Gingival Epithelial Cells Research Articles

Evaluation of the Effects of E-Cigarette Aerosol Extracts and Tobacco Cigarette Smoke Extracts on Human Gingival Epithelial Cells.

Smoking increases the risk of a number of diseases, including cardiovascular, oral, and lung diseases. E-cigarettes are gaining popularity among young people as an alternative to cigarettes, but there is debate over whether they are less harmful to the mouth than e-cigarettes. In this study, human gingival epithelial cells (HGECs) were treated with four commercially available e-cigarette aerosol condensates (ECAC) or commercially available generic cigarette smoke condensates (CSC) with different nicotine concentrations. Cell viability was determined by MTT assay. Cell apoptosis was observed by acridine orange (AO) and Hoechst33258 staining. The levels of type I collagen, matrix metalloproteinase (MMP-1, MMP-3), cyclooxygenase 2, and inflammatory factors were detected by ELISA and RT-PCR. Finally, ROS levels were analyzed by ROS staining. The different effects of CSC and ECAC on HGECs were compared. The results showed that higher nicotine concentration of CS significantly reduced the activity of HGECs. By contrast, all ECAC had no significant effect. The levels of matrix metalloproteinase, COX-2, and inflammatory factors were higher in HGECs treated with CSC than those treated with ECAC. In contrast, the level of type I collagen was higher in HGECs treated with ECAC than those treated with CSC. In conclusion, all four flavors of e-cigarettes were less toxic to HGE cells than tobacco, but further clinical studies are needed to determine whether e-cigarettes are less harmful to oral health than conventional cigarettes.

Oxidative Status Determines the Cytotoxicity of Ascorbic Acid in Human Oral Normal and Cancer Cells.

Oral squamous cell carcinoma (OSCC) can arise anywhere in the oral cavity. OSCC's molecular pathogenesis is complex, resulting from a wide range of events that involve the interplay between genetic mutations and altered levels of transcripts, proteins, and metabolites. Platinum-based drugs are the first-line treatment for OSCC; however, severe side-effects and resistance are challenging issues. Thus, there is an urgent clinical need to develop novel and/or combinatory therapeutics. In this study, we investigated the cytotoxic effects of pharmacological concentrations of ascorbate on two human oral cell lines, the oral epidermoid carcinoma meng-1 (OECM-1) cell and the Smulow-Glickman (SG) human normal gingival epithelial cell. Our study examined the potential functional impact of pharmacological concentrations of ascorbates on the cell-cycle profiles, mitochondrial-membrane potential, oxidative response, the synergistic effect of cisplatin, and the differential responsiveness between OECM-1 and SG cells. Two forms of ascorbate, free and sodium forms, were applied to examine the cytotoxic effect and it was found that both forms had a similar higher sensitivity to OECM-1 cells than to SG cells. In addition, our study data suggest that the determinant factor of cell density is important for ascorbate-induced cytotoxicity in OECM-1 and SG cells. Our findings further revealed that the cytotoxic effect might be mediated through the induction of mitochondrial reactive oxygen species (ROS) generation and the reduction in cytosolic ROS generation. The combination index supported the agonistic effect between sodium ascorbate and cisplatin in OECM-1 cells, but not in SG cells. In summary, our current findings provide supporting evidence for ascorbate to serve as a sensitizer for platinum-based treatment of OSCC. Hence, our work provides not only repurposing of the drug, ascorbate, but also an opportunity to decrease the side-effects of, and risk of resistance to, platinum-based treatment for OSCC.

Marker of gingival epithelial cell aging in patients with non-alcoholic fatty liver disease

