Proliferation Of Periodontal Ligament Stem Cells Research Articles

Vaspin facilitates the proliferation and osteogenic differentiation of periodontal ligament stem cells.

To investigate whether visceral adipose tissue-derived serine protease inhibitor (vaspin) can alleviate the inhibitory effect of high-glucose (HG) culture on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) and to preliminarily explore the underlying mechanisms. High glucose produces damage to the regeneration of periodontal tissue of PDLSCs. The expression level of vaspin in periodontal tissue is high in periodontitis patients and effectively reduced after initial therapy of periodontal diseases. However, the effect of vaspin on PDLSCs remains unknown. PDLSCs were cultured in media augmented with 5.5 or 25.0 mM concentrations of glucose to elucidate the impact and mechanism of vaspin on PDLSCs under high glucose invitro. Proliferation was measured by Cell Counting Kit-8 (CCK8) assay. Osteogenesis of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, and Alizarin Red staining. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) were used to investigate the osteo-specific markers. Then, the molecular impact of vaspin in the presence/absence of HG on PDLSCs physiology was determined with TGF-β1/Smad signaling pathway as the main focus. It was revealed that the proliferation and osteogenic differentiation (OD) of PDLSCs under HG was reduced, and by adding vaspin the anti-osteogenic impact of HG was relieved. Moreover, vaspin enhanced TGF-β1/Smad signaling pathway activity. Pretreatment with TGF-β1 inhibitor blocked vaspin-triggered TGF-β1/Smad signal activation and minimized the vaspin-induced protective effect against HG-inhibited growth and OD. In summary, vaspin observably reduces HG-mediated inhibition of PDLSCs OD by modulating the TGF-β1/Smad signaling pathway. Vaspin may be a potential therapeutic for periodontal tissue regeneration in diabetic patients.

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.

Bacterial growth stage determines the yields, protein composition, and periodontal pathogenicity of Porphyromonas gingivalis outer membrane vesicles.

P. gingivalis (W83), as the keystone pathogen in chronic periodontitis, has been found to be tightly bound to systemic diseases. Outer membrane vesicles (OMVs) produced by P. gingivalis (W83) are thought to serve key functions in bacterial virulence and pathogenicity. This study aims to comprehend the biological functions of P. gingivalis OMVs isolated from different growth stages by comparing their physicochemical properties and pathogenicity. Protein composition was analyzed via isotope-labeled relative and absolute quantification (iTRAQ). Macrophage polarization and the expression of IL-6 and IL-1β were detected. The proliferation, migration, osteogenic differentiation, and IL-1b/NLRP3 expression of periodontal ligament stem cells (PDLSCs) were evaluated. P. gingivalis/P. gingivalis OMVs-induced periodontal models were also constructed in Sprague Dawley rats. The protein composition of P. gingivalis OMVs isolated from different growth stages demonstrated obvious differences ranging from 25 KDa to 75 KDa. In the results of flow cytometry, we found that in vitro experiments the M1 subtype of macrophages was more abundant in the late-log OMVs and stationary OMVs groups which boosted the production of inflammatory cytokines more than pre-log OMVs. Compared to pre-log OMVs, late-log OMVs and stationary OMVs had more pronounced inhibitory effects on proliferation, migration, and early osteogenesis of PDLSCs. The NLRP3 inflammasome was activated to a larger extent in the stationary OMVs group. Micro-computed tomography (Micro CT), hematoxylin-eosin staining (HE), and tartrate acid phosphatase (TRAP) results showed that the periodontal damage in the stationary OMVs group was worse than that in the pre-log OMVs and late-log OMVs group, but almost equal to that in the positive control group (P. gingivalis). In general, both in vivo and in vitro experiments showed that late-log OMVs and stationary OMVs have more significant pathogenicity in periodontal disease.

A composite membrane with microtopographical morphology to regulate cellular behavior for improved tissue regeneration.

