Mechano-growth factor regulates periodontal ligament stem cell proliferation and differentiation through Fyn-RhoA-YAP signaling.

BACKGROUND: Mechano-growth factor (MGF), which is a growth factor produced specifically in response to mechanical stimuli, with potential of tissue repair and regeneration. Our previous research has shown that MGF plays a crucial role in repair of damaged periodontal ligaments by promoting differentiation of periodontal ligament stem cells (PDLSCs). However, the molecular mechanism is not fully understood. This study aimed to investigated the regulatory effect of MGF on differentiation of PDLSCs and its molecular mechanism. METHODS: Initially, we investigated how MGF impacts cell growth and differentiation, and the relationship with the activation of Fyn-p-YAPY357 and LATS1-p-YAPS127. Then, inhibitors were used to interfere Fyn phosphorylation to verify the role of Fyn-p-YAP Y357 signal after MGF stimulation; moreover, siRNA was used to downregulate YAP expression to clarify the function of YAP in PDLSCs proliferation and differentiation. Finally, after C3 was used to inhibit the RhoA expression, we explored the role of RhoA in the Fyn-p-YAP Y357 signaling pathway in PDLSCs proliferation and differentiation. RESULTS: Our study revealed that MGF plays a regulatory role in promoting PDLSCs proliferation and fibrogenic differentiation by inducing Fyn-YAPY357 phosphorylation but not LATS1-YAP S127 phosphorylation. Moreover, the results indicated that Fyn could not activate YAP directly but rather activated YAP through RhoA in response to MGF stimulation. CONCLUSION: The research findings indicated that the Fyn-RhoA-p-YAPY357 pathway is significant in facilitating the proliferation and fibrogenic differentiation of PDLSCs by MGF. Providing new ideas for the study of MGF in promoting periodontal regenerative repair.

Inflammatory Periodontal Ligament Stem Cells Drive M1 Macrophage Polarization via Exosomal miR-143-3p-Mediated Regulation of PI3K/AKT/NF-κB Signaling.

Macrophage polarization plays an important role in the progression of inflammation. Exosomes derived from stem cells are promising candidates for macrophage immunoregulation. However, how exosomes derived from periodontal ligament stem cells (PDLSCs) in an inflammatory environment influence macrophage polarization has yet to be fully elucidated. In this study, inflammatory PDLSCs were found to downregulate M2 macrophage polarization at the mRNA and protein levels in a Transwell coculture system of PDLSCs and THP-1-derived M0 macrophages. Furthermore, inflammatory PDLSC-derived exosomes shifted macrophages toward the M1 phenotype. The inhibition of inflammatory PDLSC-derived exosomes by GW4869 weakened inflammatory PDLSC-mediated M1 macrophage polarization. A miRNA microarray was used to determine the differential miRNAs shuttled by healthy and inflammatory PDLSC-derived exosomes. Compared with healthy exosomes, miR-143-3p was enriched in inflammatory PDLSC-derived exosomes, which targeted and inhibited the expression of PI3Kγ and promoted M1 macrophage polarization by suppressing PI3K/AKT signaling and activating NF-κB signaling, while an agonist of the PI3K pathway reversed this effect. Moreover, exosome-shuttled miR-143-3p from PDLSCs drove M1 macrophage polarization and aggravated periodontal inflammation in a mouse periodontitis model. In conclusion, these results demonstrate that inflammatory PDLSCs facilitate M1 macrophage polarization through the exosomal miR-143-3p-mediated regulation of PI3K/AKT/NF-κB signaling, providing a potential new target for periodontitis treatment.

The mechanism of lncRNA MALAT1 targeting the miR-124-3p/IGF2BP1 axis to regulate osteogenic differentiation of periodontal ligament stem cells.

