Healthy Periodontal Ligament Research Articles

Assessment of the viability of human periodontal ligament cells in black tea, lime juice, and passion fruit concentrate - A comparative in vitro study.

Background:Tooth avulsion is considered as a severe form of dental trauma, causing damage to the periodontium. Hence, the preservation of healthy periodontal ligament (PDL) cells in the storage medium are pivotal for the success of replantation.Aim and Objective:The aim of this study is to assess the viability of human PDL cells in black tea, lime juice, and passion fruit concentrate.Methods:Human periodontal cells were cultured and stored in three experimental media – black tea, lime juice, and passion fruit concentrate and subjected to 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay for 1 h and the cell viability was determined. Mean and standard deviation were statistically analyzed using one-way analysis of variance to identify the significant groups.Results:The human PDL cells showed 100% viability in lime juice and passion fruit concentrate, followed by 98% viability in black tea.Conclusion:Black tea, lime juice, and passion fruit concentrate can be used effectively as storage media for maintaining PDL cells viability in avulsed teeth, with 100% viability exhibited by lime juice and passion fruit concentrate.

Comparative study with the effect of stromal cell derived factor-1 on osteogenic differentiation of human healthy and inflammatory periodontal ligament stem cells

This study aims to compare the osteogenic differentiation capability of stem cells derived from human inflammatory periodontal ligament tissues (iPDLSCs) with those of stem cells derived from healthy periodontal ligament tissues (hPDLSCs). Both types of tissues were induced by stromal cell derived factor (SDF-1) in vitro. iPDLSCs and hPDLSCs were primarily cultured by tissue digestion method and purified by limited dilution cloning. The cells were passaged and identified by stem cell surface marker expression through flow cytometry. Then, we used thiazolyl blue tetrazolium bromide to detect and compare the proliferation capabilities of the iPDLSCs and hPDLSCs. Express of bone volumes were detected by alizarin red staining after SDF-1 was added to the cells. Using alkaline phosphatase, we evaluated the osteogenic differentiation capability of the cells induced by SDF-1. The expression levels of the osteogenesis-related genes of the cells induced by SDF-1 were determined by reverse transcription-polymerase chain reaction. After purification, both iPDLSCs and hPDLSCs expressed stem cell markers. hPDLCSs had a higher proliferation capability than iPDLSCs. Osteogenesis-related genes had higher expression levels in the cells induced by SDF-1 than in those without induction (P<0.05). SDF-1 at 50 and 200 ng·mL⁻¹ concentration greatly affected the differen-tiation capabilities of iPDLSCs and hPDLSCs respectively. iPDLSCs and hPDLSCs had osteogenic differentia-tion capability. The level of osteogenic differentiation in normal and inflamed periodontal ligament stem cells increases after SDF-1 induction.

Down-regulation of long non-coding RNA MEG3 suppresses osteogenic differentiation of periodontal ligament stem cells (PDLSCs) through miR-27a-3p/IGF1 axis in periodontitis.

Objective: This study aimed to investigate the roles of long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in periodontitis.Methods: Differentially expressed lncRNAs and mRNAs between periodontitis periodontal ligament tissues and healthy periodontal ligament tissues were selected out using R project. PDLSCs were identified using flow cytometry. Western blot was employed to detect pathway relative proteins. Besides, targeted relationships between lncRNA and miRNA, as well as miRNA and mRNA were verified by dual luciferase reporter gene assay. Osteogenic differentiation of PDLSCs was assessed by alkaline phosphatase (ALP) staining and Alizarin Red Staining (ARS). Markers for osteoblast (Runx2, Osterix, Osteocalcin, Colla1) were detected using western blot.Results: LncRNA MEG3 and IGF1 were both down-regulated, while miR-27a-3p was up-regulated in periodontitis samples compared with healthy samples. Overexpression of MEG3 promoted osteogenic differentiation by enhancing expression of IGF1 yet suppressing expression of miRNA-27a-3p. Meanwhile, the results of ALP and ARS staining indicated that up-regulation of lncRNA MEG3 or IGF1 promoted osteogenic differentiation in PDLSCs, which could be reversed with up-regulation of miRNA-27a-3p.Conclusion: Down-regulation of MEG3 suppressed osteogenic differentiation of PDLSCs through miR-27a-3p/IGF1 axis in periodontitis.

