Gingival Keratinocytes Research Articles

An Ayurvedic herbal extract inhibits oral epithelial cell IL-8 responses to host and bacterial agonists

BackgroundNatural products constitute a promising class of therapeutics for the treatment of gingivitis and periodontitis as well as the maintenance of oral health. However, the limited understanding behind their potential mechanisms and modes of action have hampered their incorporation into popular western therapeutics. This in vitro study characterizes an Ayurvedic herbal extract mixture, which has been clinically shown to promote gingival health and homeostasis.MethodsTelomerase immortalized gingival keratinocytes (TIGK) were infected with either Fusobacterium nucleatum cell wall, live F. nucleatum, IL-1β or TNF-α for 4 hours with and without the herbal extract. The immunomodulatory effects of the extract on host IL-8 production was measured by ELISA.ResultsIt was found that the Ayurvedic herbal extract mixture inhibited gingival epithelial cell IL-8 expression in response to both bacterial and host cytokine agonists. The herbal extract inhibited IL-8 stimulated by F. nucleatum cell wall, live F. nucleatum, IL-1β, and TNF-α in a dose-dependent manner that was not a result of host cell death. Furthermore, the extract showed significantly different ID50 doses demonstrating the differential ability to modulate both stimulated and basal IL-8 levels.ConclusionsIn vitro investigation of this herbal extract mixture revealed that it has the ability to modulate gingival epithelial cell IL-8 expression in response to stimulation by bacterial components and host pro-inflammatory signals. This data demonstrates that the reduction in the gingival epithelial cell IL-8 response may in part be responsible for the previously reported ability of the Ayurvedic herbal extract mixture to reduce gingivitis in two separate human clinical studies.

Stimulation of MMP-9 of oral epithelial cells by areca nut extract is related to TGF-β/Smad2-dependent and -independent pathways and prevented by betel leaf extract, hydroxychavicol and melatonin.

Background: There are 200-600 million betel quid (BQ) chewers in the world. BQ increases oral cancer risk. Matrix metalloproteinase-9 (MMP-9) is responsible for matrix degradation, cancer invasion and metastasis. Whether areca nut extract (ANE), a BQ component, stimulates MMP-9 secretion, and the related signaling pathways awaits investigation.Results: ANE (but not arecoline) stimulated MMP-9 production of gingival keratinocytes and SAS cancer epithelial cells. ANE stimulated TGF-β1, p-Smad2, and p-TAK1 protein expression. ANE-induced MMP-9 production/expression in SAS cells can be attenuated by SB431542 (ALK5/Smad2 inhibitor), 5Z-7-Oxozeaenol (TAK1 inhibitor), catalase, PD153035 (EGFR tyrosine kinase inhibitor), AG490 (JAK inhibitor), U0126 (MEK/ERK inhibitor), LY294002 (PI3K/Akt inhibitor), betel leaf (PBL) extract, and hydroxychavicol (HC, a PBL component), and melatonin, but not by aspirin.Conclusions: AN components contribute to oral carcinogenesis by stimulating MMP-9 secretion, thus enhancing tumor invasion/metastasis. These events are related to reactive oxygen species, TGF-β1, Smad2-dependent and –independent signaling, but not COX. These signaling molecules can be biomarkers of BQ carcinogenesis. PBL, HC and melatonin and other targeting therapy can be used for oral cancer treatment.Methods: ANE-induced MMP-9 expression/secretion of oral epithelial cells and related TGF-β1, Smad-dependent and –independent signaling were studied by MTT assay, RT-PCR, western blotting, immunofluorescent staining, and ELISA.

Endothelial Progenitor Cells inhibit jaw osteonecrosis in a rat model: A major adverse effect of bisphosphonate therapy