Shortening of periodontal telomere length can be not only a factor of physiological aging, but also be a marker of the adverse effects of non‑alcoholic fatty liver disease (NAFLD).
 Objective — to study the changes in periodontal status in patients with non‑alcoholic fatty liver disease.
 Materials and methods. 76 patients with non‑alcoholic fatty liver disease and 14 somatically healthy patients were selected. Questioning of patients, dental examination with determination of indexes, qPCR studies from scraping from the surface of the attached gum with disposable applicators were carried out.
 Results. Periodontal diseases in patients with NAFLD had an inflammatory nature of different clinical and morphological forms and were represented mainly by chronic generalized periodontitis. The length of telomeres was significantly shorter in patients with NAFLD compared with the respondents in the control group. A negative average strength relationship was found between telomere length and chronic generalized periodontitis (r = –0.590, p = 0.002), the presence of NAFLD (r = –0.506, p = 0.045), body mass index (r = –0.3068, p = 0.031), level of aspartate aminotransferase (r = –0.286, p = 0.017).
 Conclusions. Chronic inflammation and oxidative stress in the periodontal tissues in NAFLD contribute to premature cellular aging and shorten the telomere length of gingival epithelial cells. High body mass index is negatively correlated with telomere length, which also confirms premature cellular aging in patients with NAFLD.

Human gingival epithelial cells stimulate proliferation, migration, and tube formation of lymphatic endothelial cells invitro.

The aim of this study was to investigate the response of gingival epithelial cells to microbial and inflammatory signals. The gingival epithelial barrier provides the first line of defense and supports tissue homeostasis by maintaining the cross-talk between gingival epithelium, oral microbiota, and immune cells. Lymphatic vessels are essential to sustaining this homeostasis. The gingival epithelial cells have been shown to produce prolymphangiogenic factors during physiologic conditions, but their role in response to microbial and inflammatory signals is unknown. Immortalized human gingival epithelial cells (HGEC) and human dermal lymphatic microvascular endothelial cells (LEC) were cultured. HGEC were exposed to Porphyromonas gingivalis derived-LPS, human IL-1 beta/IL-1F2 protein, or recombinant human IL-6/IL-6R. Levels of vascular growth factors (VEGF-A, VEGF-C, and VEGF-D) in cell supernatants were determined by ELISA. LEC were grown to confluence, and a scratch was induced in the monolayer. Uncovered area was measured up to 48 h after exposure to conditioned medium (CM) from HGEC. Tube formation assays were performed with LEC cocultured with labelled HGEC or exposed to CM. VEGF-A, VEGF-C, and low levels of VEGF-D were constitutively expressed by HGEC. The expression of VEGF-C and VEGF-D, but not VEGF-A, was upregulated in response to proinflammatory mediators. VEGF-C was upregulated in response to P.gingivalis LPS, but not to Escherichia coli LPS. A scratch migration assay showed that LEC migration was significantly increased by CM from HGEC. Both the CM and coculture with HGEC induced significant tube formation of LEC. HGEC can regulate production of lymphangiogenic/angiogenic factors during inflammatory insults and can stimulate proliferation, migration, and tube formation of LEC in vitro in a paracrine manner.

Kaposi's Sarcoma-Associated Herpesvirus Immediate Early Proteins Trigger FOXQ1 Expression in Oral Epithelial Cells, Engaging in a Novel Lytic Cycle-Sustaining Positive Feedback Loop.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus that can replicate in oral epithelial cells to promote viral transmission via saliva. To identify novel regulators of KSHV oral infection, we performed a transcriptome analysis of KSHV-infected primary human gingival epithelial (HGEP) cells, which identified the gene coding for the host transcription factor FOXQ1 as the top induced host gene. FOXQ1 is nearly undetectable in uninfected HGEP and telomerase-immortalized gingival keratinocytes (TIGK) cells but is highly expressed within hours of KSHV infection. We found that while the FOXQ1 promoter lacks activating histone acetylation marks in uninfected oral epithelial cells, these marks accumulate in the FOXQ1 promoter in infected cells, revealing a rapid epigenetic reprogramming event. To evaluate FOXQ1 function, we depleted FOXQ1 in KSHV-infected TIGK cells, which resulted in reduced accumulation of KSHV lytic proteins and viral DNA over the course of 4 days of infection, uncovering a novel lytic cycle-sustaining role of FOXQ1. A screen of KSHV lytic proteins demonstrated that the immediate early proteins ORF45 and replication and transcription activator (RTA) were both sufficient for FOXQ1 induction in oral epithelial cells, indicating active involvement of incoming and rapidly expressed factors in altering host gene expression. ORF45 is known to sustain extracellular signal-regulated kinase (ERK) p90 ribosomal s6 kinase (RSK) pathway activity to promote lytic infection. We found that an ORF45 mutant lacking RSK activation function failed to induce FOXQ1 in TIGK cells, revealing that ORF45 uses a shared mechanism to rapidly induce both host and viral genes to sustain lytic infection in oral epithelial cells. IMPORTANCE The oral cavity is a primary site of initial contact and entry for many viruses. Viral replication in the oral epithelium promotes viral shedding in saliva, allowing interpersonal transmission, as well as spread to other cell types, where chronic infection can be established. Understanding the regulation of KSHV infection in the oral epithelium would allow for the design of universal strategies to target the first stage of viral infection, thereby halting systemic viral pathogenesis. Overall, we uncover a novel positive feedback loop in which immediate early KSHV factors drive rapid host reprogramming of oral epithelial cells to sustain the lytic cycle in the oral cavity.

