Macrophage M2 polarization promotes pulpal inflammation resolution during orthodontic tooth movement.

Mechanical force induces hypoxia in the pulpal area by compressing the apical blood vessels of the pulp, triggering pulpal inflammation during orthodontic tooth movement. However, this inflammation tends to be restorable. Macrophages are recognized as pivotal immunoreactive cells in the dental pulp. Whether they are involved in the resolution of pulpal inflammation in orthodontic teeth remains unclear. In this study, we investigated macrophage polarization and its effects during orthodontic tooth movement. It was demonstrated that macrophages within the dental pulp polarized to M2 type and actively participated in the process of pulpal inflammation resolution. Inflammatory reactions were generated and vascularization occurred in the pulp during orthodontic tooth movement. Macrophages in orthodontic pulp show a tendency to polarize towards M2 type as a result of pulpal hypoxia. Furthermore, by blocking M2 polarization, we found that macrophage M2 polarization inhibits dental pulp-secreting inflammatory factors and enhances VEGF production. In conclusion, our findings suggest that macrophages promote pulpal inflammation resolution by enhancing M2 polarization and maintaining dental health during orthodontic tooth movement.

Hypoxic dental pulp stem cells-released succinate promotes osteoclastogenesis and root resorption.

Introduction: Clinical studies have shown that endodontically-treated nonvital teeth exhibit less root resorption during orthodontic tooth movement. The purpose of this study was to explore whether hypoxic dental pulp stem cells (DPSCs) can promote osteoclastogenesis in orthodontically induced inflammatory root resorption (OIIRR). Methods: Succinate in the supernatant of DPSCs under normal and hypoxic conditions was measured by a succinic acid assay kit. The culture supernatant of hypoxia-treated DPSCs was used as conditioned medium (Hypo-CM). Bone marrow-derived macrophages (BMDMs) from succinate receptor 1 (SUCNR1)-knockout or wild-type mice were cultured with conditioned medium (CM), exogenous succinate or a specific inhibitor of SUCNR1 (4c). Tartrate-resistant acid phosphatase (TRAP) staining, Transwell assays, qPCR, Western blotting, and resorption assays were used to evaluate osteoclastogenesis-related changes. Results: The concentration of succinate reached a maximal concentration at 6 h in the supernatant of hypoxia-treated DPSCs. Hypo-CM-treated macrophages were polarized to M1 proinflammatory macrophages. Hypo-CM treatment significantly increased the formation and differentiation of osteoclasts and increased the expression of osteoclastogenesis-related genes, and this effect was inhibited by the specific succinate inhibitor 4c. Succinate promoted chemotaxis and polarization of M1-type macrophages with increased expression of osteoclast generation-related genes. SUCNR1 knockout decreased macrophage migration, M1 macrophage polarization, differentiation and maturation of osteoclasts, as shown by TRAP and NFATc1 expression and cementum resorption. Conclusions: Hypoxic DPSC-derived succinate may promote osteoclast differentiation and root resorption. The regulation of the succinate-SUCNR1 axis may contribute to the reduction in the OIIRR.

Ginsenoside Rb1 alleviates lipopolysaccharide-induced inflammation in human dental pulp cells via the PI3K/Akt, NF-κB, and MAPK signalling pathways.

AIM: Among numerous constituents of Panax ginseng, a constituent named Ginsenoside Rb1 (G-Rb1) has been studied to diminish inflammation associated with diseases. This study investigated the anti-inflammatory properties of G-Rb1 on human dental pulp cells (hDPCs) exposed to lipopolysaccharide (LPS) and aimed to determine the underlying molecular mechanisms. METHODOLOGY: The KEGG pathway analysis was performed after RNA sequencing in G-Rb1- and LPS-treated hDPCs. Reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis were used for the assessment of cell adhesion molecules and inflammatory cytokines. Statistical analysis was performed with one-way ANOVA and the Student-Newman-Keuls test. RESULTS: G-Rb1 did not exhibit any cytotoxicity within the range of concentrations tested. However, it affected the levels of TNF-α, IL-6 and IL-8, as these showed reduced levels with exposure to LPS. Additionally, less mRNA and protein expressions of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were shown. With the presence of G-Rb1, decreased levels of PI3K/Akt, phosphorylated IκBα and p65 were also observed. Furthermore, phosphorylated ERK and JNK by LPS were diminished within 15, 30 and 60 min of G-Rb1 exposure; however, the expression of non-phosphorylated ERK and JNK remained unchanged. CONCLUSIONS: G-Rb1 suppressed the LPS-induced increase of cell adhesion molecules and inflammatory cytokines, while also inhibiting PI3K/Akt, phosphorylation of NF-κB transcription factors, ERK and JNK of MAPK signalling in hDPCs.