Tissue engineering scaffolds with specific surface topographical morphologies can regulate cellular behaviors and promote tissue repair. In this study, poly lactic(co-glycolic acid) (PLGA)/wool keratin composite guided tissue regeneration (GTR) membranes with three types of microtopographies (three groups each of pits, grooves and columns, thus nine groups in total) were prepared. Then, the effects of the nine groups of membranes on cell adhesion, proliferation and osteogenic differentiation were examined. The nine different membranes had clear, regular and uniform surface topographical morphologies. The 2 µm pit-structured membrane had the best effect on promoting the proliferation of bone marrow mesenchymal stem cells(BMSCs) and periodontal ligament stem cells (PDLSCs), while the 10 µm groove-structured membrane was the best for inducing osteogenic differentiation of BMSCs and PDLSCs. Then, we investigated the ectopic osteogenic, guided bone tissue regeneration and guided periodontal tissue regeneration effects of the 10 µm groove-structured membrane combined with cells or cell sheets. The 10 µm groove-structured membrane/cell complex had good compatibility and certain ectopic osteogenic effects, and the 10 µm groove-structured membrane/cell sheet complex promoted better bone repair and regeneration and periodontal tissue regeneration. Thus, the 10 µm groove-structured membrane shows potential to treat bone defects and periodontal disease. STATEMENT OF SIGNIFICANCE: PLGA/wool keratin composite GTR membranes with microcolumn, micropit and microgroove topographical morphologies were prepared by dry etching technology and the solvent casting method. The composite GTR membranes had different effects on cell behavior. The 2 µm pit-structured membrane had the best effect on promoting the proliferation of rabbit BMSCs and PDLSCs and the 10 µm groove-structured membrane was the best for inducing the osteogenic differentiation of BMSCs and PDLSCs. The combined application ofa 10 µm groove-structured membrane and PDLSC sheet can promote better bone repair and regeneration as well as periodontal tissue regeneration. Our findings may have significant potential for guiding the design of future GTR membranes with topographical morphologies and clinical applications of the groove-structured membrane/cell sheet complex.

Dental Pulp Stem Cell-Derived Exosomes Regulate Anti-Inflammatory and Osteogenesis in Periodontal Ligament Stem Cells and Promote the Repair of Experimental Periodontitis in Rats.

Dental pulp stem cell-derived exosomes (DPSC-EXO), which have biological characteristics similar to those of metrocytes, have been found to be closely associated with tissue regeneration. Periodontitis is an immune inflammation and tissue destructive disease caused by plaque, resulting in alveolar bone loss and periodontal epithelial destruction. It is not clear whether DPSC-EXO can be used as an effective therapy for periodontal regeneration. The purpose of this study was not only to verify the effect of DPSC-EXO on reducing periodontitis and promoting periodontal tissue regeneration, but also to reveal the possible mechanism. DPSC-EXO was isolated by ultracentrifugation. Then it characterized by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA) and Western Blot. In vitro, periodontal ligament stem cells (PDLSCs) were treated with DPSC-EXO, the abilities of cell proliferation, migration and osteogenic potential were evaluated. Furthermore, we detected the expression of IL-1β, TNF-αand key proteins in the IL-6/JAK2/STAT3 signaling pathway after simulating the inflammatory environment by LPS. In addition, the effect of DPSC-EXO on the polarization phenotype of macrophages was detected. In vivo, the experimental periodontitis in rats was established and treated with DPSC-EXO or PBS. After 4 weeks, the maxillae were collected and detected by micro-CT and histological staining. DPSC-EXO promoted the proliferation, migration and osteogenesis of PDLSCs in vitro. DPSC-EXO also regulated inflammation by inhibiting the IL-6/JAK2/STAT3 signaling pathway during acute inflammatory stress. In addition, the results showed that DPSC-EXO could polarize macrophages from the M1 phenotype to the M2 phenotype. In vivo, we found that DPSC-EXO could effectively reduce alveolar bone loss and promote the healing of the periodontal epithelium in rats with experimental periodontitis. DPSC-EXO plays an important role in inhibiting periodontitis and promoting tissue regeneration. This study provides a promising acellular therapy for periodontitis.

ZEB1 interferes with human periodontal ligament stem cell proliferation and differentiation.

