Differentiation Of Periodontal Ligament Cells Research Articles

Calcitonin gene-related peptide regulates periodontal tissue regeneration

Calcitonin gene-related peptide (CGRP), a neuropeptide composed of 37 amino acids secreted from the sensory nerve endings, reportedly possesses various physiological effects, such as vasodilation and neurotransmission. Recently, there have been increasing reports of the involvement of CGRP in bone metabolism; however, its specific role in the pathogenesis of periodontitis, particularly in the repair and healing processes, remains to be elucidated. Therefore, this study aimed to investigate dynamic expression patterns of CGRP during the destruction and regeneration processes of periodontal tissues in a mouse model of experimental periodontitis. We also explored the effects of CGRP on periodontal ligament cells, which can differentiate to hard tissue-forming cells (cementoblasts or osteoblasts). Our findings demonstrated that CGRP stimulation promotes the differentiation of periodontal ligament cells into hard tissue-forming cells. Experimental results using a ligature-induced periodontitis mouse model also suggested fluctuations in CGRP expression during periodontal tissue healing, underscoring the vital role of CGRP signaling in alveolar bone recovery. The study results highlight the important role of nerves in the periodontal ligament not only in sensory reception in the periphery, as previously known, but also in periodontal tissue homeostasis and tissue repair processes.

LRP1-deficient leptin receptor-positive cells in periodontal ligament tissue reduce alveolar bone mass by inhibiting bone formation

ObjectiveLeptin receptor-positive (LepR+) periodontal ligament (PDL) cells play a crucial role in osteogenesis during tooth socket healing and orthodontic tooth movement; however, the factors regulating osteoblast differentiation remain unclear. This study aimed to demonstrate the function of low-density lipoprotein receptor-related protein 1 (LRP1) in alveolar bone formation by examining conditional knockout (cKO) mice lacking LRP1 in LepR+ cells. DesignBone mass and formation were examined via bone morphometric analysis. Bone formation and resorption activities were determined via histochemical staining. Additionally, PDL cells collected from molars were induced to differentiate into osteoblasts with the addition of BMP2 and to mineralize with the addition of osteogenic medium. Osteoblast differentiation of PDL cells was examined by measuring the expression of osteoblast markers. ResultsBone morphometry analysis revealed decreased mineral apposition rate and alveolar bone mass in cKO mice. Additionally, cKO mice showed a decreased number of osterix-positive cells in the PDL. cKO mice had a large number of osteoclasts around the alveolar bone near the root apex and mesial surface of the tooth. In the PDL cells from cKO mice, inhibition of mineralized matrix formation and decreased expression of alkaline phosphatase, osterix, bone sialoprotein, and osteocalcin were observed even when BMP2 was added to the medium. BMP2, BMP4, and osteoprotegerin expression also decreased, but RANKL expression increased dominantly. ConclusionLRP1 in LepR+ cells promotes bone formation by stimulating osteoblast differentiation. Our findings can contribute to clinical research on bone diseases and help elucidate bone metabolism in the periodontal tissue.

LncRNA JHDM1D-AS1 promotes osteogenic differentiation of periodontal ligament cells by targeting miR-532-5p to activate IGF1R signaling.

The aim of this study was to explore the mechanism underlying periodontal ligament cells (PDLCs) osteogenic differentiation. Periodontitis causes damage to tooth-supporting apparatus and eventually leads to tooth loss. PDLCs hold great promise in periodontal regeneration due to their osteogenic features. The expression of osteogenic markers, lncRNA JHDM1D-AS1, miR-532-5p and IGF1R was examined. For osteogenic differentiation, primary human PDLCs (hPDLCs) were cultured in an osteogenic medium, and it was assessed by ALP activity and Alizarin Red staining. The interaction between JHDM1D-AS1, miR-532-5p and IGF1R was analyzed via dual luciferase, RIP and RNA pull-down assays. JHDM1D-AS1 was up-regulated during osteogenic differentiation and its silencing inhibited hPDLC osteogenic differentiation. JHDM1D-AS1 worked as a miR-532-5p sponge in hPDLCs. miR-532-5p directly targeted IGF1R to suppress its expression, and miR-532-5p knockdown facilitated osteogenic differentiation of hPDLCs. Overexpression of IGF1R promoted osteogenic differentiation of hPDLCs via activating Notch/HES1 signaling in hPDLCs. JHDM1D-AS1 promotes osteogenic differentiation of hPDLCs via sponging miR-532-5p to facilitate IGF1R expression and activate Notch/HES1 signaling.

