Osteogenic Differentiation Of Periodontal Ligament Stem Cells Research Articles

Circ_0003764 Regulates the Osteogenic Differentiation of Periodontal Ligament Stem Cells

Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role. The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs. qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs. Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs. This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.

Scopolamine regulates the osteogenic differentiation of human periodontal ligament stem cells through lactylation modification of RUNX2 protein.

Periodontal ligament stem cells (PDLSCs) are important mesenchymal stem cells contributing to regenerating lost periodontal tissues and repairing bone defects. Studies on the molecular mechanism affecting the osteogenic differentiation of PDLSCs are necessary. Scopolamine (SCO) is known as a regulator of neural cell damage. The focus of the current study is on unveiling the role of SCO-mediated molecular mechanism in the osteogenic differentiation of PDLSCs. Through CCK-8 assay and LDH detection, we confirmed that SCO enhanced the viability of PDLSCs. Moreover, we determined that SCO induced the PDLSCs osteogenic differentiation, according to data of ALP activity measurement and ARS staining. Mechanistically, we performed western blot and identified that SCO could promote the lactylation of runt-related transcription factor 2 (RUNX2). We also found through rescue assays that knockdown of RUNX2 could reverse the effect of SCO treatment on the osteogenic differentiation of PDLSCs. Further mechanism investigation revealed that lactylation of RUNX2 at K176 site enhances the protein stability of RUNX2 through deubiquitination. Collectively, our present study unveils that SCO stabilizes RUNX2 to promote the osteogenic differentiation of PDLSCs through the lactylation modification of RUNX2.

Role of maternally expressed 8 small nucleolar RNA (MEG8) in osteogenic differentiation of periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) are essential for the treatment of bone diseases because of its great potential to differentiate into osteoblasts. Remarkably, increasing long-non-coding RNAs (lncRNAs) have been reported to be involved in the osteogenic differentiation of PDLSCs. Maternally expressed 8, small nucleolar RNA host gene (MEG8) is implicated in multiple diseases. This study intended to unearth the potential role of MEG8 and unveil the mechanism in PDLSCs undergoing osteoblastic differentiation. MEG8 expression was measured by quantitative real-time PCR (RT-qPCR) during osteogenic differentiation of PDLSCs into bone cells. Functional assays were used to uncover the biological function of MEG8. Besides, RNA pulldown, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assays were used to explore the molecular mechanism of MEG8. MEG8 was apparently overexpressed in osteogenically differentiated PDLSCs. Moreover, MEG8 deficiency suppressed the osteoblastic differentiation of PDLSCs. Furthermore, MEG8 modulated the expression of transcription factor 4 (TCF4) by scavenging microRNA-495-3p (miR-495-3p) and microRNA-485-3p (miR-485-3p) through the competing endogenous RNA (ceRNA) mechanism, further stimulating the Wnt/β-catenin pathway. MEG8 stimulates the capacity of PDLSCs for osteogenic differentiation through a ceRNA mode.

Xanthones Isolated from Cratoxylum cochinchinensis Reduced Oxidative Stress in Periodontal Ligament Stem Cells.

Xanthone compounds from Cratoxylum cochinchinensis (C. cochinchinensis) have demonstrated antioxidant effects and potency in treating many inflammatory diseases. However, the efficiency of the three xanthone extracts isolated from the young fruit of this plant, i.e., two geranyloxy xanthones (F6, F8) and one 1,3,7-hydroxy xanthone (F137), as antioxidants and therapeutics for periodontal disease has not been evaluated. The aim of this study was to investigate the antioxidant effects of three xanthones isolated from C. cochinchinensis on periodontal ligament stem cells (PDLSCs) and their osteogenic differentiation. The antioxidant activity of the aqueous extracts was determined using a DPPH assay, and their cytotoxicity was evaluated using an MTT assay. H2O2 was used to induce intracellular stress, and the scavenging effect of the isolated compounds against reactive oxygen species (ROS) was analyzed with a fluorescence assay. The expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was evaluated, and the effects of the three compounds on PDLSCs osteogenic differentiation were investigated. The isolated compounds reduced both extracellular and intracellular ROS in a dose-dependent manner and induced the expression of Nrf2 and HO-1 in PDLSCs. Under redox conditions, these compounds potentiated PDLSCs osteogenic differentiation. Our study demonstrated that the hydroxy xanthones from C. cochinchinensis had antioxidant effects on the Nrf2/HO-1 pathway and might be effective therapeutic substrates for damage prevention and the regeneration of damaged periodontal tissues in periodontitis patients.

