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Objectives: This study investigated the miRNA expression profile in Notch-activated human dental stem pulp stem cells (DPSCs) and validated the functions of miRNAs in modulating the odonto/osteogenic properties of DPSCs., Methods: DPSCs were treated with indirect immobilized Jagged1. The miRNA expression profile was examined using NanoString analysis. Bioinformatic analysis was performed, and miRNA expression was validated. Odonto/osteogenic differentiation was examined using alkaline phosphatase staining, Alizarin Red S staining, as well as odonto/osteogenic-related gene and protein expression., Results: Fourteen miRNAs were differentially expressed in Jagged1-treated DPSCs. Pathway analysis revealed that altered miRNAs were associated with TGF-β, Hippo, ErbB signalling pathways, FoxO and Ras signalling. Target prediction analysis demonstrated that 7604 genes were predicted to be targets for these altered miRNAs. Enrichment analysis revealed relationships to various DNA bindings. Among differentially expressed miRNA, miR-296-3p and miR-450b-5p were upregulated under Jagged1-treated conditions. Overexpression of miR-296-3p and miR-450b-5p enhanced mineralization and upregulation of odonto/osteogenic-related genes, whereas inhibition of these miRNAs revealed opposing results. The miR-296-3p and miR-450b-5p inhibitors attenuated the effects of Jagged1-induced mineralization in DPSCs., Conclusions: Jagged-1 promotes mineralization in DPSCs that are partially regulated by miRNA. The novel understanding of these miRNAs could lead to innovative controlled mechanisms that can be applied to modulate biology-targeted dental materials., (© 2024 Wiley Periodicals LLC.)

Background: The integration of stem cells, signaling molecules, and biomaterial scaffolds is fundamental for the successful engineering of functional bone tissue. Currently, the development of composite scaffolds has emerged as an attractive approach to meet the criteria of ideal scaffolds utilized in bone tissue engineering (BTE) for facilitating bone regeneration in bone defects. Recently, the incorporation of polycaprolactone (PCL) with hydroxyapatite (HA) has been developed as one of the suitable substitutes for BTE applications owing to their promising osteogenic properties. In this study, a three-dimensional (3D) scaffold composed of PCL integrated with HA (PCL/HA) was prepared and assessed for its ability to support osteogenesis in vitro. Furthermore, this scaffold was evaluated explicitly for its efficacy in promoting the proliferation and osteogenic differentiation of canine bone marrow-derived mesenchymal stem cells (cBM-MSCs) to fill the knowledge gap regarding the use of composite scaffolds for BTE in the veterinary orthopedics field., Results: Our findings indicate that the PCL/HA scaffolds substantially supported the proliferation of cBM-MSCs. Notably, the group subjected to osteogenic induction exhibited a markedly upregulated expression of the osteogenic gene osterix (OSX) compared to the control group. Additionally, the construction of 3D scaffold constructs with differentiated cells and an extracellular matrix (ECM) was successfully imaged using scanning electron microscopy. Elemental analysis using a scanning electron microscope coupled with energy-dispersive X-ray spectroscopy confirmed that these constructs possessed the mineral content of bone-like compositions, particularly the presence of calcium and phosphorus., Conclusions: This research highlights the synergistic potential of PCL/HA scaffolds in concert with cBM-MSCs, presenting a multidisciplinary approach to scaffold fabrication that effectively regulates cell proliferation and osteogenic differentiation. Future in vivo studies focusing on the repair and regeneration of bone defects are warranted to further explore the regenerative capacity of these constructs, with the ultimate goal of assessing their potential in veterinary clinical applications., (© 2024. The Author(s).)

Extracellular matrix (ECM) is an intricate structure providing the microenvironment niche that influences stem cell differentiation. This study aimed to investigate the efficacy of decellularized ECM derived from human dental pulp stem cells (dECM_DPSCs) and gingival-derived mesenchymal stem cells (dECM_GSCs) as an inductive scaffold for osteogenic differentiation of GSCs. The proteomic analysis demonstrated that common and signature matrisome proteins from dECM_DPSCs and dECM_GSCs were related to osteogenesis/osteogenic differentiation. RNA sequencing data from GSCs reseeded on dECM_DPSCs revealed that dECM_DPSCs upregulated genes related to the Hippo and Wnt signaling pathways in GSCs. In the inhibitor experiments, results revealed that dECM_DPSCs superiorly promoted GSCs osteogenic differentiation, mainly mediated through Hippo and Wnt signaling. The present study emphasizes the promising translational application of dECM_DPSCs as a bio-scaffold rich in favorable regenerative microenvironment for tissue engineering., (© 2024. The Author(s).)

