GDF15 Modulates the Zoledronic-Acid-Induced Hyperinflammatory Mechanoresponse of Periodontal Ligament Fibroblasts.

Orthodontic tooth movement (OTM) is thought to be impeded by bisphosphonate (BP) therapy, mainly due to increased osteoclast apoptosis and changes in the periodontal ligament (PdL), a connecting tissue between the alveolar bone and teeth. PdL cells, mainly fibroblasts (PdLFs), are crucial regulators in OTM by modulating force-induced local inflammatory processes. Recently, we identified the TGF-ß/BMP superfamily member GDF15 as an important modulator in OTM, promoting the pro-inflammatory mechanoresponses of PdLFs. The precise impact of the highly potent BP zoledronate (ZOL) on the mechanofunctionality of PdLFs is still under-investigated. Therefore, the aim of this study was to further characterize the ZOL-induced changes in the initial inflammatory mechanoresponse of human PdLFs (hPdLFs) and to further clarify a potential interrelationship with GDF15 signaling. Thus, two-day in vitro treatment with 0.5 µM, 5 µM and 50 µM of ZOL altered the cellular properties of hPdLFs partially in a concentration-dependent manner. In particular, exposure to ZOL decreased their metabolic activity, the proliferation rate, detected using Ki-67 immunofluorescent staining, and survival, analyzed using trypan blue. An increasing occurrence of DNA strand breaks was observed using TUNEL and an activated DNA damage response was demonstrated using H2A.X (phosphoS139) staining. While the osteogenic differentiation of hPdLFs was unaffected by ZOL, increased cellular senescence was observed using enhanced p21Waf1/Cip1/Sdi1 and ß-galactosidase staining. In addition, cytokine-encoding genes such as IL6, IL8, COX2 and GDF15, which are associated with a senescence-associated secretory phenotype, were up-regulated by ZOL. Subsequently, this change in the hPdLF phenotype promoted a hyperinflammatory response to applied compressive forces with an increased expression of the pro-inflammatory markers IL1ß, IL6 and GDF15, as well as the activation of monocytic THP1 cells. GDF15 appeared to be particularly relevant to these changes, as siRNA-mediated down-regulation balanced these hyperinflammatory responses by reducing IL-1ß and IL-6 expression (IL1B p-value < 0.0001; IL6 p-value < 0.001) and secretion (IL-1ß p-value < 0.05; IL-6 p-value < 0.001), as well as immune cell activation (p-value < 0.0001). In addition, ZOL-related reduced RANKL/OPG values and inhibited osteoclast activation were enhanced in GDF15-deficient hPdLFs (both p-values < 0.0001; all statistical tests: one-way ANOVA, Tukey's post hoc test). Thus, GDF15 may become a promising new target in the personalized orthodontic treatment of bisphosphonatepatients.

High-Glucose Media Reduced the Viability and Induced Differential Pro-Inflammatory Cytokines in Human Periodontal Ligament Fibroblasts.

Hyperglycemic condition in diabetic patients tends to exacerbate periodontitis severity. Thus, the influence of hyperglycemia on the biological and inflammatory response of periodontal ligament fibroblasts (PDLFs) needs to be elucidated. In this study, PDLFs were seeded in media containing glucose concentrations (5.5, 25, or 50 mM) and stimulated with 1 µg/mL of lipopolysaccharide (LPS). PDLFs' viability, cytotoxicity, and the migration ability were determined. The mRNA expression of Interleukin (IL)-6, IL-10, and IL-23 (p19/p40), and Toll-like receptor (TLR)-4 were analyzed; at 6 and 24 h, protein expression of IL-6 and IL-10 was also determined. PDLFs grown in 50 mM glucose medium showed lower viability. The 5.5 mM glucose led to the highest percentage of wound closure compared to 25 mM and 50 mM glucose with/without LPS. Additionally, 50 mM glucose with LPS exhibited the least migration ability among all groups. The expression of IL-6 was amplified significantly in LPS-stimulated cells in 50 mM glucose medium. IL-10 was constitutively expressed in different glucose concentrations, and LPS stimulation decreased it. IL-23 p40 was up-regulated after LPS stimulation in 50 mM glucose concentration. TLR-4 was highly expressed after LPS stimulation in all glucose concentrations. Hyperglycemic conditions limit PDLF proliferation and migration, and enhance the expression of certain pro-inflammatory cytokines to induce periodontitis.

