Human Gingival Epithelium Research Articles

Arecoline Induces ROS Accumulation, Transcription of Proinflammatory Factors, and Expression of KRT6 in Oral Epithelial Cells.

Areca nut is a major contributor to the high prevalence of oral cancer in Asia. The precise mechanisms by which areca nut stimulates mucosal cells and contributes to the progression of oral cancer urgently require clarification. The current study aimed to assess the effects of arecoline on the normal human gingival epithelium cell line S-G. Cell viability, levels of reactive oxygen species (ROS), protein expression, cellular morphology, and gene expression were evaluated using the MTT test, flow cytometry, Western blot analysis, optical or confocal microscopy, and RT-qPCR. Keratin (KRT6) analysis involved matched normal and cancer tissues from clinical head and neck specimens. The results demonstrated that 12.5 µg/mL of arecoline induced ROS production, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) mRNA expression in S-G cells. This activation of the MAPK/ERK pathway increased KRT6 expression while limiting cell migration. In head and neck cancer tissues, KRT6B gene expression exceeded that of normal tissues. This study confirms that arecoline induces ROS accumulation in normal cells, leading to the secretion of proinflammatory factors and KRT6 expression. This impedes oral mucosal healing, thereby promoting the progression of oral cancer.

Salivary microRNAs as innovative biomarkers for early diagnosis of oral diseases: a comparison of conventional cigarette smokers and tobacco heating system 2.2 users.

MicroRNAs (miRNAs) are considered valid prognostic and diagnostic biomarkers. The different miRNA expression profiles in cancer cells compared to normal cells make them potential biomarkers used for the early diagnosis of oral diseases. Following exposure to cigarette smoking, miRNA altered profile expression is associated with resistance mechanisms against anticancer therapies. Cellular models showed a reduced human gingival epithelium alteration after exposure to THS2.2 and a lower pathogenicity than 3R4F CS. The aim of the study was to compare the expression of saliva miRNA profile of THS2.2 and 3R4F CS users compared to patients not exposed to the risk factor and to identify and study the modulation of miRNAs associated with the development of oral diseases. In particular, we will focus on the analysis of a group of miRNAs know to be involved in the development of smoking-related diseases. The study will be performed in 18 months and dentists and biochemists will be involved in the different phases. To perform the study, healthy volunteers, including smokers of THS2.2 or 3R4F CS, will be enrolled. The samples will be collected from 3 experimental groups, each consisting of 30 subjects: group 1 (no smoking subjects), group 2 (subjects exposed to THS2.2), group 3 (subjects exposed to 3R4F CS). The collection of the saliva sample will be conducted in a standardized way. Following the collection, saliva will be processed. Previous studies have suggested that miRNAs are prognostic biomarkers for various smoking-related diseases. Based on the post-transcriptional regulation of some mRNAs connected to different oral pathologies, we expect a specific miRNA-mRNA interaction, which could be a starting point for the development of new possible diagnostic, therapeutic and prognostic approaches.

The Effect of Subacute Oral Folic Acid Treatment on Growth of Porphyromonas gulae in Dogs.

Periodontitis is one of the most prevalent infectious diseases in humans and animals. It is a multifactorial disease resulting in attachment loss and tooth loss. Therefore, preventive dentistry, such as daily teeth cleaning or providing dental chews from puppyhood is essential. This study aimed to find an alternative option for preventive dentistry by examining both in vitro and clinically, the antibacterial, antihalitosis, and anti-inflammatory effects of folic acid (FA) in dogs with periodontal disease. The antibacterial and antihalitosis responses of FA were evaluated in vitro using Porphyromonas gulae, a bacterium that plays a significant role in the development of periodontal disease in dogs. Anti-inflammatory responses, such as secretion of IL-1β, IL-6, and IL-8 induced by P. gulae infection in human gingival epithelium have been studied. This study used dogs with P. gulae-associated periodontal diseases and was conducted by providing a dental chew containing 0.13% FA for 28 days. The viability and halitosis production (hydrogen sulfide and methyl mercaptan) of P. gulae was significantly inhibited by FA in a dose and time-dependent manner. IL-1β, IL-6, and IL-8 secretion were also significantly suppressed by FA treatment in a dose-dependent manner. In vitro bactericidal, antihalitosis, and anti-inflammatory effects of FA were confirmed in dogs with P. gulae-associated periodontal disease. One month of oral treatment with 0.13% FA-containing dental chews significantly reduced halitosis as well as P. gulae activity. This study suggests that oral treatment with FA can be a preventive option for periodontal disease in dogs as well as humans.