Background: This study aimed to investigate the effects of various toll-like receptor (TLR) and C-type lectin receptor (CLR) ligands on osteogenic differentiation in human dental pulp stem cells (hDPSCs)., Methods: hDPSCs were cultured and treated with various concentrations (0.01, 0.1, 1.0, and 10 µg/mL) of TLR or CLR agonists (PG-LPS, E.coli LPS, poly(I:C), Pam3CSK4, Furfurman, and Zymosan). Cell viability was determined by MTT assay. The effects of TLR and CLR agonists on osteogenic differentiation of hDPSCs were measured by alkaline phosphatase (ALP) activity, Alizarin Red S staining, and Von Kossa staining. In addition, the mRNA expression of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1) was examined by RT-qPCR. A non-parametric analysis was employed for the statistical analyses. The statistically significant difference was considered when p < 0.05., Results: Treatment with TLR and CLR agonists was associated with an increase in hDPSCs' colony-forming unit ability. Compared with the control group, TLR and CLR agonists significantly inhibited the osteogenic differentiation of hDPSCs by decreasing the ALP activity, mineralised nodule formation, and mRNA expression levels of osteogenesis-related genes (ALP, COL1A1, RUNX2, OSX, OCN and DMP1). The inhibition of TRIF but not Akt signalling rescued the effects of TLR and CLR agonist attenuating hDPSCs' mineralisation., Conclusions: The activation of TLRs or CLRs exhibited an inhibitory effect on osteogenic differentiation of hDPSCs via the TRIF-dependent signalling pathway., (© 2024. The Author(s).)

Asiatic acid (AA) and asiaticoside, pentacyclic triterpenoid compounds derived from Centella asiatica, are known for their biological effects in promoting type I collagen synthesis and inducing osteogenesis of stem cells. However, their applications in regenerative medicine are limited due to their low potency and poor aqueous solubility. This work aimed to evaluate the osteogenic induction activity of AA derivatives in human periodontal ligament stem cells (hPDLSCs) in vitro. Four compounds were synthesised, namely 501, 502, 503, and 506. AA was used as the control. The 502 exhibited low water solubility, while the 506 compound showed the highest. The cytotoxicity analysis demonstrated that 503 caused significant deterioration in cell viability, while other derivatives showed no harmful effect on hPDLSCs. The dimethyl aminopropyl amine derivative of AA, compound 506, demonstrated a relatively high potency in inducing osteogenic differentiation. An elevated mRNA expression of osteogenic-related genes, BMP2, WNT3A, ALP, OSX and IBSP was observed with 506. Additionally, the expression of BMP-2 protein was enhanced with increasing dose of 506, and the effect was pronounced when the Erk signalling molecule was inhibited. The 506 derivative was proposed for the promotion of osteogenic differentiation in hPDLSCs by upregulating BMP2 via the Erk signalling pathway. The 506 molecule showed promise in bone tissue regeneration., (© 2023. Springer Nature Limited.)

Periodontal ligament stem cells (PDLSCs) play a significant role on periodontal tissue and alveolar bone homeostasis. During inflammation, interleukin (IL)-6 serves as one of key cytokine players controlling tissue reaction as well as alveolar bone tissue remodeling. It is believed that periodontal tissue inflammation causes periodontium degradation, especially alveolar bone. However, in this study, we show that an inflammatory mediator, IL-6, may serve another direction on alveolar bone homeostasis during inflammatory condition. We found that, IL-6 at 10 and 20 ng/mL was not cytotoxic and dose-dependently exerted beneficial effects on osteogenic differentiation of human PDLSCs (hPDLSCs), as demonstrated by increased alkaline phosphatase activity, mRNA expression of osteogenic markers, and matrix mineralization. The presence of physiological and inflammatory level of IL-6, the osteogenic differentiation potential by hPDLSCs was enhanced by several possible mechanisms including transforming growth factor (TGF), Wnt, and Notch pathways. After in-depth and thorough exploration, we found that Wnt pathway serves as key regulator controlling osteogenic differentiation by hPDLSCs amid the IL-6 presentation. Surprisingly, apart from other mesenchymal stem cells, distinct Wnt components are employed by hPDLSCs, and both canonical and non-canonical Wnt pathways are triggered by different mechanisms. Further validation by gene silencing, treatment with recombinant Wnt ligands, and β-catenin stabilization/translocation confirmed that IL-6 governed the canonical Wnt/β-catenin pathway via either WNT2B or WNT10B and employed WNT5A to activate the non-canonical Wnt pathway. These findings fulfill the homeostasis pathway governing periodontal tissue and alveolar bone regeneration and may serve for further therapeutic regimen design for restoring the tissues., (© 2023. The Author(s).)

