Human Gingival Tissue Research Articles

DNER and GNL2 are differentially m6A methylated in periodontitis in comparison with periodontal health revealed by m6A microarray of human gingival tissue and transcriptomic analysis

This study aims to investigate the differences in the epigenomic patterns of N6-methyladenosine (m6A) methylation in gingival tissues between patients with periodontitis (PD) and healthy controls, identifying potential biomarkers. As a multifactorial disease, PD involves multiple genetic and environmental effects. The m6A modification is the most prevalent internal mRNA modification and linked to various inflammatory diseases. However, the m6A modification pattern and m6A-related signatures in PD remain unclear. An m6A microarray of human gingival tissues was conducted in eight subjects: four diagnosed with PD and four healthy controls. Microarray analysis was performed to identify the differentially m6A methylated mRNAs (DMGs) and the differentially expressed mRNAs (DEGs). The differentially methylated and expressed mRNAs (DMEGs) were subjected to functional enrichment analysis by Metascape. The weighted gene co-expression network analysis (WGCNA) algorithm, the least absolute shrinkage and selection operator (LASSO) regression, and univariate logistic regression were performed to identify potential biomarkers. The cell type localization of the target genes was determined using single-cell RNA-seq (scRNA-seq) analysis. The m6A methylation level and gene expression of hub genes were subsequently verified by m6A methylated RNA immunoprecipitation (MeRIP) and quantitative real-time PCR (qRT-PCR). In total, 458 DMGs, 750 DEGs, and 279 DMEGs were identified based on our microarray. Pathway analyses conducted for the DMEGs revealed that biological functions were mainly involved in the regulation of stem cell differentiation, ossification, circadian rhythm, and insulin secretion pathways. Besides, the genes involved in crucial biological processes were mainly expressed in fibroblast and epithelial cells. Furthermore, the m6A methylation and expression levels of two hub biomarkers (DNER and GNL2) were validated. The current study exhibited a distinct m6A epitranscriptome, identified and verified two PD-related biomarkers (DNER and GNL2), which may provide novel insights into revealing the new molecular mechanisms and latent targets of PD.

Mural cell composition and functional analysis in the healing process of human gingiva from periodontal intrabony defects

ObjectiveTo evaluate the composition and function of mural cell populations in human gingival tissues DesignA cross-sectional study was conducted on seven periodontitis (stage Ⅲ) patients. Gingival tissues were collected two months after scaling and root planing and divided into 3 groups: 1, h_h group (horizontal bone resorption, residual pocket depth ≤3 mm); 2, v_h group (vertical bone resorption >4 mm, residual pocket depth ≤3 mm); 3, v_i group (vertical bone resorption >4 mm, residual pocket depth ≥6 mm). Single-cell RNA sequencing (10X genomics) and subsequent bioinformatics analysis were performed. Protein expression of selected genes was confirmed by histological staining. ResultsTwo mural cell clusters, RGS5+THY1+ and ACTA2+MYH11+ subpopulations, were identified and confirmed by histological staining and cross-validation with three different single-cell RNA sequencing datasets in the GEO database. RGS5+THY1+ cluster in perivascular areas possessed cellular protrusions and exhibited immunomodulatory and synthetic phenotypes. In contrast, the ACTA2+MYH11+ cluster strictly distributed around vessel walls was characterized by a contractile phenotype. Mural cells closely interacted with endothelial cells through PDGF and NOTCH3 signaling. Mural cell loss was detected in the v_i group and in hopeless periodontal teeth, which might be caused by tumor necrosis factor-alpha induced apoptosis. ConclusionsGingival mural cells can be classified into two distinct clusters according to their gene signatures and cell morphology. The loss of mural cells may indicate periodontitis progression.

Three-Dimensional Humanized Model of the Periodontal Gingival Pocket to Study Oral Microbiome.