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.

FOXO1 inhibits FSL-1 regulation of integrin β6 by blocking STAT3 binding to the integrin β6 gene promoter.

Integrin β6 (ITGB6), an epithelial-specific receptor, is downregulated in the gingival epithelium of periodontitis and is associated with inflammation response and periodontitis development. However, the transcriptional regulatory mechanism of ITGB6 downregulation in the human gingival epithelium remains unclear. Fibroblast-stimulating lipopeptide-1 (FSL-1), an oral biofilm component, promotes an epithelial cell-driven proinflammatory response in periodontitis partially by suppressing ITGB6 expression. The aim of the current study was to investigate the transcriptional regulatory mechanism of ITGB6 inhibition by FSL-1 in human epithelial cells (HaCaT and primary human gingival epithelial cells), and to delineate the transcriptional mechanism of ITGB6 suppression in periodontitis. We found that FSL-1 inhibited ITGB6 transcription through increasing forkhead box protein O1 (FOXO1) expression and inhibiting signal transducer and activator of transcription 3 (STAT3) activation. Furthermore, FOXO1 bound to STAT3 directly, leading to decreased STAT3 phosphorylation induced by FSL-1. Consequently, the binding of phosphorylated STAT3 to the ITGB6 promoter was decreased, and ITGB6 transcription was therefore downregulated following FSL-1 stimulation. The reciprocal action of STAT3 and FOXO1 on ITGB6 downregulation was also confirmed by the immunostaining of the inflammatory epithelium associated with periodontitis. Our findings suggest that the interaction of FOXO1–STAT3 may be a useful signal target for the treatment of periodontitis.

Regulation of the unfolded protein response transducer IRE1α by SERPINH1 aggravates periodontitis with diabetes mellitus via prolonged ER stress

The hyperglycemic microenvironment induced by diabetes mellitus aggravates the inflammatory response, in which the IRE1α signal transduction pathway of the unfolded protein response (UPR) participates. However, the mechanism by which hyperglycemia regulates the IRE1α signaling pathway and affects endoplasmic reticulum (ER) homeostasis in human gingival epithelium in periodontitis with diabetes mellitus remains unknown. Our current data provide evidence that diabetes mellitus causes a hyperinflammatory response in the gingival epithelium, which accelerates periodontal inflammation. Next, we assessed UPR-IRE1α signaling in periodontitis with diabetes mellitus by examining human clinical gingival epithelium samples from healthy subjects, subjects with periodontitis and subjects with periodontitis with diabetes mellitus and by in vitro challenge of human epithelial cells with a hyperglycemic microenvironment. The results showed that a hyperglycemic microenvironment inhibited the IRE1α/XBP1 axis, decreased the expression of a UPR target gene (GRP78), and ultimately impaired the UPR, causing ER stress to be prolonged or more severe in human gingival epithelium. Subsequently, RNA sequencing (RNA-seq) data was analyzed to investigate the expression of ER-related genes in human gingival epithelium. Experiments verified that the mechanism by which periodontitis is aggravated in individuals with diabetes mellitus may involve decreased SERPINH1 expression. Furthermore, experiments in SERPINH1-knockdown and SERPINH1-overexpression models established in vitro indicated that SERPINH1 might act as an activator of IRE1α, maintaining human gingival epithelium homeostasis and reducing proinflammatory cytokine expression by preventing prolonged ER stress induced by high-glucose conditions. In conclusion, regulation of the UPR transducer IRE1α by SERPINH1 alleviates periodontitis with diabetes mellitus by mitigating prolonged ER stress. This finding provides evidence for the further study of periodontitis with diabetes mellitus.