Oral Cell Lysates Reduce the Inflammatory Response of Activated Macrophages

Necrotic cell damage occurs as a consequence of invasive dental procedures. Loss of membrane integrity being the hallmark of necrotic cells leads to the release of cytoplasmic and membranous components. Macrophages are predestined to respond to lysates originating from necrotic cells. Here, we implement necrotic lysates from human gingival fibroblasts, HSC2, and TR146 oral epithelial cell lines, and RAW264.7 macrophage cell lines to be tested for their potential to modulate the inflammatory response of macrophages. To this aim, necrotic cell lysates were prepared by sonication or freezing/thawing of the respective cell suspension. Necrotic cell lysates were tested for their potential to modulate the lipopolysaccharide (LPS)-induced expression of inflammatory cytokines using RAW264.7 macrophages as a bioassay. We show here that all necrotic cell lysates, independent of the origin and the preparation way, reduced the expression of IL1 and IL6 in LPS-induced RAW264.7 macrophages, most obviously shown for TR146 cells. This finding was supported in a bioassay when macrophages were exposed to poly (I:C) HMW, an agonist of TLR-3. Consistently, all necrotic lysates from gingival fibroblasts, HSC2, TR146, and RAW264.7 cells reduced the nuclear translocation of p65 in LPS-exposed macrophages. This screening approach supports the overall concept that necrotic cell lysates can modulate the inflammatory capacity of macrophages.

Porphyromonas gingivalis Evades Immune Clearance by Regulating Lysosome Efflux

Porphyromonas gingivalis, a major periodontal pathogen, invades autophagosomes of cells, including gingival epithelial cells, endothelial cells, gingival fibroblasts, macrophages, and dendritic cells, to escape antimicrobial autophagy and lysosome fusion. However, it is not known how P. gingivalis resists autophagic immunity, survives within cells, and induces inflammation. Thus, we investigated whether P. gingivalis could escape antimicrobial autophagy by promoting lysosome efflux to block autophagic maturation, leading to intracellular survival, and whether the growth of P. gingivalis within cells results in cellular oxidative stress, causing mitochondrial damage and inflammatory responses. P. gingivalis invaded human immortalized oral epithelial cells in vitro and mouse oral epithelial cells of gingival tissues in vivo. The production of reactive oxygen species (ROS) increased upon bacterial invasion, as well as mitochondrial dysfunction-related parameters with downregulated mitochondrial membrane potential and intracellular adenosine triphosphate (ATP), upregulated mitochondrial membrane permeability, intracellular Ca2+ influx, mitochondrial DNA expression, and extracellular ATP. Lysosome excretion was elevated, the number of intracellular lysosomes was diminished, and lysosomal-associated membrane protein 2 was downregulated. Expression of autophagy-related proteins, microtubule-associated protein light chain 3, sequestosome-1, the NLRP3 inflammasome, and interleukin-1β increased with P. gingivalis infection. P. gingivalis may survive in vivo by promoting lysosome efflux, blocking autophagosome-lysosome fusion, and destroying autophagic flux. As a result, ROS and damaged mitochondria accumulated and activated the NLRP3 inflammasome, which recruited the adaptor protein ASC and caspase 1, leading to the production of proinflammatory factor interleukin-1β and inflammation.