Medication-related osteonecrosis of the jaw (MRONJ) is a serious adverse effect of antiresorptive and antiangiogenic therapies. MRONJ is identified by chronic wounds in the oral mucosa associated with exposed necrotic bone. We hypothesized that zoledronic acid (ZOL) impairs keratinocyte and fibroblast function and reduces soft tissue vascularization; therefore, treating MRONJ with proangiogenic cells may benefit MRONJ patients. The effect of ZOL and dexamethasone (DEX) on gingival fibroblasts and keratinocytes was investigated. In-vitro, ZOL inhibited fibroblast and keratinocyte proliferation, delaying scratch healing. In-vivo, exposed bone was detected at tooth extraction sites, mainly in ZOL(+)/DEX(+) rats; and was associated with significantly decreased soft tissue vascularization, serum-VEGF, and tissue-VEGF. Local injection of early and late endothelial progenitor cells (EPCs) healed 13 of 14 MRONJ lesions compared with 2/7 lesions in the mesenchymal stem cells, and 2/6, in culture-medium group. The EPCs reduced necrotic bone area, increased serum and tissue VEGF levels. EPCs engraftment was minimal, suggesting their paracrine role in MRONJ healing. The EPC-conditioned medium improved scratch healing of keratinocytes and fibroblasts via VEGF pathway and elevated mRNA of VEGFA and collagen1A1. In conclusion, a novel MRONJ treatment with EPCs, increased vascularization and improved epithelial and fibroblast functions as well as cured the lesion.

In Vitro Evaluation of Substantivity, Staining Potential, and Biofilm Reduction of Guava Leaf Extract Mouth Rinse in Combination with its Anti-Inflammatory Effect on Human Gingival Epithelial Keratinocytes.

This study aimed to assess the biofilm reduction, staining potential, and cytotoxicity of guava extract mouth rinse compared to chlorhexidine (CHX). Substantivity, staining, and antibiofilm potential were investigated by spectrophotometry, colony-forming units, and luminosity color meter, respectively. The cell viability assay was conducted using a colorimetric assay to determine nontoxic levels of guava (0.15%) and CHX in human gingival epithelial keratinocytes (HGEK-16). Cells were treated with lipopolysaccharides (LPS, 1μg/mL) and guava to assess inflammatory gene expression levels of interleukin-β1, tumor necrosis factor-α, and Prostaglandin E2. A scratch wound healing assay investigated the effects of guava on cell migration. The teeth coated in guava mouth rinse displayed 19.4% higher substantivity compared to CHX (0.2%), and the anti-biofilm reduction was observed with both guava and CHX mouth rinses (P < 0.05). The overall discoloration changes were higher with CHX and distilled water compared to guava. Also, guava significantly enhanced HGEK-16 cell viability (P < 0.05), and IL-β1, TNFα and PGE2 expression presented a 0.6-fold decrease when exposed to guava and LPS (P < 0.05). The present study showed that guava mouth rinse fulfilled the requirement for an effective and useful oral care product with desirable substantivity and anti-biofilm action. In addition, guava reduced the inflammation response in HGEK-16 and may be a potential oral rinse for oral anti-inflammatory therapies.

Nicotine Activity on Healthy Oral Cells and Pharyngeal Tumor Cells

A number of studies highlight the harmful effects of smoking and the extremely serious consequences of the majority of compounds found in cigarettes and cigarette smoke. Of these, nicotine has attracted attention due to the suspicion of a carcinogen compound. The present study investigated the effect of nicotine on primary gingival keratinocytes but also on pharyngeal tumor cells, by evaluating viability and apoptotic processes.

Nicotine Activity on Healthy Oral Cells and Pharyngeal Tumor Cells

A number of studies highlight the harmful effects of smoking and the extremely serious consequences of the majority of compounds found in cigarettes and cigarette smoke. Of these, nicotine has attracted attention due to the suspicion of a carcinogen compound. The present study investigated the effect of nicotine on primary gingival keratinocytes but also on pharyngeal tumor cells, by evaluating viability and apoptotic processes. Keywords: nicotine, cells, keratinocytes, pharynx, tumor

Determination of the effects of cinnamon bark fractions on Candida albicans and oral epithelial cells