Metformin combats high glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells via inhibition of the NPR3-mediated MAPK pathway.

BACKGROUND: High glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) has long been a challenge to periodontal regeneration for diabetic individuals. Metformin is an anti-hyperglycemic drug that exhibits abundant biological activities associated with cell metabolism and downstream tissue regeneration. However, how metformin combats damage to PDLSC osteogenic differentiation under high glucose and the underlying mechanisms remain unknown. METHODS: Osteogenic differentiation of PDLSCs was assessed by alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red staining and quantitative assay, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. RNA-seq analysis was performed to screen target genes of metformin, and the effects of target genes were confirmed using lentivirus transfection. Western blot analysis was also used to detect the protein level of underlying signaling pathways. RESULTS: We found that osteogenic differentiation of PDLSCs under high glucose was decreased, and metformin addition enhanced this capacity of differentiation. Furthermore, the results of RNA-seq analysis showed that natriuretic peptide receptor 3 (NPR3) was upregulated in PDLSCs under high glucose and downregulated after metformin addition. When the underlying pathways involved were investigated, we found that upregulation of NPR3 can compromise the metformin-enhanced PDLSC osteogenic differentiation and activate the MAPK pathway (especially the p38 MAPK and Erk1/2 pathway), and that inhibition of the NPR3-mediated p38 MAPK or Erk1/2 pathway enhanced the osteogenic differentiation of PDLSCs under high glucose. CONCLUSIONS: The present study suggests that metformin may enhance the osteogenic differentiation of PDLSCs under high glucose via downregulation of NPR3 and inhibition of its downstream MAPK pathway. This is the first report identifying the involvement of NPR3-mediated MAPK pathway in the metformin-enhanced osteogenic differentiation, indicating that NPR3 antagonists, such as metformin, may be feasible therapeutics for periodontal tissue regeneration in diabetic individuals.

LiCl-induced immunomodulatory periodontal regeneration via the activation of the Wnt/ß-catenin signaling pathway.

BACKGROUND: Growing evidence suggests that excessive inflammation hampers the regenerative capacity of periodontal ligament cells (PDLCs) and that activation of the Wnt/ß-catenin pathway is crucial in suppressing immune dysregulation. OBJECTIVE: This study aimed to establish the role of the Wnt/ß-catenin in regulating the immune microenvironment and its subsequent impact on periodontal regeneration. METHODS: Lithium chloride (LiCl, Wnt activator) was administered daily into the standard periodontal defects created in 12-week-old Lewis rats. Harvested at 1-week and 2-week post-surgery, samples were then subjected to histological and immunohistochemical evaluation of macrophage distribution and phenotype (pro-inflammatory M1 and anti-inflammatory M2). A murine macrophage cell line, RAW 264.7, was stimulated with LiCl to activate Wnt/ß-catenin. Following treatment with the conditioned medium derived from the LiCl-activated macrophages, the expression of bone- and cementum-related markers of the PDLCs was determined. The involvement of Wnt/ß-catenin in the immunoregulation and autophagic activity was further investigated with the addition of cardamonin, a commercially available Wnt inhibitor. RESULTS: A significantly increased number of macrophages were detected around the defects during early healing upon receiving the Wnt/ß-catenin signaling cue. The defect sites in week 2 exhibited fewer M1 and more M2 macrophages along with an enhanced regeneration of alveolar bone and cementum in the Wnt/ß-catenin activation group. LiCl-induced immunomodulatory effect was accompanied with the activation Wnt/ß-catenin signaling, which was suppressed in the presence of Wnt inhibitor. Exposure to LiCl could induce autophagy in a dose-dependent manner, thus maintaining macrophages in a regulatory state. The expression level of bone- and cementum-related markers was significantly elevated in PDLCs stimulated with LiCl-activated macrophages. CONCLUSION: The application of Wnt activator LiCl facilitates the recruitment of macrophages to defect sites and regulates their phenotypic switching in favor of periodontal regeneration. Suppression of Wnt/ß-catenin pathway could attenuate the LiCl-induced immunomodulatory effect. Taken together, the Wnt/ß-catenin pathway may be targeted for therapeutic interventions in periodontal diseases.