The oral cavity contains distinct microenvironments that serve as oral barriers, such as the non-shedding surface of the teeth (e.g., enamel), the epithelial mucosa and gingival tissue (attached gingiva) where microbial communities coexist. The interactions and balances between these communities are responsible for oral tissue homeostasis or dysbiosis, that ultimately dictate health or disease. Disruption of this equilibrium can lead to chronic inflammation and permanent tissue damage in the case of chronic periodontitis. There are currently no experimental tissue models able to mimic the structural, physical, and metabolic conditions present in the human oral gingival tissue to support the long-term investigation of host-pathogens imbalances. Herein, the authors report an in vitro 3D anatomical gingival tissue model, fabricated from silk biopolymer by casting a replica mold of an adult human mandibular gingiva to recreate a tooth-gum unit. The model is based on human primary cultures that recapitulate physiological tissue organization, as well as a native oxygen gradient within the gingival pocket to support human subgingival plaque microbiome with a physiologically relevant level of microbial diversity up to 24 h. The modulation of inflammatory markers in the presence of oral microbiome indicates the humanized functional response of this model and establishes a new set of tools to investigate host-pathogen imbalances in gingivitis and periodontal diseases.

A high-throughput, 28-day, microfluidic model of gingival tissue inflammation and recovery

Nearly half of American adults suffer from gum disease, including mild inflammation of gingival tissue, known as gingivitis. Currently, advances in therapeutic treatments are hampered by a lack of mechanistic understanding of disease progression in physiologically relevant vascularized tissues. To address this, we present a high-throughput microfluidic organ-on-chip model of human gingival tissue containing keratinocytes, fibroblast and endothelial cells. We show the triculture model exhibits physiological tissue structure, mucosal barrier formation, and protein biomarker expression and secretion over several weeks. Through inflammatory cytokine administration, we demonstrate the induction of inflammation measured by changes in barrier function and cytokine secretion. These states of inflammation are induced at various time points within a stable culture window, providing a robust platform for evaluation of therapeutic agents. These data reveal that the administration of specific small molecule inhibitors mitigates the inflammatory response and enables tissue recovery, providing an opportunity for identification of new therapeutic targets for gum disease with the potential to facilitate relevant preclinical drug efficacy and toxicity testing.

Surface pre-reacted glass-ionomer eluate protects gingival epithelium from penetration by lipopolysaccharides and peptidoglycans via transcription factor EB pathway.

Surface pre-reacted glass-ionomer (S-PRG) filler, produced by PRG technology for use with various dental materials, is bioactive and known to release ions from a glass-ionomer phase. We previously reported that coxsackievirus and adenovirus receptor (CXADR), a tight junction associated protein, was located in the epithelial barrier of gingival epithelium. In the present study, the tissue protective effects of an S-PRG eluate prepared with S-PRG filler were investigated using a three-dimensional human gingival epithelial tissue model. The results showed that the S-PRG eluate specifically induced CXADR expression at the transcriptional level of messenger RNA as well as the protein level, and also nuclear translocation of transcription factor EB (TFEB) in gingival epithelial cells. Furthermore, shigyakusan, a TFEB inhibitor, canceled induction of the CXADR protein by the S-PRG eluate. Additionally, gingival epithelial permeation by 40-kDa dextran, lipopolysaccharide, and peptidoglycan in the 3D-tissue models was prevented by the eluate, with those effects abrogated by knockdown of CXADR. These findings suggest that S-PRG eluate increases CXADR expression via the TFEB pathway, thus inhibiting penetration of bacterial virulence factors into subepithelial tissues.

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air–liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

Human gingival mesenchymal stem cells retain their growth and immunomodulatory characteristics independent of donor age.

Aging has been reported to deteriorate the quantity and quality of mesenchymal stem cells (MSCs), which affect their therapeutic use in regenerative medicine. A dearth of age-related stem cell research further restricts their clinical applications. The present study explores the possibility of using MSCs derived from human gingival tissues (GMSCs) for studying their ex vivo growth characteristics and differentiation potential with respect to donor age. GMSCs displayed decreased in vitro adipogenesis and in vitro and in vivo osteogenesis with age, but in vitro neurogenesis remained unaffected. An increased expression of p53 and SIRT1 with donor age was correlated to their ability of eliminating tumorigenic events through apoptosis or autophagy, respectively. Irrespective of donor age, GMSCs displayed effective immunoregulation and regenerative potential in a mouse model of LPS-induced acute lung injury. Thus, we suggest the potential of GMSCs for designing cell-based immunomodulatory therapeutic approaches and their further extrapolation for acute inflammatory conditions such as acute respiratory distress syndrome and COVID-19.