Exosomal microRNA-223 from neutrophil-like cells inhibits osteogenic differentiation of PDLSCs through the cGMP-PKG signaling pathway.

Neutrophils-derived exosomes have been shown to cause tissue inflammation in many diseases, but their role in periodontitis, a neutrophil-mediated disease, is unknown. Here, we investigated the effect of neutrophil-like cells derived exosomes on osteogenic dysfunction of periodontal ligament stem cells (PDLSCs) in periodontitis. Neutrophil-like cells were derived from HL-60 cells by dimethylsulfoxide stimulation. Exosomes were isolated by ultracentrifugation and characterized using transmission electron microscopy, nanoflow cytometry and western blot. MicroRNA-223 (miR-223) expression were analyzed by real-time PCR. Western blot, alkaline phosphatase (ALP), and alizarin red staining were conducted to assess whether exosomes could affect the osteogenic differentiation of PDLSCs. The expression of miR-223 was inhibited in PDLSCs by transfecting with miR-223 inhibitor. Cyclic guanosine monophosphate (cGMP) expression was determined by enzyme-linked immunosorbent assay. We found that miR-223 was significantly increased in neutrophils and neutrophil-like cells derived exosomes. Treatment with exosomes derived from neutrophil-like cells upregulated miR-223 expression and inhibited the osteogenic differentiation of PDLSCs, while transfection with miR-223 inhibitor significantly promoted PDLSCs osteogenic differentiation. In addition, co-treatment with KT5823, a cGMP-PKG pathway inhibitor, markedly abrogated the rescue effects of miR-223 inhibitor on the osteogenic differentiation of PDLSCs. Our findings suggest that neutrophil-like cells derived exosomes might inhibit osteogenic differentiation of PDLSCs by transporting miR-223 and regulating the cGMP-PKG signaling pathway.

Long non-coding RNA X-Inactive Specific Transcript (XIST) interacting with USF2 promotes osteogenic differentiation of periodontal ligament stem cells through regulation of WDR72 transcription.

Periodontal ligament stem cells (PDLSCs) are the most potential cells in periodontal tissue regeneration and bone tissue regeneration. Our prior work had revealed that WD repeat-containing protein 72 (WDR72) was crucial for osteogenic differentiation of PDLSCs. Here, we further elucidated its underlying mechanism in PDLSC osteogenic differentiation. Human PDLSCs, isolated and identified by flow cytometry, were prepared for osteogenic differentiation induction. Levels of WDR72, long non-coding RNA X-Inactive Specific Transcript (XIST), upstream stimulatory factor 2 (USF2), and osteogenic marker genes (Runx2, Osteocalcin, and Collagen I) in human PDLSCs and clinical specimens were detected by RT-qPCR. Protein expressions of WDR72, Runx2, Osteocalcin, and Colla1 were tested by Western blot. The interactions among the molecules were verified by RIP, RNA pull-down, ChIP, and luciferase reporter assays. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red staining (ARS). WDR72 was decreased in periodontal tissues of periodontitis patients, and overexpression reversed TNF-α-mediated suppressive effects on PDLSC osteogenic differentiation. Mechanically, XIST recruited the enrichment of USF2 to the WDR72 promoter region, thereby positively regulating WDR72. WDR72 silencing overturned XIST-mediated biological effects in PDLSCs. WDR72, regulated by the XIST/USF2 axis, enhances osteogenic differentiation of PDLSCs, implying a novel strategy for alleviating periodontitis.

LncRNA MALAT1 promotes osteogenic differentiation through the miR-93-5p/SMAD5 axis.