Histone Methylation Mechanisms Modulate the Inflammatory Response of Periodontal Ligament Progenitors.

Inflammatory conditions affect periodontal ligament (PDL) homeostasis and diminish its regenerative capacity. The complexity of biological activities during an inflammatory response depends on genetic and epigenetic mechanisms. To characterize the epigenetic changes in response to periodontal pathogens we have focused on histone lysine methylation as a relatively stable chromatin modification involved in the epigenetic activation and repression of transcription and a prime candidate mechanism responsible for the exacerbated and prolonged response of periodontal cells and tissues to dental plaque biofilm. To determine the effect of inflammatory conditions on histone methylation profiles, related gene expression and cellular functions of human periodontal ligament (hPDL) progenitor cells, a hPDL cell culture system was subjected to bacterial cell wall toxin exposure [lipopolysaccharide (LPS)]. Chromatin immunoprecipitation-on-chip analysis revealed that healthy PDL cells featured high enrichment levels for the active H3K4me3 mark at COL1A1, COL3, and RUNX2 gene promoters, whereas there were high occupancy levels for the repressive H3K27me3 marks at DEFA4, CCL5, and IL-1β gene promoters. In response to LPS, H3K27me3 enrichment increased on extracellular matrix and osteogenesis lineage gene promoters, whereas H3K4me3 enrichment increased on the promoters of inflammatory response genes, suggestive of an involvement of epigenetic mechanisms in periodontal lineage differentiation and in the coordination of the periodontal inflammatory response. On a gene expression level, LPS treatment downregulated COL1A1, COL3A1, and RUNX2 expression and upregulated CCL5, DEFA4, and IL-1β gene expression. LPS also greatly affected PDL progenitor function, including a reduction in proliferation and differentiation potential and an increase in cell migration capacity. Confirming the role of epigenetic mechanisms in periodontal inflammatory conditions, our studies highlight the significant role of histone methylation mechanisms and modification enzymes in the inflammatory response to LPS bacterial cell wall toxins and periodontal stem cell function.

Biomechanical analysis of initial incisor crowding alignment in the periodontally reduced mandible using the finite element method.

To reduce remaining plaque niches due to dental malocclusion after periodontal treatment and to avoid reinflammation of periodontitis, severe anterior crowding can be treated orthodontically. The treatment indication is motivated by aesthetic and functional needs. In this study the biomechanical behaviour of crowded lower front teeth in reduced periodontium is analysed. Using the finite element (FE) method, amodel of the mandible was constructed with an anterior crowding of 4 mm and avertical bone loss of 4 mm in the front tooth area. A0.3 mm (0.012″) round superelastic nickel titanium (NiTi) arch wire was fitted to an ideal positioned teeth set-up and was inserted into the brackets of teeth 34 to 44 in the crowded model. The premolars were used as the anchorage unit. Material parameters were adopted from previous investigations, including bone (homogenous, isotropic, E = 2GPa), teeth (E = 20GPa) and healthy periodontal ligament (PDL, bilinear elastic; E1 = 0.05 MPa; E2 = 0.2 MPa; ε12 = 7%). All simulations were compared to simulations with aphysiological periodontal model to assess the effect of bone loss at teeth 42 to 32. Additionally, the influence of three arch wire materials (nonsuperelastic NiTi, superelastic NiTi and stainless steel) were analysed in areduced model, including only brackets in position of the crowded front teeth, wire and ligatures. Wire force levels and stresses were determined to assess the influence of material variation. Initial tooth mobility is increased by afactor of 2.5 in case of amoderate periodontal defect. Front teeth with reduced attachment display increased strains in the periodontal ligament up to afactor of 2. Forces in the model with reduced periodontium were decreased by afactor of 2. Comparing different aligning arch wires, stainless steel appears to have the highest force and stress levels. Force levels of this alloy were 7.5times higher than with the superelastic NiTi wire. Force levels of nonsuperelastic NiTi appeared to be 1.8times higher than superelastic NiTi. Calculated stresses with stainless steel were 5times higher than with the nonsuperelastic NiTi and 10times higher than with superelastic NiTi. Periodontally reduced incisors 42 to 32 are associated with an increased load on periodontal tissue and increased level of tooth mobility during fixed orthodontic treatment. This has to be considered by reducing orthodontic force levels and by selecting mechanics that reduce the load to the tissue.