TRPC expression in human periodontal ligament cells and the periodontal tissue of periodontitis mice: a preliminary study

BackgroundTransient receptor potential canonical (TRPC) channels are non-selective cationic channels with permeability to Ca2+ and Na+. Despite their importance, there are currently few studies on TRPC in the periodontal ligament (PDL) and bone cells in the dental field. To provide biological information regarding TRPC in PDL cells and periodontal tissue, we evaluated TRPC channels expression in the osteoblast differentiation of PDL cells and periodontitis-induced tissue. Human PDL cells were cultured in osteogenic differentiation media for 28 days, and the expression of Runx2, osteocalcin (OCN), and TRPC1, 3, 4, and 6 was evaluated by real-time PCR. In ligature-induced periodontitis mice, the alveolar bone and osteoid areas, the osteoclast number, and the expression of Runx2, OCN, TRPC3, and TRPC6 was evaluated by H&E staining, TRAP staining, and immunohistochemistry, respectively.ResultsIn the PDL cell differentiation group, TRPC6 expression peaked on day 7 and TRPC3 expression generally increased during differentiation. During the 28 days of periodontitis progression, alveolar bone loss and osteoclast numbers increased compared to the control group during the experimental period and the osteoid area increased from day 14. TRPC6 expression in the periodontitis group increased in the PDL area and in the osteoblasts compared to the control group, whereas TRPC3 expression increased only in the PDL area on days 7 and 28.ConclusionsThese results indicate changes of TRPC3 and TRPC6 expression in PDL cells that were differentiating into osteoblasts and in periodontitis-induced tissue, suggesting the need for research on the role of TRPC in osteoblast differentiation or periodontitis progression.

The molecular weight of hyaluronic acid influences metabolic activity and osteogenic differentiation of periodontal ligament cells

ObjectiveWhile HA is present naturally in periodontal tissues, its molecular weight can vary widely in vivo. The objective of this study was to directly compare the biological reactions of periodontal ligament cells to four distinct molecular weights of hyaluronic acid (HA).Materials and methodsImmortalized human periodontal ligament cells (PDL-hTERT) were cultured for 21 days in culture medium alone (control) or enriched with osteogenic supplements (OS group). Other 4 experimental groups were cultured in OS medium with the addition of HA with different molecular weights (HMW, MMW, LMW, and ULMW). The cell morphology was examined daily. WST1 assays were performed to evaluate metabolic activity. Von Kossa staining and calcium deposition assay were used to analyze osteogenic differentiation and mineralization.ResultsCell morphology remained unaltered in all groups. Cells stimulated with OS alone or with the addition of hyaluronan showed all the typical microscopic appearance of osteogenic differentiation. Metabolic activity increased in all groups over time. Hyaluronan stimulated greater metabolic activity than the control group, with LMW HA and MMW HA showing the most significant increase. All groups showed mineral deposits and calcium deposition after 21 days of stimulation.ConclusionOur results suggest that hyaluronan can promote metabolic activity and mineralization of PDL-hTERT cells, with LMW HA being the most effective.Clinical relevanceThese results shed light on how the various molecular weight fractions of HA promote tissue regeneration and repair, as well as help to identify an optimal molecular weight range for this application in periodontal tissues.

Activation of periodontal ligament cell cytodifferentiation by juxtacrine signaling from cementoblasts.

New cementum forms from existing cementum during periodontal tissue regeneration, indicating that cementoblasts may interact with progenitor cells in the periodontal ligament to enhance cementogenesis. However, the molecular mechanisms of this process are currently unknown. This study aims to clarify the role of cell-cell interactions between cementoblasts and periodontal ligament cells in differentiation into cementoblasts. To analyze the role of human cementoblast-like cells (HCEMs) on human periodontal ligament cells (HPDLs), we mixed cell suspensions of enhanced green fluorescent protein-tagged HPDLs and HCEMs, and then seeded and cultured them in single wells (direct co-cultures). We sorted co-cultured HPDLs and analyzed their characteristics, including the expression of cementum-related genes. In addition, we cultured HPDLs and HCEMs in a non-contact environment using a culture system composed of an upper insert and a lower well separated by a semi-permeable membrane (indirect co-cultures), and similar analysis was performed. Gene expression of integrin-binding sialoprotein (IBSP) in cementoblasts was confirmed in mouse periodontal tissues. We also investigated the effect of Wingless-type (Wnt) signaling on the differentiation of HPDLs into cementoblasts. Direct co-culture of HPDLs with HCEMs significantly upregulated the expression of cementoblast-related genes in HPDLs, whereas indirect co-culture exerted no effect. Wnt3A stimulation significantly upregulated IBSP expression in HPDLs, whereas inhibition of canonical Wnt signaling suppressed the effects of co-culture. Our results suggest that direct cell interactions with cementoblasts promote periodontal ligament cell differentiation into cementoblasts. Juxtacrine signaling via the canonical Wnt pathway plays a role in this interaction.