TSG-6 Inhibits the NF-κB Signaling Pathway and Promotes the Odontogenic Differentiation of Dental Pulp Stem Cells via CD44 in an Inflammatory Environment.

In pulpitis, dentinal restorative processes are considerably associated with undifferentiated mesenchymal cells in the pulp. This study aimed to investigate strategies to improve the odonto/osteogenic differentiation of dental pulp stem cells (DPSCs) in an inflammatory environment. After pretreatment of DPSCs with 20 ng/mL tumor necrosis factor-induced protein-6 (TSG-6), DPSCs were cultured in an inflammation-inducing solution. Real-time polymerase chain reaction and Western blotting were performed to measure the expression levels of nuclear factor kappa B (NF-κB) and odonto/osteogenic differentiation markers, respectively. Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays were used to assess cell proliferation and activity. Subcutaneous ectopic osteogenesis and mandibular bone cultures were performed to assess the effects of TSG-6 in vivo. The expression levels of odonto/osteogenic markers were higher in TSG-6-pre-treated DPSCs than nontreated DPSCs, whereas NF-κB-related proteins were lower after the induction of inflammation. An anti-CD44 antibody counteracted the rescue effect of TSG-6 on DPSC activity and mineralization in an inflammatory environment. Exogenous administration of TSG-6 enhanced the anti-inflammatory properties of DPSCs and partially restored their mineralization function by inhibiting NF-κB signaling. The mechanism of action of TSG-6 was attributed to its interaction with CD44. These findings reveal novel mechanisms by which DPSCs counter inflammation and provide a basis for the treatment of pulpitis.

Enhancing Vasculogenesis in Dental Pulp Development: DPSCs-ECs Communication via FN1-ITGA5 Signaling.

BACKGROUND: Dental pulp regeneration therapy is a challenge to achieve early vascularization during treatment. Studying the regulatory mechanisms of vascular formation during human dental pulp development may provide insights for related therapies. In this study, we utilized single-cell sequencing analysis to compare the gene expression of dental pulp stem cells (DPSCs) and vascular endothelial cells (ECs) from developing and mature dental pulps. METHOD: Immunohistochemistry, Western blot, and real-time polymerase chain reaction (RT-PCR) were used to detect fibronectin 1 (FN1) expression and molecules, such as PI3K/AKT. Cell proliferation assay, scratch assay, tube formation assay and were used to investigate the effects of DPSCs on the vasculogenetic capability of ECs. Additionally, animal experiments involving mice were conducted. RESULT: The results revealed that DPSCs exist around dental pulp vasculature. FN1 expression was significantly higher in DPSCs from young permanent pulps than mature pulps, promoting HUVEC proliferation, migration, and tube formation via ITGA5 and the downstream PI3K/AKT signaling pathway. CONCLUSION: Our data indicate that intercellular communication between DPSCs and ECs mediated by FN1-ITGA5 signaling is crucial for vascularizationduring dental pulp development, laying an experimental foundation for future clinical studies.

Luteolin Is a Potential Immunomodulating Natural Compound against Pulpal Inflammation.

Aim: The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (n = 6). Periodontal bone resorption volumes were calculated for each group (nonligated-ligated), and the ratio of bone volume to tissue volume was measured. Results: Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. Conclusion: Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

Osteopontin interacts with dendritic cells and macrophages in pulp inflammation: Comprehensive transcriptomic analysis and laboratory investigations.