Expression of Toll-like receptors 2 and 4 and its association with matrix metalloproteinases in symptomatic and asymptomatic apical periodontitis.

To determine Toll-like receptors (TLR)2 and TLR4 expression levels and associate them with matrix metalloproteinases (MMPs) in asymptomatic apical periodontitis (AAP), symptomatic apical periodontitis (SAP), and healthy controls. Apical tissue/lesion samples were obtained from chronic AAP (n = 35) and SAP (n = 29), and healthy periodontal ligament (HPL, n = 10) with indication of tooth extraction, respectively. mRNA expression levels of TLR2, TLR4, MMP-1, MMP-2, MMP-8, and MMP-13 were determined by real-time reverse-transcription polymerase chain reaction. The data were analyzed with Kruskal-Wallis and Dunn's pot hoc test (p < 0.05). The correlation coefficient was obtained using the Spearman correlation (p < 0.05). TLR2, MMP-1, MMP-2, and MMP-13 mRNA levels were the highest in SAP followed by AAP and controls (p < 0.05). TLR4 and MMP-8 were over expressed in AAP and SAP compared to HPL (p < 0.05). TLR2 positively correlated with TLR4, MMP-1, MMP-8, and MMP-13 in SAP (p < 0.05). TLR2 and TLR4 are overexpressed in apical lesions versus healthy periodontal ligament and correlate with collagenolytic MMPs. Particularly, TLR2 is overexpressed in SAP in association with MMP-1, MMP-8, and MMP-13. Our results suggest that the activation of TLR2 along with MMP overexpression might contribute to SAP clinical presentation and progression. TLRs, MMPs, and their interaction can explain the clinical presentations and evolution of apical periodontitis and might represent key targets for new diagnostic and treatment approaches.

Transplant surgical templates for dental autotransplantation: a technical note

Introduction: This technical note presents the autotransplantation of an impacted third molar to replace a severely damaged homolateral molar using a surgical template of the tooth to be transplanted to prepare the recipient site based on an original protocol. Technique: Mandibular molar extraction is first performed, and autotransplantation is done after 7 days. Alveolar adjustment of the receiving socket is done using a resin replica of the tooth to be transplanted; the replica is obtained using DICOM diagnosis data. The three-dimensional (3D) replica is produced using 3D printing techniques with photopolymerizable methacrylate resin. Once the recipient site is ready, the transplant is avulsed. Immediate positioning of the tooth in the new socket will save a tremendous amount of extra-alveolar time. Cross-stitch sutures are done to obtain complete restraint, and endodontic treatment is administered after 15 days. Postoperative clinical and radiographic examination showed periodontal healing after postoperative 6 months. Comments: The use of the surgical template resulted in an extra-alveolar time of below 5 s and preservation of healthy periodontal ligament cells of the donor tooth as much as possible. Conclusion: The 3D technology, from imaging to printing, can drive the improvement of prognosis in a tooth autotransplantation protocol.

DNA methylation profiles of immune response-related genes in apical periodontitis.