OBJECTIVES: This study aimed to investigate the regulatory roles of lncRNA MALAT1, miR-124-3p, and IGF2BP1 in osteogenic differentiation of periodontal ligament stem cells (PDLSCs). MATERIALS AND METHODS: We characterized PDLSCs by employing quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses to evaluate the expression of key osteogenic markers including ALPL, SPP1, and RUNX2. Manipulation of lncRNA MALAT1 and miR-124-3p expression levels was achieved through transfection techniques. In addition, early osteogenic differentiation was assessed via Alkaline phosphatase (ALP) staining, and mineral deposition was quantified using Alizarin Red S (ARS) staining. Cellular localization of lncRNA MALAT1 was determined through Fluorescence In Situ Hybridization (FISH). To elucidate the intricate regulatory network, we conducted dual-luciferase reporter assays to decipher the binding interactions between lncRNA MALAT1 and miR-124-3P as well as between miR-124-3P and IGF2BP1. RESULTS: Overexpression of lncRNA MALAT1 robustly promoted osteogenesis in PDLSCs, while its knockdown significantly inhibited the process. We confirmed the direct interaction between miR-124-3p and lncRNA MALAT1, underscoring its role in impeding osteogenic differentiation. Notably, IGF2BP1 was identified as a direct binding partner of lncRNA MALAT1, highlighting its pivotal role within this intricate network. Moreover, we determined the optimal IGF2BP1 concentration (50 ng/ml) as a potent enhancer of osteogenesis, effectively countering the inhibition induced by si-MALAT1. Furthermore, in vivo experiments utilizing rat calvarial defects provided compelling evidence, solidifying lncRNA MALAT1's crucial role in bone formation. CONCLUSIONS: Our study reveals the regulatory network involving lncRNA MALAT1, miR-124-3p, and IGF2BP1 in PDLSCs' osteogenic differentiation. CLINICAL RELEVANCE: These findings enhance our understanding of lncRNA-mediated osteogenesis, offering potential therapeutic implications for periodontal tissue regeneration and the treatment of bone defects.

Thyrotropin inhibits osteogenic differentiation of human periodontal ligament stem cells.

BACKGROUND AND OBJECTIVE: 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. MATERIALS AND METHODS: PDLSCs were isolated and cultured from human periodontal ligament and grown in osteogenic medium (containing sodium ß-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. RESULTS: 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. CONCLUSION: Unless TSH is ≥10 mU/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.

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.

LRP6/filamentous-actin signaling facilitates osteogenic commitment in mechanically induced periodontal ligament stem cells.

BACKGROUND: Mechanotransduction mechanisms whereby periodontal ligament stem cells (PDLSCs) translate mechanical stress into biochemical signals and thereby trigger osteogenic programs necessary for alveolar bone remodeling are being deciphered. Low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt transmembrane receptor, has been qualified as a key monitor for mechanical cues. However, the role of LRP6 in the mechanotransduction of mechanically induced PDLSCs remains obscure. METHODS: The Tension System and tooth movement model were established to determine the expression profile of LRP6. The loss-of-function assay was used to investigate the role of LRP6 on force-regulated osteogenic commitment in PDLSCs. The ability of osteogenic differentiation and proliferation was estimated by alkaline phosphatase (ALP) staining, ALP activity assay, western blotting, quantitative real-time PCR (qRT-PCR), and immunofluorescence. Crystalline violet staining was used to visualize cell morphological change. Western blotting, qRT-PCR, and phalloidin staining were adopted to affirm filamentous actin (F-actin) alteration. YAP nucleoplasmic localization was assessed by immunofluorescence and western blotting. YAP transcriptional response was evaluated by qRT-PCR. Cytochalasin D was used to determine the effects of F-actin on osteogenic commitment and YAP switch behavior in mechanically induced PDLSCs. RESULTS: LRP6 was robustly activated in mechanically induced PDLSCs and PDL tissues. LRP6 deficiency impeded force-dependent osteogenic differentiation and proliferation in PDLSCs. Intriguingly, LRP6 loss caused cell morphological aberration, F-actin dynamics disruption, YAP nucleoplasmic relocation, and subsequent YAP inactivation. Moreover, disrupted F-actin dynamics inhibited osteogenic differentiation, proliferation, YAP nuclear translocation, and YAP activation in mechanically induced PDLSCs. CONCLUSIONS: We identified that LRP6 in PDLSCs acted as the mechanosensor regulating mechanical stress-inducible osteogenic commitment via the F-actin/YAP cascade. Targeting LRP6 for controlling alveolar bone remodeling may be a prospective therapy to attenuate relapse of orthodontic treatment.

Bioprinted PDLSCs with high-concentration GelMA hydrogels exhibit enhanced osteogenic differentiation in vitro and promote bone regeneration in vivo.