Role of cytochrome P450 2C8 genetic polymorphism and epoxygenase uncoupling in periodontal remodelling affecting orthodontic treatment.

In genome-wide association studies, the CYP2C8 gene locus has been reported to be associated with bisphosphonate-related osteonecrosis of the jaw, a severe devastating side effect of antiresorptive bone treatment. The aim of this study was to elucidate the putative pathomechanism explaining the association between the genetic polymorphism with the alleles CYP2C8*2 and *3 causing low CYP2C8 activity, and disturbed periodontal remodelling in periodontal fibroblasts cultured from patients undergoing orthodontic treatment. CYP2C8 activity, enzyme expression and substrate metabolism were detected in human periodontal fibroblast cultures. Zoledronic acid caused enhanced reactive oxygen species (ROS) production in periodontal fibroblasts, which was enhanced by arachidonic acid as inflammatory signal. Enhanced bisphosphonate-induced uncoupling of the CYP2C8 enzyme was detected in the variant allele (CYP2C8*3) with the result of increased H2 O2 production and lowered substrate oxidation. Conversely, substrate (amodiaquine) addition led to decreased H2 O2 production in isolated CYP2C8 enzymes, but in CYP2C8*3 enzyme, increased H2 O2 was still detected, especially in presence of arachidonic acid. CYP2C8 variants leading to decreased enzyme activity in substrate oxidation may enhance ROS production by reaction uncoupling, and thus, contribute to difficulties in orthodontic treatment and the risk of side effects of antiresorptive drugs.

miR-23b mediates TNF-α-Inhibited Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting Runx2.

Periodontitis is the most prevalent oral infection disease, which causes the destruction of periodontal supporting tissues and eventual tooth loss. This study aimed to investigate the molecular mechanism of miRNA-23b (miR-23b) in regulating the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in an inflammatory environment. Results revealed that tumor necrosis factor-α (TNF-α), a notoriously inflammatory cytokine, remarkably attenuated the osteogenic differentiation of hPDLSCs, which were partially rescued by SKL2001 (Wnt/ß-catenin agonist). We further explored the underlying roles of miRNAs involved in TNF-α-inhibited osteogenesis of hPDLSCs. The miR-23b significantly increased with TNF-α stimulation, which was abolished by SKL2001. Similar to the effect of TNF-α, miR-23b agonist (agomir-23b) dramatically reduced the expression of runt-related transcription factor 2 (Runx2) and suppressed the osteogenic differentiation of hPDLSCs. The inhibition of miR-23b significantly increased Runx2, which is the major transcription factor during osteogenesis, thereby indicating that miR-23b was an endogenous regulator of Runx2 in hPDLSCs. Bioinformatic analysis and dual luciferase reporter assays confirmed that Runx2 was a target gene of miR-23b. Furthermore, the gain function assay of Runx2 revealed that the Runx2 overexpression efficiently reversed the suppression of the osteogenic differentiation of hPDLSCs with miR-23b agonist, suggesting that the suppressing effect of miR-23b on osteogenesis was mediated by Runx2 inhibition. Our study clarified that miR-23b mediated the TNF-α-inhibited osteogenic differentiation of hPDLSCs by targeting Runx2. Therefore, the expanded function of miR-23b in the osteogenesis of hPDLSCs under inflammatory conditions. This study might provide new insights and a novel therapeutic target for periodontitis.

Mineral trioxide aggregate immersed in sodium hypochlorite reduce the osteoblastic differentiation of human periodontal ligament stem cells.

White mineral trioxide aggregate (WMTA) is a root canal treatment material, which is known to exhibit a dark brown color when in contact with sodium hypochlorite solution (NaOCl). This study aimed to investigate the effects of NaOCl on the surface properties of WMTA discs and WMTA-induced osteoblastic differentiation of periodontal ligament stem cells (PDLSCs). Mixed WMTA (ProRoot MTA) was filled into the molds to form WMTA discs. These discs were immersed in distilled water (D-WMTA) or 5% NaOCl (Na-WMTA). Their surface structures and Ca2+ release level was investigated. Moreover, they were cultured with a clonal human PDLSC line (line 1-17 cells). The main crystal structures of Na-WMTA were identical to the structures of D-WMTA. Globular aggregates with polygonal and needle-like crystals were found on D-WMTA and Na-WMTA, which included Ca, Si, Al, C and O. However, many amorphous structures were also identified on Na-WMTA. These structures consisted of Na and Cl, but did not include Ca. NaOCl immersion also reduced Ca2+ release level from whole WMTA discs. Line 1-17 cells cultured with D-WMTA formed many mineralized nodules and exhibited high expression levels of osteoblast-related genes. However, cells incubated with Na-WMTA generated a small number of nodules and showed low expression levels of osteoblast-related genes. These results indicated that NaOCl reduced Ca2+ release from WMTA by generating amorphous structures and changing its elemental distribution. NaOCl may also partially abolish the ability of WMTA to stimulate osteoblastic differentiation of PDLSCs.