To investigate the DNA methylation profiles of immune response-related genes in apical periodontitis (AP) lesions. The methylation profiles on the cytosine-phosphate-guanine (CpG) regions of 22 gene promoters involved in inflammation and autoimmunity were assessed in 60 human AP lesions and 24 healthy periodontal ligaments (controls) using a pathway-specific real-time polymerase chain reaction array (EpiTect® Methyl Signature PCR Array Human Inflammatory Response). Differentially methylated genes were subsequently assessed for their mRNA expression. Data analyses (One-way anova, Tukey's multiple comparisons tests and Mann-Whitney tests) were performed using GraphPad Prism 6 software. P values≤0.05 were considered statistically significant. Significant DNA hypermethylation was observed for CXCL3 and FADD gene promoters in AP lesions when compared to control tissues (P<0.001) and among other genes (P<0.05). In contrast, IL12B and IL4R were associated with significant hypomethylation in comparison to other genes (P<0.05). IL12B, IL4R, CXCL3 and FADD had differential mRNA expression in AP lesions and controls (P<0.001). Differential methylation profiles of immune response-related genes, such as FADD, CXCL3, IL12B and IL4R, may have an influence on individual AP susceptibility and patient treatment outcomes, through their potential contributions to altered expression of disease-relevant genes. Methylation and/or genetic variations in additional genes may also contribute to the dynamics of AP development and should be considered in future studies.

Substance P and Calcitonin gene-related peptide expression in human periodontal ligament after root canal preparation with Reciproc Blue, WaveOne Gold, XP EndoShaper and hand files.

To quantify Substance P (SP) and Calcitonin gene-related peptide (CGRP) expression in healthy human periodontal ligament from premolars after root canal preparation with Reciproc Blue, WaveOne Gold, XP EndoShaper and hand files. A total of 50 human periodontal ligament samples were obtained from healthy mandibular premolars where extraction was indicated for orthodontic reasons. Prior to extraction, 40 of these premolars were equally divided into four groups, and root canals were prepared using four different systems: Reciproc Blue, WaveOne Gold, XP EndoShaper and a hand instrumentation technique. The remaining 10 healthy premolars were extracted without treatment and served as a negative control group. All periodontal ligament samples were processed, and SP and CGRP were measured by radioimmunoassay. The Kruskal-Wallis test was used to establish significant differences between groups and LSD post hoc comparisons were also performed. Greater SP and CGRP values were found in the hand instrumentation group, followed by the XP EndoShaper, WaveOne Gold and the Reciproc groups. The lower SP and CGRP values were for the healthy periodontal ligament group. The Kruskal-Wallis test revealed significant differences between groups (P<0.05). Post hoc Least Significant Difference (LSD) tests revealed significant differences (P<0.05) in SP and CGRP expression between all the comparisons except for the Reciproc Blue and WaveOne Gold group (P>0.05). All the root canal preparation techniques tested increased SP and CGRP expression in human periodontal ligament, with hand files and XP EndoShaper instruments being associated with greater neuropeptide release compared to Reciproc Blue and WaveOne Gold files.

PR361: Neurogenic, angiogenic and osteogenic differentiation potential of healthy human periodontal ligament stem cells

PR361: Neurogenic, angiogenic and osteogenic differentiation potential of healthy human periodontal ligament stem cells

Activation of autophagy in periodontal ligament mesenchymal stem cells promotes angiogenesis in periodontitis.

