Proliferation Of Periodontal Ligament Stem Cells Research Articles

Mechanical force modulates periodontal ligament stem cell characteristics during bone remodelling via TRPV4.

ObjectivesMechanical force plays an important role in modulating stem cell fate and behaviours. However, how periodontal ligament stem cells (PDLSCs) perceive mechanical stimulus and transfer it into biological signals, and thereby promote alveolar bone remodelling, is unclear.Materials and MethodsAn animal model of force‐induced tooth movement and a compressive force in vitro was used. After force application, tooth movement distance, mesenchymal stem cell and osteoclast number, and proinflammatory cytokine expression were detected in periodontal tissues. Then, rat primary PDLSCs with or without force loading were isolated, and their stem cell characteristics including clonogenicity, proliferation, multipotent differentiation and immunoregulatory properties were evaluated. Under compressive force in vitro, the effects of the ERK signalling pathway on PDLSC characteristics were evaluated by Western blotting.ResultsMechanical force in vivo induced PDLSC proliferation, which was accompanied with inflammatory cytokine accumulation, osteoclast differentiation and TRPV4 activation; the force‐stimulated PDLSCs showed greater clonogenicity and proliferation, reduced differentiation ability, improved induction of macrophage migration, osteoclast differentiation and proinflammatory factor expression. The biological changes induced by mechanical force could be partially suppressed by TRPV4 inhibition. Mechanistically, force‐induced activation of TRPV4 in PDLSCs regulated osteoclast differentiation by affecting the RANKL/OPG system via ERK signalling.ConclusionsTaken together, we show here that TRPV4 activation in PDLSCs under mechanical force contributes to changing their stem cell characteristics and modulates bone remodelling during tooth movement.

Evaluation of conditioned medium from placenta-derived mesenchymal stem cells as a storage medium for avulsed teeth: An in vitro study.

The viability of periodontal ligament cells on the root surface is a major factor that influences the healing of replanted teeth. A suitable storage medium is necessary to preserve avulsed teeth before replantation. Conditioned medium from placenta-derived mesenchymal stem cells (PMSC-CM) contains a variety of growth factors. The aim of this study was to evaluate the effectiveness of PMSC-CM as a storage medium to maintain the cell viability of avulsed teeth. Extracted premolars from healthy humans were randomly stored in Hank's balanced salt solution (HBSS) and PMSC-CM for 6, 12 and 24hours, respectively, at room temperature, and then the ratio of apoptosis of the periodontal ligament (PDL) cells was identified by flow cytometry. Human periodontal ligament stem cells (PDLSCs) were incubated with HBSS and PMSC-CM, respectively, for 6, 12, 24 and 48hours in 5% CO2 at 37°C. Then, the cell viability of PDLSCs was determined using the cell counting kit-8 (CCK-8) and a cell cycle assay was performed. The apoptosis rate of PDL cells in PMSC-CM was significantly lower than that in HBSS at 24hours (P<.001), while the two groups showed similar cell apoptosis rates at 6 and 12hours (P>.05). The cell proliferation of PDLSCs treated with PMSC-CM significantly increased compared with the HBSS group (P<.05). The cell cycle assay revealed that the PDLSCs treated with HBSS were arrested at the G1 phase, while there was no difference between the PMSC-CM group and the control group (P>.05). Compared with HBSS, PMSC-CM showed better inhibition of apoptosis of PDL cells and promoted the proliferation of PDLSCs. Thus, PMSC-CM could be a promising storage medium for avulsed teeth.

Kaempferol promotes proliferation and osteogenic differentiation of periodontal ligament stem cells via Wnt/β-catenin signaling pathway