Thrombin-activated platelet-rich plasma enhances osteogenic differentiation of human periodontal ligament stem cells by activating SIRT1-mediated autophagy.

BACKGROUND: Platelet-rich plasma (PRP) has the potential to be used for bone regeneration. However, its effect on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its effect on cell autophagy of hPDLSCs remain unknown. In this study, we investigated the effects of PRP on cell viability and osteogenic differentiation of hPDLSCs and the underlying molecular mechanisms. METHODS: hPDLSCs were isolated and identified by morphology and flow cytometry analysis. Next, thrombin-activated PRP was used to stimulate hPDLSCs. The MTT assay was used to analyze cell viability. Osteogenic differentiation was investigated using alkaline phosphatase (ALP) activity assay, alizarin red S (ARS) staining, and gene expression analysis of osteogenic markers. Expression of the autophagic proteins was determined using western blotting. RESULTS: Thrombin-activated PRP significantly enhanced cell viability, ALP activity, osteogenic-related mRNA levels and alizarin red-mineralization activity in hPDLSCs in a dose-dependent manner. Furthermore, activated PRP dose-dependently increased LC3-II/I ratio and the expression of SIRT1 and Beclin-1. PRP treatment also enhanced the autophagic flux. It was also demonstrated that the inhibition of SIRT1 using sirtinol or suppression of autophagy by 3-methyladenine (3-MA) abrogated PRP-induced viability and osteogenic differentiation of hPDLSCs. CONCLUSION: Our study suggested that thrombin-activated PRP accelerated the viability and osteogenic differentiation of hPDLSCs via SIRT1-mediated autophagy induction.

Betulinic acid promotes the osteogenic differentiation of human periodontal ligament stem cells by upregulating EGR1.

Periodontitis is one of the most common chronic inflammations of the oral cavity, which eventually leads to tooth loss. Betulinic acid (BetA) is an organic acid that has anti-inflammatory effects and is derived from fruits and plants, but its effect on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is still unclear. This study aimed to explore the effect of BetA on the osteogenic differentiation of hPDLSCs and its mechanism. Our results revealed that BetA not only promoted the viability of hPDLSCs but also induced their osteogenic differentiation in a dose-dependent manner. In addition, RNA sequencing was used to screen the differentially expressed genes (DEGs) after hPDLSCs were treated with BetA, and 127 upregulated and 138 downregulated genes were identified. Gene Ontology enrichment analysis showed that DEGs were mainly involved in the response to lithium ions and the positive regulation of macrophage-derived foam cell differentiation. The Kyoto Encyclopedia of Genes and Genomes analysis results revealed that DEGs were enriched in the nuclear factor-κB and interleukin-17 signaling pathways. More importantly, we confirmed that early growth response gene 1 (EGR1), one of the three DEGs involved in bone formation, significantly promoted the expression of osteogenic markers and the mineralization of hPDLSCs. Knockdown of EGR1 obviously limited the effect of BetA on the osteogenic differentiation of hPDLSCs. In conclusion, BetA promoted the osteogenic differentiation of hPDLSCs through upregulating EGR1, and BetA might be a promising candidate in the clinical application of periodontal tissue regeneration.

Regenerative benefits of using growth factors in treatment of periodontal defects: A systematic review and meta-analysis with Trial Sequential Analysis on preclinical studies.