Angiogenesis alteration in tooth support tissue plays an essential role in periodontitis. Mesenchymal stem cells (MSCs) can affect vessel formation by endothelial cells (ECs) through paracrine function. Autophagy is reported to be closely related to cell secretion. Here we investigated the angiogenesis-promoting ability of MSCs that reside in the periodontal ligament (known as periodontal ligament stem cells, PDLSCs) under inflammatory conditions in order to explore the mechanism of angiogenesis alteration in periodontitis. PDLSCs were isolated from healthy and inflamed human periodontal ligament tissues (HPDLSCs and PPDLSCs, respectively). HPDLSCs were subjected to an inflammatory environment (IPDLSCs) in vitro using inflammatory cytokines. Angiogenesis-promoting cytokine expression and autophagy were evaluated in PDLSCs by quantitative reverse transcription-polymerase chain reaction and western-blot analysis before co-culturing them with ECs. The angiogenesis ability of ECs in the co-culture system was examined by a matrigel tube formation test. Rapamycin and pcDNA for Beclin-1 (cDNA-Beclin-1) were used to promote autophagy in PDLSCs and siRNA Beclin-1 (siBeclin-1) was used to repress it. The inflammatory environment increased autophagy and the expression of basic fibroblast growth factor (bFGF) and angiogenin (Ang) in PDLSCs. More tube formation was observed in ECs from the co-culture system which was pretreated with tumor necrosis factor (TNF)-α and interleukin (IL)-1β. PDLSCs treated with rapamycin or transfected with cDNA-Beclin-1 showed higher expression levels of bFGF and Ang that promoted tube formation by the co-cultured ECs. PDLSCs transfected with siBeclin-1 resulted in the opposite results. Autophagy modulates angiogenesis-promoting ability of PDLSCs, which could be increased by an inflammatory environment.

Human periodontal ligament fibroblasts synthesize C-reactive protein and Th-related cytokines in response to interleukin (IL)-6 trans-signalling.

To characterize the potential of human periodontal ligament fibroblasts (HPLF) to synthesize CRP and Th-related cytokines in response to IL-6 in periodontal health and apical inflammation. Primary HPLF stimulated with IL-6, soluble(s) IL-6 receptor (R) and controls were assayed for CRP, Th1, Th2, Th17 and Treg-related cytokines by quantitative real-time PCR and ELISA, respectively. IL-6R mRNA expression and its soluble protein levels were screened in HPLF cultures, and exvivo samples of healthy periodontal ligaments (n=5) and apical lesions (n=13). Data were analysed with ANOVA or unpaired t-test. 0.5ng mL-1 IL-6 plus 1ng mL-1 of its soluble receptor (sIL-6R) for 24h was effective in inducing CRP production. IL-6 alone had a mild dose-dependent effect; co-stimulation with sIL-6R significantly enhanced this effect, whereas it was completely abolished by the addition of IL-6R blocking antibody (P<0.05). Similarly, higher mRNA expression and protein levels of Th1, Th17 and partially Treg-related cytokines were found for IL-6 combined with its soluble receptor versus the nonstimulated group and IL-6R antibody (P<0.05). IL-6R mRNA expression was slightly induced by IL-6 compared to THP-1 cells, but sILR-6 protein could not be detected in HPLF. High sIL-6R levels were detected in apical lesions and were immunolocalized to mononuclear inflammatory cells and proliferating epithelium. IL-6 trans-signalling induced Th1 and Th17-related cytokines and represents an extra-hepatic mechanism for PCR synthesis in human periodontal ligament fibroblasts, contributing to explain the bone-destructive phenotype of apical lesions and eventually its systemic complications.

Identification and integrated analysis of differentially expressed lncRNAs and circRNAs reveal the potential ceRNA networks during PDLSC osteogenic differentiation