Aim: To investigate the effect of Wnt3a on odonto/osteogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHEDs) and reparative dentine formation in a rat model., Methodology: Stem cells isolated from human exfoliated deciduous teeth were cultured in media with Wnt3a (50-200 ng/ml). Wnt activation was confirmed by β-catenin immunocytochemistry. Colony-forming unit assay (normalized percentage area), osteogenic gene expression analysis by real-time polymerase chain reaction and mineralization assays measured by the absorption at 540 nm were performed. Tertiary dentine formation in vivo was evaluated using 8-week-old, male Wistar rats. Cavities with pinpoint pulp exposure by a sharp instrument were prepared at the mesial surface of the first molars. Teeth were divided into (n = 6): (1) distilled water (negative control), (2) phosphate-buffered saline (PBS), (3) lithium chloride in DI (20 μM), and (4) Wnt3a in PBS (200 ng/ml). Collagen sponge was used as a scaffold. The cavity was sealed with glass ionomer restoration. Four weeks later, animals were euthanized by sodium pentobarbital (120 mg/kg body weight). Hard tissue formation was evaluated using micro-computerized tomography. Sixty consecutive slides from the initial plane were analysed and calculated as bone/dentine volume per total tissue volume. Paraffin sections (2 μm) were stained with haematoxylin and eosin and Masson's trichrome for morphological evaluation. Data are presented as the mean ± standard error. Mann-Whitney U test was used for two-group comparison. Kruskal Wallis followed by pairwise comparison was employed for three or more group comparisons. Statistical analysis was performed using GraphPad Prism 7. Differences were considered significant at p < .05., Results: Wnt3a decreased SHEDs colony formation and increased OSX, BMP2, and DMP1 expression, corresponding to an increase in mineralization. Additionally, a significant increase in dentine/bone volume per total tissue volume was observed in Wnt3a treated defects. Dentine bridge formation at the exposure sites treated with Wnt3a demonstrated, while fibrous tissues were observed in the control., Conclusions: Wnt3a suppressed proliferation, increased osteogenic differentiation of SHEDs and promotes tertiary dentine formation. Wnt3a could be utilized as biological molecule for vital pulp therapy., (© 2023 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Aim: Tideglusib is a small molecule agonist of the canonical Wnt pathway. The present study investigated the influence of Tideglusib on human dental pulp stem cell (hDPSC) proliferation, apoptosis, migration and odonto/osteogenic differentiation., Methodology: hDPSCs were treated with 50, 100 nM or 200 nM Tideglusib. β-catenin accumulation was detected by immunofluorescence staining. Colony-forming unit ability was assessed by staining with Coomassie blue. Cell cycle progression and cell apoptosis were investigated using flow cytometry. Cell migration was examined using an in vitro wound-healing assay. Osteogenic differentiation was examined using alkaline phosphatase (ALP) staining, alizarin red S staining and osteogenic-related gene expression. The gene expression profile was examined using a high-throughput RNA sequencing technique. All experiments were repeated using cells derived from at least four different donors (n = 4). The Mann-Whitney U-test was used to identify significant differences between two independent group comparisons. For three or more group comparisons, statistical differences were assessed using the Kruskal-Wallis test followed by pairwise comparison. The significance level was set at 5% (p < .05)., Results: Tideglusib activated the Wnt signalling pathway in hDPSCs as demonstrated by an increase in cytoplasmic β-catenin accumulation and nuclear translocation. Tideglusib did not affect hDPSC proliferation, cell cycle progression, cell apoptosis or cell migration. In contrast, 50 and 100 nM Tideglusib significantly enhanced mineralization and osteogenic marker gene expression (RUNX2, ALP, BMP2 and DSPP; p < .05)., Conclusions: Tideglusib enhanced the odonto/osteogenic differentiation of hDPSCs. Therefore, incorporating this bioactive molecule in a pulp-capping material could be a promising strategy to promote dentine repair., (© 2022 British Endodontic Society. Published by John Wiley & Sons Ltd.)