BackgroundCandida albicans is an opportunistic pathogen that causes oral candidiasis and denture stomatitis. It has also been reported to infect oral mucositis lesions in patients who suffer from cancer affecting the head and neck and who receive chemotherapy and radiotherapy treatments. This study aimed to investigate the effects of two cinnamon bark fractions, i.e., an essential oil and an aqueous extract enriched in proanthocyanidins (Cinnulin PF®) on growth, biofilm formation, and adherence properties of C. albicans as well as on oral epithelial cells (barrier integrity, inflammatory response).MethodsA microplate dilution assay was used to determine antifungal and anti-biofilm properties. A fluorescent assay was used to determine C. albicans adherence to oral epithelial cells. Cytotoxicity toward oral epithelial cells was assessed by determination of cell metabolic activity. Tight junction integrity of gingival keratinocytes was assessed by determination of transepithelial electrical resistance. IL-6 and IL-8 secretion by TNFα-stimulated oral epithelial cells was quantified by ELISA.ResultsWhile Cinnulin PF® did not reduce C. albicans growth, the cinnamon bark oil exhibited high antifungal activity with minimum inhibitory concentrations and minimum fungicidal concentrations in the range of 0.039 to 0.078%. The cinnamon oil was also active against a pre-formed C. albicans biofilm. Interestingly, Cinnulin PF® prevented biofilm formation by C. albicans and attenuated its adherence to oral epithelial cells. At their effective concentrations, the cinnamon oil and the Cinnulin PF® displayed no significant cytotoxicity against oral epithelial cells. In an in vitro model, both cinnamon fractions reinforced the integrity of the oral epithelial barrier. Lastly, Cinnulin PF® inhibited the secretion of interleukin-6 and interleukin-8 by oral epithelial cells stimulated with TNF-α.ConclusionBy their ability to attenuate growth, biofilm formation and adherence property of C. albicans, to reinforce the epithelial barrier function, and to exert anti-inflammatory properties the two cinnamon fractions (essential oil, Cinnulin PF®) investigated in the present study may be promising agents for treating oral infections involving C. albicans.

Dose-dependent green tea effect on decrease of inflammation in human oral gingival epithelial keratinocytes: in vitro study

ObjectivesThis in vitro study aimed to analyze the anti-inflammatory and wound healing potential of green tea extract (GTE) in human gingival epithelial keratinocytes (HGEK) treated with lipopolysaccharides (LPS).Materials and methodsA cell viability assay was conducted using MTT to determine nontoxic levels of GTE on immortalized HGEK. Cells were concomitantly treated with LPS (1 μg/ml) and GTE (1 mg/ml, 2.5 mg/ml, 5 mg/ml, and 10 mg/ml) to assess inflammation. Gene expression levels of inflammatory markers IL-β1, IL-6, and TNFα were measured by RT-PCR and their protein production was assessed by ELISA. The scratch wound healing assay was used to investigate the effects of different concentrations of GTE on cell migration. We also explored the effect of GTE on the induction of the Nrf2/HO-1 pathway in the cells with or without LPS.ResultsGTE at concentrations of 2.5 mg/ml, 5 mg/ml, and 10 mg/ml significantly enhanced cell viability (p < 0.05). And IL-β1, IL-6, and TNFα gene expression presented up to 10-fold decrease compared with LPS-treated cells, which was also similarly found on the protein levels. At the same concentrations, cell migration increased.ConclusionsThe mechanism results showed that GTE produced the anti-inflammatory response by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and increasing the level of anti-oxidant protein heme oxygenase-1 (HO-1).Clinical relevanceGTE may be potentially used as oral rinse anti-inflammatory drug for treatment and prevention of oral inflammatory diseases, which is shown here by the ability to reduce the inflammation and increase in cell migration in a dose-dependent manner.

Marijuana-Derived Cannabinoids Trigger a CB2/PI3K Axis of Suppression of the Innate Response to Oral Pathogens.

