Differentiation Of Periodontal Ligament Stem Cells Research Articles

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.

Regulatory mechanisms of GCN5 in osteogenic differentiation of MSCs in periodontitis.

The regulatory mechanisms of GCN5 (General control non-repressed protein5) in the osteogenic differentiation of mesenchymal stem cells (MSCs) in periodontitis are still unclear. The purpose of this review focuses on the regulating roles of GCN5 in bone metabolism and periodontitis, discusses the potential molecular mechanism and provides targets and new ideas for the treatment of periodontitis. The integrative review methodology was used. Data sources include PubMed, Cochrane Library, and additional sources. MSCs play an important role in the osteogenesis balance of periodontal tissue. Periodontal ligament stem cells (PDLSCs) from periodontitis patients exhibited defective osteogenic differentiation capacities. Histone acetylation is important in regulating the differentiation of different types of MSCs cells and is closely related to the reduced osteogenic differentiation of PDLSCs. GCN5, one of the first histone acetyltransferase linked to gene transcriptional activation, participates in many biological processes of mesenchymal stem cells. Downregulation of GCN5 expression and lack of GCN5 caused decreased osteogenic differentiation of PDLSCs. Intercellular information exchange may be an important way for MSCs to exert their regulatory and therapeutic functions. GCN5 affects the function of cell metabolism-related genes by regulating the acetylation status of histones or non-histones, thereby regulating some important progress of MSCs such as PDLSCs' osteogenic differentiation and BMCS osteogenic differentiation.

Exendin-4 regulates the MAPK and WNT signaling pathways to alleviate the osteogenic inhibition of periodontal ligament stem cells in a high glucose environment.

Diabetes mellitus (DM) increases the destruction of periodontal tissue and impairs osteogenesis differentiation. Exendin-4 (Ex-4), a glucagon-like peptide-1 (GLP-1) analogue, can be used for treating DM and promotes bone regeneration. The aim of this study was to explore the effect and mechanism of Ex-4 on improving the osteogenesis of periodontal ligament stem cells (PDLSCs) in a high glucose environment. Alkaline phosphatase staining and alizarin red staining were used to detect the osteogenic differentiation of PDLSCs. The results showed that 10 nM Ex-4 could reduce the osteogenesis inhibition of PDLSCs induced by high glucose. RT-PCR and western blot results showed that Ex-4 increased the osteogenesis-related gene expression of ALP, Runx2, and Osx, and upregulated the phosphorylation of P38, JNK, and ERK1/2; the peak effect was observed in the range 0.5-1.0 h. Mitogen-activated protein kinase (MAPK) inhibitors PD98059, SB203580, and SP600125 blocked the effects of Ex-4 on MAPK activation and decreased the expression of ALP, Runx2, and Osx in PDLSCs. Moreover, after Ex-4 treatment, the total β-catenin, p-GSK3β, LEF, and Runx2 protein levels increased under normal or high glucose environments. In conclusion, our results indicated that Ex-4 regulates the MAPK and WNT signaling pathways to alleviate the osteogenic inhibition of PDLSCs in a high glucose environment.

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.

Human gingival mesenchymal stem cell-derived exosomes cross-regulate the Wnt/β-catenin and NF-κB signalling pathways in the periodontal inflammation microenvironment.

To examine the immunomodulatory effect of exosomes originating from gingival mesenchymal stem cells (GMSC-Exo) on periodontal bone regeneration and its role in the regulation of the nuclear-factor kappaB (NF-κB) and Wnt/β-catenin pathways in the periodontal inflammatory microenvironment. First, periodontal ligament stem cells (PDLSCs) were treated with GMSC-Exo or Porphyromonas gingivalis-derived lipopolysaccharide (P.g-LPS) in vitro. Quantitative real-time PCR (qRT-PCR) and western blot were carried out to detect the expressions of osteogenic differentiation-related factors in cells. Further, PDLSCs were treated with P.g-LPS or inhibitors. The expression of NF-κB pathway-related factors as well as of Wnt/β-catenin pathway-related factors were detected by qRT-PCR and western blot. GMSC-Exo treatment promoted the expression of osteogenic differentiation-related factors within PDLSCs in both normal and inflammatory environments. Further investigations showed that GMSC-Exo could also inhibit the P.g-LPS-induced activation of the NF-κB pathway, leading to the up-regulation of the Wnt/β-catenin pathway. When the Wnt/β-catenin signalling was blocked, the inhibitory effect of GMSC-Exo on the NF-κB pathway was abolished. GMSC-Exo could promote the osteogenic differentiation of PDLSCs. There could be mutually exclusive regulatory roles between the NF-κB and Wnt/β-catenin signalling pathways in a periodontal inflammatory environment. GMSC-Exo exhibited an effective cross-regulation ability for both pathways.