The ultimate goal in periodontal treatments is to achieve a functional and anatomical regeneration of the lost tissues. Numerous studies have in some way illustrated the beneficial effects of biologic modifiers in this process, yet they are subject to a rather large degree of diversity in their results. Thanks to the promising outcomes of bioengineering techniques in the field of periodontal regeneration, this systematic review aims to evaluate the effect of various biologic modifiers used in periodontal defects of animal models. Electronic databases (Medline, Scopus, Embase, Web of Science, and Google Scholar) were searched (March 2010-December 2020) for every study that used biomolecules for regeneration of periodontal osseous defects in animal models. Regenerated bone height or area, new cementum, new connective tissues, new regenerated periodontal ligament and the dimensions of epithelial attachment (either in mm/mm2 or percentage) were the investigated outcomes. The risk of bias of the included studies was assessed using the SYRCLE tool. In closing, there was a meta-analysis carried out on the outcomes of interest. Trial Sequential Analysis was also carried out to figure out the power of meta-analytic outcomes. From 1995 studies which were found in the initial search, 34 studies were included in this review, and 20 of them were selected for the meta-analysis. The eligible studies were categorized according to the morphology of the experimental periodontal defects as one-, two-, and three-wall intrabony defects; furcation defects, and recession-type defects. The most studied biomolecules were rhFGF-2, rhGDF-5, platelet-derived growth factor, bone morphogenetic protein-2, and enamel matrix derivative (EMD). Based on the meta-analysis findings, combined application of biomolecules with regenerative treatments could improve new bone and cementum formation near 1 mm when compared to the control groups in one, two and three-wall intrabony defect models (p < 0.001). In furcation grade II defect, the addition of biomolecules was observed to enhance bone area gain and cementum height regeneration up to almost 2 mm (p < 0.001). Trial Sequential Analysis results confirmed the significant effect in the aforementioned meta-analyses. In cases of the buccal recession model, the application of rhFGF-2 and rhGDF-5 decreased the dimension of epithelial attachments besides regenerative advantages on bone and cementum formation, but EMD deposition exerted no inhibitory effect on epithelial down-growth. Application of biologic modifiers especially FGF-2 and GDF-5, could positively improve the regeneration of periodontal tissues, particularly cementum and bone in animal models. Trial Sequential Analysis confirmed the results but the power of the evidences was high just in some subgroup meta-analyses, like bone and cementum regeneration in furcation grade II model and cementum regeneration in one-wall intrabony defects. The outcomes of this study can potentially endow clinicians with guidelines for the appropriate application of growth factors in periodontal regenerative therapies.

Role of transient receptor potential channel 6 in the osteogenesis of periodontal ligament cells.

Transient receptor potential channel 6 (TRPC6) is a receptor-operated Ca2+ channel that plays an important role in Ca2+ influx in the majority of non-excitable cells and influences calcium signalling and cellular responses. Therefore, the purpose of the present study was to gain insight into the role of TRPC6 in the osteogenesis of periodontal ligament cells (PDLCs). By western blot and immunohistochemical staining, the protein level of TRPC6 was found to be increased in a time-dependent manner during osteoblastic differentiation of PDLCs. In addition, the TRPC6 inhibitor SKF96365 was used to block the function of TRPC6 and inhibit osteoblastic differentiation of PDLCs. The TRPC6 activator hyperforin dicyclohexylammonium salt (hyperforin DCHA) was used to activate TRPC6 and promote osteoblastic differentiation of PDLCs. In vivo, wild-type mice showed better bone regeneration than TRPC6-/- mice, suggesting that TRPC6 has notable osteogenic induction properties and is important for bone defect repair. In conclusion, the current data demonstrated that TRPC6 plays a significant role in osteoblastic differentiation of PDLCs, suggesting that it may be a promising therapeutic target in osteogenesis.

Bio-Morphological Reaction of Human Periodontal Ligament Fibroblasts to Different Types of Dentinal Derivates: In Vitro Study.