Periodontitis can eventually contribute to tooth loss. Zinc finger E-box binding homeobox 1 (ZEB1) is identified as overexpressed in the gingival tissue of mice with periodontitis. This study is designed to decipher the mechanism of ZEB1's involvement in periodontitis. Human periodontal mesenchymal stem cells (hPDLSCs) were exposed to LPS to mimic the inflammation in periodontitis. Following ZEB1 silencing, FX1 (an inhibitor of Bcl-6) treatment or ROCK1 overexpression, cell viability, and apoptosis were analyzed. Alkaline phosphatase (ALP) staining, Alizarin red staining, RT-qPCR, and western blot were performed to evaluate osteogenic differentiation and mineralization. hPDLSCs were processed for luciferase reporter assay and ChIP-PCR to confirm the association between ZEB1 and ROCK1. The induction of ZEB1 silencing resulted in reduced cell apoptosis, enhanced osteogenic differentiation, and mineralization. Nevertheless, these effects were significantly blunted by FX1. ZEB1 was confirmed to bind to the promoter sites of ROCK1 and regulate the ROCK1/AMPK. Whereas ROCK1 overexpression reversed the effects of ZEB1 silencing on Bcl-6/STAT1, as well as cell proliferation and osteogenesis differentiation. hPDLSCs displayed decreased proliferation and weakened osteogenesis differentiation in response to LPS. These impacts were mediated by ZEB1 regulating Bcl-6/STAT1 via AMPK/ROCK1.

Graphene quantum dots enhance the osteogenic differentiation of PDLSCs in the inflammatory microenvironment

Background and objectiveGraphene quantum dots (GQDs), a type of carbon-based nanomaterial, have remarkable biological, physical, and chemical properties. This study investigated the biological mechanisms of the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) induced by GQDs in an inflammatory microenvironment.Materials and methodsPDLSCs were cultured in osteogenic-induced medium with various concentrations of GQDs in standard medium or medium mimicking a proinflammatory environment. The effects of GQDs on the proliferation and osteogenic differentiation activity of PDLSCs were tested by CCK-8 assay, Alizarin Red S staining, and qRT‒PCR. In addition, Wnt/β-catenin signalling pathway-related gene expression was measured by qRT‒PCR.ResultsCompared with the control group, the mRNA expression levels of ALP, RUNX2, and OCN and the number of mineralized nodules were all increased in PDLSCs after treatment with GQDs. Moreover, during the osteogenic differentiation of PDLSCs, the expression levels of LRP6 and β-catenin, which are Wnt/β-catenin signalling pathway-related genes, were upregulated.ConclusionIn the inflammatory microenvironment, GQDs might promote the osteogenic differentiation ability of PDLSCs by activating the Wnt/β-catenin signalling pathway.

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.

Hydrogen Sulphide Alleviates Senescence of Human Periodontal Ligament Stem Cells by TRPV4 Channel Mediated Calcium Flux.

To explore whether hydrogen sulphide (H2S) could protect human periodontal ligament stem cells (PDLSCs) from senescence and the possible underlying mechanisms. Cell cycle assay and Ki-67 assay were used to measure proliferation of PDLSCs. Real-time polymerase chain reaction (PCR) was used to measure cellular senescence-related p16 and p21. Calcium influx was detected by measurement of Ca2+ imaging. In addition, we analysed the possible mechanisms underlying H2S acting on PDLSCs by microarray. The cell proliferation rate of aging PDLSCs decreased significantly. The expression of cellular senescence-related p16 and p21 significantly increased in aging PDLSCs. H2S donor (GYY4137) treatment increased the proliferation rate of senescence PDLSCs. Furthermore, the donor of H2S treatment effectively prevented cell cycle arrest of PDLSCs during the aging process and inhibited the expression of cellular senescence-related markers. Mechanically, H2S donor treatment could activate the calcium influx in PDLSCs. Moreover, pretreatment with TRPV4 inhibitors significantly attenuated the calcium influx induced by H2S donor treatment in PDLSCs. It also alleviated the protective effect of H2S on the senescence of PDLSCs. H2S alleviated the senescence of human PDLSCs by TRPV4 channel mediated calcium flux. These results provide a potential strategy to deal with cell aging and may facilitate cell therapy for oral diseases.

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.

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.

Influence of Low-Level Laser Irradiation on Periodontal Ligament Stem Cell Proliferation and Migration in Diabetic Models (Invitro Study)

Influence of Low-Level Laser Irradiation on Periodontal Ligament Stem Cell Proliferation and Migration in Diabetic Models (Invitro Study)

Sodium butyrate inhibits osteogenesis in human periodontal ligament stem cells by suppressing smad1 expression

