenced by the expression of Dentin sialophosphoprotein (DSPP). Finally, DPSC induced more vessel formation around the dental discs. This study provides evidence of a cost-effective, xenofree scaffold that is compatible with either autologous or allogenic strategy for dental pulp regeneration. This attempt if successfully implemented, could make REPs treatment widely accessible, contributing in improving global health conditions.