AimsKaempferol, a type of flavonoid, is widely present in fruits, vegetables and medicinal herbs. This study was designed to investigate the effects of kaempferol on proliferation and osteogenesis of periodontal ligament stem cells (PDLSCs) and to identify the related pathway. Materials and methodsPDLSCs were isolated from extracted premolars and cultured in vitro. Cell-counting kit-8 (CCK-8) and colony formation assays were performed to determine the effect of kaempferol, at various concentrations, on the proliferation of PDLSCs. Alkaline phosphatase (ALP) activity was analyzed both quantitatively and qualitatively, and extracellular matrix mineralization was examined by alizarin red-S staining. In addition, the mRNA and protein expression levels of ALP, RUNX Family Transcription Factor 2 (RUNX2), Sp7 Transcription Factor (SP7; Osterix), Bone Gamma-Carboxyglutamate Protein (BGLAP; osteocalcin) and catenin beta 1 (CTNNB1; β-catenin) were monitored by qPCR and Western blot analysis. Additionally, the tankyrase inhibitor, XAV939, was used to determine the role of the Wnt/β-catenin pathway. Key findingsThe results illustrated that 10−6 M kaempferol markedly promoted the proliferation, ALP activity and mineral deposition of PDLSCs (P < 0.05). The expression levels of ALP, RUNX2, SP7, BGLAP and β-catenin were all upregulated (P < 0.05). After blocking the Wnt/β-catenin pathway with XAV939, the effects of kaempferol were apparently reversed. Significancekaempferol enhanced proliferation and osteogenesis of PDLSCs by activating the Wnt/β-catenin signaling, which suggests the potential application of kaempferol for periodontal tissue regeneration.

Red and Yellow Injectable Platelet-Rich Fibrin Demonstrated Differential Effects on Periodontal Ligament Stem Cell Proliferation, Migration, and Osteogenic Differentiation.

The biological benefits of using two fractions derived from injectable platelet-rich fibrin (i-PRF) in bone regeneration remain unclear. Thus, the current study examined two fractionation protocols producing yellow i-PRF and red i-PRF on periodontal ligament stem cells (PDLSCs). The i-PRF samples from five donors were harvested from two different levels, with and without a buffy coat layer, to obtain red and yellow i-PRF, respectively. The PDLSCs were isolated and characterized before their experimental use. The culture medium in each assay was loaded with 20% of the conditioned medium containing the factors released from the red and yellow i-PRF. Cell proliferation and cell migration were determined with an MTT and trans-well assay, respectively. Osteogenic differentiation was investigated using alkaline phosphatase and Alizarin red staining. The efficiency of both i-PRFs was statistically compared. We found that the factors released from the red i-PRF had a greater effect on cell proliferation and cell migration. Moreover, the factors released from the yellow i-PRF stimulated PDLSC osteogenic differentiation earlier compared with the red i-PRF. These data suggest that the red i-PRF might be suitable for using in bone regeneration because it induced the mobilization and growth of bone regenerative cells without inducing premature mineralization.

MiR-375 promotes human periodontal ligament stem cells proliferation and osteogenic differentiation by targeting transducer of ERBB2, 2

BackgroundMiRNAs have been demonstrated to be important regulators during osteogenic differentiation in multiple types of stem cells. In the study, the interaction between miR-375 and TOB2 was analyzed to identify their functions on the proliferation and osteogenic differentiation of hPDLSCs. MethodshPDLSCs were isolated from human first premolars, and hPDLSCs stably expressing and silenced miR-375 were constructed using miR-375-ago and miR-375-antago, respectively. miR-375 and RUNX2 mRNA expression levels in hPDLSCs during osteogenic differentiation were investigated using qRT-PCR. The impact of miR-375 expression on hPDLSCs proliferation and osteogenic differentiation was determined using MTT assay, ALP assay, and alizarin red S staining. The protein expression levels of COL1A1, RUNX2 and OCN were detected using Western blot. The targeting of TOB2 by miR-375 was validated using dual luciferase reporter assay. ResultsThe expression levels of miR-375 were increased in hPDLSCs during osteogenic differentiation in a time-dependant manner, and was positively correlated with RUNX2 mRNA expression. miR-375 facilitated the proliferation and osteogenic differentiation of hPDLSCs, and promoted the protein expression levels of COL1A1, RUNX2 and OCN. Moreover, TOB2 protein expression was reduced in hPDLSCs during osteogenic differentiation in a time-dependant manner, and miR-375 directly targeted TOB2 expression. In addition, targeting TOB2 expression in hPDLSCs could rescue the suppression of cell proliferation and osteogenic differentiation by miR-375-antago. ConclusionIn summary, miR-375 promotes proliferation and osteogenic differentiation of hPDLSCs by targeting TOB2, which reveals a new regulatory mechanism underlying osteogenic differentiation of hPDLSCs by miR-375/TOB2 axis.

