Oroxylin A inhibits inflammatory cytokines in periodontitis via HO­1.

Periodontal disease is a common infectious disease that can lead to the loss of teeth. Hower how to effectively suppress the inflammation with medication is unclear. The aim of the present study was to investigate the anti­inflammatory effect of Oroxylin A in periodontitis and its potential role through heme oxygenase­1 (HO­1). Primary rat gingival fibroblasts (RGFs) were cultured using the tissue block method and identified by immunofluorescence. Following lipopolysaccharide (LPS) stimulation of RGFs, Oroxylin A was administered at 50, 100, 200 or 400 µg/ml. Reverse transcription­quantitative PCR was used to assess mRNA expression of cyclooxygenase (COX)­2, TNF­α, RANKL and osteoprotegerin (OPG). Western blotting was used to detect protein expression levels of COX ­2, TNF­α, RANKL and OPG. Following HO­1 knockdown, the same treatment was performed. The expression of COX­2 in rat gingival tissue was observed by immunohistochemistry. One­way analysis of variance and Student's t test were used for statistical analysis. Oroxylin A downregulated mRNA expression of COX­2, TNF­α, RANKL and OPG in LPS­induced RGFs. With increase of Oroxylin A dose, the expression of HO­1 was gradually upregulated. When HO­1 was knocked down, Oroxylin A did not downregulate the expression of COX­2, TNF­α, RANKL and OPG in LPS­induced RGFs. Immunohistochemical results showed that expression of COX­2 was downregulated by Oroxylin A, and the expression of TNF­α, RANKL and OPG were also downregulated. Oroxylin A decreased expression of inflammatory cytokines in LPS­induced RGFs and had a good inhibitory effect on periodontitis in rats.

Novel approach to soft tissue regeneration: in vitro study of compound hyaluronic acid and horizontal platelet-rich fibrin combination.

OBJECTIVE: This study aims to develop a compound biomaterial to achieve effective soft tissue regeneration. METHODOLOGY: Compound hyaluronic acid (CHA) and liquid horizontal-platelet-rich fibrin (H-PRF) were mixed at a ratio of 1:1 to form a CHA-PRF gel. Human gingival fibroblasts (HGFs) were used in this study. The effect of CHA, H-PRF, and the CHA-PRF gel on cell viability was evaluated by CCK-8 assays. Then, the effect of CHA, H-PRF, and the CHA-PRF gel on collagen formation and deposition was evaluated by qRT‒PCR and immunofluorescence analysis. Finally, qRT‒PCR, immunofluorescence analysis, Transwell assays, and scratch wound-healing assays were performed to determine how CHA, H-PRF, and the CHA-PRF gel affect the migration of HGFs. RESULTS: The combination of CHA and H-PRF shortened the coagulation time of liquid H-PRF. Compared to the pure CHA and H-PRF group, the CHA-PRF group exhibited the highest cell proliferation at all time points, as shown by the CCK-8 assay. Col1a and FAK were expressed at the highest levels in the CHA-PRF group, as shown by qRT‒PCR. CHA and PRF could stimulate collagen formation and HGF migration, as observed by fluorescence microscopy analysis of COL1 and F-actin and Transwell and scratch healing assays. CONCLUSION: The CHA-PRF group exhibited greater potential to promote soft tissue regeneration by inducing cell proliferation, collagen synthesis, and migration in HGFs than the pure CHA or H-PRF group. CHA-PRF can serve as a great candidate for use alone or in combination with autografts in periodontal or peri-implant soft tissue regeneration.

Bioactivity and antibacterial effects of zinc-containing bioactive glass on the surface of zirconia abutments.