Cannabis use is an emergent risk factor for periodontitis, a chronic bacterial-induced disease of the supporting structures of the teeth. However, the mechanisms by which marijuana exposure predisposes to periodontal tissue destruction have yet to be elucidated. Therefore, we examined the influence of physiologically relevant doses of major marijuana-derived phytocannabinoid subtypes (cannabidiol [CBD]; cannabinol [CBN]; and tetrahydrocannabinol [THC], 1.0 μg/ml) on the interactions of three ultrastructurally variant oral pathogens, Porphyromonas gingivalis, Filifactor alocis, and Treponema denticola with the immune system. CBD, CBN, and THC each suppressed P. gingivalis-induced IL-12 p40, IL-6, IL-8, and TNF release while enhancing the anti-inflammatory cytokine, IL-10, from human innate cells. Similar phenomena were observed in F. alocis- and T. denticola-exposed human monocytes and human gingival keratinocytes. Higher phytocannabinoid doses (≥5.0 μg/ml) compromised innate cell viability and inhibited the growth of P. gingivalis and F. alocis, relative to unexposed bacteria. T. denticola, however, was resistant to all cannabinoid doses tested (up to 10.0 μg/ml). Pharmaceutical inhibition and efficient gene silencing indicated that a common CB2/PI3K axis of immune suppression is triggered by phytocannabinoids in vitro. This pathway does not appear to perpetuate through the canonical GSK3β-dependent cholinergic anti-inflammatory pathway, the predominant endogenous inflammatory control system. In a repetitive, transient oral infection model, CBD also suppressed P. gingivalis-induced innate immune markers in wild-type mice, but not in CB2−/− mice. If such phenomena occur in humans in situ, environmental cannabinoids may enhance periodontitis via direct toxic effects on specific oral bacteria; by compromising innate cell vitality; and/or through a suppressed innate response to periodontal pathogens involving a CB2/PI3K signaling lineage.

Bifunctional Bioactive Polymer Surfaces with Micrometer and Submicrometer-sized Structure: The Effects of Structure Spacing and Elastic Modulus on Bioactivity.

This study presents a comparison of two types of bifunctional structured surface that were made from the same polymer—an antimicrobial polycation (a synthetic mimic of an antimicrobial peptide, SMAMP) and a protein-repellent polyzwitterion (poly(sulfobetaines), PSB). The first type of bifunctional surface was fabricated by a colloidal lithography (CL) based process where the two polymers were immobilized sequentially onto pre-structured surfaces with a chemical contrast (gold on silicon). This enabled site-selective covalent attachment. The CL materials had a spacing ranging from 200 nm to 2 µm. The second type of structured surface (spacing: 1–8.5 µm) was fabricated using a microcontact printing (µCP) process where SMAMP patches were printed onto a PSB network, so that 3D surface features were obtained. The thus obtained materials were studied by quantitative nanomechanical measurements using atomic force microscopy (QNM-AFM). The different architectures led to different local elastic moduli at the polymer-air interface, where the CL surfaces were much stiffer (Derjaguin-Muller-Toporov (DMT) modulus = 20 ± 0.8 GPa) compared to the structured 3D networks obtained by µCP (DMT modulus = 42 ± 1.1 MPa). The effects of the surface topology and stiffness on the antimicrobial activity against Escherichia coli, the protein repellency (using fibrinogen), and the compatibility with human gingival mucosal keratinocytes were investigated. The softer 3D µCP surfaces had simultaneous antimicrobial activity, protein repellency, and cell compatibility at all spacings. For the stiffer CL surfaces, quantitative simultaneous antimicrobial activity and protein repellency was not obtained. However, the cell compatibility could be maintained at all spacings. The optimum spacing for the CL materials was in the range of 500 nm–1 µm, with significantly reduced antimicrobial activity at 2 µm spacing. Thus, the soft polymer network obtained by µCP could be more easily optimized than the stiff CL surface, and had a broader topology range of optimal or near-optimal bioactivity.

Computational Models Accurately Predict Multi-Cell Biomarker Profiles in Inflammation and Cancer

Individual computational models of single myeloid, lymphoid, epithelial, and cancer cells were created and combined into multi-cell computational models and used to predict the collective chemokine, cytokine, and cellular biomarker profiles often seen in inflamed or cancerous tissues. Predicted chemokine and cytokine output profiles from multi-cell computational models of gingival epithelial keratinocytes (GE KER), dendritic cells (DC), and helper T lymphocytes (HTL) exposed to lipopolysaccharide (LPS) or synthetic triacylated lipopeptide (Pam3CSK4) as well as multi-cell computational models of multiple myeloma (MM) and DC were validated using the observed chemokine and cytokine responses from the same cell type combinations grown in laboratory multi-cell cultures with accuracy. Predicted and observed chemokine and cytokine responses of GE KER + DC + HTL exposed to LPS and Pam3CSK4 matched 75% (15/20, p = 0.02069) and 80% (16/20, P = 0.005909), respectively. Multi-cell computational models became ‘personalized’ when cell line-specific genomic data were included into simulations, again validated with the same cell lines grown in laboratory multi-cell cultures. Here, predicted and observed chemokine and cytokine responses of MM cells lines MM.1S and U266B1 matched 75% (3/4) and MM.1S and U266B1 inhibition of DC marker expression in co-culture matched 100% (6/6). Multi-cell computational models have the potential to identify approaches altering the predicted disease-associated output profiles, particularly as high throughput screening tools for anti-inflammatory or immuno-oncology treatments of inflamed multi-cellular tissues and the tumor microenvironment.