BackgroundButyrate is a major subgingival microbial metabolite that is closely related to periodontal disease. It affects the proliferation and differentiation of mesenchymal stem cells. However, the mechanisms by which butyrate affects the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) remain unclear. Here, we investigated the effect of sodium butyrate (NaB) on the osteogenic differentiation of human PDLSCs.MethodsPDLSCs were isolated from human periodontal ligaments and treated with various concentrations of NaB in vitro. The cell counting kit-8 assay and flow cytometric analysis were used to assess cell viability. The osteogenic differentiation capabilities of PDLSCs were evaluated using the alkaline phosphatase activity assay, alizarin red staining, RT-PCR, western blotting and in vivo transplantation.ResultsNaB decreased PDLSC proliferation and induced apoptosis in a dose- and time-depend manner. Additionally, 1 mM NaB reduced alkaline phosphatase activity, mineralization ability, and the expression of osteogenic differentiation-related genes and proteins. Treatment with a free fatty acids receptor 2 (FFAR2) antagonist and agonist indicated that NaB inhibited the osteogenic differentiation capacity of PDLSCs by affecting the expression of Smad1.ConclusionOur findings suggest that NaB inhibits the osteogenic differentiation of PDLSCs by activating FFAR2 and decreasing the expression of Smad1.

Effect of Bone Morphogenetic Protein (BMP)-2, -4, and -7 on Proliferation and Osteogenic Differentiation in Human Periodontal Ligament Stem Cells (PDLSCs)

Aim: To assess the effects of bone morphogenetic protein (BMP)-2, BMP-4, and BMP-7 on human periodontal ligament stem cells (PDLSC) proliferation and osteogenic differentiation. Methods: PDLSCs were isolated by an immunomagnetic method. Cell surface antigen (CD146, CD44, and CD34) expression and pluripotency (osteogenic and adipogenic) were determined. In doseresponse and time-course experiments, cultured PDLSCs were treated with individual BMPs, a combination of BMPs in 1:1 ratios, and a mixture of all three BMPs (1:1:1 ratio). In the dose-response experiments, PDLSCs were incubated for 12 d with medium containing BMPs at a dose of 0, 10, 25, 50, or 100 ng/ml. In the time-course experiments, PDLSCs were administered 50 ng/ml BMPs for different times (0, 3, 6, 12, or 24 d). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and an enzyme kit were used to quantify cell growth and alkaline phosphatase (ALP) activity. Immunohistochemistry and western blotting were used to assess the expression of osteogenic differentiation-related proteins, i.e., osteocalcin, bone sialoprotein, collagen type I, and collagen type III. Results: PDLSCs expressed CD146 (93%) and CD44 (91.2%) but not CD34 (1.8%). All cells exhibited osteogenic and adipogenic potential. PDLSC proliferation and ALP activity in PDLSCs treated with individual BMPs and BMPs combinations increased in a dose- and time-dependent manner; proliferation and ALP activity were greater upon treatment with BMP combinations than upon treatment with individual BMPs. Compared with those in the control group, osteogenic differentiation-related proteins levels in PDLSCs treated with 50 ng/ml BMPs for 12 d were markedly increased, whereas no obvious differences were detected between the groups subjected to different BMP treatments. Conclusion: BMP-2, BMP-4, and BMP-7 alone and in combination promoted the development and osteogenic differentiation of PDLSCs, and the promotion of these cellular processes was more pronounced upon treatment with BMP combinations.

CUL4B Upregulates RUNX2 to Promote the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Epigenetically Repressing the Expression of miR-320c and miR-372/373-3p.

Mesenchymal stem cells (MSCs) within the periodontal ligament (PDL), termed periodontal ligament stem cells (PDLSCs), have a self-renewing capability and a multidirectional differentiation potential. The molecular mechanisms that regulate multidirectional differentiation, such as the osteogenic differentiation of PDLSCs, remain to be elucidated. Cullin 4B (CUL4B), which assembles the CUL4B-RING ubiquitin ligase (CRL4B) complex, is involved in regulating a variety of developmental and physiological processes including the skeletal development and stemness of cancer stem cells. However, nothing is known about the possible role of CUL4B in the osteogenic differentiation of PDLSCs. Here, we found that knockdown of CUL4B decreased the proliferation, migration, stemness and osteogenic differentiation ability of PDLSCs. Mechanistically, we demonstrate that CUL4B cooperates with the PRC2 complex to repress the expression of miR-320c and miR-372/373-3p, which results in the upregulation of RUNX2, a master transcription factor (TF) that regulates osteogenic differentiation. In brief, the present study reveals the role of CUL4B as a new regulator of osteogenic differentiation in PDLSCs.