LncRNA MAFG-AS1 regulates human periodontal ligament stem cell proliferation and Toll-like receptor 4 expression.

LncRNA MAFG-AS1 is predicted to interact with miR-146a, which can target Toll-like receptor 4 (TLR4), a key player in periodontitis. This study aimed to investigate the roles of MAFG-AS1 in periodontitis. It was observed that MAFG-AS1 was downregulated in the human periodontal ligament stem cells (PDLSCs) derived from periodontitis-affected teeth. Dual-luciferase assay revealed that co-transfection of MAFG-AS1 expression vector and miR-146a mimic showed significantly lower relative luciferase activity comparing to co-transfection of MAFG-AS1 expression vector and negative control (NC) miRNA. However, MAFG-AS1 and miR-146a failed to affect each other. Interestingly, MAFG-AS1 overexpression led to the upregulated TLR4. In addition, MAFG-AS1 overexpression also led to the inhibited proliferation of PDLSCs. Therefore, MAFG-AS1 may regulate the proliferation of PDLSCs and the expression of TLR4 to participate in periodontitis.

Effects of rutin on the oxidative stress, proliferation and osteogenic differentiation of periodontal ligament stem cells in LPS-induced inflammatory environment and the underlying mechanism.

Periodontitis can cause damage to dental support tissue and affect the function of periodontal ligament cells. Rutin, a common flavonoid, plays a key role in anti-inflammatory responses, tissue repair and bone development. The purpose of this study was to investigate the effects of rutin on the oxidative stress, proliferation, and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) in an inflammatory environment and the underlying mechanism. Lipopolysaccharide (LPS) was used to stimulate PDLSCs to mimic an inflammatory environment model. Reactive oxygen species (ROS) levels were detected by the dichlorodihydrofluorescein diacetate (DCFH-DA) probe and the oxidative stress factors were tested by an oxidative stress factor detection kit. Moreover, the proliferation of PDLSCs was evaluated by cell counting kit-8 (CCK-8) assay. In addition, the osteogenic differentiation of PDLSCs was determined by alkaline phosphatase (ALP) staining, ALP activity test, alizarin red staining, and alizarin red semi-quantitative analysis. Furthermore, the protein levels of AKT and p-AKT were detected by Western blot. The results showed that rutin inhibited the release of ROS and increased the secretion of oxidative stress factors [superoxide dismutase (SOD) and glutathione (GSH)] and promoted the proliferation of PDLSCs in an inflammatory environment. Moreover, rutin upregulated ALP activity and enhanced the number of mineralized nodules. Conversely, the use of LY294002 (an inhibitor of PI3K) blocked the activation of the PI3K/AKT signaling pathway and prevented the beneficial effects of rutin. In conclusion, rutin promoted the antioxidative stress ability, proliferation and osteogenic differentiation of PDLSCs through PI3K/AKT signaling pathway in LPS-induced inflammatory environment.

Dual-Functionalized Apatite Nanocomposites with Enhanced Cytocompatibility and Osteogenesis for Periodontal Bone Regeneration.

The development of biomimetic bone graft materials for periodontal tissue engineering is a field of topical interest. In this study, we designed a dual-functionalized apatite nanocomposite, which could integrate multiple molecular cues for manipulating the fate of periodontal ligament stem cells (PDLSCs). Briefly, inspired by mussels, a biomimetic nanohydroxyapatite was fabricated using a polydopamine structure as a template (named as tHA) and then surface-modified with bone-forming peptide-1 (BFP-1) and vascular endothelial growth factor-mimicking peptide (QK) via a single step of catechol chemistry. Our study showed that the biofunctions of tethered peptides were not compromised on the surface of apatite nanoparticles. Because of the synergistic effect of BFP-1 and QK peptides, the dual-functionalized apatite nanocomposite showed improved cytocompatibility compared to controls. Moreover, it can boost the proliferation and osteogenic differentiation of PDLSCs, indicating excellent bioactivity of tHA-BFP/QK nanoparticles on cell fate decision. More importantly, animal experiments showed that dual-functionalized apatite nanocomposites could dramatically promote the regeneration of periodontal bone. It is concluded that our work provides an instructive insight into the design of biomimetic apatite nanocomposites, which holds a great potential for applications in periodontal bone repair.