OBJECTIVES: We aimed to explore the osteogenic potential of periodontal ligament stem cells (PDLSCs) in bioprinted methacrylate gelatine (GelMA) hydrogels in vitro and in vivo. MATERIALS AND METHODS: PDLSCs in GelMA hydrogels at various concentrations (3%, 5%, and 10%) were bioprinted. The mechanical properties (stiffness, nanostructure, swelling, and degradation properties) of bioprinted constructs and the biological properties (cell viability, proliferation, spreading, osteogenic differentiation, and cell survival in vivo) of PDLSCs in bioprinted constructs were evaluated. Then, the effect of bioprinted constructs on bone regeneration was investigated using a mouse cranial defect model. RESULTS: Ten percent GelMA printed constructs had a higher compression modulus, smaller porosity, lower swelling rate, and lower degradation rate than 3% GelMA. PDLSCs in bioprinted 10% GelMA bioprinted constructs showed lower cell viability, less cell spreading, upregulated osteogenic differentiation in vitro, and lower cell survival in vivo. Moreover, upregulated expression of ephrinB2 and EphB4 protein and their phosphorylated forms were found in PDLSCs in 10% GelMA bioprinted constructs, and inhibition of eprhinB2/EphB4 signalling reversed the enhanced osteogenic differentiation of PDLSCs in 10% GelMA. The in vivo experiment showed that 10% GelMA bioprinted constructs with PDLSCs contributed to more new bone formation than 10% GelMA constructs without PDLSCs and constructs with lower GelMA concentrations. CONCLUSIONS: Bioprinted PDLSCs with high-concentrated GelMA hydrogels exhibited enhanced osteogenic differentiation partially through upregulated ephrinB2/EphB4 signalling in vitro and promoted bone regeneration in vivo, which might be more appropriate for future bone regeneration applications. CLINICAL RELEVANCE: Bone defects are a common clinical oral problem. Our results provide a promising strategy for bone regeneration through bioprinting PDLSCs in GelMA hydrogels.

Comparative proteomics analysis of human stem cells from dental gingival and periodontal ligament.

Dental stem cells isolated from oral tissues have been shown to provide with high proliferation ability and multilineage differentiation potential. Gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs), kinds of dental stem cells, can be used as substitutes for tissue repair materials because of their similar regenerative functions. In this study, we aim to explore the similarities and differences between the protein profiles of GMSCs and PDLSCs through quantitative proteomics. A total of 2821 proteins were identified and retrieved, of which 271 were upregulated and 57 were downregulated in GMSCs compared to PDLSCs. Gene Ontology (GO) analysis demonstrated that the 328 differentially abundant proteins (DAPs) were involved in the regulation of gene expression, metabolism, and signal transduction in biological process, mainly distributed in organelles related to vesicle transport, and involved in the molecular function of binding protein. And Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DAPs were committed to regulating the synthesis of proteasome and spliceosome. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that ARPC1B, PDAP1, and SEC61B can be used as special markers to distinguish GMSCs from PDLSCs. This research contributes to explaining the molecular mechanism and promoting the clinical application of tissue regeneration of GMSCs and PDLSCs.

Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs.

Periodontal ligament derived stem cells (PDLSC) are adult multipotent mesenchymal-like stem cells (MSCs) that can induce a promising immunomodulation to interact with immune cells for disease treatment. Metabolic reconfiguration has been shown to be involved in the immunomodulatory activity of MSCs. However, the underlying mechanisms are largely unknown, and it remains a challenging to establish a therapeutic avenue to enhance immunomodulation of endogenous stem cells for disease management. In the present study, RNA-sequencing (RNA-seq) analysis explores that curcumin significantly promotes PDLSC function through activation of MSC-related markers and metabolic pathways. In vitro stem cell characterization further confirms that self-renewal and multipotent differentiation capabilities are largely elevated in curcumin treated PDLSCs. Mechanistically, RNA-seq reveals that curcumin activates ERK and mTOR cascades through upregulating growth factor pathways for metabolic reconfiguration toward glycolysis. Interestingly, PDLSCs immunomodulation is significantly increased after curcumin treatment through activation of prostaglandin E2-Indoleamine 2,3 dioxygenase (PGE2-IDO) signaling, whereas inhibition of glycolysis activity by 2-deoxyglucose (2-DG) largely blocked immunomodulatory capacity of PDLSCs. Taken together, this study provides a novel pharmacological approach to activate endogenous stem cells through metabolic reprogramming for immunomodulation and tissue regeneration.

Promotion effect of apical tooth germ cell-conditioned medium on osteoblastic differentiation of periodontal ligament stem cells through regulating miR-146a-5p.