CKIP-1 mediates P. gingivalis-suppressed osteogenic/cementogenic differentiation of periodontal ligament cells partially via p38 signaling pathway

ABSTRACT Objectives Casein kinase 2 interacting protein-1 (CKIP-1) is a versatile player involved in various biological processes. However, whether CKIP-1 mediates the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) under Porphyromonas gingivalis (Pg) stimulation remains unknown. Material and Methods The effect of Pg on PDLC differentiation was first verified. CKIP-1 expression in Pg-infected PDLCs or in PDL of apical periodontitis (AP) mice was detected. The changes of CKIP-1 during PDLC differentiation was also determined. PDLC differentiation capacity in CKIP-1 knockout (KO) mice and CKIP-1-silenced PDLCs with or without Pg stimulation were further studied. Inhibitor was finally applied to verify the involvement of p38 signaling pathway in PDLC differentiation. Results The suppression effect of Pg on PDLC differentiation was demonstrated. CKIP-1 increased in the PDL of AP mice and Pg-induced PDLCs, and decreased gradually during PDLC differentiation. Increased OSX and RUNX2 expression in PDL were observed in CKIP-1 KO mice. Also, CKIP-1 silencing facilitated and rescued Pg-inhibited PDLC differentiation. Inhibitor for p38 signaling pathway blocked CKIP-1 silencing-facilitated PDLC differentiation. Conclusions CKIP-1 mediated the osteogenic/cementogenic differentiation of PDLCs partially through p38 signaling pathway, which may provide evidence for the regeneration of periodontal hard tissues damaged by Pg.

Nanosilicate-functionalized nanofibrous membrane facilitated periodontal regeneration potential by harnessing periodontal ligament cell-mediated osteogenesis and immunomodulation

Although various new biomaterials have enriched the methods for periodontal regeneration, their efficacy is still controversial, and the regeneration of damaged support tissue in the periodontium remains challenging. Laponite (LAP) nanosilicate is a layered two-dimensional nanoscale, ultrathin nanomaterial with a unique structure and brilliant biocompatibility and bioactivity. This study aimed to investigate the effects of nanosilicate-incorporated PCL (PCL/LAP) nanofibrous membranes on periodontal ligament cells (PDLCs) in vitro and periodontal regeneration in vivo. A PCL/LAP nanofibrous membrane was fabricated by an electrospinning method. The characterization of PCL/LAP nanofibrous membrane were determined by scanning electron microscopy (SEM), energy dispersive spectrum of X-ray (EDS), inductively coupled plasma mass spectrometry (ICP-MS) and tensile test. The proliferation and osteogenic differentiation of PDLCs on the PCL/LAP nanofibrous membrane were evaluated. A PDLCs and macrophage coculture system was used to explore the immunomodulatory effects of the PCL/LAP nanofibrous membrane. PCL/LAP nanofibrous membrane was implanted into rat calvarial and periodontal defects, and the regenerative potential was evaluated by microcomputed topography (micro-CT) and histological analysis. The PCL/LAP nanofibrous membrane showed good biocompatibility and bioactivity. It enhanced the proliferation and osteogenic differentiation of PDLCs. The PCL/LAP nanofibrous membrane also stimulated anti-inflammatory and pro-remodeling N2 neutrophil formation, regulated inflammatory responses and induced M2 macrophage polarization by orchestrating the immunomodulatory effects of PDLCs. The PCL/LAP nanofibrous membrane promoted rat calvarial defect repair and periodontal regeneration in vivo. LAP nanosilicate-incorporated PCL membrane is capable of mediating osteogenesis and immunomodulation of PDLCs in vitro and accelerating periodontal regeneration in vivo. It could be a promising biomaterial for periodontal regeneration therapy.