Caspase-3 and gasdermin E detection in peri-implantitis

Peri-implantitis could lead to progressive bone loss and implant failure; however, the mechanism of peri-implantitis remains unclear. Based on emerging evidence, pyroptosis, a novel proinflammatory programmed death, contributes to different oral infectious diseases. In the present study, we investigated the involvement of cleaved caspase-3 and gasdermin E (GSDME) in peri-implantitis and established a pyroptosis model in vitro. By collecting and examining the inflamed biopsies around peri-implantitis, we found that the pyroptosis-related markers (caspase-3, GSDME, and IL-1β) were enhanced relative to levels in control individuals. Furthermore, human gingival epithelium cells (HGECs) induced by tumor necrosis factor-α (TNF-α) exhibited pyroptosis morphological changes (cell swelling and balloon-shaped bubbles) and upregulated expression of pyroptosis-related markers. Pretreated with Ac-DEVD-CHO (a caspase-3 inhibitor) or GSDME small interference RNA (siRNA) were found to attenuate pyroptosis in HGECs. In conclusion, our findings revealed a high expression of caspase-3 and GSDME in the inflamed biopsies of peri-implantitis and confirmed that the caspase-3/GSDME pathway mediates TNF-α-triggered pyroptosis in human gingival epithelium cells, which provides a new target for peri-implantitis treatment.

Inhibitory effects of curcumin against cytotoxicity of Porphyromonas gingivalis outer membrane vesicles

ObjectiveThe purpose of this study was to examine whether curcumin, a turmeric root extract, protects human gingival epithelial (HGE) cells from the cytotoxic effects ofPorphyromonas gingivalis outer membrane vesicles (OMVs). DesignOMVs were prepared fromP. gingivalis OMZ314 and used to stimulate human gingival epithelial (HGE) cells. The effects of curcumin on cellular expression of inflammatory cytokines were evaluated using real-time reverse transcription-polymerase chain reaction assays, while those on cellular migration were examined with a scratch wound assay. Furthermore, HGE cells were incubated with OMVs in the presence or absence of curcumin, then intracellular invasion by OMVs was observed with confocal laser scanning microscopy. Also, the effects of curcumin on cellular apoptotic death was examined. ResultsGene expressions of IL-6, IL-1β, and TNF-α in HGE cells stimulated with OMVs were significantly suppressed by curcumin in a dose-dependent manner, with suppressed protein production also noted. Moreover, curcumin inhibited the cytotoxic effects of OMVs on cellular migration. Finally, curcumin inhibited OMV adherence to and entry of cells, as well as cellular apoptotic death in a dose-dependent manner. ConclusionsCurcumin showed marked inhibitory effects against the cytotoxic actions of P. gingivalis OMVs, indicating possible potency for preventing periodontal disease.

Repurposing Napabucasin as an Antimicrobial Agent against Oral Streptococcal Biofilms.

Objectives Disruption of microbial biofilms is an effective way to control dental caries. Drug resistance and side effects of the existing antimicrobials necessitate the development of novel antibacterial agents. The current study was aimed at investigating the antibacterial activities of the repurposed natural compound napabucasin against oral streptococci. Methods The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibition concentration, and minimum biofilm reduction concentration of Streptococcus mutans, Streptococcus gordonii, and Streptococcus sanguinis were examined by a microdilution method. Cytotoxicity of napabucasin against human oral keratinocytes, human gingival epithelia, and macrophage RAW264.7 was evaluated by CCK8 assays. The dead/live bacterium and exopolysaccharide in the napabucasin-treated multispecies biofilms were evaluated by confocal laser scanning microscopy. Microbial composition within the napabucasin-treated biofilms was further visualized by fluorescent in situ hybridization and qPCR. And the cariogenicity of napabucasin-treated biofilms was evaluated by transverse microradiography. Results Napabucasin exhibited good antimicrobial activity against oral streptococcal planktonic cultures and biofilms but with lessened cytotoxicity as compared to chlorhexidine. Napabucasin reduced the cariogenic S. mutans and increased the proportion of the commensal S. gordonii in the multispecies biofilms. More importantly, napabucasin significantly reduced the demineralization capability of biofilms on tooth enamels. Conclusion Napabucasin shows lessened cytotoxicity and comparable antimicrobial effects to chlorhexidine. Repurposing napabucasin may represent a promising adjuvant for the management of dental caries.