BACKGROUND: MicroRNAs (miRNAs) play an important role in gene regulation that controls stem cells differentiation. Periodontal ligament stem cells (PDLSCs) could differentiate into osteo-/cementoblast-like cells that secretes cementum-like matrix both in vitro and in vivo. Whether miRNAs play key roles in osteoblastic differentiation of PDLSCs triggered by a special microenviroment remains elusive. In this study, we aimed to investigate potential miRNA expression changes in osteoblastic differentiation of PDLSCs by the induction of apical tooth germ cell-conditioned medium (APTG-CM). METHODS AND RESULTS: First, we analyzed the ability of APTG-CM to osteogenically differentiate PDLSCs. The results exhibited an enhanced mineralization ability, higher ALP activity and increased expression of osteogenic genes in APTG-CM-induced PDLSCs. Second, we used miRNA sequencing to analyze the miRNA expression profile of PDLSCs derived from three donors under 21-day induction or non-induction of APTG-CM. MiR-146a-5p was found to be up-regulated miRNA in induced PDLSCs and validated by RT-qPCR. Third, we used lentivirus-up/down system to verify the role of miR-146a-5p in the regulation of osteoblastic differentiation of PDLSCs. CONCLUSIONS: In conclusion, our results demonstrated that miR-146a-5p was involved in the promotion effect of APTG-CM on osteoblastic differentiation of PDLSCs, and suggested that miR-146a-5p might be a novel way in deciding the direction of PDLSCs differentiation.

MiR-153-3p inhibits osteogenic differentiation of periodontal ligament stem cells through KDM6A-induced demethylation of H3K27me3.

BACKGROUND AND OBJECTIVE: Periodontal ligament stem cells (PDLSCs) have potential for osteogenic differentiation and show a great foreground in treating bone diseases. Histone three lysine 27 (H3K27) demethylase lysine demethylase 6A (KDM6A) is a critical epigenetic modifier and plays an important role in regulating osteogenic differentiation. Multiple microRNAs have been found to play important roles in osteogenesis. The aim of this study was to explore the mechanisms underlying the roles of miR-153-3p and KDM6A in PDLSC osteogenesis. METHODS: The levels of the osteogenic markers alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN) were measured by western blotting. Osteoblast activity and mineral deposition were detected by ALP and Alizarin red S (ARS) staining. The levels of miR-153-3p and KDM6A were measured by quantitative real-time PCR (qRT-PCR). A luciferase reporter assay was used to confirm the interaction between KDM6A and miR-153-3p. Gain-of-function and loss-of-function assays were performed to identify the roles of miR-153-3p and KDM6A in the osteogenic differentiation of PDLSCs. RESULTS: In osteogenic PDLSCs, the expression of KDM6A, ALP, Runx2, and OPN was upregulated, whereas that of miR-153-3p was downregulated. miR-153-3p downregulation or KDM6A overexpression promoted the osteogenic differentiation of PDLSCs, as demonstrated by increases in ALP activity, matrix mineralization, and ALP, Runx2, and OPN expression. KDM6A was confirmed to be a target of miR-153-3p, and KDM6A overexpression reversed the inhibitory effect of miR-153-3p mimic on PDLSC osteogenesis. KDM6A promoted ALP, Runx2, and OPN expression through the demethylation of H3K27me3 on the promoter regions of these genes. CONCLUSION: miR-153-3p inhibited PDLSC osteogenesis by targeting KDM6A and inhibiting ALP, Runx2, and OPN transcription. These findings provide latent hope for PDLSCs application in periodontal therapy.

Klotho inhibits H2 O2 -induced oxidative stress and apoptosis in periodontal ligament stem cells by regulating UCP2 expression.