Solution NMR structure of cementum protein 1 derived peptide (CEMP1-p1) and its role in the mineralization process.

We report the characterization of the three-dimensional structure of the CEMP1-p1 peptide [MGTSSTDSQQAQHRRCSTSN: corresponding to residues 1-20 of the N-terminus of cementum protein 1 (CEMP1)]. This peptide imitates the capacity of CEMP1 to stimulate hydroxyapatite (HA) crystal nucleation and growth, and promotes the differentiation of periodontal ligament cells into a cementoblastic phenotype. Additionally, in experimental models of critical-sized calvarial defects in Wistar rats, CEMP1-p1 has shown osteogenic properties that enhanced the physiological deposition and maturation of newly formed bone. In this work, studies of CEMP1-p1 by circular dichroism (CD) and nuclear magnetic resonance (NMR) were performed in trifluoroethanol D2 (TFED2) and aqueous solution to determine the 3D structure of the peptide. Using the 3D model, experimental data from HA crystals formation and calcium fluorescence emission, we explain the biological mechanisms involved in CEMP1-p1 activity to promote calcium recruitment and its affinity to HA crystals. This information is valuable because it proposes, for the first time, a plausible molecular mechanism during the mineralization process, from a specific cementum protein-derived peptide.

Intracellular glucose starvation inhibits osteogenic differentiation in human periodontal ligament cells

Periodontal ligament cells (PDLCs), as mesenchymal cells in the oral cavity, are closely linked to periodontal tissue regeneration. However, the effect of local glucose deficiency on periodontal tissue regeneration, such as immediately post-surgery, remains unknown. In the present study, we investigated the effect of a low-glucose environment on the proliferation and osteogenic differentiation of PDLCs. We used media with five glucose concentrations (100, 75, 50, 25, and 0mg/dL) and focused on the effects of a low-glucose environment on the proliferation, osteogenic differentiation, and autophagy of PDLCs. Additionally, we focused on changes in lactate production in a low-glucose environment and investigated the involvement of lactate with AZD3965, a monocarboxylate transporter-1 (MCT-1) inhibitor. The low-glucose environment inhibited PDLCs proliferation, migration, and osteogenic differentiation, and induced the expression of the autophagy-related factors LC3 and p62. Lactate and ATP production were decreased under low-glucose conditions. The addition of AZD3965 (MCT-1 inhibitor) in normal glucose conditions caused a similar trend as in low-glucose conditions on PDLCs. Our results suggest lactate production through glucose metabolism in the osteogenic differentiation of PDLCs. A low-glucose environment decreased lactate production, inhibiting cell proliferation, migration, and osteogenic differentiation and inducing autophagy in PDLCs.

3D-printed mesoporous bioactive glass/GelMA biomimetic scaffolds for osteogenic/cementogenic differentiation of periodontal ligament cells.

Integrated regeneration of periodontal tissues remains a challenge in current clinical applications. Due to the tunable physical characteristics and the precise control of the scaffold microarchitecture, three-dimensionally (3D) printed gelatin methacryloyl (GelMA)-based scaffold has emerged as a promising strategy for periodontal tissue regeneration. However, the optimization of the printing biomaterial links the formulation and the relationship between the composition and structures of the printed scaffolds and their comprehensive properties (e.g. mechanical strength, degradation, and biological behaviors) remains unclear. Here, in this work, a novel mesoporous bioactive glass (BG)/GelMA biomimetic scaffold with a large pore size (∼300μm) was developed by extrusion-based 3D printing. Our results showed that the incorporation of mesoporous bioactive glass nanoparticles (BG NPs) significantly improved shape fidelity, surface roughness, and bioactivity of 3D-printed macroporous GelMA scaffolds, resulting in the enhanced effects on cell attachment and promoting osteogenic/cementogenic differentiation in human periodontal ligament cells. The excellent maintenance of the macropore structure, the visibly improved cells spreading, the release of bioactive ions (Si4+, Ca2+), the upregulation of gene expressions of osteogenesis and cementogensis, and the increase in alkaline phosphatase (ALP) activity and calcium nodules suggested that BG NPs could endow GelMA-based scaffolds with excellent structural stability and the ability to promote osteogenic/cementogenic differentiation. Our findings demonstrated the great potential of the newly formulated biomaterial inks and biomimetic BG/GelMA scaffolds for being used in periodontal tissue regeneration and provide important insights into the understanding of cell-scaffold interaction in promoting the regeneration of functional periodontal tissues.