Experimental and numerical responses of fibroblast and epithelial cells to the frequency of electric toothbrush

In the oral environment, fibroblast and gingival epithelial cells undergo distinct forces. Chewing, brushing, or force interactions with dental materials like implants can produce these forces. The behavior and response of these cells to forces are determined by their stiffness. Additionally, this behavior can be crucial in mechanosensory and tissue development. In this study, after being cultured using nanomagnet materials, fibroblast and epithelial cells were subjected to magnetic tweezers cytometry testing, and the viscoelastic model was used to determine their stiffness. The reaction of single gingival cells was modeled by determining the stiffness of cells at Gel Point frequencies and the operating frequency of electric toothbrushes and employing the Finite Element Method (FEM). Epithelial cell and fibroblast gel points took place at frequencies of 5Hz and 3Hz, respectively. At these frequencies, the behavior of cells is both quasi-solid and fluid. In addition, the findings of the finite element analysis demonstrated that the cells undergo a greater degree of deformation at the Gel point frequency compared to the operating frequency of toothbrushes. This quantity was approximately 331 times greater in epithelial cells, which reached a maximum of 7.114 μm. Additionally, the maximal fibroblast cell deformation at 3Hz frequency was determined to be 2.981 μm, which is roughly 117 times that at 150Hz frequency.

Specific microRNA Signature Kinetics in Porphyromonas gingivalis-Induced Periodontitis.

Porphyromonas gingivalis is one of the major bacteria constituting the subgingival pathogenic polymicrobial milieu during periodontitis. Our objective is to determine the global microRNA (miRNA, miR) expression kinetics in 8- and 16-weeks duration of P. gingivalis infection in C57BL/6J mice and to identify the miRNA signatures at specific time-points in mice. We evaluated differential expression (DE) miRNAs in mandibles (n = 10) using high-throughput NanoString nCounter® miRNA expression panels. The bacterial colonization, alveolar bone resorption (ABR), serum immunoglobulin G (IgG) antibodies, and bacterial dissemination were confirmed. In addition, all the infected mice showed bacterial colonization on the gingival surface, significant increases in ABR (p < 0.0001), and specific IgG antibody responses (p < 0.05-0.001). The miRNA profiling showed 26 upregulated miRNAs (e.g., miR-804, miR-690) and 14 downregulated miRNAs (e.g., miR-1902, miR-1937a) during an 8-weeks infection, whereas 7 upregulated miRNAs (e.g., miR-145, miR-195) and one downregulated miR-302b were identified during a 16-weeks infection. Both miR-103 and miR-30d were commonly upregulated at both time-points, and all the DE miRNAs were unique to the specific time-points. However, miR-31, miR-125b, miR-15a, and miR-195 observed in P. gingivalis-infected mouse mandibles were also identified in the gingival tissues of periodontitis patients. None of the previously identified miRNAs reported in in vitro studies using cell lines (periodontal ligament cells, gingival epithelial cells, human leukemia monocytic cell line (THP-1), and B cells) exposed to P. gingivalis lipopolysaccharide were observed in the in vivo study. Most of the pathways (endocytosis, bacterial invasion, and FcR-mediated phagocytosis) targeted by the DE miRNAs were linked with bacterial pathogen recognition and clearance. Further, eighteen miRNAs were closely associated with the bacterial invasion of epithelial cells. This study highlights the altered expression of miRNA in gingiva, and their expression depends on the time-points of infection. This is the first in vivo study that identified specific signature miRNAs (miR-103 and miR-30d) in P. gingivalis invasion of epithelial cells, establishes a link between miRNA and development of periodontitis and helping to better understand the pathobiology of periodontitis.