BackgroundResearchers have been exploring the molecular mechanisms underlying the control of periodontal ligament stem cell (PDLSC) osteogenic differentiation. Recently, long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) were shown to function as competitive endogenous RNAs (ceRNAs) to regulate the effect of microRNAs (miRNAs) on their target genes during cell differentiation. However, comprehensive identification and integrated analysis of lncRNAs and circRNAs acting as ceRNAs during PDLSC osteogenic differentiation have not been performed.ResultsPDLSCs were derived from healthy human periodontal ligament and cultured separately with osteogenic induction and normal media for 7 days. Cultured PDLSCs were positive for STRO-1 and CD146 and negative for CD31 and CD45. Osteo-induced PDLSCs showed increased ALP (alkaline phosphatase) activity and up-regulated expression levels of the osteogenesis-related markers ALP, Runt-related transcription factor 2 and osteocalcin. Then, a total of 960 lncRNAs and 1456 circRNAs were found to be differentially expressed by RNA sequencing. The expression profiles of eight lncRNAs and eight circRNAs were measured with quantitative real-time polymerase chain reaction and were shown to agree with the RNA-seq results. Furthermore, the potential functions of lncRNAs and circRNAs as ceRNAs were predicted based on miRanda and were investigated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. In total, 147 lncRNAs and 1382 circRNAs were predicted to combine with 148 common miRNAs and compete for miRNA binding sites with 744 messenger RNAs. These mRNAs were predicted to significantly participate in osteoblast differentiation, the MAPK pathway, the Wnt pathway and the signaling pathways regulating pluripotency of stem cells. Among them, lncRNAs coded as TCONS_00212979 and TCONS_00212984, as well as circRNA BANP and circRNA ITCH, might interact with miRNA34a and miRNA146a to regulate PDLSC osteogenic differentiation via the MAPK pathway.ConclusionsThis study comprehensively identified lncRNAs/circRNAs and first integrated their potential ceRNA function during PDLSC osteogenic differentiation. These findings suggest that specific lncRNAs and circRNAs might function as ceRNAs to promote PDLSC osteogenic differentiation and periodontal regeneration.

Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes

IntroductionUnderstanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets. MethodsPeriapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant. ResultsProteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG. ConclusionsOur data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory response in apical periodontitis. Additional functional studies should be performed to further characterize the role of these molecules during the development/progression of apical periodontitis.

Clinical and radiological analysis of the causes for endodontic treatment failure

Summary Introduction Development of inflammatory lesions or their persistence after primary treatment is considered endodontic failure. The reason for failure can be complex anatomy of the canal system and numerous iatrogenic factors. The objective of this study was to analyze, clinically and radiographically, the causes of primary endodontic treatment failure and assess possibilities for retreatment of teeth with failed endodontic treatment. Method The study included 79 teeth (36 multirooted and 43 singlerooted tooth) indicated for repeated endodontic treatment. Based on the radiographic assessment of the status of periapical structures, teeth were divided into two groups. The first group included teeth without periapical lesions, i.e. the healthy periodontal tissues (PAI score of 1 and 2) in which retreatment was required for prosthodontic reason due to the poor quality of obturation (28 teeth), and the second group included teeth with visible signs of periapical tissue damage (PAI scores 3, 4 and 5) (51 teeth). In both groups, quality of obturation, coronal sealing and the presence or absence of clinical symptoms was analyzed. Results The most common radiographic finding of definitive obturation was short filling (65.8% of cases); “forgotten” canals (25.3%); non-homogeneous obturation with correct length (5.1%) and fractured instrument (3.8%). There was significant difference between healthy periodontal ligament and adequate restoration (P &lt; 0.001). In 95% of patients with symptoms, changes in the periapical tissue were observed. Also, there was significant difference in the presence of symptoms after primary treatments, between the teeth with healthy apical periodontal tissue and teeth with periapical lesions (P = 0.019). Conclusion The outcome of the root canal treatment is significantly affected by the quality (density) of obturation and the presence and quality of coronal restoration. In patients with symptoms there were changes in the periapical tissue.

MicroRNA-335-5p Plays Dual Roles in Periapical Lesions by Complex Regulation Pathways