PRDX6 alleviates lipopolysaccharide-induced inflammation and ferroptosis in periodontitis

Objective Periodontitis is a progressive and inflammatory oral disease and results in the damage of the supporting tissues of teeth. Peroxiredoxin 6 (PRDX6) is an antioxidant enzyme identified as a regulator in ferroptosis. This study aimed to investigate whether PRDX6 could protect human gingival fibroblasts (HGFs) from lipopolysaccharide (LPS)-induced inflammation and its mechanisms. Material and methods Both inflamed and non-inflamed human gingival tissues were collected to assess the expression of PRDX6 and nuclear factor erythropoietin 2-related factor 2 (NRF2) by Immunohistochemistry and Western blotting. Furthermore, the molecular mechanisms of PRDX6 have been clarified in PRDX6 silenced cells. The inflammatory cytokines in HGFs were measured by RT-qPCR and ELISA. The lipid hydroperoxide (LOOH) was detected by C11-BODIPY. Results The expression of PRDX6 and NRF2 were decreased in gingival tissues of severe periodontitis patients. The increased LPS-induced LOOH and inflammatory cytokines were found in PRDX6 knockdown HGFs. Besides, the inhibition of ferroptosis or PRDX6 phospholipase A2 activity (PLA2) alleviated LPS-induced inflammatory cytokines and LOOH. However, inhibiting NRF2 signalling upregulated those in HGFs. Conclusions Therefore, this study provided a new mechanistic insight that PRDX6, regulated by the NRF2 signalling, alleviates LPS-induced inflammation and ferroptosis in human gingival fibroblasts.

MZB1 targeted by miR-185-5p inhibits the migration of human periodontal ligament cells through NF-κB signaling and promotes alveolar bone loss.

To explore the role of Marginal Zone B and B-1 Cell-Specific Protein (MZB1), a novel molecule associated with periodontitis, in migration of human periodontal ligament cells (hPDLCs) and alveolar bone orchestration. MZB1 is an ER-localized protein and its upregulation has been found to be associated with a variety of human diseases. However, few studies have investigated the effect and mechanism of MZB1 on hPDLCs in periodontitis. Gene expression profiles in human gingival tissues were acquired from the Gene Expression Omnibus (GEO) database, and candidate molecules were then selected through bioinformatic analysis. Subsequently, we identified the localization and expression of MZB1 in human gingival tissues, mice, and hPDLCs by immunofluorescence, RT-qPCR, and Western blot. Dual-luciferase reporter assay was applied to assess the binding of miR-185-5p to MZB1. Furthermore, the effects of MZB1 on cell migration, proliferation, and apoptosis in vitro were investigated by wound-healing assay, transwell assay, CCK-8 assay, and flow cytometry analysis. Finally, Micro-CT analysis and H&E staining were performed to examine the effects of MZB1 on alveolar bone loss in vivo. Bioinformatic analysis discovered that MZB1 was one of the most significantly increased genes in periodontitis patients. MZB1 was markedly increased in the gingival tissues of periodontitis patients, in the mouse models, and in the hPDLCs treated with lipopolysaccharide of Porphyromonas gingivalis (LPS-PG). Furthermore, in vitro experiments showed that MZB1, as a target gene of miR-185-5p, inhibited migration of hPDLCs. Overexpression of MZB1 specifically upregulated the phosphorylation of p65, while pretreatment of MZB1-overexpressed hPDLCs with PDTC (NF-κB inhibitor) notably reduced the p-p65 level and promoted cell migration. In addition, the mRNA expression levels of alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2) were inhibited in MZB1-overexpressed hPDLCs and miR-185-5p inhibitor treated hPDLCs, respectively. In vivo experiments showed that knockdown of MZB1 alleviated the loss of alveolar bone. As a target gene of miR-185-5p, MZB1 plays a crucial role in inhibiting the migration of hPDLCs through NF-κB signaling pathway and deteriorating alveolar bone loss.