Invasion of Gingival Epithelial Cells by Porphyromonas gingivalis.

Porphyromonas gingivalis is a major pathogen responsible for severe and chronic manifestations of periodontal disease, which is one of the most common infectious disorders of humans. Although human gingival epithelium prevents intrusions by periodontal bacteria, P. gingivalis is able to invade gingival epithelial cells. To study the dynamics and the fate of intracellular P. gingivalis, confocal laser scanning microscopy (CLSM) is a method of choice. Information gained with CLSM contains not only the number of P. gingivalis associated with gingival epithelial cells but also the bacterial localization on/inside the host cells, morphological change of host cells, and physical interaction between the bacteria and host organelle. In this chapter, we describe the protocols for microscopy techniques to morphologically study gingival epithelial cells infected by P. gingivalis.

Maintaining barrier function of infected gingival epithelial cells by inhibition of DNA methylation.

Infection and inflammation induce epigenetic changes that alter gene expression. In periodontal disease, inflammation, and microbial dysbiosis occur, which can lead to compromised barrier function of the gingival epithelia. Here, we tested the hypotheses that infection of cultured human gingival epithelial (HGEp) cells with Porphyromonas gingivalis disrupts barrier function by inducing epigenetic alterations and that these effects can be blocked by inhibitors of DNA methylation. Primary HGEp cells were infected with P. gingivalis either in the presence or absence of the non-nucleoside DNA methyltransferase (DNMT) inhibitors RG108, (-) epigallocatechin-3-gallate (EGCG), or curcumin. Barrier function was assessed as transepithelial electrical resistance (TEER). DNA methylation and mRNA abundance were quantified for genes encoding components of three cell-cell junction complexes, CDH1, PKP2, and TJP1. Cell morphology and the abundance of cell-cell junction proteins were evaluated by confocal microscopy. Compared to non-infected cells, P. gingivalis infection decreased TEER (P<0.0001) of HGEp cells; increased methylation of the CDH1, PKP2, and TJP1 (P<0.0001); and reduced their expression (mRNA abundance) (P<0.005). Pretreatment with DNMT inhibitors prevented these infection-induced changes in HGEp cells, as well as the altered morphology associated with infection. Pathogenic infection induced changes in DNA methylation and impaired the barrier function of cultured primary gingival epithelial cells, which suggests a mechanism for systemic consequences of periodontal disease. Inhibition of these events by non-nucleoside DNMT inhibitors represents a potential strategy to treat periodontal disease.

Immunomodulation‐Based Strategy for Improving Soft Tissue and Metal Implant Integration and Its Implications in the Development of Metal Soft Tissue Materials

AbstractThe difficulties associated with metal implants and soft tissue integration have significantly affected the applications of metal implants in soft‐tissue‐related areas. Prompted by the close association between soft tissue integration and the immune response, an immunomodulation‐based strategy is proposed to manipulate the immune microenvironment and improve metal implant–soft tissue integration. Considering their vital roles in soft tissue responses to metal implants, macrophages are used and the cytokines fingerprints of M1 and M2 macrophage immune microenvironments are evaluated for their potential modulatory effects on metal implant–soft tissue integration. The modulatory effects of different immune microenvironments on model soft tissue cells (human gingival epithelium cells) cultured on model metal implants (titanium alloy disks) are then described, with the underlying possible mechanism FAK‐AKT‐mTOR signaling unveiled. As further proof of concept, IL‐4/PDA (polydopamine)‐coated titanium alloy implants, aiming at modulating M2 macrophage polarization, are prepared and found to improve the in vivo metal implant‐soft tissue integration. It is the authors' ambition that this immunomodulation‐based strategy will change the negative perception and encourage the active development of metal materials with favorable soft tissue integration properties, thus improving the success rates of perforating metal implants and broadening their application in soft‐tissue‐related areas.