AIM: To investigate novel diagnostic markers for pulpitis and validate by clinical samples from normal and inflamed pulp. To explore the relationship between diagnostic markers and immune cells or their phenotypes during pulp inflammation. METHODOLOGY: Two microarray datasets, GSE77459 and GSE92681, and identified differential expression genes were integrated. To understand immune features, gene functions, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO) and ImmuneSigDB Gene Set Enrichment Analysis (GSEA) were analysed. For predictive purposes, machine learning techniques were applied to detect diagnostic markers. Immune infiltration in inflamed pulp was studied using CIBERSORT. The relationship between diagnostic markers and immune cells was investigated and validated their gene expression in clinical samples from the normal or inflamed pulp by qRT-PCR. Finally, the correlation between one marker, secreted phosphoprotein 1 (SPP1), encoding osteopontin (OPN), and dendritic cells (DCs)/macrophages was identified via HE staining and multiplex immunohistochemistry. An in vitro inflammatory dental pulp microenvironment model of THP-1 macrophages cocultured with dental pulp cells derived conditioned media (DPCs-CM) to investigate OPN production and macrophage phenotypes was established. RESULTS: Analysis revealed unique immunologic features in inflamed pulp. Three diagnostic markers for pulpitis: endothelin-1 (EDN1), SPP1, and purine nucleoside phosphorylase (PNP), and validated them using qRT-PCR were predicted. Multiplex immunohistochemistry demonstrated OPN co-localized with activated DCs and M2 macrophages during pulp inflammation. In vitro experiments showed that THP-1 macrophages produced the highest levels of OPN when stimulated with DPCs-CM derived from the 20 µg/mL LPS pre-conditioned group, suggesting an M2b-like phenotype by increasing surface marker CD86 and expression of IL6, TNFα, IL10, and CCL1 but not CCL17 and MerTK. Levels of CCL1 and IL10 elevated significantly in the macrophages' supernatant from the 20 µg/mL LPS pre-conditioned CM group. OPN was proven co-localizing with CD86 in the inflamed pulp by immunofluorescence. CONCLUSIONS: The current findings suggest that OPN can serve as a promising biomarker for pulpitis, correlated with DCs and macrophages. OPN+ macrophages in the inflamed pulp are associated with M2b-like phenotypes. These insights offer the potential for improved diagnosis and targeted therapy.

Lipopolysaccharide-mediated ATP signaling regulates interleukin-6 mRNA expression via the P2-purinoceptor in human dental pulp cells.

Dental pulp cells play a crucial role in maintaining the balance of the pulp tissue. They actively respond to bacterial inflammation by producing proinflammatory cytokines, particularly interleukin-6 (IL-6). While many cell types release adenosine triphosphate (ATP) in response to various stimuli, the mechanisms and significance of ATP release in dental pulp cells under inflammatory conditions are not well understood. This study aimed to investigate ATP release and its relationship with IL-6 during the inflammatory response in immortalized human dental pulp stem cells (hDPSC-K4DT) following lipopolysaccharide (LPS) stimulation. We found that hDPSC-K4DT cells released ATP extracellularly when exposed to LPS concentrations above 10 µg/mL. ATP release was exclusively attenuated by N-ethylmaleimide, whereas other inhibitors, including clodronic acid (a vesicular nucleotide transporter inhibitor), probenecid (a selective pannexin-1 channel inhibitor), meclofenamic acid (a selective connexin 43 inhibitor), suramin (a nonspecific P2 receptor inhibitor), and KN-62 (a specific P2X7 antagonist), did not exhibit any effect. Additionally, LPS increased IL-6 mRNA expression, which was mitigated by the ATPase apyrase enzyme, N-ethylmaleimide, and suramin, but not by KN-62. Moreover, exogenous ATP induced IL-6 mRNA expression, whereas ATPase apyrase, N-ethylmaleimide, and suramin, but not KN-62, diminished ATP-induced IL-6 mRNA expression. Overall, our findings suggest that LPS-induced ATP release stimulates the IL-6 pathway through P2-purinoceptor, indicating that ATP may function as an anti-inflammatory signal, contributing to the maintenance of dental pulp homeostasis.

Senescence on Dental Pulp Cells: Effects on Morphology, Migration, Proliferation, and Immune Response.