Rutin promotes osteogenic differentiation of periodontal ligament stem cells through the GPR30-mediated PI3K/AKT/mTOR signaling pathway.

Rutin is one of the flavonoids found in fruits and vegetables. Recent reports have revealed that rutin is a major player in proliferation and bone development. However, data on how rutin regulates the proliferation of periodontal ligament stem cells (PDLSCs), as well as the differentiation of osteogenic cells are scanty. Here, our findings showed that rutin enhanced PDLSCs proliferation, increased ALP activity, and matrix mineralization. Moreover, rutin significantly promoted the expression of osteogenic genes and elevated phosphorylated AKT and mTOR. Treatment with LY294002 reversed these effects by inhibiting PI3K. We also found that the expression levels of GPR30 were increased by rutin. Interestingly, this upregulation was not altered after the addition of LY294002. In addition, G15, a selective antagonist of GPR30, could reduce the beneficial effects induced by rutin and interfere with the modulation of PI3K/AKT/mTOR signal transduction. Collectively, our findings revealed that rutin increased proliferation and osteogenic differentiation of PDLSCs through GPR30-mediated PI3K/AKT/mTOR signal transduction. Therefore, it could be deduced that rutin as a certain flavonoid possesses therapeutic value for periodontal bone regeneration and tissue engineering. Impact statement In our study, the effects and mechanisms of rutin on the osteogenic differentiation and proliferation of PDLSCs were investigated. Our findings might provide basic knowledge and guidance to understand and use rutin in the bioengineering of the periodontal tissues and regeneration of bones. The following is a short description of the main findings: rutin promotes the osteogenic differentiation and proliferation of PDLSCs; PI3K/AKT/mTOR signal pathway mediates the effects of rutin on PDLSCs; rutin activates PI3K/AKT/mTOR signal pathway via GPR30.

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

A simvastatin-releasing scaffold with periodontal ligament stem cell sheets for periodontal regeneration.

Simvastatin (SIM) has been documented to induce the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). To establish an efficient release system for periodontal regeneration, a polycaprolactone (PCL) membrane scaffold containing SIM was electrospun and evaluated. The obtained PCL-SIM membrane scaffold showed sustained release up to 28 days, without deleterious effect on proliferation of PDLSCs on the scaffolds. PDLSCs were seeded onto scaffolds and their osteogenic differentiation was evaluated. After 21 days, expressions of collagen type I, alkaline phosphatase and bone sialoprotein genes were significantly upregulated and mineralized matrix formation was increased on the PCL-SIM scaffolds compared with the PCL scaffolds. In a heterotopic periodontal regeneration model, a cell sheet-scaffold construct was assembled by placement of multilayers of PDLSC sheets on PCL or PCL-SIM scaffolds, and these were then placed between dentin and ceramic bovine bone for subcutaneous implantation in athymic mice. After 8 weeks, the PCL-SIM membrane showed formation of significantly more ectopic cementum-like mineral on the dentin surface. These findings demonstrated that the PCL-SIM membrane scaffold promotes cementum-like tissue formation by sustained drug release, suggesting the feasibility of its therapeutic use with PDLSC sheets to improve periodontal regeneration.

Nicotinamide-induced silencing of SIRT1 by miR-22-3p increases periodontal ligament stem cell proliferation and differentiation.

Dental stem cell proliferation and osteoblast differentiation are key cellular processes involved in periodontitis diseases. Researchers have found that SIRT1 (sirtuin 1, silent mating type information regulation 2 homolog 1) and microRNAs play a pivotal role in the process, but a clear underlying mechanism has not been determined. In this study, the has-miR-22-3p that target SIRT1 was predicted by TargetScan. Luciferase reporter assay was used to confirm that SIRT1 is the direct target of miR-22-3p. Importantly, miR-22-3p was revealed to control SIRT1 in periodontal ligament stem cell (PDLSC) and to regulate the proliferation and differentiation of PDLSC by SIRT1 silencing. Furthermore, we detected the induction of miR-22-3p expression by nicotinamide treatment on PDLSC. Induction of PDLSC proliferation and differentiation by nicotinamide treatment was blocked by miR-22-3p knockdown. These results suggested that the effect of nicotinamide on PDLSC is through miR-22-3p. In addition, miR-22-3p also upregulated the expression levels of the inflammatory cytokines tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-8 in PDLSC through SIRT1 pathway and downregulated the expression of TLR-2 and TLR-4. miR-22-3p is a new target either for the treatment of periodontitis or the improvement of inflammation caused by orthodontics.