Preparation of three-dimensional matrices of human gingival tissue and comparison of induction of mesenchymal and blastema stem cell behaviors in prepared scaffolds.

This study aimed to compare the behavior of rat bone marrow mesenchymal stem cells and rabbit auricle blastema cells implanted in non-cellular gingival tissue scaffold of human. In this regard, the tissues obtained from gingival surgeries in the dental clinic were de-cellulated using two detergents of sodium dodecyl sulfate and triton 100-X. After washing and sterilization, they were used as a scaffold for culture with bone marrow mesenchymal stem cells of rats. Using light and electron microscopy, these scaffolds were examined before and after 1, 2, and 4 weeks of cell culture. Also, the prepared three-dimensional scaffold was placed in the blastema ring obtained from the rabbit earlobe punch. The samples were evaluated 1, 2, and 4 weeks after culture based on histology techniques. The results showed that the study of scaffolds by electron microscopy showed preserving the epithelium matrix and the collagen fibers in the tissue. Structures similar to the epithelium were created in both samples. In addition, induction of cellular secretion was observed in scaffold cells migrating to the scaffold. In general, scaffolds made from human gums can be a good platform for studying cellular behaviors. Of course, further experiments to determine the nature of differentiated cells can help advance our knowledge of matrix cell interactions.

Periodontitis promotes the expression of gingival transmembrane serine protease 2 (TMPRSS2), a priming protease for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

ObjectivesThe oral cavity is one of the main entry sites for SARS-CoV-2. Gingival keratinocytes express transmembrane serine protease 2 (TMPRSS2), responsible for priming the SARS-CoV-2 spike protein. We investigated whether periodontitis increased the expression of TMPRSS2. MethodsTo investigate gene expression in periodontitis, we analyzed the expression of specific genes from (1) the Gene Expression Omnibus (GEO) dataset of 247 human gingival tissues and (2) an experimentally-induced periodontitis mouse model. Human gingival tissues with or without periodontitis were immunohistochemically stained using an anti-TMPRSS2 antibody. Analysis of the TMPRSS2 promoter was performed using a ChIP-Atlas dataset. TMPRSS2 expression was detected in cultured human keratinocytes using quantitative reverse transcription (qRT)-PCR and Western blot analysis. ResultsGEO dataset analysis and an experimentally-induced periodontitis model revealed increased expression of TMPRSS2 in periodontitis gingiva. The keratinocyte cell membrane in periodontitis gingiva was strongly immunohistochemically stained for TMPRSS2. Using ChIP-Atlas and GEO datasets, we screened for transcription factors that bind to the TMPRSS2 promoter region. We found one candidate, estrogen receptor 1 (ESR1), highly expressed in periodontitis gingiva. Analysis of the GEO dataset revealed a correlation between ESR1 and TMPRSS2 expression in gingival tissues. An ESR1 ligand induced TMPRSS2 expression in cultured keratinocytes. ConclusionsPeriodontitis increases TMPRSS2 expression in the cell membrane of gingival keratinocytes.

Non-Invasive Physical Plasma Generated by a Medical Argon Plasma Device Induces the Expression of Regenerative Factors in Human Gingival Keratinocytes, Fibroblasts, and Tissue Biopsies.