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.

Thyrotropin inhibits osteogenic differentiation of human periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) are derived from the periodontal ligament and have the characteristics of pluripotent differentiation, including osteogenesis, and are one of the important seed cells in oral tissue engineering. Thyrotropin (TSH) has been shown to regulate bone metabolism independently of thyroid hormone, including the fate of osteoblasts and osteoclasts, but whether it affects osteogenic differentiation of PDLSCs is unknown. PDLSCs were isolated and cultured from human periodontal ligament and grown in osteogenic medium (containing sodium β-glycerophosphate, ascorbic acid, and dexamethasone). Recombinant human TSH was added to the culture medium. Osteogenic differentiation of PDLSCs was assessed after 14 days by staining with alkaline phosphatase and alizarin red and by detection of osteogenic differentiation genes. Differentially expressed genes (DEGs) in PDLSCs under TSH were detected by high-throughput sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyzed the biological functions and signaling pathways involved in DEGs. We found that osteogenic differentiation of PDLSCs was significantly inhibited in the presence of TSH: including decreased calcium nodule formation, decreased alkaline phosphatase levels, and decreased collagen synthesis. Using high-throughput sequencing, we found changes in the expression of some osteogenesis-related genes, which may be the reason that TSH inhibits osteogenic differentiation of PDLSCs. Unless TSH is ≥10mU/L, patients with subclinical hypothyroidism usually do not undergo thyroxine supplementation therapy. However, in this work, we found that elevated TSH inhibited the osteogenic differentiation of PDLSCs. Therefore, correction of TSH levels in patients with subclinical hypothyroidism may be beneficial to improve orthodontic, implant, and periodontitis outcomes in these patients.

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.

Long non-coding RNA AC018926.2 regulates palmitic acid exposure-compromised osteogenic potential of periodontal ligament stem cells via the ITGA2/FAK/AKT pathway.

Although obesity has been proposed as a risk factor for periodontitis, the influence of excessive fat accumulation on the development of periodontitis and periodontal recovery from disease remains largely unknown. This study investigated the cellular response of periodontal ligament stem cells (PDLSCs) to elevated levels of a specific fatty acid, namely, palmitic acid (PA). The mechanism by which PA exposure compromises the osteogenic potential of cells was also explored. It was found that exposure of PDLSCs to abundant PA led to decreased cell osteogenic differentiation. Given that long non-coding RNAs (lncRNAs) play a key role in the stem cell response to adverse environmental stimuli, we screened the lncRNAs that were differentially expressed in PDLSCs following PA exposure using lncRNA microarray analysis, and AC018926.2 was identified as the lncRNA that was most sensitive to PA. Next, gain/loss-of-function studies illustrated that AC018926.2 was an important regulator in PA-mediated osteogenic differentiation of PDLSCs. Mechanistically, AC018926.2 upregulated integrin α2 (ITGA2) expression and therefore activated ITGA2/FAK/AKT signalling. Further functional studies revealed that inactivation of ITGA2/FAK/AKT signalling by silencing ITGA2 counteracted the pro-osteogenic effect induced by AC018926.2 overexpression. Moreover, the results of bioinformatics analysis and RNA immunoprecipitation assay suggested that AC018926.2 might transcriptionally regulate ITGA2 expression by binding to PARP1 protein. Our data suggest that AC018926.2 may serve as a therapeutic target for the management of periodontitis in obese patients.

Epigenetic regulation of osteogenic differentiation of periodontal ligament stem cells in periodontitis.