A novel junctional epithelial cell line, mHAT-JE01, derived from incisor epithelial cells

ObjectivesJunctional epithelium (JE) connects the tooth surface and gingival epithelium and adheres directly to the tooth enamel. JE plays an important role as a barrier preventing the invasion of exogenous bacteria and substances. However, the cellular characteristics of this epithelium have not been adequately described, because no useful in vitro experimental model exists for JE. MethodsWe generated a novel JE cell line, mHAT-JE01, using naturally immortalized dental epithelium derived from incisor labial cervical cells and by selecting cells that adhered to apatite. mHAT-JE01 was characterized by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction and compared with the gingival epithelial cell line, mOE-PE01. ResultsThe mHAT-JE01 cells had a higher capacity for producing JE-specific markers than oral mucous epithelial cells. In addition, the presence of lipopolysaccharides from Porphyromonas gingivalis downregulated the expression of JE protein markers in mHAT-JE01 cells. ConclusionsThis cell line is stable and presents the opportunity to characterize JE efficiently, which is essential for the prevention and treatment of periodontal disease.

Selenomonas sputigena Interactions with Gingival Epithelial Cells That Promote Inflammation.

Increased prevalence and abundance of Selenomonas sputigena have been associated with periodontitis, a chronic inflammatory disease of tooth-supporting tissues, for more than 50 years. Over the past decade, molecular surveys of periodontal disease using 16S and shotgun metagenomic sequencing approaches have confirmed the disease association of classically recognized periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia while highlighting previously underappreciated organisms such as Filifactor alocis and S. sputigena. Despite abundant clinical association between S. sputigena and periodontal disease, we have little to no understanding of its pathogenic potential, and virulence mechanisms have not been studied. In this study, we sought to characterize the response of gingival epithelial cells to infection with S. sputigena. Here, we show that S. sputigena attaches to gingival keratinocytes and induces expression and secretion of cytokines and chemokines associated with inflammation and leukocyte recruitment. We demonstrate that S. sputigena induces signaling through Toll-like receptor 2 (TLR2) and TLR4 but evades activation of TLR5. Cytokines released from S. sputigena-infected keratinocytes induced monocyte and neutrophil chemotaxis. These results show that S. sputigena-host interactions have the potential to contribute to bacterially driven inflammation and tissue destruction, the hallmark of periodontitis. Characterization of previously unstudied pathogens may provide novel approaches to develop therapeutics to treat or prevent periodontal disease.

Calcitriol-enhanced autophagy in gingival epithelium attenuates periodontal inflammation in rats with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM)-associated periodontitis is a common disease with high prevalence, associated with persistent infection and complicated manifestations. Calcitriol (1 alpha, 25-dihydroxyvitamin D3, 1,25D) is the active form of vitamin D that plays a protective role in immune regulation, bone metabolism, and inflammatory response. In this study, we constructed a T2DM model in rats by combining a high-fat diet with low-dose streptozotocin. The periodontitis model in rats was developed by ligation and Porphyromonas gingivalis (ATCC 33277) inoculation. Rats were randomly divided into five groups: non-diabetic blank, diabetic blank, diabetes with calcitriol treatment, diabetes with 3-methyladenine (3-MA) treatment, or diabetes with calcitriol and 3-MA treatment. The diabetic rats exhibited an intense inflammatory response and decreased autophagy compared with the non-diabetic rats. Intraperitoneal injection of calcitriol and autophagy inhibitor (3-MA) allowed us to explore the effect of calcitriol on inflammation in the gingival epithelium and the role of autophagy in this process. Treatment with calcitriol resulted in the decreased expression of NFκB-p65, p62/SQSTM1 and inflammatory response and increased expression of LC3-II/LC3-I. Application of 3-MA significantly suppressed autophagy, which was apparently retrieved by calcitriol. Antibacterial peptide (LL-37) is the only antimicrobial peptide in the cathelicidin family that is found in the human body, and it exhibits a broad spectrum of antibacterial activity and regulates the immune system. In the present study, our findings indicated that calcitriol-enhanced autophagy may attenuated periodontitis and the decrease of LL-37 was rescued by calcitriol treatment in the gingival epithelial cells of T2DM rats. Our study provides evidence for the application of calcitriol as an adjunctive treatment for T2DM-associated periodontitis.

Trichomonas tenax induces barrier defects and modulates the inflammatory cytotoxicity of gingival and pulmonary epithelial cells.