IntroductionMicroRNA-335-5p has been reported to regulate osteogenic and chondrogenic differentiations of mesenchymal stem cells. The aim of this study was to explore the function and regulation mechanism of miR-335-5p in apical periodontitis (AP). MethodsTotal RNAs were extracted from human periodontal ligament fibroblasts (HPDLFs), 10 AP tissues, and 6 healthy periodontal ligament tissues using lysis buffer. Gene expression was detected using real-time polymerase chain reaction. The Dual Luciferase Assay (Promega, Madison, WI) was used to test miR-335-5p directly targeted urokinase-type plasminogen activator receptor (uPAR) and the receptor activator of nuclear factor kappa-B ligand (RANKL). Western Blot was used to detect protein expressions of RANKL, uPAR, and the fragile X–related 1 gene (FXR1). The enzyme-linked immunosorbent assay was used to detect the secretions of interleukin 6, tumor necrosis factor alpha, and RANKL. Data were analyzed using the Student t test. ResultsmiR-335-5p acted as a positive mediator in HPDLF inflammation (P < .05). Two targets of miR-335-5p, uPAR and RANKL, were identified. Interestingly, uPAR was repressed by miR-335-5p at the basal level, but it can be relieved from miR-335-5p–mediated repression, which is called derepression, when HPDLFs were subjected to lipopolysaccharide stimulation. miR-335-5p promoted RANKL in HPDLFs regardless of whether or not it was under inflammatory conditions (P < .05). We proved FXR1 was responsible for the derepression of uPAR from miR-335-5p (P < .01). Both FXR1 and uPAR were positive mediators in HPDLF inflammation (P < .05). miR-335-5p, uPAR, RANKL, and FXR1 had the same expression profiles in HPDLF inflammation and AP tissues (P < .05). ConclusionsOur data showed that miR-335-5p may play dual roles in AP, and it might be considered as a target for therapeutic potency in clinical applications.

In vivo and In vitro Identification of Endocannabinoid Signaling in Periodontal Tissues and Their Potential Role in Local Pathophysiology.

The endocannabinoid system (ECS) with its binding receptors CB1 and CB2 impacts multiple pathophysiologies not only limited to neuronal psychoactivity. CB1 is assigned to cerebral neuron action, whereas CB2 is mainly expressed in different non-neuronal tissues and associated with immunosuppressive effects. Based on these tissue-selective CB receptor roles, it was the aim of this study to analyze potential expression in periodontal tissues under physiological conditions and inflammatory states. In vivo, CB receptor expression was investigated on human periodontal biopsies with or without bacterial inflammation and on rat maxillae with or without sterile inflammation. In vitro analyses were performed on human periodontal ligament (PDL) cells at rest or under mechanical strain via qRT-PCR, Western blot, and immunocytochemistry. P<0.05 was set statistical significant. In vivo, CB1 expression was significantly higher in healthy PDL structures compared to CB2 (13.5%±1.3 of PDL tissues positively stained; 7.1%±0.9). Bacterial inflammation effected decrease in CB1 (9.7%±2.4), but increase in CB2 (14.7%±2.5). In contrast, sterile inflammation caused extensive CB1 (40%±1.9) and CB2 (41.7%±2.2) accumulations evenly distributed in the tooth surrounding PDL. In vitro, CB2 was ubiquitously expressed on gene and protein level. CB1 was constitutively expressed on transcriptional level (0.41%±0.09), even higher than CB2 (0.29%±0.06), but undetectable on protein level. Analyses further revealed expression changes of both receptors in mechanically loaded PDL cells. CB1 and CB2 are varyingly expressed in periodontal tissues, both adjusted by different entities of periodontal inflammation and by mechanical stress. This indicates potential ECS function as regulatory tool in controlling of periodontal pathophysiology.

Bone resorptive activity in symptomatic and asymptomatic apical lesions of endodontic origin