OBJECTIVES: This study aimed to enhance gingival fibroblast function and to achieve antibacterial activity around the implant abutment by using a zinc (Zn)-containing bioactive glass (BG) coating. METHODS: 45S5 BG containing 0, 5, and 10 wt.% Zn were coated on zirconia disks. The release of silica and Zn ions in physiological saline and their antibacterial effects were measured. The effects of BG coatings on human gingival fibroblasts (hGFs) were assessed using cytotoxicity assays and by analyzing the gene expression of various genes related to antioxidant enzymes, wound healing, and fibrosis. RESULTS: BG coatings are capable of continuous degradation and simultaneous ion release. The antibacterial effect of BG coatings increased with the addition of Zn, while the cytotoxicity remained unchanged compared to the group without coatings. BG coating enhances the expression of angiogenesis genes, while the Zn-containing BG enhances the expression of antioxidant genes at an early time point. BG coating enhances the expression of collagen genes at later time points. CONCLUSIONS: The antibacterial effect of BG improved with the increase in Zn concentration, without inducing cytotoxicity. BG coating enhances the expression of angiogenesis genes, and Zn-containing BG enhances the expression of antioxidant genes at an early time point. BG coating enhances the expression of collagen genes at later time points. CLINICAL SIGNIFICANCE: Adding 10 wt% Zn to BG could enhance the environment around implant abutments by providing antibacterial, antioxidant, and anti-fibrotic effects, having potential for clinical use.

An in vitro assessment of teething gels' effects on human gingival mesenchymal stem cells.

BACKGROUND: The aim of this study is to examine the cytotoxic effects of dental gels with different contents, which are frequently used during teething, on gingival mesenchymal stem cells (G-MSCs). METHOD: The teething gels used in this study were Dentinox, Gengigel, Osanite, and Jack and Jill. The human gingival mesenchimal stem cells (hG-MSCs) were incubated with these teething gel solutions (0.1%, 50% and 80% concentrations). Reproductive behavior of G-MSCs was monitored in real time for 72 h using the xCELLigence real-time cell analyzer (RTCA) system. Two-way repeated Anova test and post hoc Bonferroni test were used to evaluate the effect of concentration and dental gel on 0-hour and 72-hour viability. Significance was evaluated at p < 0.05 level. RESULTS: Teething gels prepared at 50% concentration are added to the G-MSC culture, the "cell index" value of G-MSCs to which Dentinox brand gel is added is significantly lower than all other groups (p = 0.05). There is a statistically significant difference between the concentrations in terms of cell index values at the 72nd hour compared to the 0th hour (p = 0.001). CONCLUSIONS: The local anesthetic dental gels used in children have a more negative effect on cell viability as concentration increases.

Cytotoxic effect of dental luting cement on human gingival mesenchymal stem cell and evaluation of cytokines and growth factor release - An in vitro study.

AIM: In routine dental care, various dental luting cements are utilized to cement the dental prosthesis. Thus, the aim of the current study was to assess the Cytotoxic effect of three different dental luting cements on human gingival mesenchymal stem cell and evaluation of cytokines and growth factors release. SETTINGS AND DESIGN: Cytotoxicity of glass ionomer cement (GIC), resin modified glass ionomer cement (RMGIC) and resin cement (RC) on the human gingival mesenchymal stem cells (HGMSCs) was evaluated. Amongst the cements tested, least cytotoxic cement was further tested for the release of cytokines and growth factors. MATERIALS AND METHODS: MTT test was used to evaluate the cytotoxicity of the dental luting cements at 1 h, 24 h, and 48 h on HGMSCs. Cytokines such as interleukin (IL) 1α & IL 8 and growth factors such as platelet derived growth factor & transforming growth factor beta release from the least cytotoxic RC was evaluated using flow cytometry analysis. STATISTICAL ANALYSIS USED: The mean absorbance values by MTT assay and cell viability at various time intervals between four groups were compared using a one way analysis of variance test and Tukey's post hoc test. The least cytotoxic RC group and the control group's mean levels of cytokines and growth factors were compared using the Mann-Whitney test. RESULT: As exposure time increased, the dental luting cement examined in this study were cytotoxic. RC was the least cytotoxic, RMGIC was moderate and glass ionomer cement showed the highest cytotoxic effect. Concomitantly, a significant positive biological response of gingival mesenchymal stem cells with the release of ILs when exposed to the RC was observed. CONCLUSION: For a fixed dental prosthesis to be clinically successful over the long term, it is imperative that the biocompatibility of the luting cement be taken into account in order to maintain a healthy periodontium surrounding the restoration.