INTRODUCTION: Evidence indicates that senescence can affect essential dental pulp functions, such as defense capacity and repair, consequently affecting the successes of conservative endodontic treatments. This study aims to evaluate the effects of senescence on the morphology, migration, proliferation, and immune response of human dental pulp cells. METHODS: Cells were treated with doxorubicin to induce senescence, confirmed by ß-galactosidase staining. Morphological changes, cellular proliferation, and migration were evaluated by scanning electron microscopy, trypan blue cells, and the scratch method, respectively. Modifications in the immune response were evaluated by measuring the genes for pro-inflammatory cytokines tumor necrosis factor alpha and interleukin (IL)-6 and anti-inflammatory cytokines transforming growth factor beta 1 and IL-10 using the real time polymerase chain reaction assay. RESULTS: An increase in cell size and a decrease in the number of extensions were observed in senescent cells. A reduction in the proliferative and migratory capacity was also found in senescent cells. In addition, there was an increase in the gene expression of inflammatory cytokines tumor necrosis factor alpha and IL-6 and a decrease in the gene expression of IL-10 and transforming growth factor beta-1, suggesting an exacerbated inflammatory situation associated with immunosuppression. CONCLUSIONS: Cellular senescence is possibly a condition that affects prognoses of conservative endodontic treatments, as it affects primordial cellular functions related to this treatment.

Increased Purinergic Signaling in Human Dental Pulps With Inflammatory Pain is Sex-Dependent.

An enhanced understanding of neurotransmitter systems contributing to pain transmission aids in drug development, while the identification of biological variables like age and sex helps in the development of personalized pain management and effective clinical trial design. This study identified enhanced expression of purinergic signaling components specifically in painful inflammation, with levels increased more in women as compared to men. Inflammatory dental pain is common and potentially debilitating; as inflammation of the dental pulp can occur with or without pain, it provides a powerful model to examine distinct pain pathways in humans. In control tissues, P2X3 and P2X2 receptors colocalized with PGP9.5-positive nerves. Expression of the ecto-nucleotidase NTPDase1 (CD39) increased with exposure to extracellular adenosine triphosphate (ATP), implying CD39 acted as a marker for sustained elevation of extracellular ATP. Both immunohistochemistry and immunoblots showed P2X2, P2X3, and CD39 increased in symptomatic pulpitis, suggesting receptors and the ATP agonist were elevated in patients with increased pain. The increased expression of P2X3 and CD39 was more frequently observed in women than men. In summary, this study identifies CD39 as a marker for chronic elevation of extracellular ATP in fixed human tissue. It supports a role for increased purinergic signaling in humans with inflammatory dental pain and suggests the contribution of purines shows sexual dimorphism. This highlights the potential for P2X antagonists to treat pain in humans and stresses the need to consider sex in clinical trials that target pain and purinergic pathways. PERSPECTIVE: This article demonstrates an elevation of ATP-marker CD39 and of ATP receptors P2X2 and P2X3 with inflammatory pain and suggests the rise is greater in women. This highlights the potential for P2X antagonists to treat pain and stresses the consideration of sexual dimorphism in studies of purines and pain.

LIF Aggravates Pulpitis by Promoting Inflammatory Response in Macrophages.

Leukemia inhibitory factor (LIF) has been recognized as a novel inflammatory modulator in inflammation-associated diseases. This study aimed to investigate the modulation of LIF in dental pulp inflammation. Experimental pulpitis was established in wild-type (WT) and Lif-deficient (Lif-/-) mice. Histological and immunostaining analyses were conducted to assess the role of LIF in the progression of pulpitis. Mouse macrophage cell line (RAW264.7) was treated with LPS to simulate an inflammatory environment. Exogenous LIF was added to this system to examine its modulation in macrophage inflammatory response in vitro. Primary bone marrow-derived macrophages (BMDMs) from WT and Lif-/- mice were isolated and stimulated with LPS to confirm the effect of Lif deletion on macrophage inflammatory response. Supernatants from LIF and LPS-treated human dental pulp cells (hDPCs) were collected and added to macrophages. Macrophage chemotaxis was assessed using transwell assays. The results showed an increased expression of LIF and LIFR with the progression of pulpitis, and LIFR was highly expressed in macrophages. Lif deficiency alleviated experimental pulpitis with the reduction of pro-inflammatory cytokines and macrophage infiltration. Exogenous LIF promoted inflammatory response of LPS-induced macrophages through a STAT3/p65-dependent pathway. Consistently, Lif deletion inhibited macrophage inflammatory response in vitro. Supernatants of LIF-treated hDPCs enhanced macrophage migration in LPS-induced inflammatory environment. Our findings demonstrated that LIF aggravates pulpitis by promoting macrophage inflammatory response through a STAT3/p65-dependent pathway. Furthermore, LIF plays a crucial role in driving the recruitment of macrophages to inflamed pulp tissue by promoting chemokine secretion in DPCs.

