Mechanism of CGF in osteogenic differentiation of periodontal ligament stem cells through the Wnt pathway

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

BackgroundPeriodontal ligament stem cells (PDLSCs) are promising seed cells for bone tissue engineering and periodontal regeneration applications. However, the mechanism underlying the osteogenic differentiation process remains largely unknown. Previous reports showed that prolactin-induced protein (PIP) was upregulated after PDLSCs osteogenic induction. However, few studies have reported on the function of PIP in osteogenic differentiation. The purpose of the present study was to investigate the effect of PIP on osteogenic differentiation of PDLSCs.Material/MethodsThe expression pattern of PIP during PDLSCs osteogenic differentiation was detected and the effect of each component in the osteogenic induction medium on PIP was also tested by qRT-PCR. Then, the PIP knockdown cells were established using lentivirus. The knockdown efficiency was measured and the proliferation, apoptosis, and osteogenic differentiation ability were examined to determine the functional role of PIP on PDLSCs.ResultsQRT-PCR showed that PIP was sustainedly upregulated during the osteogenic induction process and the phenomenon was mainly caused by the stimulation of dexamethasone in the induction medium. CCK-8 and flow cytometer showed that knocking down PIP had no influence on proliferation and apoptosis of PDLSCs. ALP staining and activity, Alizarin Red staining, and western blot analysis demonstrated PIP knockdown enhanced the osteogenic differentiation and mineralization of PDLSCs.ConclusionsPIP was upregulated after osteogenic induction; however, PIP knockdown promoted PDLSCs osteogenic differentiation. PIP might be a by-product of osteogenic induction, and downregulating of PIP might be a new target in bone tissue engineering applications.

Circ_0003764 Regulates the Osteogenic Differentiation of Periodontal Ligament Stem Cells

This research aimed to examine the impact of M2 Macrophages-derived exosomes (M2-exo) on the osteogenic differentiation of Periodontal Ligament Stem Cells (PDLSCs). Human monocytes THP-1 were used to induce the M1 and M2 polarization. The PDLSCs were co-cultured with M1 and M2 type THP-1, and subjected to M2-exo treatment, respectively. The osteogenic differentiation of PDLSCs was analyzed through ALP and ARS staining. The mRNA expression of biomarkers of M1 and M2 macrophages, OPN, Runx2, ALP and CCL18 was evaluated by RT-qPCR assay. The findings suggested that M1 type THP-1 inhibited the steogenic differentiation of PDLSCs and M2 type THP-1 promoted it. M2-exo also promoted steogenic differentiation of PDLSCs and increased the OPN, Runx2 and ALP mRNA levels. Furthermore, CCL18 levels were increased in M2 type THP-1 and M2-exo treated PDLSCs. After CCL18 knockdown, the role of M2-exo in PDLSCs was reversed. In conclusion, this study confirmed that M2-exo derived exosomes facilitated PDLSCs' osteogenic differentiation through secreting CCL18.

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.

This study aims to investigate the mechanism of K (lysine) acetyltransferase 2A (KAT2A) regulation and control on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). The expression levels of KAT2A in PDLSCs were compared from each generation of the normal (H-PDLSCs) and periodontitis tissues (P-PDLSCs). The influences of KAT2A gene interference on the osteogenic differentiation of PDLSCs were also detected. In addition, the influences of the KAT2A gene interference to the canonical Wnt pathway and ligands were detected. The upstream and down-stream relationships between KAT2A and canonical Wnt pathway were also determined. The decreased expression of KAT2A in PDLSCs from the inflammatory tissue in each generation was compared with that in PDLSCs from the healthy tissue, and the difference was statistically significant (P<0.05). When the KAT2A gene was disrupted, the osteogenesis ability of PDLSC was declined, and the difference was statistically significant (P<0.05). The canonical Wnt pathway was activated, and the antagonist Dickkopf-1 (DKK-1) was reduced. After the DKK-1 addition, the osteogenic differentiation of the disturbed PDLSCs was recovered, and KAT2A was unaffected. The KAT2A expression in PDLSCs was decreased because of perio-dontitis. The classical Wnt pathway was activated to inhibit the osteogenic differentiation of the cells.

BackgroundLong non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs.MethodsMicroarray was used to observe the different expression of lncRNAs in differentiated and undifferentiated PDLSCs. And then osteogenic-related lncRNA, DANCR was screened out. Its effects on proliferation and osteogenic differentiation was explored by constructing an overexpression and inhibition model. qRT-PCR was used to detect the mRNA expression of osteogenesis related genes. MTT assay was performed to assess the effects of DANCR on cell growth curve. To quantify the effects of DANCR on osteogenic differentiation of PDLSCs, ALP staining and alizarin red was performed in basic culture medium and osteogenic medium. Data were statistically processed.ResultsCompared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of lncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers’ upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs.ConclusionsThe osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs’ osteogenic differentiation.

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.

Exendin-4 regulates Wnt and NF-κB signaling in lipopolysaccharide-induced human periodontal ligament stem cells to promote osteogenic differentiation