Ropinirole suppresses LPS-induced periodontal inflammation by inhibiting the NAT10 in an ac4C-dependent manner.

BACKGROUND: Periodontitis is a chronic osteolytic inflammatory disease, where anti-inflammatory intervention is critical for restricting periodontal damage and regenerating alveolar bone. Ropinirole, a dopamine D2 receptor agonist, has previously shown therapeutic potential for periodontitis but the underlying mechanism is still unclear. METHODS: Human gingival fibroblasts (HGFs) treated with LPS were considered to mimic periodontitis in vitro. The dosage of Ropinirole was selected through the cell viability of HGFs evaluation. The protective effects of Ropinirole on HGFs were evaluated by detecting cell viability, cell apoptosis, and pro-inflammatory factor levels. The molecular docking between NAT10 and Ropinirole was performed. The interaction relationship between NAT10 and KLF6 was verified by ac4C Acetylated RNA Immunoprecipitation followed by qPCR (acRIP-qPCR) and dual-luciferase reporter assay. RESULTS: Ropinirole alleviates LPS-induced damage of HGFs by promoting cell viability, inhibiting cell apoptosis and the levels of IL-1ß, IL-18, and TNF-α. Overexpression of NAT10 weakens the effects of Ropinirole on protecting HGFs. Meanwhile, NAT10-mediated ac4C RNA acetylation promotes KLF6 mRNA stability. Upregulation of KLF6 reversed the effects of NAT10 inhibition on HGFs. CONCLUSIONS: Taken together, Ropinirole protected HGFs through inhibiting the NAT10 ac4C RNA acetylation to decrease the KLF6 mRNA stability from LPS injury. The discovery of this pharmacological and molecular mechanism of Ropinirole further strengthens its therapeutic potential for periodontitis.

Viability and Proliferation Assessment of Gingival Fibroblasts Cultured on Silver Nanoparticle-Doped Ti-6Al-4V Surfaces.

PURPOSE: To investigate the biocompatibility of silver nanoparticle (AgNP)-doped Ti-6Al-4V surfaces by evaluating the viability and proliferation rate of human gingival fibroblasts (HGFs)-as the dominant cells of peri-implant soft tissues-seeded on the modified surfaces. MATERIALS AND METHODS: AgNPs (sizes 8 nm and 30 nm) were incorporated onto Ti-6Al-4V specimen surfaces via electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations of 100 ppm, 200 ppm, and 300 ppm. One control and six experimental groups were included in the study: (1) control (Ti-6Al-4V), (2) 8 nm/100 ppm, (3) 8 nm/200 ppm, (4) 8 nm/300 ppm, (5) 30 nm/100 ppm, (6) 30 nm/200 ppm, and (7) 30 nm/300 ppm. HGF cell primary cultures were isolated from periodontally healthy donor patients and cultured in direct contact with the group specimens for 24 and 72 hours. The cytotoxicity of AgNP-doped Ti-6Al-4V specimens toward HGF was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and BrdU (5-bromo-2'-deoxyuridine) assay tests. Calcein AM and ethidium homodimer (EthD-1) fluorescent stains were used to determine the live and dead cells. The morphology and attachment properties of the HGFs were determined via scanning electron microscopy (SEM). RESULTS: Energy dispersive x-ray (EDX) analysis confirmed the presence of AgNPs on the specimens. The MTT test revealed that AgNPs of both sizes and all concentrations presented a decreased cellular metabolic activity compared to the control discs. All concentrations of both sizes of AgNPs affected the cell proliferation rate compared to the control group, as revealed by the BrdU assay. Overall, cytotoxicity of the modified Ti-6Al-4V surfaces depended on cell exposure time. Observation via confocal microscopy confirmed the results of the MTT and BrdU assay tests. Specifically, most cells remained alive throughout the 72-hour culture period. SEM images revealed that adjacent cells form bonds with each other, creating confluent layers of conjugated cells. CONCLUSIONS: The findings of the present study indicate that Ti-6Al-4V surfaces modified with 8 nm and 30 nm AgNPs at concentrations of 100 ppm, 200 ppm, and 300 ppm do not produce any serious cytotoxicity toward HGFs. The initial arrest of the HGF proliferation rate recovered at 72 hours. These results on the antibacterial activity against common periodontal pathogens, in combination with the results found in a previous study by the same research group, suggest that AgNP-doped Ti-6Al-4V surfaces are potential candidates for use in implant abutments for preventing peri-implant diseases.