ObjectivesThe aim of this study is to assess the levels and diagnostic accuracy of a set of bone resorption biomarkers, including TRAP-5, RANKL, and OPG in symptomatic and asymptomatic apical lesions and controls.Materials and methodsApical tissues from symptomatic and asymptomatic apical periodontitis patients and periodontal ligaments from healthy teeth extracted for orthodontic reasons were processed for tissue homogenization and the levels of TRAP-5, RANKL, and OPG were determined by multiplex assay. Marker levels were analyzed by Kruskal Wallis test, and diagnostic accuracy was analyzed with ROC curves.ResultsHigher levels of RANKL, OPG, and RANKL/OPG ratio were determined in both types of apical lesions compared to healthy periodontal ligament, whereas higher TRAP-5 levels were found only in symptomatic apical lesions (p < 0.05). OPG, RANKL, and RANKL/OPG ratio showed diagnostic potential to identify apical lesions versus healthy controls (AUC = 0.69, p < 0.05); while TRAP-5 showed a potential to discriminate symptomatic versus asymptomatic apical periodontitis (AUC = 0.71, p < 0.05) and healthy controls (AUC = 0.83, p < 0.05).ConclusionsApical lesions showed higher RANKL and OPG levels than healthy tissues. TRAP-5 levels were the highest in symptomatic apical lesions, suggesting that these represent a progressive state, and showed diagnostic potential.Clinical relevanceClinically symptomatic apical periodontitis might represent biologically progressive apical lesions based on TRAP5 levels. TRAP5 has diagnostic potential to identify these lesions, representing a candidate prognostic biomarker.

In vitro comparative evaluation of different storage media (hank's balanced salt solution, propolis, Aloe vera, and pomegranate juice) for preservation of avulsed tooth.

Objectives:Prognosis of the avulsed teeth is mostly affected by extraoral dry period and storage medium used to store teeth before reimplantation. However, ability of storage media can affect cell viability and success of treatment. Various storage media were tried with some success. The present study was undertaken to comparatively evaluate the efficacy of hank's balanced salt solution (HBSS), propolis, Aloe vera, and pomegranate juice (PJ) in preserving the vitality of periodontal ligament (PDL) cells of avulsed teeth.Materials and Methods:Fifty orthodontically extracted sound teeth with healthy PDL were selected for the present study. Selected teeth were randomly divided into study groups (10 in each) and 5 each as positive and negative control groups. All the teeth were immersed immediately after extraction into respective storage media. Data were statistically analyzed using IBM SPSS software for Windows, Version 19.0., IBM Corp., Armonk, NY, USA. Analysis of variance and multiple range were done using Tukey's honestly significant difference with level of significance at 5% (P > 0.05).Results:Propolis (285,000 viable cells with standard deviation 4.11028 and standard error of 1.38097) showed more viable PDL cells followed by HBSS, A. vera, and PJ.Conclusion:Propolis, A. vera, and PJ can be used as an alternative tooth storage media.

Involvement of interleukin-23 induced by Porphyromonas endodontalis lipopolysaccharide in osteoclastogenesis

Periapical lesions are characterized by the destruction of periapical bone, and occur as a result of local inflammatory responses to root canal infection by microorganisms including Porphyromonas endodontalis (P. endodontalis). P. endodontalis and its primary virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical lesions and alveolar bone loss. Interleukin-23 (IL-23) is critical in the initiation and progression of periodontal disease via effects on peripheral bone metabolism. The present study investigated the expression of IL-23 in tissue where a periapical lesion was present, and the effect of P. endodontalis LPS on the expression of IL-23 in periodontal ligament (PDL) cells. Reverse transcription- quantitative polymerase chain reaction and immunohistochemistry revealed increased levels of IL-23 expression in tissue with periapical lesions compared with healthy PDL tissue. Treatment with P. endodontalis LPS increased the expression of IL-23 in the SH-9 human PDL cell line. BAY11-7082, a nuclear factor κB inhibitor, suppressed P. endodontalis LPS-induced IL-23 expression in SH-9 cells. Treatment of RAW264.7 cells with conditioned medium from P. endodontalis LPS-treated SH-9 cells promoted osteoclastogenesis. By contrast, RAW264.7 cells treated with conditioned medium from IL-23-knockdown SH-9 cells underwent reduced levels of osteoclastogenesis. The results of the present study indicated that the expression of IL-23 in PDL cells induced by P. endodontalis LPS treatment may be involved in the progression of periapical lesions via stimulation of the osteoclastogenesis process.