High glucose disrupts autophagy lysosomal pathway in gingival epithelial cells via ATP6V0C

Hyperglycemic micro-environment induced by diabetes could regulate the response of periodontal tissues to pathogenic microorganisms in which disruption of autophagy lysosomal pathway (ALP) may participate. This study aimed to explore the mechanisms underlying how high glucose (HG) regulates ALP in gingival epithelial cells (GECs). Human gingival tissues in healthy group (C), periodontitis group (P), diabetes group (DM), and diabetes + periodontitis group (DP) were collected and were applied to observe the fusion of autophagy and lysosome. Diabetic mouse model with periodontitis was established and RNA-seq was applied to investigate the expression of ALP-associated genes in gingival epithelium. To explore the key role of ATPase transmembrane v0 domain, subunit c (ATP6V0C) in the disruption of ALP by HG, human gingival epithelial cells (HGECs) were cultured in 5.5mM/25mM glucose medium for 48hours and followed by Porphyromonas gingivalis stimulation for 0, 6, and 12hours. HBLV-h-ATP6V0C was transfected in HGECs that were stimulated by 25mM HG condition. Immunofluorescence double staining exhibited the disruption of ALP in human gingival epithelium in diabetes groups and HGECs under 25mM glucose condition, accompanied with significantly downregulated lysosomal acidity. RNA-seq of mouse gingival epithelium screened out Atp6v0c. Compared with HGECs in normal culture medium, ATP6V0C expression and LC3-II/I expression ratio were significantly downregulated, with an upregulated expression of P62, IL-1β in HGECs under HG condition. Over-expression of ATP6V0C rescued HG-induced disruption of ALP in HBLV-h-ATP6V0C transfected HGECs, with significantly upregulation of LC3-II/I and downregulation of P62, IL-1β. ATP6V0C mediates HG-induced ALP disruption in HGECs, eventually exacerbates periodontal inflammation.

Bismuth drugs tackle Porphyromonas gingivalis and attune cytokine response in human cells.

Periodontitis is the leading cause of severe tooth loss and edentulism in adults worldwide and is closely linked to systemic conditions such as diabetes and cardiovascular disease. Porphyromonas gingivalis is the key pathogen in periodontitis. Herein, we provided the first evidence that bismuth drugs suppress P. gingivalis in its planktonic, biofilm, and intracellular states. In total, 42 bismuth-associated proteins were identified including its major virulent factors (e.g., gingipains, hemagglutinin HagA, and fimbriae). Bismuth perturbed its iron acquisition, disturbed the energy metabolism and virulence, and deactivated multiple key enzymes (e.g., superoxide dismutase and thioredoxins). Moreover, bismuth inhibited its biofilm formation and disrupted the 3-day matured biofilms. Notably, the internalized P. gingivalis in various human cells (e.g., human gingival epithelium progenitors, HGEPs) was oppressed by bismuth but not the commonly used antibiotic metronidazole. Importantly, bismuth drugs enabled the counteraction of immuno-inflammatory responses in different host cells perturbed by P. gingivalis. The production of IL-6 and IL-8 attenuated by P. gingivalis in both of native and IL-1β-stimulated HGEPs was restored, while the bacterium-enhanced expression of IL-6, IL-1β, and TNFα in THP-1 macrophages was alleviated. This proof-of-concept study brings prospects for the potential reposition of the routinely used anti-Helicobacter pylori bismuth drugs to better manage inflammatory diseases such as periodontitis and P. gingivalis-related complex systemic disorders.

Human Gingival Epithelial Growth In Vitro on a Polymer-Infiltrated Ceramic Network Restorative Material.