Understanding the biological and morphological reactions of human cells towards different dentinal derivate grafting materials is fundamental for choosing the type of dentin for specific clinical situations. This study aimed to evaluate human periodontal ligament fibroblasts (hPLF) cells exposed to different dentinal derivates particles. The study design included the in vitro evaluation of mineralized dentine (SG), deproteinized and demineralized dentine (DDP), and demineralized dentine (TT) as test materials and of deproteinized bovine bone (BIOS) as the positive control material. The materials were kept with the hPLF cell line, and the evaluations were made after 24 h, 72 h, and 7 days of in vitro culture. The evaluated outcomes were proliferation by using XTT assays, the morphological characteristics by light microscopy (LM) and by the use of scanning electron microscopy (SEM), and adhesion by using confocal microscopy (CLSM). Overall, the experimental materials induced a positive response of the hPLFs in terms of proliferation and adhesion. The XTT assay showed the TT, and the SG induced significant growth compared to the negative control at 7 days follow-up. The morphological data supported the XTT assay: the LM observations showed the presence of densely packed cells with a modified shape; the SEM observations allowed the assessment of how fibroblasts exposed to DDP and TT presented cytoplasmatic extensions; and SG and BIOS also presented the thickening of the cellular membrane. The CLMS observations showed the expression of the proliferative marker, as well as and the expression of cytoskeletal elements involved in the adhesion process. In particular, the vinculin and integrin signals were stronger at 72 h, while the actin signal remained constantly expressed in all the follow-up of the sample exposed to SG material. The integrin signal was stronger at 72 h, and the vinculin and actin signals were stronger at 7 days follow-up in the sample exposed to DDP material. The vinculin and integrin signals were stronger at 72 h follow-up in the sample exposed to TT material; vinculin and integrin signals appear stronger at 24 h follow-up in the sample exposed to BIOS material. These data confirmed how dentinal derivates present satisfying biocompatibility and high conductivity and inductivity properties fundamental in the regenerative processes. Furthermore, the knowledge of the effects of the dentin's degree of mineralization on cellular behavior will help clinicians choose the type of dentine derivates material according to the required clinical situation.

Quercetin Prevents Oxidative Stress-Induced Injury of Periodontal Ligament Cells and Alveolar Bone Loss in Periodontitis.

PURPOSE: Emerging evidence has indicated that oxidative stress (OS) contributes to periodontitis. Periodontal ligament cells (PDLCs) are important for the regeneration of periodontal tissue. Quercetin, which is extracted from fruits and vegetables, has strong antioxidant capabilities. However, whether and how quercetin affects oxidative damage in PDLCs during periodontitis remains unknown. The aim of this study was to assess the effects of quercetin on oxidative damage in PDLCs and alveolar bone loss in periodontitis and underlying mechanisms. MATERIALS AND METHODS: The tissue block culture method was used to extract human PDLCs (hPDLCs). First, a cell counting kit 8 (CCK-8) assay was used to identify the optimal concentrations of hydrogen peroxide (H2O2) and quercetin. Subsequently, a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, RT-qPCR, Western blotting and other methods were used to explore the effects of quercetin on OS in hPDLCs and the underlying mechanism. Finally, quercetin was administered to mice with periodontitis through gavage, and the effect of quercetin on the level of OS and alveolar bone resorption in these mice was observed by immunofluorescence, microcomputed tomography (micro-CT), hematoxylin and eosin staining (H&E) staining and so on. RESULTS: Quercetin at 5 µM strongly activated NF-E2-related factor 2 (NRF2) signaling, alleviated oxidative damage and enhanced the antioxidant capacity of hPDLCs. In addition, quercetin reduced cellular senescence and protected the osteogenic ability of hPDLCs. Finally, quercetin activated NRF2 signaling in the periodontal ligaments, reduced the OS level of mice with periodontitis, and slowed the absorption of alveolar bone in vivo. CONCLUSION: Quercetin can increase the antioxidant capacity of PDLCs and reduce OS damage by activating the NRF2 signaling pathway, which alleviates alveolar bone loss in periodontitis.

Hyperlipidemic Conditions Impact Force-Induced Inflammatory Response of Human Periodontal Ligament Fibroblasts Concomitantly Challenged with P. gingivalis-LPS.

In obese patients, enhanced serum levels of free fatty acids (FFA), such as palmitate (PA) or oleate (OA), are associated with an increase in systemic inflammatory markers. Bacterial infection during periodontal disease also promotes local and systemic low-grade inflammation. How both conditions concomitantly impact tooth movement is largely unknown. Thus, the aim of this study was to address the changes in cytokine expression and the secretion of human periodontal ligament fibroblasts (HPdLF) due to hyperlipidemic conditions, when additionally stressed by bacterial and mechanical stimuli. To investigate the impact of obesity-related hyperlipidemic FFA levels on HPdLF, cells were treated with 200 µM PA or OA prior to the application of 2 g/cm2 compressive force. To further determine the additive impact of bacterial infection, HPdLF were stimulated with lipopolysaccharides (LPS) obtained from Porphyromonas gingivalis. In mechanically compressed HPdLF, PA enhanced COX2 expression and PGE2 secretion. When mechanically stressed HPdLF were additionally stimulated with LPS, the PGE2 and IL6 secretion, as well as monocyte adhesion, were further increased in PA-treated cultures. Our data emphasize that a hyperlipidemic condition enhances the susceptibility of HPdLF to an excessive inflammatory response to compressive forces, when cells are concomitantly exposed to bacterial components.