Phytocannabinoids and gingival inflammation: Preclinical findings and a placebo-controlled double-blind randomized clinical trial with cannabidiol.

OBJECTIVE: The aim of this study was to: (1) evaluate the anti-inflammatory effects of cannabidiol (CBD) on primary cultures of human gingival fibroblasts (HGFs) and (2) to clinically monitor the effect of CBD in subjects with periodontitis. BACKGROUND: The use of phytocannabinoids is a new approach in the treatment of widely prevalent periodontal disease. MATERIALS AND METHODS: Cannabinoid receptors were analyzed by western blot and interleukin production detected using enzyme immunoassay. Activation of the Nrf2 pathway was studied via monitoring the mRNA level of heme oxygenase-1. Antimicrobial effects were determined by standard microdilution and 16S rRNA screening. In the clinical part, a placebo-control double-blind randomized study was conducted (56 days) in three groups (n = 90) using dental gel without CBD (group A) and with 1% (w/w) CBD (group B) and corresponding toothpaste (group A - no CBD, group B - with CBD) for home use to maintain oral health. Group C used dental gel containing 1% chlorhexidine digluconate (active comparator) and toothpaste without CBD. RESULTS: Human gingival fibroblasts were confirmed to express the cannabinoid receptor CB2. Lipopolysaccharide-induced cells exhibited increased production of pro-inflammatory IL-6 and IL-8, with deceasing levels upon exposure to CBD. CBD also exhibited antimicrobial activities against Porphyromonas gingivalis, with an MIC of 1.5 µg/mL. Activation of the Nrf2 pathway was also demonstrated. In the clinical part, statistically significant improvement was found for the gingival, gingival bleeding, and modified gingival indices between placebo group A and CBD group B after 56 days. CONCLUSIONS: Cannabidiol reduced inflammation and the growth of selected periodontal pathogenic bacteria. The clinical trial demonstrated a statistically significant improvement after CBD application. No adverse effects of CBD were reported by patients or observed upon clinical examination during the study. The results are a promising basis for a more comprehensive investigation of the application of non-psychotropic cannabinoids in dentistry.

Dipotassium glycyrrhizate and hinokitiol enhance macrophage efferocytosis by regulating recognition, uptake, and metabolism of apoptotic cells in vitro.