Promoting the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is a way to regenerate periodontal bone. This study aimed to determine whether lncRNA MALAT1 promotes the osteogenic differentiation of human PDLSCs in vitro. Human PDLSCs were extracted from the human periodontal ligament, and after osteogenic differentiation was induced using osteogenic medium, the human PDLSCs were transfected with siRNA-MALAT1, miR-93-5p mimics, and miR-93-5p inhibitors. The expression of osteogenesis-related genes was assessed by RT-qPCR and western blotting, alkaline phosphatase (ALP) activity was assessed by ALP activity assay, and the formation of mineralized nodules was assessed by alizarin red S (ARS) staining. RNA immunoprecipitation (RIP) and luciferase assays were performed to assess the binding of MALAT1, miR-93-5p, and SMAD5. The expression of lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was upregulated, while that of miR-93-5p was downregulated after PDLSC osteogenic differentiation. Knockdown of MALAT1 inhibited the osteogenic differentiation of PDLSCs, and MALAT1 expression negatively correlated with miR-93-5p expression. miR-93-5p inhibited the osteogenic differentiation of human PDLSCs by specifically binding to SMAD5. MALAT1 regulates human PDLSC differentiation by regulating the miR-93-5p/SMAD5 axis.

NADPH-dependent ROS accumulation contributes to the impaired osteogenic differentiation of periodontal ligament stem cells under high glucose conditions.

Diabetes mellitus is an established risk factor for periodontal disease that can aggravate the severity of periodontal inflammation and accelerate periodontal destruction. The chronic high glucose condition is a hallmark of diabetes-related pathogenesis, and has been demonstrated to impair the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), leading to delayed recovery of periodontal defects in diabetic patients. Reactive oxygen species (ROS) are small molecules that can influence cell fate determination and the direction of cell differentiation. Although excessive accumulation of ROS has been found to be associated with high glucose-induced cell damage, the underlying mechanisms remain unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) is an important electron donor and functions as a critical ROS scavenger in antioxidant systems. It has been identified as a key mediator of various biological processes, including energy metabolism and cell differentiation. However, whether NADPH is involved in the dysregulation of ROS and further compromise of PDLSC osteogenic differentiation under high glucose conditions is still not known. In the present study, we found that PDLSCs incubated under high glucose conditions showed impaired osteogenic differentiation, excessive ROS accumulation and increased NADPH production. Furthermore, after inhibiting the synthesis of NADPH, the osteogenic differentiation of PDLSCs was significantly enhanced, accompanied by reduced cellular ROS accumulation. Our findings demonstrated the crucial role of NADPH in regulating cellular osteogenic differentiation under high glucose conditions and suggested a new target for rescuing high glucose-induced cell dysfunction and promoting tissue regeneration in the future.

FoxO1 agonists promote bone regeneration in periodontitis by protecting the osteogenesis of periodontal ligament stem cells.

Protecting the function of periodontal ligament stem cells (PDLSCs) is crucial for bone regeneration in periodontitis. Forkhead box protein O1 (FoxO1) has been previously reported as a crucial mediator in bone homeostasis, providing a favorable environment for osteoblast proliferation and differentiation. Here, we investigated the effect and mechanism of FoxO1 agonists on the osteogenesis of PDLSCs under inflammatory conditions. Here, we screened FoxO1 agonists by detecting their effects on the osteogenic differentiation of PDLSCs. Then, the function of these agonists in bone regeneration was analyzed in the periodontitis model. We found that hyperoside or 2-furoyl-LIGRLO-amide trifluoroacetate salt (2-Fly) promoted osteogenic differentiation under inflammation by simultaneously inhibiting nuclear factor κB (NF-κB) activation, β-catenin expression, and reactive oxygen species (ROS) production. Moreover, local injection of hyperoside or 2-Fly rescued the expression of FoxO1 and runt-related transcription factor 2 (Runx2) in vivo, alleviating alveolar bone loss and periodontal ligament damage. These findings suggested that FoxO1 agonists exerted a protective effect on osteogenesis in PDLSCs, as a result, facilitating bone formation under inflammatory conditions. Taken together, FoxO1 might serve as a therapeutic target for bone regeneration in periodontitis by mediating multiple signaling pathways.

Effect of lipoxin A4 on the osteogenic differentiation of periodontal ligament stem cells under lipopolysaccharide-induced inflammatory conditions.