Trichomonas tenax is a single-cell flagellated anaerobic organism, commensal in the human oral cavity. Although a previous study indicated that T. tenax could cause cell damage and phagocytose host epithelial cells, its pathological effects on gum cells remain unknown. Furthermore, several case reports have detected T. tenax in several patients with empyema and/or pleural effusion, which may have been aspirated from the oral cavity. However, the cytotoxic effects and immune responses of alveolar cells are unknown. Therefore, we aimed to determine the cytotoxic and immune effects of T. tenax on gums and pulmonary cell lines. The cytopathic effect and lactate dehydrogenase (LDH) cytotoxicity assays were used to determine the level of cell damage in gum and lung epithelial cells. Western blot was used to determine the disruption of cell junctions. Finally, epithelial cell cytokines were measured using ELISA to elucidate the immune response to T. tenax. We found that T. tenax induced a cytotoxic effect on gum epithelial cells by disrupting cell junctions; however, it hardly triggered cellular damage in alveolar A549 cells and mucoepidermoid NCI-H292 cells. Furthermore, T. tenax induced the production of IL-6 at a low multiplicity of infection (MOI) in gum, A549, and NCI-H292 cells. Our results suggest that T. tenax can trigger gingival cell cytotoxicity, disrupt cell junctions, and induce IL-6 production in gingival and pulmonary cell lines.

Ropinirole inhibits inflammatory cytokine production in gingival epithelial cells and suppresses alveolar bone loss in an experimental rat model of periodontitis.

The present study explored whether the dopamine 2-like receptor agonist, ropinirole, a drug used for treating Parkinson's disease, suppresses neutrophilic inflammation and alveolar bone loss in an experimental rat model of periodontitis. Periodontitis is a neutrophilic inflammatory disease caused by periodontal pathogens. An excessive T helper (Th)17 immune response is involved in the progression of periodontitis, and interleukin (IL)-17 promotes the exacerbation of inflammation and alveolar bone destruction. Recent evidence has suggested that dopamine signaling plays a key role in Th17 cell differentiation, and that dopamine 2-like receptor agonists suppress cytokine production from Th17 cells. We previously demonstrated that tannic acid, which is a dopamine 2-like receptor agonist, inhibits alveolar bone resorption in an experimental model of periodontitis. The present study used a carrageenan-induced rat model of periodontitis with or without ropinirole. Micro-computed tomography analysis was performed. Cells of the murine gingival epithelial cell line GE1 were stimulated with carrageenan and IL-17A in the presence or absence of ropinirole. The anti-inflammatory effect of ropinirole was analyzed using reverse transcription- quantitative PCR and enzyme-linked immunosorbent assay. Subsequently, in the carrageenan-induced rat model of periodontitis, alveolar bone resorption was observed in the maxillary second molar by micro-computed tomography analysis. Intriguingly, ropinirole suppressed the alveolar bone destruction. The expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-17 receptor A (IL-17RA) in GE1 cells were increased by carrageenan, and CXCL1 expression in GE1 cells was upregulated under IL-17A stimulation. Moreover, ropinirole inhibited CXCL1 and IL-17RA expression in GE1 cells in the presence of IL-17A and carrageenan. Finally, haloperidol promoted CXCL1 expression in GE1 cells in the presence of carrageenan. Overall, these findings suggested that ropinirole suppressed neutrophilic inflammation and alveolar bone destruction in periodontitis by inhibiting CXCL1 expression in gingival epithelial cells through the dopamine 2-like receptor. Thus, ropinirole shows promise as a drug for the treatment of periodontitis.

Relationship between periodontal disease and vitamin D

Vitamin D is a hormone synthesized by human skin cells or consumed through diet with immunomodulatory, anti-inflammatory, and antiproliferative effects. Vitamin D deficiency may increase the risk of periodontal disease by causing decreased bone mineral density, osteoporosis, progression of periodontal diseases, and resorption of the jawbone. In addition, vitamin D is important for bone metabolism, alveolar bone resorption, and the prevention of tooth loss. It increases the antibacterial defense of gingival epithelial cells, reduces gingival inflammation, accelerates postoperative wound healing after periodontal surgery, and is a key supplement functioning as a prophylaxis in periodontology. The present review study aims to highlight the role of vitamin D in periodontal disease.