CircSKIL promotes osteoblastic differentiation of periodontal ligament cells by sponging miR-532-5p to activate Notch signaling.

Periodontal ligament cells (PDLCs) possess the capacity to differentiate into a variety of cell types to benefit periodontal regeneration. In this study, we examined the circSKIL/miR-532-5p/Notch1 axis in controlling the osteoblastic differentiation of PDLCs. Primary human PDLCs (hPDLCs) were isolated and induced to differentiate into osteoblasts. Osteogenic responses were assessed for the expressions of osteoblast-related marker proteins (including alkaline phosphatase (ALP), osteocalcin (OCN), bone morphogenetic protein-2 (BMP2), and runt-related transcription factor 2 (RUNX2) by RT-PCR. The formation of mineralized nodules was examined by Alizarin Red S (ARS) staining and ALP activity. Expressions of circSKIL, miR-532-5p, and Notch1 were measured by RT-PCR and western blotting, and their regulations by combining bioinformatic analysis and luciferase reporter assay. Notch signaling was assessed for the expressions of hairy and enhancer of split-1 (HES1) and Notch intracellular domain (NICD). During osteoblastic differentiation of hPDLCs, circSKIL, and Notch1 were up-regulated, while miR-532-5p down-regulated. By sponging miR-532-5p, circSKIL activated Notch signaling, increasing levels of Notch1, HES1, and NICD. Functionally, knocking down circSKIL or overexpressing miR-532-5p inhibited osteoblastic differentiation of PDLCs, down-regulating ALP, OCN, BMP2, and RUNX2, and reducing ARS staining or ALP activity. The impacts of circSKIL knockdown were rescued by miR-532-5p inhibitor or overexpressing Notch1, while those caused by up-regulating miR-532-5p were reversed by overexpressing Notch1. By targeting miR-532-5p and up-regulating Notch1, circSKIL critically controls osteoblastic differentiation of hPDLCs. Therefore, modulating this axis may maximize the differentiation of PDLCs into osteoblasts and benefit periodontal regeneration.

Circ_0099630 Participates in SPRY1-Mediated Repression in Periodontitis

BackgroundPeriodontitis is chronic inflammation that causes damage to periodontal tissues and cementum. It has been reported that circular RNA hsa_circ_0099630 (circ_0099630) was overexpressed in gingival samples from patients with periodontitis. However, the function of circ_0099630 on the osteogenic differentiation of periodontal ligament cells (PDLCs) in periodontitis remains unclear. MethodsPeriodontal ligaments from patients with periodontitis and third molars (termed wisdom teeth) were utilised to isolate inflamed PDLCs (iPDLCs) and healthy PDLCs (hPDLCs). Expression levels of circ_0099630 in isolated PDLCs were assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Effects of circ_0099630 overexpression and silencing on iPDLC viability, proliferation, and cycle progression were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2’-deoxyuridine (EdU), and flow cytometry assays. The osteogenic differentiation was detected by analysing the alkaline phosphatase (ALP) activity, mineralisation amount, and osteogenic markers osterix (OSX), ALP, and RUNX2 in iPDLCs. The regulatory mechanism of circ_0099630 was predicted by bioinformatics analysis and validated by dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. ResultsCirc_0099630 was underexpressed in iPDLCs compared to hPDLCs. Overexpression of circ_0099630 repressed iPDLC proliferation and osteogenic differentiation, but circ_0099630 silencing exerted an opposing effect. Mechanically, circ_0099630 sponged miR-212-5p to block the inhibiting effect of miR-212-5p on SPRY1. Elevated expression of SPRY1 partly reversed the promoting effect of circ_0099630 knockdown on iPDLC proliferation and osteogenic differentiation. ConclusionsCirc_0099630 curbed PDLC proliferation and osteogenic differentiation through elevating SPRY1 expression via sponging miR-212-5p in periodontitis.