Extracellular Vesicles Carrying RUNX3 Promote Differentiation of Dental Pulp Stem Cells.

BACKGROUND: This study aims to clarify the mechanism underlying dental pulp cells-extracellular vesicles (DPC-EVs) carrying runt-related transcription factor 3 (RUNX3) in mediating odontogenic differentiation of dental pulp stem cells (DPSCs) with the involvement of miR-30a-5p-regulated NOTCH1. METHODS: Extracellular vesicles (EVs) were isolated from human DPSCs, and identified using transmission electron microscopy, and nanoparticle tracking analysis. PBS, EVs, or EV inhibitor GW4869 was added to DPSCs for co-culture, whilst odontogenic differentiation was assessed in terms of ratio of mineralized nodules and expression odontoblast differentiation markers. Dual luciferase reporter gene assay and chromatin immunoprecipitation for binding relation among RUNX3, miR-30a-5p and NOTCH1were employed to evaluate their roles in odontogenic differentiation was determined. Animal experiment was established to confirm the effect of DPC-EVs-loaded RUNX3 on dental pulp. RESULTS: In vitro finding demonstrated that EVs delivered RUNX3 to DPSCs, thereby activated miR-30a-5p expression and inhibited NOTCH1 expression, which was reversed by addition of GW4869. RUNX3 upregulation promoted miR-30a-5p while miR-30a-5p targeted and inhibited NOTCH1. Silencing of RUNX3 in EVs decreased expression of those differentiation markers, downregulated miR-30a-5p and upregulated NOTCH1. CONCLUSION: DPSC-EVs can carry RUNX3 to the DPSCs, promote the transcription of miR-30a-5p, and then inhibit the expression of NOTCH1, and finally promote the odontogenic differentiation of DPSCs.

Inhibitory effects of the nanoscale lysate derived from xenogenic dental pulp stem cells in lung cancer models.

BACKGROUND: Lung cancer is a highly prevalent malignancy and has the highest mortality rate among all tumors due to lymph node metastasis. Bone marrow and umbilical cord-derived mesenchymal stem cells (MSCs) have demonstrated tumor-suppressive effects on lung cancer. This study investigated the effects of DPSC lysate on proliferation, apoptosis, migration and invasion of cancer cells were studied in vivo and in vitro. METHODS: The proliferation, apoptosis, and migration/metastasis were evaluated by cell counting kit-8 assay, Annexin-V and propidium iodide staining, and the transwell assay, respectively. The expression levels of apoptosis-, cell cycle-, migration-, and adhesion-related mRNA and proteins were measured by qRT-PCR and western blot. The level and mRNA expression of tumor markers carcino embryonic antigen (CEA), neuron-specific enolase (NSE), and squamous cell carcinoma (SCC) were measured by Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR. Finally, a tumor-bearing mouse model was constructed to observe the tumor-suppressive effect of DPSC lysate after intraperitoneal injection. RESULTS: DPSC lysate decreased the viability of A549 cells and induced apoptosis in lung cancer cells. Western blot confirmed that levels of Caspase-3, Bax, and Bad were increased, and Bcl-2 protein levels were decreased in A549 cells treated with DPSC lysate. In addition, DPSC lysate inhibited the migration and invasion of A549 cells; downregulated key genes of the cell cycle, migration, and adhesion; and significantly suppressed tumor markers. Xenograft results showed that DPSC lysate inhibited tumor growth and reduced tumor weight. CONCLUSIONS: DPSC lysate inhibited proliferation, invasion, and metastasis; promoted apoptosis in lung cancer cells; and suppressed tumor growth- potentially providing a cell-based alternative therapy for lung cancer treatment.

Electroacupuncture exerts prolonged analgesic and neuroprotective effects in a persistent dental pain model induced by multiple dental pulp injuries: GABAergic interneurons-astrocytes interaction.