Periodontitis is an inflammatory disease characterized by alveolar bone loss. Periodontal ligament stem cells (PDLSCs) have osteogenic differentiation potential, which can be influenced by epigenetics regulation in periodontitis. Therefore, this review aimed to shed light on the role of different epigenetic mechanisms in the osteogenic differentiation of PDLSCs and to consider the prospects of their possible therapeutic applications in periodontitis. Databases MEDLINE (through PubMed) and Web of Science were searched for the current knowledge of epigenetics in osteogenic differentiation of PDLSCs using the keywords "periodontal ligament stem cells", "epigenetic regulation", "epigenetics", "osteogenic differentiation", and "osteogenesis". All studies introducing epigenetic regulation and PDLSCs were retrieved. This review shows that epigenetic factors like DNMT, KDM6A, HDACi, some miRNAs, and lncRNAs can induce the osteogenic differentiation of PDLSCs in the noninflammatory microenvironment. However, the osteogenic differentiation of PDLSCs is inhibited in the inflammatory microenvironment through the upregulated DNA methylation of osteogenesis-related genes and specific changes in histone modification and noncoding RNA. Epigenetics of osteogenic differentiation of PDLSCs in inflammation exhibits the contrary effect compared with a noninflammatory environment. The application of epigenetic drugs to regulate the abnormal epigenetic status in periodontitis and focus on alveolar bone regeneration is promising.

N6-methyladenosine promotes osteogenic differentiation of PDLSCs from periodontitis patients.

This study aimed to investigate the mechanism of N6-methyladenosine (m6A) in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) from periodontitis patients. Differentially m6A-methylated lncRNA/mRNA profiles were detected by a m6A epitranscriptomic microarray. Bioinformatics analysis was performed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The transfection efficiency of the lentivirus was detected. The osteogenic activity of PDLSCs from periodontitis patients (PPDLSCs) was assessed. The microarray results showed that 275 lncRNAs and 1292 mRNAs were significantly differentially methylated between PPDLSCs and PDLSCs from healthy people. Among those lncRNAs, lncRNA4114 (transcript_ID: ENST00000444114) showed both reduced m6A methylation levels and expression levels in PPDLSCs. Further bioinformatics analysis predicted that the differentially methylated mRNAs were mainly involved in cell interaction, stem cell pluripotency, and osteogenic differentiation signals. Then, overexpression of methyltransferase like 3 (METTL3) promoted the osteogenic differentiation of PPDLSCs, while knocking down METTL3 showed an inhibitory effect. Furthermore, METTL3 overexpression promotes the stability of lncRNA4114 to upregulate the expression level. Moreover, lncRNA4114 overexpression promoted the osteogenic differentiation of PPDLSCs. METTL3 promotes the osteogenic differentiation of PPDLSCs by regulating the stability of lncRNA4114.

Comparative analysis of proliferative and multilineage differentiation potential of human periodontal ligament stem cells from maxillary and mandibular molars.

Clinical experience indicates that wounds in alveolar bone and periodontal tissue heal faster and more efficiently in the maxilla compared with the mandible. Since stem cells are known to have a decisive influence on wound healing and tissue regeneration, the aim of this study was to determine whether differences in proliferation and differentiation of periodontal ligament stem cells (PDLSC) from upper (u-PDLSC) and lower jaw (l-PDLSC) contribute to the enhanced wound healing in the maxilla. u-PDLSC and l-PDLSC from the same donor were harvested from the periodontal ligament of extracted human maxillary and mandibular third molars. Cell differentiation potential was assessed by analyzing stem cell markers, proliferation rate, and multilineage differentiation among each other and bone marrow-derived mesenchymal stem cells (MSC). Successful differentiation of PDLSC and MSC toward osteoblasts, adipocytes, and chondrocytes was analyzed via reverse transcriptase-quantitative polymerase chain reaction and histochemical staining (Alizarin Red, Oil Red O, Toluidine Blue). u-PDLSC and l-PDLSC expressed the MSC-markers CD73+ , CD90+ , and CD105+ and lacked expression of CD34- and CD45- . Proliferation was significantly higher in u-PDLSC than in l-PDLSC, regardless of the culture conditions. Osteogenic (ALP, RunX2, and osteocalcin) and chondrogenic (SOX9 and ACAN) related gene expression as well as staining intensities were significantly higher in u-PDLSC than in l-PDLSC. No difference in adipogenic differentiation was observed. u-PDLSC showed a significantly higher proliferative and differentiation potential than l-PDLSC, offering a possible cell-based explanation for the differences in periodontal wound healing efficacy between maxilla and mandible.