Potential donor-dependent regulative effects of endogenous sclerostin expression and mineralization potential in primary human PDL cells in vitro

ObjectivesThe glycoprotein sclerostin is mostly expressed in osteocytes and plays a central role in human bone metabolism. However, sclerostin and the corresponding SOST gene have been found in periodontal ligament cells under mineralizing conditions as well. The present study aimed to investigate, whether there was a correlation between endogenous SOST expression, the corresponding gene, and mineralization potential in human periodontal ligament cells and to identify different sclerostin expression and secretion patterns in cells derived from individual donors. Material and methodsPrimary human periodontal ligament cells of three different donors were cultivated under control or mineralizing conditions for 6, 13, 15 and 18 days, respectively. Calcium deposits were stained with alizarin red and quantified afterwards. Quantitative expression analysis of the SOST gene encoding sclerostin was performed using quantitative reverse transcription polymerase chain reaction (RT-PCR). Additionally, intracellular sclerostin expression was analyzed using Western blotting and extracellular sclerostin secretion was quantified using Enzyme-linked Immunosorbent Assay (ELISA). ResultsAlizarin red staining identified calcium deposits in periodontal ligament cells under mineralizing conditions beginning from day 13, relative SOST expression occurred on day 6. Whereas staining continued to increase in donor 1 on day 15, it remained stable in donors 2 and 3. Conversely, baseline SOST expression was significantly lower in donor 1 compared to donors 2 and 3. Western blotting and ELISA revealed increased intra- and extracellular sclerostin expression at day 13 under mineralizing conditions. Donor 3 exhibited the highest overall sclerostin levels. ConclusionsOur data emphasize donor-specific characteristics in differentiation potential and sclerostin expression patterns in primary human periodontal ligament cells. Sclerostin might play a central role in modulating osteogenic differentiation in periodontal ligament cells as part of a negative feedback mechanism in avoiding excessive mineralization.

A vitronectin-derived peptide prevents and restores alveolar bone loss by modulating bone re-modelling and expression of RANKL and IL-17A.

This study investigated whether a vitronectin-derived peptide (VnP-16) prevents and/or reverses alveolar bone resorption induced by ligature-induced periodontitis in rodents and identified the underlying mechanism. We evaluated the effects of VnP-16 on osteogenic differentiation in human periodontal ligament cells (hPDLCs), lipopolysaccharide-induced inflammatory responses in gingival fibroblasts, and immune response in T lymphocytes. Ligature-induced periodontitis was induced by ligating the bilateral mandibular first molars for 14 days in rats and for 7 days in mice (n= 10/group). VnP-16 (100 μg/10μl) was applied topically into the gingival sulcus of rats via intra-sulcular injection, whereas the peptide (50 μg/5μl) was administered directly into the gingiva of mice via intra-gingival injection. To evaluate the preventive and therapeutic effects of VnP-16, micro-computed tomography analysis and histological staining were then performed. VnP-16 promoted osteogenic differentiation of periodontal ligament cells and inhibited the production of lipopolysaccharide-induced inflammatory mediators in gingival fibroblasts. Concomitantly, VnP-16 modulated the host immune response by reducing the number of receptor activator of NF-κB ligand (RANKL)-expressing lipopolysaccharide-stimulated CD4+ and CD8+ T cells, and by suppressing RANKL and interleukin (IL)-17A production. Furthermore, local administration of VnP-16 in rats and mice significantly prevented and reversed alveolar bone loss induced by ligature-induced periodontitis. VnP-16 enhanced osteoblastogenesis and simultaneously inhibited osteoclastogenesis and suppressed RANKL and IL-17A expression in vivo. Our findings suggest that VnP-16 acts as a potent therapeutic agent for preventing and treating periodontitis by regulating bone re-modelling and immune and inflammatory responses.

Expression analysis of Wnt signaling pathway related lncRNAs in periodontitis: A pilot case-control study