Pain within the trigeminal system, particularly dental pain, is poorly understood. This study aimed to determine whether single or multiple dental pulp injuries induce persistent pain, its association with trigeminal central nociceptive pathways and whether electroacupuncture (EA) provides prolonged analgesic and neuroprotective effects in a persistent dental pain model. Models of single dental pulp injury (SDPI) and multiple dental pulp injuries (MDPI) were used to induce trigeminal neuropathic pain. The signs of dental pain-related behavior were assessed using the mechanical head withdrawal threshold (HWT). Immunofluorescence and western blot protocols were used to monitor astrocyte activation, changes in apoptosis-related proteins, and GABAergic interneuron plasticity. SDPI mice exhibited an initial marked decrease in HWT from days one to 14, followed by progressive recovery from days 21 to 42. From days 49 to 70, the HWT increased and returned to the control values. In contrast, MDPI mice showed a persistent decrease in HWT from days one to 70. MDPI increased glial fibrillary acidic protein (GFAP) and decreased glutamine synthetase (GS) and glutamate transporter-1 (GLT1) expression in the Vi/Vc transition zone of the brainstem on day 70, whereas no changes in astrocytic markers were observed on day 70 after SDPI. Increased expression of cleaved cysteine-aspartic protease-3 (cleaved caspase-3) and Bcl-2-associated X protein (Bax), along with decreased B-cell lymphoma/leukemia 2 (Bcl-2), were observed at day 70 after MDPI but not after SDPI. The downregulation of glutamic acid decarboxylase (GAD65) expression was observed on day 70 only after MDPI. The effects of MDPI-induced lower HWT from days one to 70 were attenuated by 12 sessions of EA treatment (days one to 21 after MDPI). Changes in astrocytic GFAP, GS, and GLT-1, along with cleaved caspase-3, Bax, Bcl-2, and GAD65 expression observed 70 days after MDPI, were reversed by EA treatment. The results suggest that persistent dental pain in mice was induced by MDPI but not by SDPI. This effect was associated with trigeminal GABAergic interneuron plasticity along with morphological and functional changes in astrocytes. EA exerts prolonged analgesic and neuroprotective effects that might be associated with the modulation of neuron-glia crosstalk mechanisms.

Stem cells in clinical dentistry.

BACKGROUND: Stem cells are present in most of the tissues in the craniofacial complex and play a major role in tissue homeostasis and repair. These cells are characterized by their capacity to differentiate into multiple cell types and to self-renew to maintain a stem cell pool throughout the life of the tissue. TYPES OF STUDIES REVIEWED: The authors discuss original data from experiments and comparative analyses and review articles describing the identification and characterization of stem cells of the oral cavity. RESULTS: Every oral tissue except enamel, dentin, and cementum contains stem cells for the entire life span. These stem cells self-renew to maintain a pool of cells that can be activated to replace terminally differentiated cells (for example, odontoblasts) or to enable wound healing (for example, dentin bridge in pulp exposures and healing of periodontal tissues after surgery). In addition, dental stem cells can differentiate into functional blood vessels and nerves. Initial clinical trials have shown that transplanting dental pulp stem cells into disinfected necrotic teeth has allowed for the recovery of tooth vitality and vertical and horizontal root growth in immature teeth with incomplete root formation. PRACTICAL IMPLICATIONS: As a consequence of these groundbreaking discoveries, stem cell banks are now offering services for the cryopreservation of dental stem cells. The future use of stem cell-based therapies in the clinic will depend on the collaboration of clinicians and researchers in projects designed to understand whether these treatments are safe, efficacious, and clinically feasible.

In vitro effects of wool-derived keratin on human dental pulp-derived stem cells for endodontic applications.