After oral surgery, intraoral wound healing and tissue regeneration is an important factor for the success of the entire therapy. In recent years, non-invasive medical plasma (NIPP) has been shown to accelerate wound healing, which would be particularly beneficial for patients with wound healing disorders. Since the application of NIPP in dentistry has not been sufficiently understood, the aim of the present study was to investigate the effect of a medical argon plasma device on gingival cells. Human gingival fibroblasts, keratinocytes, and tissue biopsies were treated with NIPP for different durations. Crucial markers associated with wound healing were examined at the mRNA and protein levels by real-time PCR, ELISA and immunohistochemistry. NIPP treatment led to an increase in Ki67 and MMP1 at mRNA and protein levels. NIPP application lasting longer than 60 s resulted in an increase in apoptotic genes at mRNA level and superficial damage to the epithelium in the tissue biopsies. Overall, our experimental setup demonstrated that NIPP application times of 30 s were most suitable for the treatment of gingival cells and tissue biopsies. Our study provides evidence for potential use of NIPP in dentistry, which would be a promising treatment option for oral surgery.

Understanding the role of endotoxin tolerance in chronic inflammatory conditions and periodontal disease.

This review aims to present the current understanding of endotoxin tolerance (ET) in chronic inflammatory diseases and explores the potential connection with periodontitis. Subsequent exposure to lipopolysaccharides (LPS) triggers ET, a phenomenon regulated by different mechanisms and pathways, including toll-like receptors (TLRs), nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB), apoptosis of immune cells, epigenetics, and microRNAs (miRNAs). These mechanisms interconnect ET with chronic inflammatory diseases including periodontitis. While the direct correlation between ET and periodontal destruction has not been fully elucidated, emerging reports point towards the potential tolerization of human periodontal ligament cells (hPDLCs) and gingival tissues with a significant reduction of TLR levels. There is a potential link between ET and periodontal diseases. Future studies should explore the crucial role of ET in the pathogenesis of periodontal diseases, as evidence of a tolerized oral mucosa may represent an intrinsic mechanism capable of regulating the oral immune response. A clear understanding of this host immune regulatory mechanism might lead to effective and more predictable therapeutic strategies to treat chronic inflammatory diseases and periodontitis.

Mixed lineage kinase domain-like pseudokinase-mediated necroptosis aggravates periodontitis progression.

Necroptosis is a form of cell death that is reportedly involved in the pathogenesis of periodontitis. The role of Mlkl-involved necroptosis remains unclear. Herein, this project aimed to explore the role of MLKL-mediated necroptosis in periodontitis in vitro and in vivo. Expression of RIPK3, MLKL, and phosphorylated MLKL was observed in gingival tissues obtained from healthy subjects or patients with periodontitis. The cell viability of Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells was detected. In wild type or Mlkl deficiency mice with ligature-induced periodontitis, alveolar bone loss and osteoclast activation were assessed. mRNA levels of inflammatory cytokines in bone marrow-derived macrophages were tested by qRT-PCR. Increased expression of RIPK3, MLKL, and phosphorylated MLKL was observed in gingival tissues obtained from patients with periodontitis. Porphyromonas gingivalis lipopolysaccharide (LPS-Pg)-treated cells developed necroptosis after caspase inhibition and negatively regulated the NF-κB signaling pathway. In mice with ligature-induced periodontitis, Mlkl deficiency reduced alveolar bone loss and weakened osteoclast activation. Furthermore, genetic ablation of Mlkl in LPS-Pg-treated bone marrow-derived macrophages increased the mRNA levels of tumor necrosis factor-α, interleukin (Il)-1β, Il-6, cyclooxygenase 2, matrix metalloproteinase 9, and receptor activator of nuclear factor kappa-B ligand. Our data indicated that MLKL-mediated necroptosis aggravates the development of periodontitis in a Mlkl-deficient mouse. This will provide a new sight for the understanding of etiology and therapies of periodontitis. KEY MESSAGES: MLKL expression was up-regulated in inflamed human gingival tissue. Mlkl deficiency affected the progression of periodontitis. Necroptosis played a major role in mice periodontitis model. Knockout of Mlkl had a significant effect on inflammatory responses.

Downregulation of protein and mRNA levels of vimentin in periodontitis – A potential biomarker candidate for periodontal severity?