The Effect of Liposomal Curcumin as an Anti-Inflammatory Strategy on Lipopolysaccharide e from Porphyromonas gingivalis Treated Endothelial Committed Neural Crest Derived Stem Cells: Morphological and Molecular Mechanisms.

Curcumin, a yellow polyphenol extracted from the turmeric root is used as a diet supplement. It exhibits anti-inflammatory, antioxidant, and antitumor properties by modulating different intracellular mechanisms. Due to their low solubility in water, the curcumin molecules must be encapsulated into liposomes to improve the bioavailability and biomedical potential. For the periodontal tissue and systemic health, it is essential to regulate the local inflammatory response. In this study, the possible beneficial effect of liposomes loaded with curcumin (CurLIP) in neural crest-derived human periodontal ligament stem cells (hPDLSCs) and in endothelial-differentiated hPDLSCs (e-hPDLSCs) induced with an inflammatory stimulus (lipopolysaccharide obtained from Porphyromonas gingivalis, LPS-G) was evaluated. The CurLIP formulation exhibited a significant anti-inflammatory effect by the downregulation of Toll-like receptor-4 (TLR4)/Myeloid differentiation primary response 88 (MyD88)/nuclear factor kappa light chain enhancer of activated B cells (NFkB)/NLR Family Pyrin Domain Containing 3 (NLRP3)/Caspase-1/Interleukin (IL)-1ß inflammation cascade and reactive oxygen species (ROS) formation. Moreover, the exposure to LPS-G caused significant alterations in the expression of epigenetic modifiers, such as DNA Methyltransferase 1 (DNMT1) and P300, while the CurLIP treatment showed physiological expression. Overall, our in vitro study provides novel mechanistic insights into the intracellular pathway exert by CurLIP in the regulation of inflammation and epigenetic modifications.

3D-Printed Collagen-Based Waveform Microfibrous Scaffold for Periodontal Ligament Reconstruction.

Reconstruction of the periodontal ligament (PDL) to fulfill functional requirement remains a challenge. This study sought to develop a biomimetic microfibrous system capable of withstanding the functional load to assist PDL regeneration. Collagen-based straight and waveform microfibers to guide PDL cell growth were prepared using an extrusion-based bioprinter, and a laminar flow-based bioreactor was used to generate fluidic shear stress. PDL cells were seeded on the respective microfibers with 0 or 6 dynes/cm2 fluidic shear stress for 1-4 h. The viability, morphology, adhesion pattern, and gene expression levels of PDL cells were assessed. The results revealed that upon bioprinting optimization, collagen-based microfibers were successfully fabricated. The straight microfibers were 189.9 ± 11.44 µm wide and the waveform microfibers were 235.9 ± 11.22 µm wide. Under 6 dynes/cm2 shear stress, PDL cells were successfully seeded, and cytoskeleton expansion, adhesion, and viability were greater. Cyclin D, E-cadherin, and periostin were upregulated on the waveform microfibers. In conclusion, 3D-printed collagen-based waveform microfibers preserved PDL cell viability and exhibited an enhanced tendency to promote healing and regeneration under shear stress. This approach is promising for the development of a guiding scaffold for PDL regeneration.

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.

Responses of canine periodontal ligament cells to bubaline blood derived platelet rich fibrin in vitro.