Microporous Bio-orthogonally Annealed Particle Hydrogels for Tissue Engineering and Regenerative Medicine.

Microporous annealed particle (MAP) hydrogels are an emerging class of biomaterials with the potential to improve outcomes in tissue repair and regeneration. Here, a new MAP hydrogel platform comprising poly(ethylene) glycol (PEG) hydrogel microparticles that are annealed in situ using bio-orthogonal tetrazine click chemistry is reported (i.e., TzMAP hydrogels). Briefly, clickable PEG-peptide hydrogel microparticles with extracellular matrix mimetic peptides to permit cell adhesion and enzymatic degradation were fabricated via submerged electrospraying and stoichiometrically controlled thiol-norbornene click chemistry. Subsequently, unreacted norbornene groups in the microparticles were leveraged for functionalization with bioactive proteins as well as annealing into TzMAP hydrogels via the tetrazine-norbornene click reaction, which is highly selective and proceeds spontaneously without requiring an initiator or catalyst. The results demonstrate that the clickable particles can be easily applied to a tissue-like defect and then annealed into an inherently microporous structure in situ. In addition, the ability to produce TzMAP hydrogels with heterogeneous properties by incorporating multiple types of hydrogel microspheres is demonstrated, first with fluorophore-functionalized hydrogel microparticles and then with protein-functionalized hydrogel microparticles. For the latter, tetrazine-modified alkaline phosphatase was conjugated to PEG hydrogel microparticles, which were mixed with nonfunctionalized microparticles and used to produce TzMAP hydrogels. A biomimetic mineralized/nonmineralized interface was then produced upon incubation in calcium glycerophosphate. Finally, platelet-derived growth factor-BB (PDGF-BB) and human periodontal ligament stem cells (PDLSC) were incorporated into the TzMAP hydrogels during the annealing step to demonstrate their potential for delivering regenerative therapeutics, specifically for periodontal tissue regeneration. In vitro characterization revealed excellent PDGF-BB retention as well as PDLSC growth and spreading. Moreover, PDGF-BB loading increased PDLSC proliferation within hydrogels by 90% and more than doubled the average volume per cell. Overall, these results demonstrate that TzMAP hydrogels are a versatile new platform for the delivery of stem cells and regenerative factors.

Downregulation of Linc-RNA activator of myogenesis lncRNA participates in FGF2-mediated proliferation of human periodontal ligament stem cells.

Accelerated proliferation of human periodontal ligament stem cells (PDLSCs) is present in periodontitis. It is known that fibroblast growth factor 2 (FGF2) regulates the proliferation of PDLSCs, while the function of FGF2 in myogenic cell differentiation is mediated by Linc-RNA Activator of Myogenesis (Linc-RAM) lncRNA. Therefore, Linc-RAM lncRNA may also participate in periodontitis. This study included 28 patients with periodontitis (patient group) and 22 patients without periodontitis but received orthodontic treatment (control group) in the stomatological hospital of Sun Yat-Sen university. Gingival biopsies were obtained from participants. RT-qPCR, cell transfection, cell proliferation assay and western blot were carrying out to analyze the samples. We found that FGF2 mRNA was upregulated, while Linc-RAM was downregulated in PDLSCs derived from periodontitis-affected teeth than in healthy teeth. FGF2 mRNA and Linc-RAM were inversely correlated in both types of PDLSCs. FGF2 overexpression led to inhibited Linc-RAM expression in PDLSCs derived from periodontitis-affected teeth and promoted the proliferation of PDLSCs. Linc-RAM overexpression failed to significantly affect FGF2 expression but attenuated the enhancing effects of FGF2 overexpression on the proliferation of PDLSCs. Therefore, downregulation of Linc-RAM lncRNA may participate in FGF-2 mediated- proliferation of human PDLSCs.