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.

Baricitinib alleviates lipopolysaccharide‑induced human periodontal ligament stem cell injury and promotes osteogenic differentiation by inhibiting JAK/STAT signaling.

Periodontitis is the chronic inflammation of the periodontal tissue. The present study aimed to investigate the role of baricitinib, a Janus kinase (JAK)1/2 inhibitor, in periodontitis by using a lipopolysaccharide (LPS)-induced human periodontal ligament stem cell (PDLSC) model. The viability of PDLSCs stimulated by LPS was assessed in the presence of baricitinib by Cell Counting Kit-8 assay. The induction of oxidative stress was evaluated by detecting the intracellular reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity and glutathione (GSH) content. ELISA and reverse transcription-quantitative PCR were used to determine the levels of inflammatory factors TNF-α, IL-1β and IL-6. Alkaline phosphatase (ALP) activity and alizarin red staining were used to assess the osteogenic differentiation of PDLSCs. The expression levels of osteogenic differentiation- and JAK/signal transducer and activator of transcription (STAT) signaling-associated proteins were estimated with western blotting. RO8191, an agonist of the JAK/STAT pathway, was used to treat PDLSCs to investigate the regulatory mechanism of baricitinib. The results indicated that baricitinib elevated the LPS-induced decrease in cell viability. LPS-triggered oxidative stress and inflammation were inhibited by baricitinib, as demonstrated by the decreased levels of ROS, TNF-α, IL-1β, IL-6 and increased levels of SOD and GSH. In addition, baricitinib caused a marked elevation in ALP activity and mineralization ability of PDLSCs, as determined by the upregulated osteocalcin and Runt-related transcription factor 2 expression. Moreover, the expression levels of phosphorylated (p)-JAK1, p-JAK2 and p-STAT3 were downregulated by baricitinib in a dose-dependent manner. Furthermore, addition of RO8191 restored the effect of baricitinib on the induction of oxidative stress, inflammation and osteogenic differentiation of PDLSCs exposed to LPS. Collectively, these findings suggested that baricitinib alleviated oxidative stress and inflammation and promoted osteogenic differentiation of LPS-induced PDLSCs by inhibiting JAK/STAT signaling.

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.

Sinomenine Inhibits Orthodontic Tooth Movement and Root Resorption in Rats and Enhances Osteogenic Differentiation of PDLSCs.

PurposeTo investigate the effects of sinomenine on orthodontic tooth movement and root resorption in rats, as well as the effect of sinomenine on the osteogenesis of periodontal ligament stem cells (PDLSCs).MethodsFifty-four male Wistar rats were randomly divided into 3 groups: control group, 20 mg/kg sinomenine group and 40 mg/kg sinomenine group. Fifty-gram orthodontic force was applied to all groups. Each group was injected intraperitoneally with corresponding concentration of sinomenine every day. After 14 days, all rats were sacrificed. Micro-computed tomography (micro-CT) scan was used to analyze tooth movement, root resorption and alveolar bone changes. The effect on periodontal tissue was analyzed by Masson, tartrate-resistant acid phosphatase (TRAP) and immunohistochemical staining. In vitro, PDLSCs were extracted and identified. The effect of sinomenine on proliferation was determined by cell-counting kit-8. The effect of sinomenine on osteogenesis was investigated by alkaline phosphatase (ALP) activity and alizarin red staining. qPCR and Western blotting were performed to explore the effects of sinomenine on the expression levels of ALP, runt-related transcription factor 2 (RUNX2), receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG).ResultsThe tooth movement and root resorption of sinomenine groups were reduced. Sinomenine decreased trabecular spacing on compression side and increased alveolar bone volume and trabecular thickness on tension side. TRAP-positive cells in sinomenine groups decreased significantly. The expressions of TNF-α and RANKL were decreased, while the expressions of OPG, RUNX2 and osteocalcin were up-regulated. In vitro, 0.1 M and 0.5 M sinomenine enhanced ALP activity, mineral deposition and the expression of ALP, RUNX2 and OPG, and reduced the expression of RANKL.ConclusionSinomenine could inhibit tooth movement, reduce root resorption, and exert a positive effect on bone formation in rats. Moreover, sinomenine promoted the osteogenesis of PDLSCs.