Abstract 2800: Oral cancer cell-derived extracellular vesicles can modulate an immunosuppressive microenvironment through M2 phenotype polarization

Abstract Oral squamous cell carcinoma (OSCC) is the most prevalent malignant tumor among head and neck cancers, with 300,000 new cases and 145,000 deaths around the world per year, with less than 50% survival rate of advanced cases after five years of diagnosis. The molecular mechanisms involved in immunosurveillance and oral cancer development are poorly understood and, neither molecular markers nor immunotherapy response predictors are available. However, recent findings in molecular biology and immunology of cancer cell have shown that tumor-stroma communication promotes intense suppressive signaling in the microenvironment, which may contribute to failure of therapy. Tumor-stroma communication is not an unidirectional process, driven only by cancer cells. Other resident cells, like cancer-associated fibroblasts (CAFs), immune cells as macrophages and lymphocytes, endothelial cells and tumor stem cells, which are in an intense cross-talk by cell-cell contact, secreted factors and other components of the extracellular matrix (ECM), can alter and be altered by the microenvironment. In this context, the cancer-derived extracellular vesicles (EVs) acquire a significant pathological role in cell-cell communication by changing the phenotype of the recipient cells, promoting tumor growth and metastasis. Here, we explored the role of three different source of EVs, originated from normal gingival keratinocyte (HMK) and from less (SCC9) and high (HSC3) aggressive OSCC cell lines in the modulation of monocyte-derived macrophage M1 or M2 phenotypes, and their ability to contribute to the suppressive phenotype in the tumor microenvironment. CD163 expression, a marker of M2 macrophages, was significantly increased in CD14+ monocytes stimulated by HSC3-EVs, in comparison with non-stimulated monocytes, HMK- and SCC9-EVs-stimulated cells. In addition, CD64+/CD86+/CD80+ M1 macrophage subset, differentiated by GM-CSF/IFN-gamma, showed a decreased expression of the T-cell co-stimulatory molecule, CD86 after stimulus with HSC3-EVs. Furthermore, the mass spectrometry analysis of HSC3-EV protein contents identified a set of up- and down-regulated candidate proteins that may be involved in immunoregulation and suppressive pathways. Since the M2 macrophage phenotype is associated with increased angiogenesis, immunological tolerance and development of neoplastic cells, the M1 subset, the classically activated macrophages, may recognize and eliminate cancer cells. Thus, our results indicate that EVs from aggressive cells HSC3 may contribute to immunosuppressive tumor microenvironment in oral cancer by deviating macrophages into a tumor-promoting instead of a tumor-controlling phenotype. Citation Format: Ana Karina Oliveira, Thiago Andrade Patente, Rodrigo Nalio Ramos, Ariane Busso Lopes, Romênia Ramos Domingues, Mariane Tami Amano, Eliana Blini Marengo, Jose Alexandre Barbuto, Adriana Franco Squina. Oral cancer cell-derived extracellular vesicles can modulate an immunosuppressive microenvironment through M2 phenotype polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2800.

Resveratrol attenuates the pathogenic and inflammatory properties of Porphyromonas gingivalis

Porphyromonas gingivalis has been strongly associated with chronic periodontitis, which affects tooth-supporting tissues. This Gram-negative anaerobic bacterium produces a repertoire of virulence factors that modulate tissue destruction directly or indirectly by the induction of inflammatory processes. The aim of this study was to investigate the effects of resveratrol, a major polyphenol found in grapes and wine, on the growth and virulence properties of P.gingivalis as well as on gingival keratinocyte tight junction integrity and the host inflammatory response. Resveratrol exhibited antibacterial activity that may result from damage to the bacterial cell membrane. Resveratrol also killed a pre-formed P.gingivalis biofilm and reduced bacterial adherence to matrix proteins. In addition, resveratrol had a protective effect on the integrity of the keratinocyte tight junctions by inhibiting its breakdown by P.gingivalis. This may be related to the ability of resveratrol to inhibit the protease activities of P.gingivalis. Lastly, resveratrol reduced P.gingivalis-mediated activation of the NF-κB signaling pathway and attenuated TREM-1 gene expression as well as soluble TREM-1 secretion in monocytes. The effect on NF-κB activation likely results from the ability of resveratrol to act as a PPAR-γ agonist. In summary, the antibacterial, anti-adherence, and antiprotease properties of resveratrol, as well as its ability to protect the gingival keratinocyte barrier and attenuate the inflammatory response in monocytes suggest that it may be a promising novel therapeutic agent for treating periodontal disease.