Periodontitis, a human chronic inflammatory disease, has affected the lives of millions of individuals. Periodontal ligament stem cells (PDLSCs), derived from the periodontal ligament, exhibit tissue specificity and impaired differentiation ability and are closely associated with tissue regeneration in periodontitis. Klotho, a single-pass transmembrane protein, has been reported to positively affect H2 O2 -induced oxidative stress and inflammation in PDLSCs. The ultimate damage of oxidative stress stimulation in PDLSCs was cell apoptosis, which was also the major lesion in periodontitis. Thus, the present study aimed to figure out the effect of klotho on H2 O2 -injured PDLSCs and its underlying mechanism to provide new therapeutic targets in periodontitis. The expression of klotho and uncoupling protein 2 (UCP2) was investigated in the gingival tissues, gingival crevicular fluid (GCF), and periodontal ligament stem cells (PDLSCs) in patients with chronic periodontitis. Then, under klotho treatment, oxidative stress was evaluated by measuring SOD and GSH-PX levels. Cell apoptosis and cell necrosis were also detected by measuring the cell death-relevant proteins, including Caspase-3, BAX, Bcl, MLKL, RIP1, and RIP3. Finally, a rescue assay was performed by inhibiting the expression of UCP2. The results showed that klotho and UCP2 were downregulated in patients with chronic periodontitis. In addition, klotho upregulated the production of UCP2 in H2 O2 -treated PDLSCs. Klotho inhibited H2 O2 -induced oxidative stress and cellular loss in PDLSCs, moreover, the rescue assay suggested that UCP2 knockdown suppressed the effects of klotho on PDLSCs. In conclusion, this study showed that klotho inhibits H2 O2 -induced oxidative stress and apoptosis in PDLSCs by regulating UCP2 expression. This novel discovery might provide a potential target for chronic periodontitis treatment.

SHED-derived conditioned exosomes enhance the osteogenic differentiation of PDLSCs via Wnt and BMP signaling in vitro.

The exosomes from human exfoliated deciduous teeth (SHED-Exos) have exhibited potential therapeutic role in dental and oral disorders. The biological effects of exosomes largely depend on cellular origin and physiological status of donor cell. In the present study, we explored the influence of conditioned exosomes from SHED with osteogenic induction on periodontal ligament stem cells (PDLSCs) in vitro. Conditioned SHED-Exos from a 3-day osteogenic supernatant were applied during PDLSCs osteogenic differentiation. We found that conditioned SHED-Exos had no cytotoxicity on PDLSCs viability assessed by CCK-8 assay. These SHED-Exos promoted PDLSCs osteogenic differentiation with deep Alizarin red staining, high alkaline phosphatase (ALP) activity and upregulated osteogenic gene expression (RUNX2, OPN and OCN). We further found BMP/Smad signaling and Wnt/ß-catenin were activated by enhanced Smad1/5/8 phosphorylation and increased nuclear ß-catenin protein expression. Inhibiting these two signaling pathways with specific inhibitors (cardamonin and LDN193189) remarkably weakened the enhanced osteogenic differentiation. Furthermore, Wnt3a and BMP2 were upregulated in SHED and SHED-Exos. Silencing Wnt3a and BMP2 in SHED-Exos partially counteracts the enhanced osteogenic differentiation. Our findings indicate that conditioned SHED-Exos-enhanced PDLSCs osteogenic differentiation was partly due to its carrying Wnt3a and BMP2. These data provide new insights into the use of SHED-Exos in periodontitis-induced bone defects therapy.

Irradiation with red light-emitting diode enhances proliferation and osteogenic differentiation of periodontal ligament stem cells.

This study aimed to evaluate the effects of low-energy red light-emitting diode (LED) irradiation on the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs). PDLSCs were derived from human periodontal ligament tissues of premolars and were irradiated with 0 (control group), 1, 3, or 5 J/cm2 red LED in osteogenic induction medium. Cell proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Osteogenic differentiation activity was evaluated by monitoring alkaline phosphatase (ALP) activity, alizarin red staining, and real-time polymerase chain reaction (RT-PCR) results. Osteoblast-associated proteins (Runx2, OCN, OPN, and BSP) were detected using western blotting. The results of the MTT assay indicated that PDLSCs in the irradiation groups exhibited a higher proliferation rate than those in the control group (P < 0.05). ALP results showed that after 7 days of illumination, only 5 J/cm2 promoted the expression of ALP of PDLSCs. However, after 14 days of illumination, the irradiation treatments did not increase ALP activity. The results of alizarin red staining showed that red LED promoted osteogenic differentiation of the PDLSCs. The real-time polymerase chain reaction (RT-PCR) results demonstrated that red LED upregulated the expression levels of osteogenic genes. Expression of the proteins BSP, OPN, OCN, and Runx2 in the irradiation groups was higher than that in the control group. Our results confirmed that low-energy red LED at 1, 3, and 5 J/cm2 promotes proliferation and osteogenic differentiation of PDLSCs.

Downregulation of lncRNA DANCR promotes osteogenic differentiation of periodontal ligament stem cells.

BACKGROUND: Long 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. METHODS: Microarray 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. RESULTS: Compared 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. CONCLUSIONS: The 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.