Platelet-rich fibrin (PRF) promotes wound healing by providing the release of growth factors. Here, the influence of Thai and Murrah bubaline blood derived PRF on canine periodontal ligament cells (cPDLs) was investigated. PRF was prepared from Thai and Murrah buffaloes with single centrifugation. Results demonstrated that Thai bubaline blood derived PRF exhibited fiber-mesh like morphology and contained more platelet entrapment than Murrah bubaline blood derived PRF. Both bubaline PRFs were able to degrade in vitro under condition with trypsin. Thai but not Murrah bubaline blood derived PRF promoted cPDLs proliferation in serum free and 2% serum culture conditions. Correspondingly, the significant upregulation of KI67 mRNA expression was observed in those cells treated with Thai bubaline blood derived PRF. However, both Thai and Murrah bubaline blood derived PRF accelerated cell migration in an in vitro wound healing assay and facilitated cell spreading. Further, cPDLs cultured in osteogenic induction medium supplemented with Thai bubaline blood derived PRF exhibited the increased mineral deposition in vitro. Frozen Thai bubaline blood derived PRF also promoted cell proliferation, KI67 mRNA expression, cell migration, and cell spreading in cPDLs. Taken these evidence together, bubaline blood derived PRF could provide potential benefits for canine periodontal tissue healing.

Response of the periodontal tissues to ß-adrenergic stimulation.

AIMS: Stimulation of ß-adrenergic receptors (ßAR) in osteoblasts by isoproterenol (ISO) was shown to induce Vascular Endothelial Growth Factor (VEGF) and angiogenesis in long bones. We thus aimed to determine the vascular response of mandibular tissues to ßAR stimulation regarding blood vessel formation. MAIN METHODS: Six-week-old wild-type C57BL6 female mice received daily intraperitoneal injections of ISO or phosphate buffered saline (PBS) for 1 month. Hemimandibles and tibias were collected for immunolocalization of endomucin, tyrosine hydroxylase (TH), neuropeptide Y (NPY) and norepinephrine transporter (NET). Moreover, Vegfa, Il-1 ß, Il-6, Adrb2 and Rankl mRNA expression was assessed in mandibles and tibias 2 h after PBS or ISO treatment. KEY FINDINGS: Despite similar sympathetic innervation and Adrb2 expression between mandibular tissues and tibias, with TH and NPY+ nerve fibers distributed around blood vessels, ISO treatment did not increase endomucin+ vessel area or the total number of endomucin+ vessels in any of the regions investigated (alveolar bone, periodontal ligament, and dental pulp). Consistent with these results, the expression of Vegfα, Il-6, Il-1ß, and Rankl in the mandibular molar region did not change following ISO administration. We detected high expression of NET by immunofluorescence in mandible alveolar osteoblasts, osteocytes, and periodontal ligament fibroblasts, in addition to significantly higher Net expression by qPCR compared to the tibia from the same animals. SIGNIFICANCE: These findings indicate a differential response to ßAR agonists between mandibular and tibial tissues, since the angiogenic potential of sympathetic outflow observed in long bones is absent in periodontal tissues.

Visfatin regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway.

Periodontitis is a widespread chronic infectious-inflammatory disease associated with multiple systemic diseases. Visfatin is an adipokine-enzyme that can be locally produced by human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). It can upregulate proinflammatory cytokines and matrix metalloproteinases (MMPs) in various types of cells. However, the effects of visfatin on healthy and inflammatory human periodontal cells as well as the underlying molecular mechanisms remain unclear. This study firstly demonstrated visfatin expression was highly elevated in inflamed human gingiva and Pg LPS-treated hPDLCs. Moreover, recombinant visfatin significantly upregulated the expression of proinflammatory cytokines (TNF-α, IL-1ß and IL-6) and prodegradative factors (EMPPRIN, MMP1, MMP3 and MMP13) in hPDLCs. Next, we found the levels of proinflammatory and prodegradative cytokines were significantly increased in visfatin-overexpressing hPDLCs, and decreased in visfatin-silencing inflammatory hGFs (iGFs) when treated with Pg LPS. In the absence of Pg LPS, visfatin silencing failed to affect the expression of these factors in iGFs, and overexpression of visfatin upregulated MMPs but no other factors in hPDLCs. Furthermore, marked NF-κB pathway activation with increased phosphorylation of p65 was observed in visfatin-overexpressing hPDLCs. BAY11-7082, a specific inhibitor of NF-κB, partially reversed the upregulation proinflammatory and prodegradative factors induced by visfatin overexpression. Taken together, this study showed that visfatin critically regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway. The findings suggest that visfatin is closely involved in the development of periodontitis, and may serve as a promising novel biomarker and therapeutic target for periodontitis management.