In this study we examine the influence of wool-derived keratin intermediate filament proteins (kIFPs) on human dental pulp-derived stem cells (hDPSCs). kIFPs were diluted (10 mg/mL to 0.001 mg/mL) in cell culture media. Effects on hDPSCs proliferation were measured using Alamar blue assay. Keratin concentrations of 1 mg/mL and 0.1 mg/mL were tested for odontogenic differentiation and mineralisation. Alkaline phosphatase (ALP) quantification (7th, 14th, and 21st days), alizarin red S (AR-S) staining and calcium quantification (21st day), reverse transcription polymerase chain reaction (RT-PCR, collagen expression), and immunocytochemical staining for dentin matrix protein (DMP) were performed. hDPSCs showed higher proliferation with kIFPs of 0.1 mg/mL or less (p < 0.0001). The 0.1 mg/mL keratin concentration promoted odontogenic differentiation, confirmed by increased ALP activity, significant calcium deposits (AR-S staining, p < 0.05), up-regulated collagen expression (RT-PCR, p < 0.05), and positive DMP staining. These results suggest that kIFPs could be a potential biomaterial for pulp-dentin regeneration.

Vitamin D3-incorporated chitosan/collagen/fibrinogen scaffolds promote angiogenesis and endothelial transition via HIF-1/IGF-1/VEGF pathways in dental pulp stem cells.

Effective vascularization during wound healing remains a critical challenge in the regeneration of skin tissue. On the other hand, mesenchymal stem cell (MSC) to endothelial phenotype transition (MEnDoT) is a potential phenomenon grossly underexplored in vascularized skin tissue engineering. Vitamin D3 has a proven role in promoting MEnDoT. Hence, a D3-incorporated scaffold made with biocompatible materials such as chitosan, collagen and fibrinogen should be able to promote endothelial lineage transition in vitro for tissue engineering purposes. In this study, we developed vitamin D3 incorporated chitosan-collagen-fibrinogen (CCF-D3) scaffolds physically crosslinked under UV and conducted thorough physicochemical and biological assays on it compared to a control scaffold without vitamin D3. Our study for the first time reports the potential vascularization property of the CCF-D3 scaffold by inducing the transitions of dental pulp MSC to endothelial lineage via the HIF-1/IGF-1/VEGF pathways. MSC seeded on UV-exposed CCF-D3 scaffolds had higher cell viability and transitioned towards endothelial lineage was observed by elevated proliferative and endothelial-specific gene expressions and flow cytometric analysis of SCA-1+ antibody. The difference in VEGF-A and α-SMA expressions was also observed in the D3-CCF scaffold compared to the scaffolds without D3.

HnRNP A1 Suppresses the Odontogenic Differentiation and the Inclusion of RUNX2 Exon 5 of Dental Mesenchymal Cells.

BACKGROUND: RUNX2 (Runt-related transcription factor 2) acts as a key regulator in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). Moreover, the inclusion of exon 5 is important for RUNX2 function. Our previous study showed that Y-Box Binding Protein 1 (YBX1) promoted RUNX2 exon 5 inclusion and mineralization of hDPSCs. However, the regulatory mechanism of RUNX2 exon 5 alternative splicing needed further exploration. METHODS: The expression level of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) during the odontogenic differentiation of hDPSCs was analyzed by RT-PCR and Western blot. The roles of hnRNP A1 in the alternative splicing of RUNX2 exon 5 and the odontogenic differentiation of dental mesenchymal cells were analyzed by gain- and loss-of-function experiments. RESULTS: Surprisingly, we found an alternative splicing factor, hnRNP A1, which had an opposite role to YBX1 in regulating RUNX2 exon 5 inclusion and odontogenic differentiation of hDPSCs. Through gain- and loss-of-function assay, we found that hnRNP A1 suppressed the inclusion of RUNX2 exon 5, resulting in the inhibition of odontoblastic differentiation. The overexpression of hnRNP A1 can inhibit the expression of ALP (alkaline phosphatase) and OCN (osteocalcin), and the formation of mineralized nodules during the odontogenic differentiation of both hDPSCs and mouse dental papilla cells (mDPCs), whereas the opposite results were obtained with an hnRNP A1 knockdown preparation. CONCLUSIONS: The present study indicated that hnRNP A1 suppressed RUNX2 exon 5 inclusion and reduced the odontogenic differentiation ability of hDPSCs and mDPCs.

[Characteristics and microRNA expression profile of exosomes derived from odontogenic dental pulp stem cells].