Depletion of PRDM9 enhances proliferation, migration and chemotaxis potentials in human periodontal ligament stem cells.

PURPOSE: Periodontal ligament mesenchymal stem cells (PDLSCs) are important for periodontal tissue regeneration, but how these cells are regulated remains unclear. PRDM (PRDI-BF1 and RIZ homology domain containing) genes play key roles in cell proliferation and differentiation. The present study aimed to investigate the role of one PRDM gene, PRDM9, in the proliferation, migration and chemotaxis potential of PDLSCs. MATERIALS AND METHODS: Cell proliferation was examined on the basis of the cell doubling time, cell counting kit-8 (CCK8) assays, and flow cytometry analysis of the cell cycle. Gene expression was detected by Western blotting and real-time RT-PCR. Scratch migration and Transwell chemotaxis assays were used to analyse cell migration and chemotaxis abilities. Microarray analysis and ChIP assays were used to examine the downstream genes of PRDM9 and the corresponding mechanism. RESULTS: The results showed that knock-down of PRDM9 enhanced cell proliferation by promoting cell cycle progression and rapid transition from the G1 to S phase via downregulation of p21 and p27 and upregulation of cyclin E. Additionally, depletion of PRDM9 increased the migration and chemotaxis potential of PDLSCs. Microarray results showed that 13 genes, including IGFBP5, IFI44L, and POSTN, were upregulated and 34 genes, including PIP, were downregulated after the depletion of PRDM9. Furthermore, we observed that the depletion of PRDM9 promoted the transcription of IGFBP5 by increasing H3K4me3 methylation in the IGFBP5 promoter. CONCLUSION: These discoveries indicated that depletion of PRDM9 increased the cell proliferation, migration and chemotaxis potential of PDLSCs and revealed important downstream genes.

PDLSCs Regulate Angiogenesis of Periodontal Ligaments via VEGF Transferred by Exosomes in Periodontitis.

Abnormal angiogenesis is one of the significant features in periodontitis leading to progressive inflammation, but angiogenic changes of periodontal ligaments under inflammatory condition were rarely reported. Periodontal ligament stem cells (PDLSCs) were a kind of dental stem cells associated with vascularization. Here we investigated the alteration of angiogenesis of periodontal ligament in periodontitis, and revealed an exosome-mediated pathway to support the effect of PDLSCs on angiogenic improvement. Vascular specific marker CD31 and VEGFA were found to be highly expressed in periodontal ligaments of periodontitis. The VEGFA expression was up-regulated in inflamed PDLSCs compared to control, meanwhile the tube formation of HUVECs was improved when co-cultured with inflamed PDLSCs. Exosomes secretion of PDSLCs was augmented by inflammation, and promoted angiogenesis of HUVECs, whereas blocking secretion of exosomes led to degenerated angiogenesis of HUVECs. Exosome-trasferred VEGFA was proven to be the crucial communicator between PDLSCs and HUVECs. Inflammation inhibited miR-17-5p expression of PDLSCs and relieved its target VEGFA. However, overexpression of miR-17-5p blocked the pro-angiogenic ability of inflamed PDLSCs. In conclusion, the findings indicated that vascularization of periodontal ligaments was enhanced, and inflammatory micro-environment of periodontitis facilitated pro-angiogenesis of PDLSCs through regulating exosome-mediated transfer of VEGFA, which was targeted by miR-17-5p.

Identification and characterization of circular RNAs involved in mechanical force-induced periodontal ligament stem cells.

Circular RNAs (circRNAs) play critical roles in signal transduction during cell proliferation, differentiation, and apoptosis in a posttranscriptional manner. Recently, circRNAs have been proved to be a large class of animal RNAs with regulatory potency. However, whether circRNAs can respond to mechanical force (MF) and impact on human periodontal ligament stem cells (PDLSCs) and the orthodontic tooth movement (OTM) process remain unknown. Here, we investigated the circRNAs expression patterns in PDLSCs induced by MF and found that circRNAs were responsive to the MF in PDLSCs. Through the valid reads' distribution analysis, we found that the majority of reads in both the control PDLSCs and the MF-induced PDLSCs were distributed in exons. Then we analyzed Gene Ontology terms of genes that overlap with or are neighbors of the stress-responsive circRNAs and found unique enrichment patterns in biological processes, molecular function, and cellular component of PDLSCs. Next, we predicted the possible functions of circRNAs through circRNAs-miRNAs networks. We found that one circRNA may regulate one or several miRNA/miRNAs and one miRNA may interact with one or multiple circRNA/circRNAs. Importantly, a number of circRNAs were predicted to directly or indirectly regulate miRNAs-mediated osteogenic differentiation in mesenchymal stem cells. For instance, circRNA3140 was highly and widely associated with microRNA-21, which plays a critical role in MF-induced osteogenic differentiation of PDLSCs. Taken together, these findings reveal a previously unrecognized mechanism that MF can induce the expression changes of circRNAs in PDLSCs, which may modulate the OTM process and the alveolar bone remodeling.