Lipoxin A4 (LXA4) has been identified as the braking signal of inflammation, but the specific role of LXA4 in regulating the regenerative potential of periodontal ligament stem cells (PDLSCs) remains unclear. The aim of this study was to investigate whether and, if so, how LXA4 improves the osteogenic differentiation of PDLSCs in a lipopolysaccharide (LPS)-induced inflammatory environment. We detected the effects of LXA4 on the osteogenic differentiation of PDLSCs in vitro and explored the bone regenerative potential of LXA4-treated inflammatory PDLSCs in vivo using a calvarial critical sized defect model in male rats. RNA sequencing, real-time PCR and western blot were performed to elucidate the relevant potential mechanisms. Results showed that LXA4 promoted the proliferation, migration and osteogenic differentiation of PDLSCs in vitro, and effectively improved the impaired osteogenic capacity of PDLSCs induced by LPS both in vitro and in vivo. Mechanistically, LXA4 significantly promoted the PI3K/AKT phosphorylation under inflammatory conditions. Additionally, LY294002 (a PI3K inhibitor) blocked the effect of LXA4, suggesting that the PI3K/AKT pathway is a key signaling pathway that mediates the effect of LXA4 on the osteogenesis of inflammatory PDLSCs. These findings indicate LXA4 may be a promising strategy for periodontal regeneration using inflammatory PDLSCs.

Gold nanoparticles enhance proliferation and osteogenic differentiation of periodontal ligament stem cells by PINK1-mediated mitophagy

ObjectiveEvidence suggests that gold nanoparticles (AuNPs) improve osteogenic differentiation of periodontal ligament stem cells (PDLSCs), PTEN-induced putative kinase 1 (PINK1) dependent mitophagy modulates inter-clonal communication among PDLSCs with osteogenic heterogeneity, but the mechanism remains vague. Therefore, the current research assessed the influence of AuNPs on proliferation, osteogenic differentiation and mitophagy of PDLSCs and the potential mechanism was analyzed. MethodsGold nanospheres with a diameter of 5, 10, 20, 40, and 80 nm were synthesized and characterized through transmission electron microscopy, and rat PDLSCs were isolated using flow sorting. Next, PDLSCs were treated with AuNPs or PINK1 lentivirus to obtain its overexpression or suppression. Proliferation and osteogenic differentiation were evaluated by CCK-8, ALP staining, ARS staining, and immunoblotting of OCN, OPN, RUNX2, ALP, BMP2, and COL1. Mitochondrial quality, homeostasis and quantity were assessed though JC-1 staining, immunoblotting of Tom20, Tim23 and HSP60 and mitochondrial ROS detection. PINK1, Parkin, Beclin1 and LC3 expression was quantified to investigate mitophagy, using RT-qPCR and immunoblotting and the formation of RFP-GFP-LC3-labeled autophagosomes were also measured. ResultsThe proliferation ability of PDLSCs almost reached the maximum under 20 nm AuNPs for 24 h. AuNPs enhanced the proliferation and osteogenic differentiation of PDLSCs, improved mitochondrial quality and homeostasis as well as attenuated mitochondrial quantity. Additionally, mitophagy was enhanced by PDLSCs. Activation of PINK1 synergistically enhanced AuNPs-mediated mitophagy, mitochondrial quality, homeostasis and osteogenic differentiation in PDLSCs, obtaining opposite effects when PINK1 was suppressed. ConclusionAuNPs enhance proliferation and osteogenic differentiation of PDLSCs through PINK1-mediated mitophagy.

Recombinant Irisin Protects Against Alveolar Bone Destruction During Orthodontic Tooth Movement

Patients with periodontitis have higher risk of alveolar bone loss when seek for orthodontic therapy. Inflammation and osteogenesis are key factors in alveolar bone destruction in periodontitis and orthodontic tooth movement (OTM). Here we evaluated the effects of irisin on alveolar bone destruction in rats with periodontitis and OTM. We isolated and cultured human periodontal ligament stem cells (PDLSCs). Irisin was administrated to PDLSCs. Cell proliferations, osteogenic differentiation, expression of RUNX2 and ALP, and the expression of OPG and RANKL were measured. We induced periodontitis and OTM in rats and treated rats with irisin. The alveolar bone loss, inflammatory cytokine levels, and expression of OPG and RANKL in gingival tissues were measured. Irisin promoted the cell proliferation and osteogenic differentiation of PDLSCs. Irisin elevated the expression of RUNX2, ALP, and OPG while decreased the expression of RANKL in PDLSCs. Irisin ameliorated the alveolar bone loss, suppressed cytokine levels, and increased OPG/RANKL expression ratio in rat with periodontitis and orthodontic tooth movement. Irisin prevented alveolar bone destruction during OTM in rats with periodontitis.