The indirect immobilisation of Jagged-1 (Jagged-1) promoted osteogenic differentiation of human dental pulp cells (hDPs). Furthermore, the analysis of the Reactome pathway of RNA sequencing data indicates the upregulated genes involved with the extracellular matrix (ECM). Hence, our objective was to investigate the effects of Jagged-1 on proteomic profiles of human dental pulp stem cells (hDPSC). hDPSCs were cultured on the surface coated with human IgG Fc fragment (hFc) and the surface coated with rhJagged1/Fc recombinant protein-coated surface. Cells were differentiated to the osteogenic lineage using an osteogenic differentiation medium (OM) for 14 days, and cells cultured in a growth medium were used as a control. The protein component of the cultured cells was extracted into the cytosol, membrane, nucleus, and cytoskeletal compartment. Subsequently, the proteomic analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS). Metascape gene list analysis reported that Jagged-1 stimulated the expression of the membrane trafficking protein (DOP1B), which can indirectly improve osteogenic differentiation. hDPSCs cultured on Jagged-1 surface under OM condition expressed COL27A1, MXRA5, COL7A1, and MMP16, which played an important role in osteogenic differentiation. Furthermore, common matrisome proteins of all cellular components were related to osteogenesis/osteogenic differentiation. Additionally, the gene ontology categorised by the biological process of cytosol, membrane, and cytoskeleton compartments was associated with the biomineralisation process. The gene ontology of different culture conditions in each cellular component showed several unique gene ontologies. Remarkably, the Jagged-1&#95;OM culture condition showed the biological process related to odontogenesis in the membrane compartment. In conclusion, the Jagged-1 induces osteogenic differentiation could, mainly through the regulation of protein in the membrane compartment.

6-bromoindirubin-3'-oxime (BIO) is a candidate small molecule that effectively modulates Wnt signalling owing to its stable property. The present study investigated the influence of BIO on the odonto/osteogenic differentiation of human dental pulp stem cells (hDPSCs). hDPSCs were treated with 200, 400, or 800 nM BIO, and the effects on hDPSC responses and osteogenic differentiation were assessed. BIO-mediated Wnt activation was confirmed by β-catenin nuclear translocation detected by immunofluorescence staining. BIO attenuated colony formation and cell migration determined by in vitro wound-healing assay. BIO increased early apoptotic cell population evaluated using flow cytometry. For osteogenic induction, BIO promoted alkaline phosphatase (ALP) activity and mineralisation in a dose-dependent manner. ALP , RUNX2 , OCN , OSX , ANKH , DMP1 , and DSPP mRNA expression were significantly upregulated. The OPG/RANKL expression ratio was also increased. Further, BIO attenuated adipogenic differentiation as demonstrated by decreased lipid accumulation and adipogenic-related gene expression. Bioinformatic analysis of RNA sequencing data from the BIO-treated hDPSCs revealed that BIO modulated pathways related to autophagy and actin cytoskeleton regulation. These findings demonstrated that BIO treatment promoted hDPSC osteogenic differentiation. Therefore, this small molecule is a strong candidate as a bioactive molecule to enhance dentin repair.