ObjectiveTo identify a differentially expressed structural protein in moderate and severe periodontitis and to quantify its corresponding mRNA levels. The secondary objective is to find correlation, if any, between Porphyromonas gingivalis load and mRNA levels of the protein. Materials and methodsTotal protein was extracted from 30 human gingival tissues (10 each from healthy, moderate and severe periodontitis) and subjected to 2D-gel electrophoresis. Protein identification was done by LC-MS/MS. The mRNA levels of protein and load of Porphyromonas gingivalis were further quantified by qRT-PCR. Results2D-gel Image Master 2D Platinum 7.0 analysis revealed a significant decrease (p ≤ 0.02) in the protein of MW 53.6 kDa in severe periodontitis compared to moderate periodontitis and healthy controls. LC-MS/MS analysis of the protein by MASCOT identified the protein as vimentin. Further, a three-fold decrease in mRNA expression of vimentin in periodontitis was obtained. A weak negative correlation was observed between Porphyromonas gingivalis load and mRNA levels of vimentin. ConclusionDifferentially expressed protein in 2D gel was identified as structural protein Vimentin and it was significantly downregulated at gene and protein level in periodontitis. This suggests the participation of vimentin in the pathogenesis of periodontitis.

Autophagy upregulates inflammatory cytokines in gingival tissue of patients with periodontitis and lipopolysaccharide-stimulated human gingival fibroblasts.

BackgroundPeriodontitis is an inflammatory disease caused by multiple disease‐associated bacterial species in periodontal tissues. Autophagy is known to modulate various inflammation‐driven diseases and inflammatory responses, but the role of autophagy related to the pathogenesis of periodontitis is not fully established. We investigated whether autophagic flux regulated the expression of inflammatory cytokines in the gingiva of periodontitis patients and lipopolysaccharide (LPS)‐stimulated human gingival fibroblasts (HGFs) and the underlying mechanism.MethodsThe mRNA and protein expression of proinflammatory cytokines was assessed in human gingival tissues collected from patients with periodontitis and HGFs treated with LPS. The expression of signaling molecules related to autophagy was evaluated by immunofluorescence and Western blot analyses.ResultsThe expression of interleukin (IL)‐6, tumor necrosis factor‐α (TNF‐α), cyclooxygenase‐2 (COX‐2), and intercellular adhesion molecule‐1 (ICAM‐1) was increased in the gingival tissues of patients with periodontitis. LC3B‐positive cells, a typical autophagic marker, were increased in the gingival tissues of periodontitis patients and LPS‐treated HGFs. The conversion ratio of LC3‐I to LC3‐II was higher in the gingival tissues associated with periodontitis and LPS‐treated HGFs compared to the controls. The autophagy inhibitor 3‐methyladenine (3MA) significantly abrogated the LPS‐sustained inflammatory effect by reducing the expression of IL‐6, TNF‐α, COX‐2, and ICAM‐1 in HGFs. The phosphorylation of protein kinase B (AKT) and protein S6K1 (S6), signals involved in the mTOR‐dependent mechanism, was decreased in gingiva derived from periodontitis patients and LPS‐treated HGFs.ConclusionsAutophagy augmented the production of inflammatory cytokines by mTOR inactivation via the AKT signaling pathway in the gingival tissues of patients with periodontitis and LPS‐stimulated HGFs. These findings would provide a better understanding of the mechanism by which autophagy regulates the inflammatory response associated with periodontal pathogenesis.

Clinically Healthy Human Gingival Tissues Show Significant Inter-individual Variability in GCF Chemokine Expression and Subgingival Plaque Microbial Composition.