LncRNAs are involved in the modulation of several signaling pathways which have a crucial effect in the differentiation of periodontal ligament cells and the induction of cementum regeneration. Autophagy and Wnt signaling are two important pathways with a wide range of interrelationships associated with periodontitis. We chose four lncRNAs based on their potential interaction with these two pathways. We examined the expression of FOXD2-AS1, NNT-AS1, GAS8-AS1, and CCAT1 lncRNAs in tissues and blood specimens of patients with periodontitis and unaffected controls using qRT-PCR. Expression amounts of FOXD2-AS1 were lower in blood of cases compared with controls (relative expression (RE) = 0.08, P value<0.001). Tissue expression of FOXD2-AS1 was similar between cases and controls. Difference in blood levels of FOXD2-AS1 was significant among female subjects (RE = 0.07, P-value = 0.01), but not among male subjects. In addition, we reported lower levels of NNT-AS1 in blood samples of cases versus controls (RE = 0.25, P value = 0.05). But such a pattern was not seen in tissue samples. Expression of GAS8-AS1 was lower in both tissue and blood specimens of patients compared with control (RE = 0.49, P value = 0.02 and RE = 0.32, P value = 0.03 for tissue and blood specimens, respectively). Moreover, the differences in blood expression of GAS8-AS1 were significant in female patients compared with female controls (RE = 0.25, P-value = 0.04). Expression of CCAT1 was significantly lower in both gingival tissue and blood samples of patients compared with control (RE for tissue samples = 0.36, P-value = 0.02 and RE for blood samples = 0.33, P-value = 0.02). This pattern of expression was also detected in tissue specimens obtained from male persons (RE = 0.23, P value = 0.02) and blood samples acquired from female patients compared with corresponding control samples (RE = 0.21, P value<0.001). FOXD2-AS1 showed the best AUC values for distinguishing periodontitis in blood samples (AUC = 0.78). Combination of transcript levels of all four lncRNAs increased the diagnostic power to 0.81 and 0.74 in the gingival tissues and blood specimens, respectively. Our study demonstrates dysregulation of several lncRNAs in the periodontitis. These transcripts might partake in the pathogenesis of periodontitis and could have a possible correlation with autophagy and Wnt signaling pathways.

Comparative Effects of Concentrated Growth Factors on the Biological Characteristics of Periodontal Ligament Cells and Stem Cells from Apical Papilla

IntroductionDuring cell-free regenerative endodontic therapy, both stem cells from apical papilla (SCAPs) and periodontal ligament cells (PDLCs) are possible cell sources because of their proximity. Nonetheless, the regenerative ability of PDLCs and SCAPs under the induction of concentrated growth factors (CGFs) remains unclear. MethodsPDLCs and SCAPs were treated with various concentrations of CGF-conditioned medium (CCM). The effects of CCM with or without Porphyromonas gingivalis lipopolysaccharide (LPS) on cell migration, odonto/osteogenic differentiation, and the expression of inflammatory cytokines were assessed. Dentin matrix transplants composed of PDLCs or SCAPs cell sheets coupled with CGF were put subcutaneously in immunocompromised mice for 8 weeks to explore their regenerative characteristics in vivo. ResultsCCM dose dependently enhanced the migration, proliferation, and odonto/osteogenic differentiation of PDLCs and SCAPs. CCM alleviated LPS-inhibited odonto/osteogenic differentiation of PDLCs and SCAPs as well as the LPS-induced up-regulation of inflammatory cytokines. In vivo, the newly regenerated tissue and microvessels formed by PDLCs and SCAPs were significantly increased under the induction of CGF. SCAPs mainly regenerated pulp/dentinlike tissues and a large number of microvessels, whereas PDLCs mainly formed bone/cementumlike structures. ConclusionsOverall, PDLCs excelled in cell proliferation, migration, and osteogenic differentiation, whereas SCAPs outperformed PDLCs in terms of angiogenic and odontogenic differentiation. The biological differences between PDLCs and SCAPs provided a possible theoretical basis for the formation of bone/cementum/periodontal ligament–like tissues after cell-free regenerative endodontic therapy.

MicroRNA-203 mediates Porphyromonas gingivalis LPS-induced inflammation and differentiation of periodontal ligament cells.

In this study, we aimed to explore the effects of microRNA-203 (miR-203) on Porphyromonas gingivalis lipopolysaccharide (P.g. LPS)-stimulated periodontal ligament cells (PDLCs) and identify potential molecular targets for periodontitis treatment. Periodontal ligament cells were stimulated by P.g. LPS, followed by quantification of miR-203 and AP-1 expression. Next, loss- and gain-of-function experiments were applied in P.g. LPS-induced PDLCs. The proliferation, apoptosis, and differentiation of PDLCs were determined, and mineralized nodule numbers were counted. Functional assays were used to identify interactions among miR-203, activator protein-1 (AP-1), and intercellular adhesion molecule-1 (ICAM-1). In addition, expression of osteogenesis-related genes and release of proinflammatory factors were analyzed. miR-203 was found to be downregulated while AP-1 was upregulated in PDLCs stimulated by P.g. LPS. The overexpression of miR-203 promoted P.g. LPS-stimulated PDLC proliferation and differentiation, inhibited apoptosis, and increased the number of mineralized nodules. miR-203 was verified to downregulate AP-1/ICAM-1 axis. miR-203 overexpression reduced the secretion of proinflammatory factors while increasing the expression of osteogenesis-related genes in P.g. LPS-stimulated PDLCs, which was reversed by overexpressing AP-1 and ICAM-1. These experimental data demonstrated the potential inhibitory effects of overexpressed miR-203 on periodontitis development by promoting PDLC differentiation and suppressing inflammatory responses through AP-1/ICAM-1 axis.