Aim: To investigate whether TGF-β/BMP signalling participates in Jagged1-induced osteogenic differentiation in human dental pulp cells (hDPs)., Methodology: Bioinformatic analysis of publicly available RNA sequencing data of Jagged1-treated hDPs was performed using NetworkAnalyst. The mRNA expression was validated using real-time polymerase chain reaction. hDPs were seeded on Jagged1 immobilized surfaces in the presence or absence of TGF-β or BMP inhibitor. Osteogenic differentiation was evaluated using alkaline phosphatase staining, osteogenic marker gene expression and mineralization assay. Statistical analyses were performed using a Kruskal-Wallis test, followed by a pairwise comparison for more than three group comparison. Mann-Whitney U-test was employed for two group comparison. The statistical significance was considered at p < .05., Results: Jagged1 treatment in growth medium significantly promoted TGFB1, TGFB2 and TGFB3 whilst significantly inhibited BMP2, BMP4 and BMP6 mRNA expression (p < .05). In osteogenic induction medium, Jagged1 significantly up-regulated TGFB1, TGFB2 and TGFB3 at days 1 and 3 (p < .05). Pre-treatment with TGF-β1, TGF-β2 or TGF-β3 prior to osteogenic induction resulted in the significant increase of osteogenic marker gene expression, collagen type 1 protein expression, alkaline phosphatase enzymatic activity and mineral deposition (p < .05). However, TGF-β signalling inhibition with SB431542 (4 μmol L -1 ) or SB505124 (47 and 129 nmol L -1 ) failed to attenuate the effect of Jagged1-induced osteogenic differentiation in hDPs. Dorsomorphin (4 and 8 μmol L -1 ) treatment significantly abolished the effect of Jagged1 on mineralization by hDPs (p < .05)., Conclusion: Notch signalling activation by Jagged1 modulated TGF-β and BMP ligand expression. Dorsomorphin, but not TGF-β receptor inhibitor, attenuated Jagged1-induced osteogenic differentiation in hDPs., (© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Objectives: Crosstalk between Notch and other cell signaling molecules has been implicated to regulate the osteogenic differentiation. Understanding the interaction between Notch and IL15 is essential to reveal molecular mechanism. Thus, the objective of the present study was to investigate whether IL15 participates in the Notch signaling-induced mineral deposition in human dental pulp cells (hDPs)., Methods: hDPs were explanted from dental pulp tissues. To activate Notch signaling, the cells were seeded on Jagged1-immobilized surfaces. The mRNA expression was evaluated using real-time polymerase chain reaction. hDPs were treated with 5-50 ng/mL IL15. Cell viability and proliferation were determined using an MTT assay. Mineral deposition was examined using alizarin red s and Von Kossa staining. In some experiments, the cells were pretreated with a JAK inhibitor prior to stimulation., Results: Jagged1 induced IL15 and IL15RA expression in hDPs. IL15 treatment significantly increased mineral deposition at 14 d and upregulated ALP, OCN, OSX, ANKH, and ENPP1 mRNA expression. IL15-induced mineralization was attenuated by JAK inhibitor pretreatment. Further, JAK inhibitor pretreatment inhibited the effect of Jagged1 on hDP mineral deposition., Conclusion: IL15 promoted the osteogenic differentiation in hDPs. Moreover, IL15 participated in the Jagged1-induced mineralization in hDPs., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Platelet-rich fibrin (PRF) promotes wound healing by providing the release of growth factors. Here, the influence of Thai and Murrah bubaline blood derived PRF on canine periodontal ligament cells (cPDLs) was investigated. PRF was prepared from Thai and Murrah buffaloes with single centrifugation. Results demonstrated that Thai bubaline blood derived PRF exhibited fiber-mesh like morphology and contained more platelet entrapment than Murrah bubaline blood derived PRF. Both bubaline PRFs were able to degrade in vitro under condition with trypsin. Thai but not Murrah bubaline blood derived PRF promoted cPDLs proliferation in serum free and 2% serum culture conditions. Correspondingly, the significant upregulation of KI67 mRNA expression was observed in those cells treated with Thai bubaline blood derived PRF. However, both Thai and Murrah bubaline blood derived PRF accelerated cell migration in an in vitro wound healing assay and facilitated cell spreading. Further, cPDLs cultured in osteogenic induction medium supplemented with Thai bubaline blood derived PRF exhibited the increased mineral deposition in vitro. Frozen Thai bubaline blood derived PRF also promoted cell proliferation, KI67 mRNA expression, cell migration, and cell spreading in cPDLs. Taken these evidence together, bubaline blood derived PRF could provide potential benefits for canine periodontal tissue healing.