To investigate the growth of primary human gingival epithelial (HGE) cells on polymer-infiltrated ceramic network (PICN) material (Vita Enamic) with different surface roughnesses. PICN material specimens were polished with either silica carbide paper (grit-polished) or the manufacturer's polishing wheels (wheel-polished), and the surface roughness (Ra ) measured. HGE cells were seeded and grown for 1, 3, or 6 days. Growth on tissue culture plastic was used as a control. Non-linear regression analysis was used to examine the effect of surface roughness on cell growth. HGE cell growth on tissue culture plastic fitted an exponential growth model over the 6-day experimental period (R2 = 0.966). Through day 6, cell density on PICN decreased with increasing surface roughness, with a fit to an exponential decay model (R2 = 0.666). A threshold Ra value of 0.254 μm (95% CI 0.177-0.332) was determined as an upper limit for exponential growth. Cell growth was greatest on the group of specimens with Ra value below 0.127μm. Specimens polished by the manufacturer's method produced surface roughness of 0.118 μm and below. PICN material polished to a smooth surface (Ra < 0.254 μm) resulted in exponential growth of HGE cell growth compared to rough surfaces. Polishing PICN material as smooth as possible (below a Ra of 0.127 μm) was found to maximize epithelial cell growth on the PICN material surface. The manufacturer's polishing method achieved a sufficiently smooth surface. These results are contrary to previous research regarding surface roughness of transgingival implant restoration components. The study results suggest that smoother restorative material surfaces could improve peri-implant soft tissue health.

Safety Assessment of Nano-Hydroxyapatite as an Oral Care Ingredient according to the EU Cosmetics Regulation

Hydroxyapatite nanoparticles (HAP-NP) are incorporated in oral care products such as toothpastes and mouthwashes to treat dental sensitivity or to promote enamel remineralisation. Despite the good performance of HAP-NP in this application, it is important to ensure its safety for consumers. For that reason, the Scientific Committee on Consumer Safety (SCCS) evaluated the safety of HAP-NP as an oral care ingredient, but the issued opinion was not completely conclusive and the SCCS recommended that additional tests should be performed. Here, we used a commercially available human gingival epithelium (HGE) as a non-animal alternative and MTT cell viability, LDH activity, and IL-1alpha production were evaluated after 3.1% HAP-NP treatment for 10 min, 1 h, and 3 h. Moreover, the absorption of HAP-NP in the gingival tissue was assessed by transmission electron microscopy (TEM) analysis. Finally, the dissolution behaviour of HAP-NP in simulated gastric fluid was also investigated. No deleterious effect was observed for HGE tissues incubated with HAP-NP for all time-points and parameters evaluated. Moreover, a complete dissolution of 3.1% HAP-NP in simulated gastric fluid was observed after 7.5 min at 37 °C. In conclusion, our results evidence the safety of HAP-NP for oral care products with the use of an in vitro replacement alternative for human gingival epithelium and a simulated gastric fluid assay.

Antifungal effect of tissue conditioners containing poly(acryloyloxyethyltrimethyl ammonium chloride)-grafted chitosan on Candida albicans growth in vitro

Background/purposeDenture stomatitis is a pathological condition affecting the mucosa underneath ill-fitting dentures, and Candida albicans is considered its main etiologic factor. Tissue conditioners are temporary lining materials often applied to dentures to treat inflamed tissues. However, tissue conditioners do not exert antifungal activity, and the soft surface texture harbors C. albicans easily. The aim of this study was to examine the antifungal activity of tissue conditioners modified with chitosan (CS) or a quaternized chitosan (QCS), which was synthesized by grafting 2-[(acryloyloxy)ethyl] trimethyl ammonium chloride onto CS. Materials and methodsTissue conditioners containing varying weight percentages of CS or QCS were prepared as experimental discs 10 mm in diameter and 1 mm in thickness. Samples were co-cultured with C. albicans and the number of colony forming units was recorded. Other evaluations included cell toxicity and tensile bond strength to the resin denture base. ResultsIt was found significantly fewer fungal colonies in tissue conditioners modified with CS or QCS, and none when the weight percentage of QCS exceeded 5%. CS and QCS did not affect the viability of human gingival epithelium cells or fibroblasts, and tensile bond strength did not differ between control and modified tissue conditioners. ConclusionThis study provides a foundation for the development of QCS as a novel and safe antifungal agent applied to tissue conditioners in clinical practice.