Aim: Clinically healthy gingival tissue is maintained through controlled regulation of host defense mechanisms against plaque biofilm overgrowth. One key component is the transit of neutrophils from the vasculature into gingival tissue where the expression of different neutrophil chemokines are tightly regulated. This cross-sectional study examines the inter-individual variability in chemokine profiles within gingival crevicular fluid (GCF) in relation to the subgingival bacterial community in a state of gingival health.Methods: Gingival crevicular fluid and subgingival plaque samples were collected from mesiobuccal surfaces of all six Ramfjord teeth of 20 systemically healthy individuals (14.55 ± 1.67 years). A multiplex immunoassay was carried out to quantify the expression of 40 different chemokines in the healthy gingival tissue. Neutrophils were assessed indirectly by myeloperoxidase (MPO) in GCF using traditional ELISA. Characterization of healthy subgingival plaque was conducted with the Illumina Miseq targeting the 16S rRNA gene.Results: In health, there are distinct variations within individual gingival crevicular fluid chemokine expression profiles, as well as in the concentration of neutrophils, that divided the participants into high or low chemokine expressing groups. Specifically, key differences were identified within MIF (2683.54 ± 985.82 pg per 30-s sample), IL-8/CXCL8 (170.98 ± 176.96 pg per 30-s sample), Gro-α/CXCL1 (160.42 ± 94.21 pg per 30-s sample), ENA-78/CXCL5 (137.76 ± 76.02 pg per 30-s sample), IL-1β (51.39 ± 37.23 pg per 30-s sample), TNF-α (1.76 ± 1.79 pg per 30-s sample), and IFN-γ (0.92 ± 0.54 pg per 30-s sample). Of these identified chemokines, the highest correlation was associated between IL-8/CXCL8 and neutrophils (r = 0.54, p = 0.014). Furthermore, species characterization of healthy subgingival plaque revealed significant inter-individual variability that identified two unique groups unrelated to the previously identified chemokine groups.Conclusion: The lack of concordance between the microbial composition and chemokine profile during health may be a reflection of the unique microbial composition of each individual coupled with variations within their host response, emphasizing the vast complexity of the defense mechanisms in place to maintain gingival health.

Increased Sphingosine-1-Phosphate Serum Concentrations in Subjects with Periodontitis: A Matter of Inflammation.

PurposePeriodontitis is an inflammatory disease of the oral cavity with an alarmingly high prevalence within the adult population. The signaling lipid sphingosine-1-phosphate (S1P) plays a crucial role in inflammatory and immunomodulatory responses. In addition to cardiovascular disease, sepsis and tumor entities, S1P has been recently identified as both mediator and biomarker in osteoporosis. We hypothesized that S1P may play a role in periodontitis as an inflammation-prone bone destructive disorder. The goal of our study was to evaluate associations between periodontitis and S1P serum concentrations in the Study of Health in Pomerania (SHIP)-Trend cohort. In addition, we investigated the expression of S1P metabolizing enzymes in inflamed gingival tissue.Patients and MethodsWe analyzed data from 3371 participants (51.6% women) of the SHIP-Trend cohort. Periodontal parameters and baseline characteristics were assessed. Serum S1P was measured by liquid chromatography tandem mass spectrometry. The expression of S1P metabolizing enzymes was determined by immunofluorescence staining of human gingival tissue.ResultsS1P serum concentrations were significantly increased in subjects with both moderate and severe periodontitis, assessed as probing depth and clinical attachment loss. In contrast, no significant association of S1P was seen with caries variables (number and percentage of decayed or filled surfaces). S1P concentrations significantly increased with increasing high-sensitivity C-reactive protein (hs-CRP) levels. Interestingly, inflamed compared to normal human gingival tissue exhibited elevated expression levels of the S1P-generating enzyme sphingosine kinase 1 (SphK1).ConclusionWe report an intriguingly significant association of various periodontal parameters with serum levels of the inflammatory lipid mediator S1P. Our data point towards a key role of S1P during periodontitis pathology. Modulation of local S1P levels or its signaling properties may represent a potential future therapeutic strategy to prevent or to retard periodontitis progression and possibly reduce periodontitis-related tooth loss.