Necroptosis is a programmed necrosis, regulated by receptor interacting protein kinase 1(RIP1) and receptor interacting protein kinase 3(RIP3), and could be inhibited by necrostatin-1(Nec-1) specifically. This study aims to evaluate the effect of Nec-1 on LPS-treated periodontal ligament stem cells (PDLSCs). In the research, three groups were established: normal cultured PDLSCs, Porphyromonas gingivalis (Pg)-LPS stimulated PDLSCs and Pg-LPS+Nec-1 treated PDLSCs. The expression of RIP1 and RIP3 and osteogenic differentiation of PDLSCs in three groups were analyzed. Then, we constructed cell aggregates (CA) using PDLSCs, then PDLSCs-CA were combined with Bio-Oss in three groups were transplanted subcutaneously in nude mice to assess their potentials of periodontal tissue regeneration. The results showed that RIP1 and RIP3 were fully expressed in Pg-LPS stimulated PDLSCs and the level increased significantly. Nec-1 inhibited RIP1-RIP3 interaction, and further inhibited necroptosis of PDLSCs in inflammatory state. Moreover, Nec-1 pretreatment ameliorates the osteogenic differentiation of LPS-treated PDLSCs and can effectively promote the cementum like structure ectopic regenerative ability of PDLSCs in nude mice. These findings show RIP1/RIP3-mediated necroptosis is an important mechanism of cell death in PDLSCs. Nec-1 has a protective effect in reducing cell death and promotes ectopic periodontal tissue like structure regeneration by inhibiting necroptosis. Nec-1 is a hopeful therapeutic agent which protects cells from necroptosis and ameliorates functional outcome.

The osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is important for periodontal tissue repair and regeneration. Long non-coding RNAs (lncRNAs) are key regulators of diverse biological processes. However, their roles in PDLSC osteogenic differentiation are still largely unknown. This study explored the effect of LINC00707 and its mechanism on the osteogenic differentiation of human PDLSCs. Results showed an increase in LINC00707 and forkhead box O1 (FOXO1) but a decrease in miR-490-3p during PDLSC osteogenic differentiation. LINC00707 and FOXO1 promoted osteogenic differentiation as evidenced by the formation of calcium nodules and the increase in osteogenic markers such as alkaline phosphatase, osteocalcin (OCN), and runt-related transcription factor 2 (Runx2). LINC00707 and FOXO1 knockdown exhibited opposite effects. Dual-luciferase reporter assay and qRT-PCR showed that LINC00707 can specially bind to miR-490-3p, which reversed the effect of LINC00707 on PDLSCs. MiR-490-3p inhibitor relieved the inhibiting effect of sh-LINC00707 on osteogenic differentiation. Further investigation revealed that LINC00707 can promote osteogenic differentiation by regulating FOXO1 expression through miR-490-3p sponging. Thus, the LINC00707/miR-490-3p/FOXO1 axis modulated PDLSC osteogenic differentiation and might be a promising therapeutic target for periodontal diseases.

The osteogenic potential of periodontal ligament stem cells (PDLSCs) is crucial for periodontal tissue regeneration. Prolonged and excessive oxidative stress (OS) impairs the osteogenic function of PDLSCs. Recently, Semaphorin 3A (Sema3A) has been reported to have multiple roles in bone protection. This study aimed to investigate the protective effect of Sema3A on the osteogenic differentiation of PDLSCs under OS conditions induced by hydrogen peroxide (H2O2). PDLSCs were subjected to H2O2 treatment to induce OS. The OS indices in PDLSCs were evaluated by analyzing levels of reactive oxygen species (ROS), cell viability, and expression of antioxidant factors using relevant assay kits. A small molecule inhibitor, XAV-939, was employed to block the Wnt/β-catenin pathway. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) activity staining and Alizarin Red S (ARS) staining for mineralized nodules. Expression levels of osteogenic gene markers and β-catenin were determined via real-time quantitative polymerase chain reaction (RT-qPCR) or western blot (WB) analysis. The stimulation of H2O2 induced OS in PDLSCs, resulting in a downregulation of Sema3A expression and a decrease in osteogenic markers, including ALP activity, mineralized nodule formation, and the expression of osteogenic genes (RUNX2 and ALP). However, the application of recombinant human Sema3A (rhSema3A) counteracted H2O2-induced OS and restored these osteogenic markers in PDLSCs under OS induced by H2O2. Mechanistic studies revealed that these effects were associated with an upregulation of β-catenin levels. Moreover, inhibiting β- catenin expression compromised the protective effect of Sema3A on osteogenesis in PDLSCs under OS. Sema3A exerts a protective effect against H2O2-induced OS and activates the Wnt/β- catenin pathway to restore osteogenic differentiation impaired by OS in PDLSCs.

The osteogenic differentiation of PDLSCs is mediated through MEK/ERK and p38 MAPK signalling under hypoxia

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.

During periodontitis and orthodontic tooth movement, periodontal vasculature is severely impaired, leading to a hypoxic microenvironment of periodontal cells. However, the impact of hypoxia on periodontal cells is poorly defined. The present study investigates responses of cocultured endothelial cells (ECs) and periodontal ligament stem cells (PDLSCs) to hypoxia. Osteogenic differentiation, molecular characterization, and various behaviors of PDLSCs and human umbilical venous ECs under hypoxia were assessed by quantitative real-time reverse-transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Moreover, the effect of ECs on PDLSC osteogenic differentiation was tested using NS398 (cyclooxygenase 2 blocker), SU5416 (vascular endothelial growth factor [VEGF] receptor inhibitor), AH6809, L-798106, and L-161982 (EP1/2/3/4 antagonists). First, hypoxia promoted osteogenic differentiation in PDLSCs and enhanced EC migration, whereas PD98059 (extracellular signal-regulated protein kinase [ERK] inhibitor) blocked, and cocultured ECs further enhanced, hypoxia-induced osteogenic differentiation. Second, NS398 impaired EC migration and prostaglandin E2 (PGE2)/VEGF release, whereas cocultured PDLSCs and exogenous PGE2 partially reversed it. Third, NS398 (pretreated ECs) decreased PGE2/VEGF concentrations. NS398-treated ECs and AH6809/SU5416-treated PDLSCs impaired cocultured EC-induced enhancement of PDLSC osteogenic differentiation. Hypoxia enhances ERK-mediated osteogenic differentiation in PDLSCs. Coculture with EC further augments PDLSC osteogenic differentiation via cyclooxygenase-2/PGE2/VEGF signaling.