Construction of Vascularized Oral Mucosa Equivalents Using a Layer-by-Layer Cell Coating Technology.

There have been many advances in tissue engineering with respect to in vitro and in vivo models of oral mucosa equivalents (OMEs). To apply in vitro reconstructed oral mucosa models to regenerative medicine and alternatives to animal testing, it is necessary to develop the technology of reconstructing different types of oral tissues, such as control of epithelial differentiation and introduction of appendages. We previously reported that functional three-dimensional (3D) tissue models could be quickly constructed by using a layer-by-layer (LbL) cell coating technique that assembles extracellular matrix (ECM) nanofilms to a cell surface. In this study, 3D human OMEs composed of lamina-propria, keratinized or non-keratinized epithelium, and blood capillaries were constructed by using the LbL cell coating technology. Human oral mucosal fibroblasts (HOMFs) were coated with ECM nanofilms and accumulated for the construction of oral mucosal lamina-propria. To construct OMEs with keratinized or non-keratinized epithelium, human oral keratinocytes isolated from gingiva (human oral gingival keratinocytes: HOGKs) or human oral keratinocytes isolated from oral mucosa (human oral mucosal keratinocytes: HOMKs) were used in this study. We further studied the construction of epithelialized OMEs with density- and size-controlled blood capillary networks by using human umbilical vein endothelial cells (HUVECs). It was revealed that these constructions had barrier functions in accordance with their histological characterization. The OMEs with keratinization (K-OMEs) showed higher transepithelial electrical resistance (TEER) values compared with OMEs with non-keratinization (N-OMEs). The constructed epithelialized OMEs with blood capillaries are useful for in vitro/ex vivo research models and regenerative medicine as in oral tissue regeneration. The results suggest that OMEs with oral tissue appendages are more promising alternatives to animal testing and can be applied to the design of in vitro oral models that mimic human tissue organs.

In Vitro Effect of Modified Polyetheretherketone (PEEK) Implant Abutments on Human Gingival Epithelial Keratinocytes Migration and Proliferation.

Improving soft tissue attachment to implant abutments is a crucial factor for enduring health and maintenance of soft peri-implant tissue health. In this in vitro study we aimed to compare the biocompatibility of three different abutment surfaces: titanium, zirconia and modified polyetheretherketone (PEEK). Surface topography, roughness and wettability were investigated with scanning electron microscopy, profilometer and contact angle meter, respectively. Human gingival epithelial keratinocytes were examined for viability, morphology, proliferation and migration by using tetrazolium salt colorimetric assay, scanning electron microscopy imaging, immunofluorescence bromodeoxyuridine analysis and scratch wound healing assays. Roughness measurements revealed differences between the investigated surfaces. Keratinocytes cultured on all examined surfaces indicated adhesion and attachment by means of scanning electron microscopy imaging. Cell viability assays showed no significant differences between the groups (p > 0.05). The modified PEEK surface similarly improved surface roughness in comparison to titanium and zirconia, which resulted in greater and equivalent cell proliferation and migration. The study methodology showed here may emphasize the importance of cell interactions with different abutment materials, which in part increases the changes of implant success. PEEK, titanium and zirconia surface types used in this study showed mostly similar epithelial biological responses.

Regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes.