OBJECTIVE: To investigate the characteristics of exosomes derived from dental pulp stem cells (DPSCs) in the direction of odontogenic differentiation, to analyze the differences in microRNA expression profile between exosomes derived from undifferentiated and odontogenic DPSCs, and to analyze their possible signal transduction pathways. METHODS: (1) DPSCs were cultured in α minimum Eagle' s medium (α-MEM), and odontogenic DPSCs were cultured in odontogenic differentiation medium for 21 days, using alizarin red staining and alkaline phosphatase staining to identify the odontogenic differentiation. Exosomes from the cell supernatant were isolated respectively, named as dental pulp stem cells-exosomes (DPSCs-Exo) and dental pulp stem cells-odontogenic-exosomes (DPSCs-OD-Exo). The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and Western blot. (2) The microRNA expression profiles of DPSCs-Exo and DPSCs-OD-Exo were investigated by microRNA microarray. To validate the result of the microRNA microarray, real-time quantitative polymerase chain reaction (real-time PCR) assay was applied on 3 most significantly differential expressed microRNA. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNA. RESULTS: (1) The DPSCs were isolated and cultured in vitro showed typical fibroblast-like morphology. The odontogenic differentiated DPSCs were spindle-shaped, polygonal, and uniform in size. Odontogenic differentiation group showed a large number of dark deposits in alizarin red staining and the cells were darkly stained in alkaline phosphatase staining, while the cells in normal culture medium group did not show obvious dyeing. The DPSCs-Exo and DPSCs-OD-Exo had the same morphology, both showed bilayer membrane and cup-shape. The peak sizes of DPSCs-Exo and DPSCs-OD-Exo were (114.67±9.07) nm and (134.00±8.54) nm, respectively. The difference between the two was statistically significant. DPSCs-Exo and DPSCs-OD-Exo both expressed the markers of exosomes, tumor susceptibility gene (TSG)101 and CD63. (2) microRNA microarray results showed that the expression profiles of DPSCs-Exo and DPSCs-OD-Exo were different. Nineteen increased by more than two times, and one decreased by 64%. Real-time PCR results showed that the expression levels of microRNA-1246, microRNA-1246-100-5p and microRNA-1246-494-3p in DPSCs-OD-Exo were significantly up-regulated. The difference was statistically significant. microRNA target prediction database and gene signaling pathway database were used to analyze differentially expressed microRNA, and it was predicted that differentially expressed microRNA could target axis inhibition protein 2(AXIN2) gene and Wnt/ß-catenin signaling pathway. CONCLUSION: DPSCs-OD-Exo and DPSCs-Exo had differences in their microRNA expression profile. Those differentially expressed microRNA may be involved in the regulation of DPSCs odontogenic differentiation.

PER2 Promotes Odontoblastic/Osteogenic Differentiation of Dental Pulp Stem Cells by Modulating Mitochondrial Metabolism.

Human dental pulp stem cells (hDPSCs) possess remarkable self-renewal and multilineage differentiation ability. PER2, an essential circadian molecule, regulates various physiological processes. Evidence suggests that circadian rhythm and PER2 participate in physiological functions of DPSCs. However, the influence of PER2 on DPSCs' differentiation remains largely unknown. This study aimed to explore the effect and potential mechanism of PER2 on hDPSCs' differentiation. Dental pulp tissues were extracted, and hDPSCs were cultured for in vitro and in vivo experiments. Dorsal subcutaneous transplantation was performed in 6-week-old male BALB/c mice. The hDPSCs' odontoblastic/osteogenic differentiation was assessed, and mitochondrial metabolism was evaluated. The results indicated PER2 expression increasing during hDPSCs' odontoblastic/osteogenic differentiation. Gain- and loss-of function studies confirmed that PER2 promoted alkaline phosphatase (ALP) activity, mineralized nodules deposition, mRNA expression of DSPP, DMP1, COL1A1 and protein expression of DSPP and DMP1 in hDPSCs. Furthermore, PER2 enhanced collagen deposition, osteodentine-like tissue formation and DSPP expression in vivo. Mitochondrial metabolic evaluation aimed to investigate the mechanism of PER2-mediated hDPSC odontoblastic/osteogenic differentiation, which showed that PER2 increased ATP synthesis, elevated mitochondrial membrane potential and changed expression of proteins regulating mitochondrial dynamics. This study demonstrated that PER2 promoted hDPSCs' odontoblastic/osteogenic differentiation, which involved mitochondrial metabolic change.