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

A major feature of chronic periodontitis (CP) is the damage and destruction of alveolar bone. Periodontal ligament stem cells (PDLSCs) can differentiate into bone and improve CP. Exendin-4 (Ex-4) has been shown to have anti-inflammatory mechanisms and can promote bone regeneration. However, the effects of Ex-4 on the osteogenic differentiation of PDLSCs in the inflammatory microenvironment remains uncharacterized. In this study, we assessed the effects of Ex-4 on PDLSCs stimulated with lipopolysaccharide (LPS) to mimic the inflammatory environment. PDLSCs proliferation was assessed through CCK-8 assays and osteogenic differentiation was measured using Alizarin Red staining. The anti-inflammatory and osteogenic mechanisms of Ex-4 were assessed by western blot, RT-PCR, ELISA and immunofluorescence. We found that LPS treatment promoted the proliferative capacity of PDLSCs and inhibited their osteogenic differentiation. However, Ex-4 reversed these effects through suppressing PDLSCs proliferation and promoting osteogenic differentiation. Ex-4 increased Runx2, ALP, and Osx levels and decreased TNF-α and IL-6 expression. Ex-4 also reduced the expression of IκBα and p-IκBα, and inhibited the nuclear translocation of NF-κB/p65. The expression of β-catenin decreased in nucleus after co-treatment of Ex-4 with LPS. Taken together, these data demonstrate that Ex-4 promotes PDLSCs osteogenic differentiation in the inflammatory microenvironment through regulating NF-κB and Wnt signaling.

Metformin facilitates the proliferation, migration, and osteogenic differentiation of periodontal ligament stem cells in vitro.

Periodontitis is one of the main causes of tooth loss and has been confirmed as the sixth complication of diabetes. Metformin promotes the osteogenic differentiation of stem cells. Periodontal ligament stem cells (PDLSCs) are the best candidate stem cells for periodontal tissue regeneration. Herein, we aimed to identify the effects of metformin on the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. PDLSCs were isolated by limiting dilution, and their characteristics were assessed by colony formation assay and flow cytometry. Cell counting and migration assays were used to investigate the effects of metformin on proliferation and migration. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene and protein levels of osteogenesis-related markers were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Metformin treatment at 10 μM did not affect PDLSC proliferation, while at 50 and 100 μM, metformin time-dependently enhanced PDLSC proliferation and significantly increased cell numbers after 5 and 7 days of stimulation (P < 0.05). In addition, 50 μM metformin exhibited a maximal effect on migration, ALP activity, and mineral deposition (P < 0.05). Furthermore, 50 μM metformin significantly upregulated the gene expression levels of ALP, BSP, OPN, OCN, and Runx2 and the protein expression of ALP and Runx2 (P < 0.05). In summary, our study confirms that metformin facilitates the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro and could be used as a new strategy for periodontal tissue regeneration.

MALAT1 overexpression promotes the proliferation of human periodontal ligament stem cells by upregulating fibroblast growth factor 2.

Fibroblast growth factor 2 (FGF2) has been revealed to promote human periodontal ligament stem cell (PDLSC) proliferation. The abnormal proliferation of PDLSCs has also been associated with the pathogenesis of periodontitis. The long non-coding RNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), has been demonstrated to regulate FGF2 secretion. Therefore, MALAT1 may also be associated with periodontitis. The aim of the present study was to investigate the effect of MALAT1 overexpression on the proliferation of PDLSCs. In the current study, PDLSCs derived from healthy and periodontitis-affected teeth were collected. MALAT1 and FGF2 mRNA expression in PDLSCs was detected using reverse transcription-quantitative PCR. PDLSCs overexpressing MALAT1 were subsequently generated. PDLSC proliferation was analyzed using a Cell Counting kit-8 assay. FGF2 protein expression was detected using western blot analysis. The results revealed that MALAT1 and FGF2 mRNA were significantly upregulated in PDLSCs derived from periodontitis-affected teeth when compared with PDLSCs derived from healthy teeth. PDLSCs derived from periodontitis-affected teeth also demonstrated a significantly higher proliferation rate than PDLSCs derived from healthy teeth. MALAT1 and FGF2 mRNA expression were positively correlated in both PDLSC groups. MALAT1 overexpression promoted the proliferation of healthy and periodontitis-affected PDLSC groups and upregulated FGF2 protein expression. The present study concluded that MALAT1 overexpression promoted the proliferation of human PDLSC potentially via upregulating FGF2.