Vitamin D reduces the inflammatory response by Porphyromonas gingivalis infection by modulating human β-defensin-3 in human gingival epithelium and periodontal ligament cells

Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process. Porphyromonas gingivalis, a Gram-negative black-pigmented anaerobe, is a major pathogen in the initiation and progression of periodontitis; it produces several virulence factors that stimulate human gingival epithelium (HGE) cells and human periodontal ligament (HPL) cells to produce various inflammatory mediators. A variety of substances, such as vitamin D, have growth-inhibitory effects on some bacterial pathogens and have shown chemo-preventive and anti-inflammatory activity. We used a model with HGE and HPL cells infected with P. gingivalis to determine the influence of vitamin D on P. gingivalis growth and adhesion and the immunomodulatory effect on TNF-α, IL-8, IL-12 and human-β-defensin 3 production. Our results demonstrated, firstly, the lack of any cytotoxic effect on the HGE and HPL cells when treated with vitamin D; in addition, vitamin D inhibited P. gingivalis adhesion and infectivity in HGE and HPL cells. Our study then showed that vitamin D reduced TNF-α, IL-8, IL-12 production in P. gingivalis-infected HGE and HPL cells. In contrast, a significant upregulation of the human-β-defensin 3 expression in HGE and HPL cells induced by P. gingivalis was demonstrated. Our results indicate that vitamin D specifically enhances the production of the human-β-defensin 3 antimicrobial peptide and exerts an inhibitory effect on the pro-inflammatory cytokines, thus suggesting that vitamin D may offer possible therapeutic applications for periodontitis.

Promise of Combining Antifungal Agents in Denture Adhesives to Fight Candida Species Infections.

Several complications may arise in patients wearing complete prosthetic appliances, including denture-associated infections and mucosal stomatitis due to Candida species. This study evaluated the activity of anti-Candida agents in denture adhesive and the cytotoxicities of these preparations for primary human gingival epithelial (GE) keratinocytes. The anti-Candida activities of antimicrobial peptides, antimicrobial lipids, and antifungal agents against C. albicans ATCC 64124 or HMV4C were assessed in microdilution assays containing water or 1% denture adhesive. The minimal inhibitory concentrations (MIC) and the minimal bactericidal concentrations (MBC) were determined. The cytotoxicities of denture adhesive compounded with these agents were assessed in 1.0 × 105 primary GE keratinocytes in LGM-3 media with resazurin. Lactoferricin B, SMAP28, sphingosine, dihydrosphingosine, and phytosphingosine in 1% denture adhesive lost antimicrobial activity for C. albicans (p < 0.05). Amphotericin B, chlorhexidine dihydrochloride, chlorhexidine gluconate, fluconazole, and nystatin in 1% denture adhesive or compounded directly into denture adhesive and then diluted to 1% adhesive, did not lose antimicrobial activity. Compounded formulations were not cytotoxic (LD50 > 100.0 μg/ml) against primary human GE keratinocytes. Antimicrobial peptides and antimicrobial lipids had diminished activities in 1% adhesive, suggesting that components in adhesives may inactivate local innate immune factors in the oral cavity, possibly predisposing denture wearers to Candida species infections. More importantly, antifungal agents retained their anti-C. albicans activities in denture adhesive, strongly suggesting that antifungal agents could be candidates for inclusion in adhesive formulations and used as prescribed topical treatments for individuals with denture stomatitis.

Cellular Interactions and Formation of an Epithelial "Nanocoating-Like Barrier" with Mesoporous Silica Nanoparticles.

Oral mucosa as the front-line barrier in the mouth is constantly exposed to a complex microenvironment with multitudinous microbes. In this study, the interactions of mesoporous silica nanoparticles (MSNs) with primary human gingival epithelial cells were analyzed for up to 72 h, and their diffusion capacity in the reconstructed human gingival epithelia (RHGE) and porcine ear skin models was further assessed at 24 h. It was found that the synthesized fluorescent mesoporous silica nanoparticles (RITC-NPs) with low cytotoxicity could be uptaken, degraded, and/or excreted by the human gingival epithelial cells. Moreover, the RITC-NPs penetrated into the stratum corneum of RHGE in a time-dependent manner, while they were unable to get across the barrier of stratum corneum in the porcine ear skins. Consequently, the penetration and accumulation of RITC-NPs at the corneum layers of epithelia could form a “nanocoating-like barrier”. This preliminary proof-of-concept study suggests the feasibility of developing nanoparticle-based antimicrobial and anti-inflammatory agents through topical application for oral healthcare.