BACKGROUND AND OBJECTIVE: Efferocytosis is a process whereby macrophages remove apoptotic cells, such as neutrophils, that have accumulated in tissues, which is required for resolution of inflammation. Efferocytosis is impaired in individuals with increasing age and in those with various systemic diseases. Recently, efferocytosis has been reported to be related to the pathogenesis and progression of periodontitis, and enhancement of efferocytosis, especially in the subjects with impaired efferocytosis, was suggested to lead to periodontitis prevention and care. Various anti-inflammatory ingredients are used in oral care products, but their effect on efferocytosis is unclear. Here, we aimed to identify ingredients contained in oral care products that are effective for efferocytosis regulation. METHODS: The ability of dead cells to induce inflammation in human gingival fibroblast (HGF) cells were evaluated by measuring IL-6 secretion. Six ingredients in oral care products used as anti-inflammatory agents were evaluated for their effect on efferocytosis using flow cytometry. The expression of various efferocytosis-related molecules, such as MERTK and LRP1 involved in recognition, and LXRα and ABCA1 that function in metabolism, were measured in RAW264.7 cells with or without ingredient treatment. Rac1 activity, which is related to the uptake of dead cells, was measured using the G-LISA kit. RESULTS: Dead cells elicited IL-6 secretion in HGF cells. Among the six ingredients, GK2 and hinokitiol enhanced efferocytosis activity. GK2 and hinokitiol significantly increased the expression of MERTK and LRP1, and also enhanced LXRα and ABCA1 expression after efferocytosis. Furthermore, they increased Rac1 activity in the presence of dead cells. CONCLUSION: Among the six ingredients tested, GK2 and hinokitiol promoted efferocytosis by regulating apoptotic cell recognition, uptake, and metabolism-related molecules. Efferocytosis upregulation may be one of the mechanisms of GK2 and hinokitiol in the treatment of inflammatory diseases, such as periodontitis.

Resveratrol May Reduce the Degree of Periodontitis by Regulating ERK Pathway in Gingival-Derived MSCs.

Gingival-derived mesenchymal stem cells (GMSCs) have strong self-renewal, multilineage differentiation, and immunomodulatory properties and are expected to be applied in anti-inflammatory and tissue regeneration. However, achieving the goal of using endogenous stem cells to treat diseases and even regenerate tissues remains a challenge. Resveratrol is a natural compound with multiple biological activities that can regulate stem cell immunomodulation when acting on them. This study found that resveratrol can reduce inflammation in human gingival tissue and upregulate the stemness of GMSCs in human gingiva. In cell experiments, it was found that resveratrol can reduce the expression of TLR4, TNFα, and NFκB and activate ERK/Wnt crosstalk, thereby alleviating inflammation, promoting the proliferation and osteogenic differentiation ability of GMSCs, and enhancing their immunomodulation. These results provide a new theoretical basis for the application of resveratrol to activate endogenous stem cells in the treatment of diseases in the future.

Effect of nano zinc oxide on proliferation and toxicity of human gingival cells.

BACKGROUND: Periodontal dressing is used to cover the gum surface and protect the wound after periodontal surgery. Nanomaterials have been widely applied in dentistry in recent years. Zinc oxide (ZnO) is one of the main components of periodontal dressing. AIM: This study aims to explore the toxicity ZnO nanoparticles (ZnO NPs) causes to human gingival fibroblast cells (HGF-1) and its effect on cell proliferation. METHODS: First, we identified and analyzed HGF-1, including cell morphology, growth curve, and immunohistochemistry staining. Then, we treated HGF-1 with ZnO NP. Cell viability, the integrity of the cell membrane, oxidative damage, and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, fluorescent probe, and flow cytometry. Furthermore, the expression of murine double minute 2 (MDM2) and p53 was determined by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. We finally overexpressed MDM2 in HGF-1 to verify the relationship between MDM2 and cell proliferation. RESULTS: Our research indicated ZnO NPs did not affect cell proliferation at low concentrations. However, high-concentration ZnO NP inhibited cell proliferation, destroyed the integrity of cell membranes, and induced oxidative stress and apoptosis. In addition, high concentration of ZnO NPs inhibited the proliferation of HGF-1 by regulating the expression of MDM2 and p53. CONCLUSION: High concentration of ZnO NP caused toxicity to HGF-1 cells and inhibited cell proliferation by regulating MDM2 and p53 expression.

Antibacterial and Antioxidant Effects of Magnesium Alloy on Titanium Dental Implants.