Periodontal bacterial supernatants modify differentiation, migration and inflammatory cytokine expression in human periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSC) play an important role in periodontal tissue homeostasis/turnover and could be applied in cell-based periodontal regenerative therapy. Bacterial supernatants secreted from diverse periodontal bacteria induce the production of cytokines that contribute to local periodontal tissue destruction. However, little is known about the impact of whole bacterial toxins on the biological behavior of PDLSC. Therefore this study investigated whether proliferation, migration, inflammatory cytokines expression and transcriptional profile would be affected by exposure to endotoxins from bacterial species found in the subgingival plaque. PDLSC were cultured with the following bacterial supernatants: S. mutans, S. anginosus, P. intermedia, F. nucleatum, P. gingivalis and T. denticola. These supernatants were prepared in dilutions of 1:1000, 1:500, 1:300 and 1:50. Using quantitative RT-PCR, gene expression of selected inflammatory cytokines (IL-6, IL-8 and IL-1β) and cell-surface receptors (TLR2, TLR4) showed upregulation of ≈2.0- to 3.0-fold, when exposed to P. intermedia, F. nucleatum, P. gingivalis and T. denticola. However, supernatants did not affect proliferation (MTT) and migration (wound scratch assays) of PDLSC. Next generation RNA sequencing confirmed modified lineage commitment of PDLSC by stimulating chondrogenesis, adipogenesis and inhibition of osteogenesis under P. gingivalis supernatant treatment compared to control. Taken together, this study shows stem cell immunomodulatory response to different periodontal bacteria supernatant and suggests that stem cell transcriptional capacity, migration/proliferation and osteogenesis may differ in the presence of those pathogens. These results bring into question stem cell contribution to periodontal tissue regeneration and onset of inflammation.

LncRNA papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) regulates the proliferation of human periodontal ligament stem cells and toll-like receptor 4 (TLR4) expression to improve periodontitis

BackgroundLncRNA PTCSC3 is a known tumor suppressor, while its roles in other human diseases are unclear.MethodsAll participants were admitted by The second Affiliated Hospital, School of Medicine, Zhejiang University from January 2016 to January 2018. RT-qPCR, Vectors, cell transfection, In vitro cell proliferation assay and western blot were using to carry out the experiment.ResultsIn this study we found that PTCSC3 was downregulated, while toll-like receptor 4 (TLR4) was upregulated in periodontal ligament stem cells (PDLSCs) isolated from periodontitis affected teeth that in PDLSCs isolated from healthy teeth. Expression levels of PTCSC3 and TLR4 were only significantly and inversely correlated in PDLSCs isolated from periodontitis affected teeth but not in PDLSCs isolated from healthy teeth. PTCSC3 overexpression led to the downregulation of TLR4 in PDLSCs isolated from periodontitis affected teeth, while TLR4 overexpression failed to significantly affect PTCSC3. PTCSC3 overexpression also led to the inhibited proliferation of periodontitis-affected PDLSCs.ConclusionsTherefore, lncRNA PTCSC3 may regulate the proliferation of PDLSCs and TLR4 expression to improve periodontitis.

Long non-coding RNAs mortal obligate RNA transcript regulates the proliferation of human periodontal ligament stem cells and affects the recurrence of periodontitis

ObjectiveThe aim of this study is to investigate the role of long non-coding RNAs (lncRNA), mortal obligate RNA transcript (MORT), in human periodontal ligament stem cells. DesignPeriodontal ligament tissues were collected from 48 periodontitis patients underwent tooth extraction and 38 people in similar age and gender distributions who experienced orthodontic treatment. After treatment, periodontitis patients were followed up for 2 years. MORT in periodontal ligament stem cells (PDLSCs) was detected by qRT-PCR. Proliferation of PDLSCs was detected by CCK-8 assay. ResultsThe proliferation rate of PDLSCs isolated from periodontitis-affected teeth was significantly higher than PDLSCs from healthy teeth. Overexpression of MORT inhibited the proliferation of both types of periodontal ligament stem cells. After treatment, periodontitis patients were followed up for 2 years and patients with high level of MORT expression showed relatively lower recurrence rate comparing to low expression group. ConclusionMORT is involved in the proliferation of human PDLSCs and affects the recurrence of periodontitis.