The Bio-Aging of Biofilms on Behalf of Various Oral Status on Different Titanium Implant Materials.

The properties of titanium implants are affected by bio-aging due to long-term exposure to the oral microenvironment. This study aimed to investigate probable changes in titanium plates after different biofilm bio-aging processes, representing various oral status. Titanium plates with different surface treatments were used, including polish, sandblasted with large grit and acid etched (SLA), microarc oxidation (MAO), and hydroxyapatite coating (HA). We established dual-species biofilms of Staphylococcus aureus (S. aureus)-Candida albicans (C. albicans) and saliva biofilms from the healthy and patients with stage III-IV periodontitis, respectively. After bio-aging with these biofilms for 30 days, the surface morphology, chemical composition, and water contact angles were measured. The adhesion of human gingival epithelial cells, human gingival fibroblasts, and three-species biofilms (Streptococcus sanguis, Porphyromonas gingivalis, and Fusobacterium nucleatum) were evaluated. The polished specimens showed no significant changes after bio-aging with these biofilms. The MAO- and SLA-treated samples showed mild corrosion after bio-aging with the salivary biofilms. The HA-coated specimens were the most vulnerable. Salivary biofilms, especially saliva from patients with periodontitis, exhibited a more distinct erosion on the HA-coating than the S. aureus-C. albicans dual-biofilms. The coating became thinner and even fell from the substrate. The surface became more hydrophilic and more prone to the adhesion of bacteria. The S. aureus-C. albicans dual-biofilms had a comparatively mild corrosion effect on these samples. The HA-coated samples showed more severe erosion after bio-aging with the salivary biofilms from patients with periodontitis compared to those of the healthy, which emphasized the importance of oral hygiene and periodontal health to implants in the long run.

Glutamine promotes the proliferation of epithelial cells via mTOR/S6 pathway in oral lichen planus.

Although abnormal cell proliferation and apoptosis are associated with the pathogenesis of oral lichen planus (OLP), the exactly mechanism of which is not yet known. It has been reported that glutamine (Gln) can promote cell proliferation and inhibit apoptosis of various tumor cells. This study aims to evaluate the effect of Gln metabolism on the balance of proliferation and apoptosis in epithelial cells of OLP. Thirty human OLP specimens and 11 normal controls were stained by immunohistochemistry to detect the levels of proliferation and Gln metabolism related proteins. Then, the critical role of Gln in cell proliferation and apoptosis was determined by Gln deprivation or treatment with glutaminase inhibitor (CB-839) to intervene Gln metabolism in human gingival epithelial cells. Cell proliferation was detected using CCK8, p-mTOR and p-S6 proteins were detected using Western Blot, cell apoptosis and cell cycle were detected using flow cytometry, and cell stress was detected using immunofluorescence. Compared with normal controls, OLP specimens showed higher levels of Ki-67 and Gln metabolism-related proteins, including Gln transporter (ASCT2), glutaminase (GLS), and pathway proteins (p-mTOR and p-S6). In vitro, Gln promoted cell proliferation and simultaneously upregulated the activity of mTOR/S6 pathway. Moreover, rapamycin, an mTOR pathway inhibitor, could effectively block the Gln-induced cell proliferation. MHY1485, an mTOR pathway agonist, could effectively reverse the decline of cell proliferation under Gln deprivation. In addition, inhibiting Gln metabolism caused the accumulation of intracellular radical oxygen species (ROS) and induced cell apoptosis. However, N-acetylcysteine reversed this state and then decreased cell apoptosis by eliminating intracellular ROS. Gln metabolism is essential to maintain the balance of proliferation and apoptosis in oral epithelial cells, and inhibition of Gln metabolism may have a beneficial effect on OLP treatment.

Identification of a new probiotic strain, Lactiplantibacillus plantarum VHProbi® V38, and its use as an oral health agent.