Circ_0138959/miR-495-3p/TRAF6 axis regulates proliferation, wound healing and osteoblastic differentiation of periodontal ligament cells in periodontitis

Background/purposePeriodontitis is a chronic inflammatory disease, and periodontal ligament cells (PDLCs) are pivotal for osteogenesis. Circular RNAs (circRNAs) can regulate disease progression via targeting miRNA/mRNA axis. The purposes of this study were to explore the function and mechanism of circ_0138959 in periodontitis. Materials and methodsPeriodontitis cell model was established by lipopolysaccharide (LPS) treatment in PDLCs. RNA expression was determined by quantitative reverse transcription-polymerase chain reaction assay. Cell proliferation was detected using 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide assay. Wound healing and cell apoptosis were examined by wound healing assay and flow cytometry. Inflammatory cytokines were measured via Enzyme-linked immunosorbent assay. Osteogenic differentiation was assessed by Alkaline phosphatase and Alizarin red S staining assays. Western blot was used for protein detection. The target interaction was validated by dual-luciferase reporter assay. ResultsCirc_0138959 was overexpressed in periodontitis tissues and LPS-treated PDLCs. Downregulation of circ_0138959 attenuated LPS-induced inhibition of proliferation, wound healing and osteogenic differentiation but promotion of apoptosis and inflammation. Circ_0138959 acted as a miR-495-3p sponge, and the regulatory role of circ_0138959 in LPS-induced cell injury was achieved by sponging miR-495-3p. Additionally, miR-495-3p targeted TNF Receptor Associated Factor 6 (TRAF6) and miR-495-3p protected against LPS-induced cell dysfunction by targeting TRAF6. Circ_0138959 upregulated TRAF6 level via inhibiting miR-495-3p. ConclusionThis study suggested that circ_0138959 upregulated the TRAF6 expression by binding to miR-495-3p, consequently aggravating LPS-induced cell damages in PDLCs. Circ_0138959 might be a probable target for treatment of periodontitis.

The Expression and Regulatory Roles of Long Non-Coding RNAs in Periodontal Ligament Cells: A Systematic Review.

Periodontal ligament (PDL) cells play a pivotal role in periodontal and bone homeostasis and have promising potential for regenerative medicine and tissue engineering. There is compelling evidence that long non-coding RNAs (lncRNAs) are differentially expressed in PDL cells compared to other cell types and that these lncRNAs are involved in a variety of biological processes. This study systematically reviews the current evidence regarding the expression and regulatory functions of lncRNAs in PDL cells during various biological processes. A systematic search was conducted on PubMed, the Web of Science, Embase, and Google Scholar to include articles published up to 1 July 2021. Original research articles that investigated the expression or regulation of lncRNAs in PDL cells were selected and evaluated for a systematic review. Fifty studies were ultimately included, based on our eligibility criteria. Thirteen of these studies broadly explored the expression profiles of lncRNAs in PDL cells using microarray or RNA sequencing. Nineteen studies investigated the mechanisms by which lncRNAs regulate osteogenic differentiation in PDL cells. The remaining 18 studies investigated the mechanism by which lncRNAs regulate the responses of PDL cells to various stimuli, namely, lipopolysaccharide-induced inflammation, tumor necrosis factor alpha-induced inflammation, mechanical stress, oxidative stress, or hypoxia. We systematically reviewed studies on the expression and regulatory roles of lncRNAs in diverse biological processes in PDL cells, including osteogenic differentiation and cellular responses to inflammation, mechanical stress, and other stimuli. These results provide new insights that may guide the development of lncRNA-based therapeutics for periodontal and bone regeneration.