CoCl2 induces apoptosis via a ROS-dependent pathway and Drp1-mediated mitochondria fission in periodontal ligament stem cells.

Oxygen deficiency is associated with various oral diseases, including chronic periodontitis, age-related alveolar bone loss, and mechanical stress-linked cell injury from orthodontic appliances. Nevertheless, our understanding of the impact of hypoxia on periodontal tissues and its biochemical mechanism is still rudimentary. The purpose of this research was to elucidate the effects of hypoxia on the apoptosis of human periodontal ligament stem cells (PDLSCs) in vitro and the underlying mechanism. Herein, we showed that cobalt chloride (CoCl2) triggered cell dysfunction in human PDLSCs in a concentration-dependent manner and resulted in cell apoptosis and oxidative stress overproduction and accumulation in PDLSCs. In addition, CoCl2 promoted mitochondrial fission in PDLSCs. Importantly, CoCl2 increased the expression of dynamin-related protein 1 (Drp1), the major regulator in mitochondrial fission, in PDLSCs. Mitochondrial division inhibitor-1, pharmacological inhibition of Drp1, not only inhibited mitochondrial fission but also protected against CoCl2-induced PDLSC dysfunction, as shown by increased mitochondrial membrane potential, increased ATP level, reduced reactive oxygen species (ROS) level, and decreased apoptosis. Furthermore, N-acety-l-cysteine, a pharmacological inhibitor of ROS, also abolished CoCl2-induced expression of Drp1 and protected against CoCl2-induced PDLSC dysfunction, as shown by restored mitochondrial membrane potential, ATP level, inhibited mitochondrial fission, and decreased apoptosis. Collectively, our data provide new insights into the role of the ROS-Drp1-dependent mitochondrial pathway in CoCl2-induced apoptosis in PDLSCs, indicating that ROS and Drp1 are promising therapeutic targets for the treatment of CoCl2-induced PDLSC dysfunction.

Long noncoding RNA ANCR suppresses bone formation of periodontal ligament stem cells via sponging miRNA-758.

Long noncoding RNAs (lncRNAs) were proposed to be important regulators influencing various differentiation processes. Yet, the molecular mechanisms of lncRNAs governing osteogenic differentiation of Periodontal Ligament Stem Cells (PDLSCs) remain unclear. Here, PDLSCs were isolated from normal periodontal ligament of human (PDL) whereas P-PDLSCs were isolated from periodontitis affected PDL. Quantitative real-time PCR (qRT-PCR) was performed to examine the relative expression level of lncRNA-ANCR and of Osterix (OSX), Alkaline Phosphatase (ALP) as well as Runt-related transcription factor 2 (RUNX2) in PDLSCs. Gain- and loss-of- function experiments was performed to study the role of lncRNA-ANCR. Alizarin Red staining was used to evaluate the function of lncRNA-ANCR and miRNA-758 on osteogenic differentiation. In addition, via dual luciferase reporter assay and RNA immunoprecipitation the microRNA sponge potential of lncRNA-ANCR was assessed. A luciferase reporter assay identified the correlation between miR-758 and Notch2. Our results showed that the expression of ALP, RUNX2 and OSX were increased whereas lncRNA-ANCR was decreased during the process of differentiation in PDLSCs. Overexpression of lncRNA-ANCR decreased the expression of ALP, RUNX2 and OSX as confirmed by Alizarin red staining. Overexpression of lncRNA-ANCR resulted in reduction of the miR-758 expression level. Furthermore, RNA immunoprecipitation proved that lncRNA-ANCR targets miR-758 directly. The results of dual luciferase reporter assay also demonstrated that miR-758 regulated Notch2 expression by targeting 3'-UTR of Notch2. In conclusion, the novel pathway lncRNA-ANCR/miR-758/Notch2 plays an important role in the process of regulating osteogenic differentiation of PDLSCs.