Probiotics can be used to treat oral diseases such as dental caries, gingivitis, periodontitis, and halitosis. This study screened for strains capable of inhibiting Streptococcus mutans,one of the primary pathogenic bacteria responsible for dental caries by agar diffusion in different samples. Strain identification was performed by 16S rDNA sequencing and the API 50CH system. The potential functions of the strains in terms of oral health properties were also tested by agglutination assays, growth inhibition assays, adhesion assays, biofilm removal assays and inhibition of adhesion in human primary gingival epithelial (HPGE) cells assays. This study identified a probiotic strain from fermented cabbages that has a strong inhibitory effect on Streptococcus mutans. The API 50CH system and 16S rDNA sequencing verified that this was a new strain and it was given the name, Lactiplantibacillus plantarum VHProbi®V38. Agglutination, growth inhibition and adhesion, and biofilm removal tests indicated that L. plantarum VHProbi® V38 inhibited and reduced S. mutans. This probiotic was shown to have a broad antibacterial spectrum, simultaneously inhibiting the growth of periodontal pathogenic bacteria such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. After 2 hours of co-cultivation with these pathogens, L. plantarum VHProbi® V38 was able to significantly reduce pathogens adhesion on human primary gingival epithelial (HPGE) cells. These findings suggest that L. plantarum VHProbi® V38 could potentially prevent and treat periodontal diseases caused by these pathogenic bacteria. L. plantarum VHProbi® V38 also adheres strongly to HPGE cells and thus has potential as an oral probiotic. This study describes new methods that can be used to aid the screening and identification of oral probiotics.

Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death.

Dental plaque bacteria produce high concentrations of short-chain fatty acids (SCFAs), as bacterial metabolites. SCFA-treated gingival epithelial cells undergo cell death. Our previous reports demonstrated that butyrate-induced cell death depends on autophagy and reactive oxygen species (ROS). However, the precise mechanisms underlying SCFA-induced gingival epithelial cell death is poorly understood. Butyrate is a strong histone deacetylase (HDAC) inhibitor. Therefore, we determined the involvement of HDAC inhibitory activity in SCFA-induced gingival epithelial cells. Ca9-22 cells were used as an in vitro counterpart of gingival epithelial cells. Ca9-22 cells were treated with HDAC inhibitors in the presence or absence of C646, aP300 histone acetyltransferase (HAT) inhibitor, and compared the number of dead cells, which are measured using SYTOX Green dye. Acetylation levels of histone H3 were examined using western blotting. Changes in transcriptomes during the butyrate and C646 treatment were examined using RNA sequencing analysis. The butyrateor propionate-treatment of Ca9-22 cells induced acetylation of histone H3, while the C646 treatment strongly reduced the elevated acetylation levels. Accordingly, butyrate or propionate-induced cell death was inhibited by the C646 treatment. Similar results were obtained when other HDAC inhibitors were used. Whole transcriptome analysis revealed that the expression of numerous genes was altered by butyrate-induced histone acetylation. Moreover, some autophagy and ROS-related genes found in the altered genes might induce cell death. This study suggests the need for HDAC-inhibitory activity of bacterial metabolites to induce cell death, and the effects might enhance autophagy and ROS production.

The nature of changes in cell renewal and apoptosis of gingival epithelium in postmenopausal patients with chronic generalized periodontitis

Objective: to identify the nature of proliferation and apoptosis process disorders in the gingival epithelium in postmenopausal women with varying degrees of bone mineralization for early diagnosis and optimization of combination therapy. Materials and Methods. We examined 80 women aged 55–60 years with chronic generalized periodontitis (CGP) and reduced bone mineral density (BMD). All patients underwent a standard dental examination, including an index assessment of periodontal tissues. BMD was quantified via a densitometric analysis of the bone tissue condition. The investigation of periodontal epithelial cell renewal was performed using immunohistochemical studies. Results. We discovered that in postmenopausal patients with CGP and BMD disorders, there was a reduction in the proliferation of gingival epithelial cells with activation of their apoptosis (Iapt=0.73±0.03%, Iki-67=11.77±0.27%) vs. the patients with intact periodontium (Iapt 0.29±0.04%, Iki-67=9.88±0.09%), p=0.04. Conclusion. We discovered that CGP in postmenopausal women occurs with enlarged activity of apoptosis of gingival epithelial cells against the background of their reduced proliferation. The activity of inflammatory destructive processes in periodontal tissues is higher in patients with osteopenia vs. those without BMD impairment.