LncRNA CYTOR Facilitates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating SOX11 via Sponging miR-6512-3p.

Periodontal ligament stem cells (PDLSCs) are considered ideal cell sources for the regeneration of periodontal and alveolar bone tissue. Cytoskeleton Regulator RNA (CYTOR), a newly discovered long noncoding RNA, has been reported to function as competing endogenous RNA (ceRNA) and to be involved in many biological processes. However, its roles in PDLSC osteogenic differentiation remain unclear. Here, we firstly found CYTOR was mainly sublocalized in the cytoplasm of PDLSCs and CYTOR expression was increased during osteogenic differentiation of PDLSCs. By employing gain- and loss-of-function approaches, we then identified CYTOR overexpression promoted osteogenic differentiation of PDLSCs while CYTOR knockdown inhibited this process. Furthermore, bioinformatics analysis was utilized to show that both CYTOR and SOX11 mRNA contained the same seed sites for miR-6512-3p, which was further confirmed by dual luciferase reporter assay and RNA-binding protein immunoprecipitation. Notably, CYTOR conferred its functions by directly binding to miR-6512-3p and an inverse correlation between CYTOR and miR-6512-3p on the level on SOX11 and osteogenic differentiation of PDLSCs was obtained. Additionally, miR-6512-3p could bind to SOX11 mRNA 3' UTR and repressed SOX11 expression. Moreover, level of SOX11 was significantly increased during osteogenic differentiation of PDLSCs. Knockdown of SOX11 attenuated the increasing effect of CYTOR overexpression on osteogenic differentiation of PDLSCs. Collectively, these data supported that CYTOR positively modulated the expression of SOX11 through competitively binding to miR-6512-3p, thus promoting osteogenic differentiation of PDLSCs. The CYTOR/miR-6512-3p/SOX11 axis could be a novel therapeutic target for periodontal regeneration medicine.

Osteogenic effect of crocin in human periodontal ligament stem cells via Wnt/β-catenin signaling.

Crocin is a major class of medicinal components in saffron. This study aimed to determine whether crocin directly promotes the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) in vitro and in vivo. CCK8 cell proliferation assay, reverse transcription quantitative polymerase chain reaction (RT-qPCR), Western blot analysis and Alizarin Red staining were performed in PDLSCs using crocin as a stimulant. DKK1 was used to selectively inhibit Wnt/β-catenin signaling, and Western blotting was performed to investigate the underlying mechanism. The PDLSCs were mixed with calcium phosphate cement and implanted into nude mice subcutaneously to study the effect of crocin on PDLSC osteogenic differentiation in vivo. The CCK-8 assay showed that crocin did not promote the proliferation of PDLSCs. Treatment with 400 μM crocin significantly promoted PDLSC mRNA levels of ALP, COL1 and OCN; RUNX2 and BMP2 protein expression; mineralized nodule formation in vitro and in vivo; and ALP activity in tissues in vivo. In addition, crocin significantly promoted the phosphorylation of β-catenin and cyclin D1. DKK1 inhibits Wnt/β-catenin activation and partially reverses crocin-mediated promotion of PDLSC osteogenic differentiation. Crocin may contribute to the regeneration of periodontal bone tissue.

Mitochondria transfer reverses the inhibitory effects of low stiffness on osteogenic differentiation of human mesenchymal stem cells

Microenvironment biophysical factors such as matrix stiffness can noticeably affect the differentiation of mesenchymal stem cells (MSCs). In this mechanobiology transduction process, mitochondria are shown to be an active participant. The present study aims to systematically elucidate the phenotypic and functional changes of mitochondria during the stiffness-mediated osteogenic differentiation. Additionally, the effect of mitochondria transfer on the osteogenesis of impaired MSCs caused by stiffness was investigated. Human periodontal ligament stem cells (PDLSCs) were used as model cells in the current study. Low stiffness restrained the cell spreading and significantly inhibited the proliferation and osteogenic differentiation of PDLSCs. Mitochondria of PDLSCs cultured on low stiffness exhibited shorter length, rounded shape, fusion/fission imbalance, ROS and mitophagy level increase, and ATP production reduction. The inhibited mitochondria function and osteogenic differentiation capacity were recovered to near-normal levels after transferring the mitochondria of PDLSCs cultured on the high stiffness. This study indicated that low matrix stiffness altered the mitochondrial morphology and induced systematical mitochondrial dysfunction during the osteogenic differentiation of MSCs. Mitochondria transfer was proved to be a feasible technique for maintaining MSCs function in vitro by reversing the osteogenesis ability.