Utilization of canine mesenchymal stem cells (cMSCs) for regenerating incorrigible bone diseases has been introduced. However, cMSCs harvested from different sources showed distinct osteogenicity. To clarify this, comparative proteomics-based systems biology analysis was used to analyze osteogenic differentiation behavior by cMSCs harvested from bone marrow and dental pulp. The results illustrated that canine dental pulp stem cells (cDPSCs) contained superior osteogenicity comparing with canine bone marrow-derived MSCs (cBM-MSCs) regarding alkaline phosphatase activity, matrix mineralization, and osteogenic marker expression. Global analyses by proteomics platform showed distinct protein clustering and expression pattern upon an in vitro osteogenic induction between them. Database annotation using Reactome and DAVID revealed contrast and unique expression profile of osteogenesis-related proteins, particularly on signaling pathways, cellular components and processes, and cellular metabolisms. Functional assay and hierarchical clustering for tracking protein dynamic change confirmed that cBM-MSCs required the presences of Wnt, transforming growth factor (TGF)-beta, and bone-morphogenetic protein (BMP) signaling, while cDPSCs mainly relied on BMP signaling presentation during osteogenic differentiation in vitro. Therefore, these findings illustrated the comprehensive data regarding an in vitro osteogenic differentiation behavior by cBM-MSCs and cDPSCs which is crucial for further mechanism study and the establishment of cMSC-based bone tissue engineering (BTE) for veterinary practice.

Jagged1 activates Notch signaling and subsequently promotes osteogenic differentiation in human periodontal ligament cells (hPDLs). The present study investigated the participation of the Notch receptor, NOTCH2, in the Jagged1-induced osteogenic differentiation in hPDLs. NOTCH2 and NOTCH4 mRNA expression levels increased during hPDL osteogenic differentiation. However, the endogenous NOTCH2 expression levels were markedly higher compared with NOTCH4. NOTCH2 expression knockdown using shRNA in hPDLs did not dramatically alter their proliferation or osteogenic differentiation compared with the shRNA control. After seeding on Jagged1-immobilized surfaces and maintaining the hPDLs in osteogenic medium, HES1 and HEY1 mRNA levels were markedly reduced in the shNOTCH2-transduced cells compared with the shControl group. Further, shNOTCH2-transduced cells exhibited less alkaline phosphatase enzymatic activity and in vitro mineralization than the shControl cells when exposed to Jagged1. MSX2 and COL1A1 mRNA expression after Jagged1 activation were reduced in shNOTCH2-transduced cells. Endogenous Notch signaling inhibition using a γ-secretase inhibitor (DAPT) attenuated mineralization in hPDLs. DAPT treatment significantly promoted TWIST1, but decreased ALP, mRNA expression, compared with the control. In conclusion, Notch signaling is involved in hPDL osteogenic differentiation. Moreover, NOTCH2 participates in the mechanism by which Jagged1 induced osteogenic differentiation in hPDLs.

Mechanical force regulates periodontal ligament cell (PDL) behavior. However, different force types lead to distinct PDL responses. Here, we report that pretreatment with an intermittent compressive force (ICF), but not a continuous compressive force (CCF), promoted human PDL (hPDL) osteogenic differentiation as determined by osteogenic marker gene expression and mineral deposition in vitro. ICF-induced osterix (OSX) expression was inhibited by cycloheximide and monensin. Although CCF and ICF significantly increased extracellular adenosine triphosphate (ATP) levels, pretreatment with exogenous ATP did not affect hPDL osteogenic differentiation. Gene-expression profiling of hPDLs subjected to CCF or ICF revealed that extracellular matrix (ECM)-receptor interaction, focal adhesion, and transforming growth factor beta (TGF-β) signaling pathway genes were commonly upregulated, while calcium signaling pathway genes were downregulated in both CCF- and ICF-treated hPDLs. The TGFB1 mRNA level was significantly increased, while those of TGFB2 and TGFB3 were decreased by ICF treatment. In contrast, CCF did not modify TGFB1 expression. Inhibiting TGF-β receptor type I or adding a TGF-β1 neutralizing antibody attenuated the ICF-induced OSX expression. Exogenous TGF-β1 pretreatment promoted hPDL osteogenic marker gene expression and mineral deposition. Additionally, pretreatment with ICF in the presence of TGF-β receptor type I inhibitor attenuated the ICF-induced mineralization. In conclusion, this study reveals the effects of ICF on osteogenic differentiation in hPDLs and implicates TGF-β signaling as one of its regulatory mechanisms.