All-trans retinoic acid inhibits the osteogenesis of periodontal ligament stem cells by promoting IL-1β production via NF-κB signaling

All-trans retinoic acid (ATRA), a main derivative of vitamin A, has been shown to affect the osteogenic differentiation of mesenchymal stem cells (MSCs). Periodontal ligament stem cells (PDLSCs) possess characteristics of MSC and show strong potential for use in periodontal tissue restoration. However, the effect of ATRA on the osteogenic differentiation of PDLSCs remains unclear. In this study, we explored the effect of ATRA on the PDLSCs osteogenic differentiation. PDLSCs were harvested from the periodontalmembrane and treated with or without ATRA. CCK-8 and cell cycle analysis were used to evaluate PDLSC proliferation. PDLSC migration was assessed by scratch tests. qRT-PCR, western blotting, alkaline phosphatase staining, alizarin red staining and calcium quantification were performed to estimate the PDLSCs osteogenic differentiation capability and RNA sequencing to select differentially expressed genes (DEGs). Expression and activation of signaling elements were assessed by qRT-PCR, western blotting and immunofluorescence. Finally, we discovered that ATRA repressed the migration, proliferation, and osteogenesis ability of PDLSCs. RNA sequencing revealed 493 DEGs. Levels of interleukin-1β (IL-1β) were increased at varied time points after ATRA treatment. The inhibitive influence of ATRA on the osteogenesis of PDLSCs was partially reversed after neutralizing IL-1β. In addition, IL-1β levels were significantly attenuated by nuclear factor-κB (NF-κB) inhibitor BAY11-7082 and NLRP3 inhibitor MCC950. Taken together, our results demonstrate that ATRA disrupts the osteogenesis and mineralizationof PDLSCs by promoting IL-1β expression via activating NF-κB signaling and NLRP3 inflammasome, which may offer a new method for improving the ATRA-induced disruption of osteoblast differentiation.

MiR-589-3p promoted osteogenic differentiation of periodontal ligament stem cells through targeting ATF1

BackgroundAn increasing number of studies have shown that dysregulated miR-589-3p is associated with multiple diseases. However, the role of miR-589-3p in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) remains unknown. This study aimed to explore the biological function and potential molecular mechanism of miR-589-3p in osteogenic differentiation of PDLSCs.MethodsGSE159508 was downloaded from Gene Expression Omibus (GEO, http://www.ncbi.nlm.nih.gov/geo/). Differentially expressed miRNAs between osteogenic induction PDLSCs versus non-induction PDLSCs were obtained by R software. miR-589-3p mimic and miR-589-3p inhibitor and corresponding negative control were obtained and to identify the role of miR-589-3p in osteogenic differentiation of PDLSCs. ALP staining and ARS were used to evaluate ALP activity and mineralization, respectively. The targeted binding relationship between miR-589-3p and ATF1 was predicted and verified by target prediction analysis and dual-luciferase assay. Furthermore, the functional mechanism based on miR-589-3p and ATF1 in osteogenic differentiation of PDLSCs was further investigated through rescue experiments.ResultsAccording to the cut-off criteria with log 2 FC > 1.0 and P < 0.05, 514 differentially expressed miRNAs were identified between osteogenic induction and non-induction PDLSCs, including 309 upregulated miRNAs and 205 downregulated miRNAs. Compared with control PDLSCs, miR-589-3p expression level was notably increased in PDLSCs that underwent osteogenic induction. The overexpression of miR-589-3p promoted the cell viability of PDLSCs, while the low expression of miR-589-3p had the opposite effect. The dual luciferase reporter assay verified that ATF1 was proved to be a direct target of miR-589-3p in PDLSCs. And overexpressed miR-589-3p reduced the expression of ATF1. Overexpression of miR-589-3p enhanced the osteogenic capacity of PDLSCs, as demonstrated by increases in ALP activity, matrix mineralization, and RUNX2, OCN and OSX expression. In addition, the rescue experiments confirmed that overexpressed ATF1 restored the effects of overexpressed miR-589-3p on cell proliferation and osteogenic differentiation of PDLSCs.ConclusionmiR-589-3p could down-regulate the expression of ATF1, thereby promote the proliferation and osteogenic differentiation of PDLSCs. This finding may provide a new therapeutic target for molecular therapy of periodontitis.