Dental implant material has an impact on adhesion and spreading of oral mucosal cells on its surface. Platelet-rich fibrin (PRF), a second-generation platelet concentrate, can enhance cell proliferation and adhesion. The aim was to examine the regulatory effects of PRF and titanium surfaces on cellular adhesion, spread, and cytokine expressions of gingival keratinocytes. Human gingival keratinocytes were cultured on titanium grade 4, titanium grade 5 (Ti5), and HA discs at 37°C in a CO2 incubator for 6h and 24h, using either elutes of titanium-PRF (T-PRF) or leukocyte and platelet-rich fibrin (L-PRF), or mammalian cell culture medium as growth media. Cell numbers were determined using a Cell Titer 96 assay. Interleukin (IL)-1β, IL-1Ra, IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) expression levels were measured using the Luminex® xMAP™ technique, and cell adhesion and spread by scanning electron microscopy. Epithelial cell adhesion and spread was most prominent to Ti5 surfaces. L-PRF stimulated cell adhesion to HA surface. Both T-PRF and L-PRF activated the expressions of IL-1 β, IL-8, IL-1Ra, MCP-1, and VEGF, T-PRF being the strongest activator. Titanium surface type has a regulatory role in epithelial cell adhesion and spread, while PRF type determines the cytokine response.

Gingipains impair attachment of epithelial cell to dental titanium abutment surfaces.

The study investigated in vitro the effect of Porphyromonas gingivalis and its cysteine proteases (gingipains) on epithelial cell adhesion to titanium-zirconium alloy surfaces. Titanium-zirconium discs with a standard machined (M) or chemically modified hydrophilic surface (modM) were coated with lamin-5 and incubated with telomerase-inactivated gingival keratinocytes (TIGK). Three P. gingivalis strains or gingipains were either added simultaneously with TIGK or after TIGK cells were already attached to the disks. Adhered TIGK cells were counted at 24 h. All P. gingivalis strains clearly inhibited adhesion of TIGK cells to M and modM surfaces. Compared with bacteria/gingipain-free TIGK cell cultures, the number of attached TIGK cells was reduced by about 80% and 60% when P. gingivalis was added simultaneously or after TIGK cells were already attached to the disks (each p < 0.01), respectively. Counts of attached cells were similarly reduced when only gingipains were used. Adhesion molecules of TIGK cells, in particular E-cadherin, were cleaved by P. gingivalis. In conclusion, P. gingivalis and gingipains interfere with the adhesion of epithelial cells to titanium-zirconium alloy surfaces by cleaving adhesion molecules, while a chemically modified hydrophilic titanium-zirconium alloy surface did not yield any protection. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2549-2556, 2019.

Functional assessment of peptide-modified PLGA nanoparticles against oral biofilms in a murine model of periodontitis

The interaction of the periodontal pathogen Porphyromonas gingivalis (Pg) with commensal streptococci promotes Pg colonization of the oral cavity. Previously, we demonstrated that a peptide (BAR) derived from Streptococcus gordonii (Sg) potently inhibited adherence of Pg to streptococci and reduced Pg virulence in a mouse model of periodontitis. Thus, BAR may represent a novel therapeutic to control periodontitis by preventing Pg colonization of the oral cavity. However, while BAR inhibited the initial formation of Pg/Sg biofilms, much higher concentrations of peptide were required to disrupt an established Pg/Sg biofilm. To improve the activity of the peptide, poly(lactic-co-glycolic acid) (PLGA) nanoparticles were surface-modified with BAR and shown to more potently disrupt Pg/Sg biofilms relative to an equimolar amount of free peptide. The goal of this work was to determine the in vivo efficacy of BAR-modified NPs (BNPs) and to assess the toxicity of BNPs against human gingival epithelial cells. In vivo efficacy of BNPs was assessed using a murine model of periodontitis by measuring alveolar bone resorption and gingival IL-17 expression as outcomes of Pg-induced inflammation. Infection of mice with Pg and Sg resulted in a significant increase in alveolar bone loss and gingival IL-17 expression over sham-infected animals. Treatment of Pg/Sg infected mice with BNPs reduced bone loss and IL-17 expression almost to the levels of sham-infected mice and to a greater extent than treatment with an equimolar amount of free BAR. The cytotoxicity of the maximum concentration of BNPs and free BAR used in in vitro and in vivo studies (1.3 and 3.4 μM), was evaluated in telomerase immortalized gingival keratinocytes (TIGKs) by measuring cell viability, cell lysis and apoptosis. BNPs were also tested for hemolytic activity against sheep erythrocytes. TIGKs treated with BNPs or free BAR demonstrated >90% viability and no significant lysis or apoptosis relative to untreated cells. In addition, neither BNPs nor free BAR exhibited hemolytic activity. In summary, BNPs were non-toxic within the evaluated concentration range of 1.3–3.4 μM and provided more efficacious protection against Pg-induced inflammation in vivo, highlighting the potential of BNPs as a biocompatible platform for translatable oral biofilm applications.