Objectives: The present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined., Methods: Gene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively., Results: From bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells., Conclusion: Notch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Notch signaling is involved in osteogenic differentiation; however, its role differs depending on cell type and differentiation stage. Here, we investigated the involvement of Notch signaling in the osteogenic differentiation of retinoic acid-treated embryoid bodies derived from mouse gingival fibroblast-derived induced pluripotent stem cells (mGF-iPSCs). When cultured in osteogenic media, mGF-iPSCs showed an increase in their expression of osteogenic marker genes and deposited a mineralized matrix. Furthermore, increased levels of mRNA for Notch1, Notch2, and Hey1 were observed. In the presence of DAPT, a Notch signaling inhibitor, during osteogenic induction, mRNA levels for osteogenic marker genes were significantly decreased; however, no difference was noted in mineral deposition. Moreover, activation of Notch signaling using Jagged1-immobilized surfaces resulted in a slight increase of in vitro mineralization on days 3 and 7 of osteogenic induction. Significant upregulation of Dlx5, Bsp, and Col I mRNA expression was observed in mGF-iPSCs cultured on Jagged1 surfaces. In conclusion, inhibition and activation of Notch signaling was shown to decrease and increase mGF-iPSC osteogenic differentiation, respectively. However, the responses were not robust, suggesting the involvement of additional signaling pathways.

Lithium Chloride (LiCl) has been used as a canonical Wnt pathway activator due to its ability to inhibit a glycogen synthase kinase-3. The aim of the present study was to investigate the effect of LiCl on cell proliferation and osteogenic differentiation in stem cells isolated from human exfoliated deciduous teeth (SHEDs). SHEDs were isolated and cultured in media supplemented with LiCl at 5, 10, or 20mM. The results demonstrated that LiCl significantly decreased SHEDs colony forming unit ability in a dose dependent manner. LiCl significantly enhanced the percentage of cells in the sub G0 phase, accompanied by a reduction of the percentage of cells in the G1 phase at day 3 and 7 after treatment. Further, LiCl markedly decreased OSX and DMP1 mRNA expression after treating SHEDs in an osteogenic induction medium for 7 days. In addition, no significant difference in alkaline phosphatase enzymatic activity or mineral deposition was found. Together, these results imply that LiCl influences SHEDs behavior., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth., Design: Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively., Results: The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control., Conclusion: The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model., (Copyright © 2016. Published by Elsevier Ltd.)

Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation of Amelx was associated with enhanced osteogenic differentiation, especially in the early stage of biomineralization.

Objective: Low oxygen tension is one of the crucial factors of the stem-cell niche. However, the long-term hypoxic culture of stem cells is difficult and requires special equipment. In this study, we investigated whether mimicking hypoxia using cobalt chloride (CoCl2) could maintain human periodontal ligament (HPDL) cell stemness., Methods: HPDL cells were treated with either 50 or 100 μM CoCl2. Cell proliferation was determined by an MTT assay. The mRNA expression of stem-cell marker and osteogenic associated genes were analyzed by RT-PCR and Real-time PCR. Osteogenic differentiation was determined by assaying alkaline phosphatase activity and in vitro mineralization., Results: The results showed that the CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 resulted in a decrease in alkaline phosphatase activity, down-regulation of osteogenic associated gene expression, and suppression of mineralization. The use of Apigenin, an HIF-1α inhibitor, indicated that CoCl2 might inhibit osteogenic differentiation through an HIF-1α- dependent mechanism., Conclusion: This study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

D-Glucose serves many roles in cellular functions, but its role in human periodontal ligament-derived mesenchymal stem cells (hPSLSCs) is yet unknown. Here, the roles of high glucose concentration on neurogenic differentiation by hPDLSCs were investigated. Two-stage neurogenic induction protocol was employed. Cells were maintained in normal neurogenic induction medium, high glucose condition, or high mannose condition. The results showed that high glucose attenuated neurosphere formation efficiency by hPDLSCs in terms of morphology, neurogenic marker expression, without a deleterious effect on cell viability. Contrastingly, neurosphere-derived cells matured in high glucose condition exhibited normal neuronal characteristics compared to the control. During neurosphere formation in high glucose, glucose transporters (GLUTs) mRNA levels were significantly decreased, corresponding with the deprivation of cellular glucose uptake. Further, a glucose uptake inhibitor, cytochalasin B, was used to confirm the deleterious effects of glucose uptake deprivation during neurosphere formation. The results demonstrated that deprivation of glucose uptake attenuated neurosphere formation efficiency by hPDLSCs. Together, the results illustrated that high glucose condition attenuated the efficiency of neurosphere formation but not neuronal maturation, which may occur through the downregulation of GLUTs and the reduction of glucose uptake., (© 2013 Wiley Periodicals, Inc.)