Cytotoxicity determination of nano-zinc oxide eugenol on human gingival fibroblast cells

Zinc oxide eugenol (ZOE) is composed of approximately 70% ZnO and approximately 30% eugenol. ZOE has been used in dentistry as a temporary filler, cavity liner, and base for tooth cavities. It is used to form a thin layer of cement to protect the pulp from any influx of bacteria or irritants present in the tooth cavity. Such bacteria and irritants can lead to the formation of an abscess or pus in the gums or periapical tissues. As ZnO has antibacterial effects, it is appropriate to use ZOE as a temporary filler, liner, or base. On the other hand, eugenol alone is acidic and may leach out to have adverse effects on the periapical tissue. Therefore, the toxicity level of ZOE is important to avoid adverse effects or unnecessary dosing. Thus, in the present study, we investigated the potential toxicity of ZOE leaching toward human gingival fibroblast tissue to verify its safety and efficacy by using ZnO particles with different morphologies and sizes. Three types of ZnO powder were used in this study: ZnO-A and ZnO–B (which were produced using the French process) and ZnO–K, which are commercial particles used for ZOE temporary fillers. All three types of ZnO were mixed with eugenol to form ZOE. Then, all three types were characterised to determine their morphology, particle size, and specific surface area. Each type of ZnO was then mixed with resin-bonded eugenol to obtain ZOE pellets. Subsequently, the toxicology of the ZOE leachings was tested on human gingival fibroblast cells using a calorimetric assay for 24 and 48 h. The results show that ZnO-A, ZnO–B, and ZnO–K have particle sizes of 22.3, 48.33, and 1196 nm, respectively. The specific surface areas were 4.44, 3.89, and 0.74 m2/g, respectively. Moreover, the toxicology of the ZOE leachings revealed a lower percentage of viable cells with an increase in the leaching concentration. After 24 and 48 h of cytotoxicity tests, the percentage of viable cells decreased slightly with leaching concentrations of 10%–40% and decreased considerably with leaching concentrations of 50%–100%. This shows that the viable cell percentage depends on the leaching concentration and duration of the exposure. Moreover, the results show that smaller particles have larger specific surface areas and higher percentages of viable cells. This means that the smaller particles released smaller amounts of eugenol and Zn2+ during the toxicity evaluation, which led to less reactive oxygen species (ROS) production and influenced the number of cells that survived after 24 and 48 h.

English

BACKGROUND Sutures require specific physical characteristics and properties such as good tensile strength, dimensional stability, lack of memory, knot security and sufficient flexibility to avoid damage to the oral mucosa. The strength and adherence of the sutured tissue increases over time. Tissue reaction characteristics involve varying degree of inflammatory reactions. Therefore, the purpose of this study was to assess clinically and histologically human gingival tissue reaction to silk & m- polytetrafluoroethylene (m-PTFE) sutures in periodontal surgical procedures. METHODS The present study is a prospective clinical randomised split mouth study. A total of 15 patients of both sexes were included in the study. All the patients were provided with thorough scaling and root planing before the commencement of the study. The patients were re-evaluated at 4 weeks and sutures were placed in the surgical area where there was a need for excision of gingival tissue as a part of routine periodontal surgery. After placement of sutures, the change of bite, change of slack, clinical changes in plaque index, modified gingival index & sulcus bleeding index and the histological changes in mean thickness of the peri sutural epithelial sleeve, proportion of inflammatory cells to peri sutural epithelial cells, diameter of the connective tissue inflammatory infiltrate (measured in mm) from the periphery of the epithelialization of the sutures from the insertion day (day 0) to the day of scheduled surgery (day 7) were recorded. RESULTS Silk sutures presented an increase in the change of bite and change of slack of the suture loop than m-PTFE. Diameter of the connective tissue infiltrate was greater around the multifilament suture (SILK) as compared to the monofilament suture (mPTFE) though the difference was not statistically significant. (P-value = 0.321). Monofilament suture (m-PTFE) showed the least amount of inflammatory infiltrate around the suture track. The proportion of inflammatory cells to epithelial cells was statistically not significant between the two suture materials (P-value = 0.82). CONCLUSIONS Though the clinical and histological parameters are statistically non-significant, mpolytetrafluoroethylene has shown better results compared to silk. Silk sutures cause a more extensive inflammatory tissue reaction in an environment characterized by moisture and infectious potential compared to m-PTFE sutures. KEY WORDS PTFE, Inflammatory Response, Sutures, Gingiva