Dasatinib regulates the proliferation and osteogenic differentiation of PDLSCs through Erk and EID3 signals.

Periodontal ligament stem cells (PDLSCs) are important candidate seed cells for alveolar bone tissue engineering. Dasatinib is a tyrosine kinase inhibitor, and its influence on the osteogenic differentiation of mesenchymal stem cells is a controversial topic. The present study explored the effects of different concentrations of dasatinib on the proliferation and osteogenic differentiation of PDLSCs and tentatively revealed the related mechanism. The results of CCK8 showed that low concentrations of dasatinib (1 nM) did not affect proliferation, while high concentrations of dasatinib significantly inhibited the proliferative activity of PDLSCs. This could be related to the inhibiting effects of dasatinib on Erk signals. ALP staining, alizarin red staining, and western blot proved that low concentrations of dasatinib (1 nM) promoted the osteogenic differentiation of PDLSCs, while high concentrations of dasatinib inhibited it. The negative effects of dasatinib on osteogenic differentiation were reversed when EID3 was knocked down, suggesting that EID3 mediates the regulation of dasatinib on the osteo-differentiation of PDLSCs. Taken together, high concentrations of dasatinib inhibited the proliferation and osteogenic differentiation of PDLSCs through Erk and EID3 signals, while low concentrations of dasatinib could be a potential method to enhance the bone regeneration ability of PDLSCs.

CTGF Promotes the Osteoblast Differentiation of Human Periodontal Ligament Stem Cells by Positively Regulating BMP2/Smad Signal Transduction.

Objective This work is aimed at revealing the role and the molecular mechanism of connective tissue growth factor 2 (CTGF) in the osteoblast differentiation of periodontal ligament stem cells (PDLSCs). Methods The osteogenic differentiation of PDLSCs was induced by osteogenic induction medium (OM), and the expression level of osteogenic related proteins ALP, RUNX2, OCN, and CTGF was estimated using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting analysis. We constructed cell lines with CTGF overexpression or knockdown to verify the role of CTGF in the osteoblast differentiation of PDLSCs. Alkaline phosphatase (ALP) staining was introduced to measure the osteoblasts activity, and alizarin red S (ARS) staining was employed to test matrix mineralization. The interaction between CTGF and bone morphogenetic protein-2 (BMP-2) was determined by endogenous coimmunoprecipitation (Co-IP). Results The expression level of CTGF was increased during the osteogenic induction of PDLSCs. Additionally, CTGF overexpression effectively maintained the stemness and facilitated the osteoblast differentiation in PDLSCs, and CTGF knockdown exerted opposite effects. Moreover, at molecular mechanism, CTGF increased the activity of BMP-2/Smad signaling pathway. Conclusion This investigation verified that CTGF promotes the osteoblast differentiation in PDLSCs at least partly by activating BMP-2/Smad cascade signal.

Integrative proteomics and phosphoproteomics profiling on osteogenic differentiation of periodontal ligament stem cell.

This study aims to elucidate the phosphorylated profile of periodontal ligament stem cells (PDLSCs) osteogenic differentiation, which contributes to the promotion of periodontium regeneration. PDLSCs cultured in the osteogenic induction medium for 14days were analyzed by proteomics and phosphoproteomics. Potential functions of phosphorylated differentially expressed proteins (DEPs) were annotated and enriched based on Gene Ontology (GO). Furtherly, overlapped DEPs were identified and conducted protein-protein interaction (PPI) network united with the top 20 up/downregulated phosphorylated DEPs. Hub phosphorylated DEPs were analyzed by Cytoscape, and the protein kinase phosphorylation network was predicted by iGPS. Proteomics identified 87 upregulated and 227 downregulated DEPs. Phosphoproteomics identified 460 upregulated and 393 downregulated phosphorylated DEPs, and they were primarily enriched in mitochondrial function and ion-channel related terms. Furthermore, 63 overlapped DEPs were recognized for more accurate predictions. Among the top 10 hub phosphorylated DEPs, only Integrin alpha-5 (ITGA5) expressed upregulated phosphorylation, and half of them belonged to extracellular matrix (ECM) proteins. In addition, numerous kinases corresponding to four interactive hub phosphorylated DEPs were predicted, including Collagen alpha-2(I) (COL1A2), Syndecan-1 (SDC1), Fibrillin-1 (FBN1), and ITGA5. Our findings established a basis for further elucidation of the phosphorylation of PDLSCs osteogenic differentiation, and COL1A2/SDC1/ITGA5/FBN1 phosphorylated network may dominate this process.