Asiaticoside, an active ingredient extracted from Centella asiatica, has been widely used to promote wound healing. In this study, the effects of asiaticoside on proliferation, protein synthesis, and osteogenic differentiation in human periodontal ligament cells (HPDLs) were investigated. HPDLs were treated with asiaticoside at concentrations of 25, 50, and 100 µg/mL. Cell number was determined by MTT assay. The mRNA expression was analyzed by reverse transcription-polymerase chain reaction. Western blot analysis and immunocytochemistry were used to confirm protein synthesis. Osteogenic differentiation was determined by alkaline phosphatase activity, osteoblast marker gene expression, and in vitro mineralization. The results showed that asiaticoside treatment, ranging from 25 to 100 mg/mL, had no effect on cytotoxicity or cell proliferation. When HPDLs were treated with asiaticoside in serum-free medium, dose-dependent increases in the levels of fibronectin and collagen type I mRNA and protein were observed at 72 h. Moreover, asiaticoside attenuated matrix metalloproteinase-1 but enhanced tissue inhibitor of metalloproteinase-1 mRNA expression. The addition of asiaticoside to osteogenic medium resulted in an increase in alkaline phosphatase enzymatic activity, up-regulation of osteoblast marker gene mRNA expression, and enhancement of mineralization by HPDLs. These results suggest the potential application of asiaticoside for enhancing periodontal tissue healing., (Copyright © 2012 John Wiley & Sons, Ltd.)

Notch signaling plays critical roles in various cell types by regulating cell fate determination and differentiation. Here, we investigated the ability to control differentiation of human periodontal ligament derived mesenchymal stem cells using modified surfaces containing the affinity immobilized Notch ligand, Jagged-1. After seeding human periodontal ligament derived mesenchymal stem cells (HPDLs) on Jagged-1 modified surfaces, expression of Notch signaling target genes, Hes-1 and Hey-1, was higher than those exposed to soluble Jagged-1 or control surfaces. Upregulation of Notch signaling target genes was attenuated after treatment with the γ secretase inhibitor. Upon seeding the cells on Jagged-1 immobilized surface and maintained in osteogenic medium, alkaline phosphatase enzymatic activity and mineralization as well as mRNA expression of alkaline phosphatase (ALP), collagen type I (COL I) and osteopontin (OPN) were significantly increased compared to those of controls. However, osteocalcin (OCN) mRNA expression level was decreased when cells were exposed to Jagged-1 modified surfaces. HPDLs on Jagged-1 modified surfaces expressed lower TWIST2 mRNA levels than the control, suggesting that the mechanism whereby Jagged-1 enhances osteogenic differentiation of HPDLs may occur through Notch signaling and TWIST regulation. In summary, an alteration of biomaterial interface using Notch ligands illustrates a promising system to control HPDLs differentiation toward osteogenic lineage., (Copyright © 2012 Wiley Periodicals, Inc.)

The fibrotic response of the body to synthetic polymers limits their success in tissue engineering and other applications. Though porous polymers have demonstrated improved healing, difficulty in controlling their pore sizes and pore interconnections has clouded the understanding of this phenomenon. In this study, a novel method to fabricate natural polymer/calcium phosphate composite scaffolds with tightly controllable pore size, pore interconnection, and calcium phosphate deposition was developed. Microporous, nanofibrous fibrin scaffolds were fabricated using sphere-templating methods. Composite scaffolds were created by solution deposition of calcium phosphate on fibrin surfaces or by direct incorporation of nanocrystalline hydroxyapatite (nHA). The SEM results showed that fibrin scaffolds exhibited a highly porous and interconnected structure. Osteoblast-like cells, obtained from murine calvaria, attached, spread and showed a polygonal morphology on the surface of the biomaterial. Multiple cell layers and fibrillar matrix deposition were observed. Moreover, cells seeded on mineralized fibrin scaffolds exhibited significantly higher alkaline phosphatase activity as well as osteoblast marker gene expression compared to fibrin scaffolds and nHA incorporated fibrin scaffolds (0.25 and 0.5g). All types of scaffolds were degraded both in vitro and in vivo. Furthermore, these scaffolds promoted bone formation in a mouse calvarial defect model and the bone formation was enhanced by addition of rhBMP-2.