MTOR is involved in LRP5-induced osteogenic differentiation of normal and aged periodontal ligament stem cells in vitro.

Periodontal ligament stem cells (PDLSCs) plays an important role in tissue engineering. As the age increased, the cell viability and osteogenic differentiation of PDLSCs all decreased. Low density lipoprotein receptor related protein 5 (LRP5) was found to promote bone marrow mesenchymal stem cells osteogenic differentiation. Therefore, our study explored the effect of LRP5 on normal and aged PDLSCs and relative mechanism. Here, we found that the expression of LRP5 in PDLSCs of 24week-old mice was decreased compared with PDLSCs of 5week-old mice (n = 5). . LRP5 overexpression in PDLSCs increased the intensity of alkaline phosphatase and alizarin red staining, accompanied with upregulated the levels of RUNX family transcription factor 2, collagen type I, and β-Catenin. LRP5 knockdown displayed the opposite results in PDLSCs in vitro.LRP5 overexpression in aged PDLSCs restored part ability of osteogenic differentiation. Meantime, LRP5 increased the protein expression of phosphorylation of mammalian target of rapamycin (p-mTOR) in normal and aged PDLSCs. Immunofluorescence showed that LRP5 increased the accumulation of p-mTOR nucleus. The effect of LRP5 in promoting osteogenic differentiation of PDLSCs can be antagonized by mTOR inhibitor rapamycin. These findings suggest that LRP5 positively regulate osteogenic differentiation of normal and aged PDLSCs and may be a potential target for enlarging the application of PDLSCs in tissue regeneration.

HIF-1α drives the transcription of NOG to inhibit osteogenic differentiation of periodontal ligament stem cells in response to hypoxia

Osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is limited in hypoxia, and HIF-1α is key to the response to hypoxia. However, its mechanisms remain largely unknown. This study discovered an osteogenesis-related gene sensitive to hypoxia in PDLSCs, and investigated the molecular mechanisms between HIF-1α and the gene. NOG, a gene that negatively regulates osteogenesis, was discovered by RNA-seq. Under normoxic conditions, HIF-1α overexpression led to enhanced expression of NOG/Noggin and inhibited the expression of osteogenesis-related genes, while inhibition of HIF-1α reversed this effect. The expression of HIF-1α, NOG/Noggin and the osteogenesis-related genes were detected by qRT-PCR or Western blot. Mechanistically, we verified that HIF-1α binds to the hypoxia response element (−1505 to −1502) in the promotor of NOG to enhance secretion of Noggin by chromatin immunoprecipitation and a dual-luciferase reporter assay. IHC staining findings in an animal model verified that Noggin-associated osteogenic differentiation was inhibited in hypoxia. NOG displayed a concordant relationship with HIF-1α, and secreted more with increasing of HIF-1α. Hypoxia stabilized HIF-1α, which bound to the HRE (−1505 to −1502) of the NOG promotor to enhance NOG transcription resulted in inhibiting osteogenic differentiation of PDLSCs. This study offers a promising therapy for periodontitis.

Periodontal ligament fibroblasts-derived exosomes induced by PGE2 inhibit human periodontal ligament stem cells osteogenic differentiation via activating miR-34c-5p/SATB2/ERK

Several studies have confirmed that exosomes containing microRNAs (miRNAs) from the aseptic inflammatory microenvironment play an important role in bone remodeling. But the mechanism that induces changes in the osteogenic ability of periodontal ligament stem cells (PDLSCs) is still unclear. In the present study, the osteogenic function of periodontal ligament fibroblasts-derived exosomes induced by PGE2 on PDLSCs was detected by real-time PCR, alizarin red assay and alkaline phosphatase staining. High-throughput miRNAs sequencing was used to reveal that miR-34c-5p in exosomes-PGE2 was upregulated compared it in exosomes-normal. Real-time PCR and western blotting assay verified that overexpression of miR-34c-5p inhibited osteogenic differentiation, and reduced phosphorylation of ERK1/2. In addition, dual-luciferase reporter assay revealed that miR-34c-5p targeted special AT-rich sequence-binding protein 2 (SATB2). It was shown that exosomal miR-34c-5p inhibited osteogenic differentiation of PDLSCs via SATB2/ERK pathway.