Metformin combats high glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells via inhibition of the NPR3-mediated MAPK pathway

BackgroundHigh glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) has long been a challenge to periodontal regeneration for diabetic individuals. Metformin is an anti-hyperglycemic drug that exhibits abundant biological activities associated with cell metabolism and downstream tissue regeneration. However, how metformin combats damage to PDLSC osteogenic differentiation under high glucose and the underlying mechanisms remain unknown.MethodsOsteogenic differentiation of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red staining and quantitative assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. RNA-seq analysis was performed to screen target genes of metformin, and the effects of target genes were confirmed using lentivirus transfection. Western blot analysis was also used to detect the protein level of underlying signaling pathways.ResultsWe found that osteogenic differentiation of PDLSCs under high glucose was decreased, and metformin addition enhanced this capacity of differentiation. Furthermore, the results of RNA-seq analysis showed that natriuretic peptide receptor 3 (NPR3) was upregulated in PDLSCs under high glucose and downregulated after metformin addition. When the underlying pathways involved were investigated, we found that upregulation of NPR3 can compromise the metformin-enhanced PDLSC osteogenic differentiation and activate the MAPK pathway (especially the p38 MAPK and Erk1/2 pathway), and that inhibition of the NPR3-mediated p38 MAPK or Erk1/2 pathway enhanced the osteogenic differentiation of PDLSCs under high glucose.ConclusionsThe present study suggests that metformin may enhance the osteogenic differentiation of PDLSCs under high glucose via downregulation of NPR3 and inhibition of its downstream MAPK pathway. This is the first report identifying the involvement of NPR3-mediated MAPK pathway in the metformin-enhanced osteogenic differentiation, indicating that NPR3 antagonists, such as metformin, may be feasible therapeutics for periodontal tissue regeneration in diabetic individuals.

ANGPTL4 regulates the osteogenic differentiation of periodontal ligament stem cells

The molecular mechanism of mechanical force regulating the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) has not been clearly elucidated. In this study, two mRNA-seqs, GSE106887 and GSE109167, which contained several samples of PDLSCs under mechanical force, were downloaded from Gene Expression Omnibus. Differential expression analysis was firstly taken between GSE106887 and GSE109167, then the common 84 up-regulated genes and 26 down-regulated genes were selected. Function enrichment analysis was used to identify the key genes and pathways in PDLSCs subjected to the tension and compression force. PDLSCs were isolated from human periodontal ligament tissues. The effects of ANGPTL4 knockdown with shRNA on the osteogenic differentiation of PDLSCs were studied in vitro. Then, the orthodontic tooth movement (OTM) rat model was used to study the expression of HIF-1α and ANGPTL4 in alveolar bone remodeling in vivo. ANGPTL4 and the HIF-1 pathway were identified in PDLSCs subjected to the tension and compression force. alizarin red staining, alcian blue staining, and oil red O staining verified that PDLSCs had the ability of osteogenic, chondrogenic, and adipogenic differentiation, respectively. Verification experiment revealed that the expression of ANGPTL4 in PDLSCs significantly increased when cultured under osteogenic medium in vitro. While ANGPTL4 was knocked down by shRNA, the levels of ALPL, RUNX2, and OCN decreased significantly, as well as the protein levels of COL1A1, ALPL, RUNX2, and OCN. During the OTM, the expression of HIF-1α and ANGPTL4 in periodontal ligament cells increased on the tension and compression sides. We concluded the positive relationship between ANGPTL4 and osteogenic differentiation of PDLSCs.