Tea polyphenols protect gingival keratinocytes against TNF-α-induced tight junction barrier dysfunction and attenuate the inflammatory response of monocytes/macrophages

Tea, an aromatic beverage prepared with the leaves of the Camellia sinensis plant, is well known to contain bioactive polyphenols. Green tea contains mainly catechins such as epigallocatechin-3-gallate (EGCG), while black tea is characterized by the presence of theaflavins. TNF-α, which is a pro-inflammatory cytokine that activates the endogenous inflammatory cascade, plays a key role in periodontitis. In the present study, we investigated the ability of tea compounds to attenuate TNF-α-mediated activation of the host inflammatory response in monocytes/macrophages as well as the protective effect of green and black tea polyphenols on gingival keratinocyte barrier dysfunction induced by TNF-α. Tea compounds inhibited both the activation of NF-κB and caspase-1 as well as IL-1β secretion by monocytes/macrophages. TNF-α time-dependently damaged keratinocyte tight junction barrier integrity, as determined by changes in transepithelial electrical resistance and FITC-dextran transport. Green tea extract, EGCG, theaflavins, and to a lesser extent, black tea extract protected keratinocytes against the TNF-α-mediated breakdown of barrier integrity. The treatment of keratinocytes with tea polyphenols markedly mitigated the morphological changes of tight junction proteins such as zonula occludens-1 and occludin compared to cells exposed only to TNF-α, as determined by immunofluorescence. Tea polyphenols also time-dependently decreased the paracellular flux of TNF-α-treated keratinocytes. In conclusion, the ability of tea polyphenols to exert an anti-inflammatory effect and to attenuate the gingival epithelial barrier dysfunction induced by TNF-α supports their potential for the prevention and treatment of periodontal disease.

Vitamin D maintains E-cadherin intercellular junctions by downregulating MMP-9 production in human gingival keratinocytes treated by TNF-α.

PurposeDespite the well-known anti-inflammatory effects of vitamin D in periodontal health, its mechanism has not been fully elucidated. In the present study, the effect of vitamin D on strengthening E-cadherin junctions (ECJs) was explored in human gingival keratinocytes (HGKs). ECJs are the major type of intercellular junction within the junctional epithelium, where loose intercellular junctions develop and microbial invasion primarily occurs.MethodsHOK-16B cells, an immortalized normal human gingival cell line, were used for the study. To mimic the inflammatory environment, cells were treated with tumor necrosis factor-alpha (TNF-α). Matrix metalloproteinases (MMPs) in the culture medium were assessed by an MMP antibody microarray and gelatin zymography. The expression of various molecules was investigated using western blotting. The extent of ECJ development was evaluated by comparing the average relative extent of the ECJs around the periphery of each cell after immunocytochemical E-cadherin staining. Vitamin D receptor (VDR) expression was examined via immunohistochemical analysis.ResultsTNF-α downregulated the development of the ECJs of the HGKs. Dissociation of the ECJs by TNF-α was accompanied by the upregulation of MMP-9 production and suppressed by a specific MMP-9 inhibitor, Bay 11-7082. Exogenous MMP-9 decreased the development of ECJs. Vitamin D reduced the production of MMP-9 and attenuated the breakdown of ECJs in the HGKs treated with TNF-α. In addition, vitamin D downregulated TNF-α-induced nuclear factor kappa B (NF-κB) signaling in the HGKs. VDR was expressed in the gingival epithelium, including the junctional epithelium.ConclusionsThese results suggest that vitamin D may avert TNF-α-induced downregulation of the development of ECJs in HGKs by decreasing the production of MMP-9, which was upregulated by TNF-α. Vitamin D may reinforce ECJs by downregulating NF-κB signaling, which is upregulated by TNF-α. Strengthening the epithelial barrier may be a way for vitamin D to protect the periodontium from bacterial invasion.