OBJECTIVES: In this study, a new type of dental implant by covering the surface of the titanium (Ti) implant with zinc-magnesium (Zn-Mg) alloy was designed, to study the antibacterial and antioxidant effects of Mg alloy on titanium (Ti) implants in oral implant restoration. METHODS: Human gingival fibroblasts (HGFs), S. sanguinis, and F. nucleatum bacteria were used to detect the bioactivity and antibacterial properties of Mg alloy-coated Ti implants. In addition, B6/J mice implanted with different materials were used to further detect their antibacterial and antioxidant properties. RESULTS: The results showed that Mg alloy could better promote the adhesion and proliferation and improve the alkaline phosphatase (ALP) activity of HGFs, which contributed to better improved stability of implant osseointegration. In addition, Mg alloy could better inhibit the proliferation of S. sanguinis, while no significant difference was found in the proliferation of F. nucleatum between the two implants. In the mouse model, the peripheral inflammatory reaction and oxidative stress of the Mg alloy implant were significantly lower than those of the Ti alloy implant. CONCLUSIONS: Zn-Mg alloy-coated Ti implants could better inhibit the growth of Gram-positive bacteria in the oral cavity, inhibit oxidative stress, and facilitate the proliferation activity of HGFs and the potential of osteoblast differentiation, thus, better increasing the stability of implant osseointegration.

Reduction of oxidative stress blunts the NLRP3 inflammatory cascade in LPS stimulated human gingival fibroblasts and oral mucosal epithelial cells.

The therapeutic armamentarium for the treatment of oral mucositis is very poor. Catechin and baicalin are two natural flavonoids that have been individually reported to have a curative potential. Flavocoxid is a mixed extract containing baicalin and catechin showing antioxidant effects and anti-inflammatory activity mainly due to a dual inhibition of inducible cyclooxygenase (COX-2), 5-lipoxygenase (5-LOX) and NLRP3 pathway. The aim of this study was to evaluate the anti-inflammatory and anti-oxidant effects of flavocoxid in an "in vitro" model of oral mucositis induced by triggering an inflammatory phenotype in human gingival fibroblasts (GF) and human oral mucosal epithelial cells (EC). GF and EC were challenged with lipopolysaccharide (LPS 2 µg/ml) alone or in combination with flavocoxid (32 µg/ml). Flavocoxid increased Nrf2, prompted a marked reduction in malondialdehyde levels and reduced the expression of COX-2 and 5-LOX together with PGE2, and LTB4 levels. Flavocoxid caused also a great decrease in the expression of NF-κB and turned off NLRP3 inflammasome and its downstream effectors signal, as caspase-1, IL-1ß and IL-18 in both GF and EC cells stimulated with LPS. These results suggest a correlation between oxidative stress and NLRP3 activation and indicate that flavocoxid suppresses the inflammatory storm that accompanies oral mucositis. This preclinical evidence deserves to be confirmed in a clinical setting.

Quercetin Preserves Oral Cavity Health by Mitigating Inflammation and Microbial Dysbiosis.

Failure to attenuate inflammation coupled with consequent microbiota changes drives the development of bone-destructive periodontitis. Quercetin, a plant-derived polyphenolic flavonoid, has been linked with health benefits in both humans and animals. Using a systematic approach, we investigated the effect of orally delivered Quercetin on host inflammatory response, oral microbial composition and periodontal disease phenotype. In vivo, quercetin supplementation diminished gingival cytokine expression, inflammatory cell infiltrate and alveolar bone loss. Microbiome analyses revealed a healthier oral microbial composition in Quercetin-treated versus vehicle-treated group characterized by reduction in the number of pathogenic species including Enterococcus, Neisseria and Pseudomonas and increase in the number of non-pathogenic Streptococcus sp. and bacterial diversity. In vitro, Quercetin diminished inflammatory cytokine production through modulating NF-κB:A20 axis in human macrophages following challenge with oral bacteria and TLR agonists. Collectively, our findings reveal that Quercetin supplement instigates a balanced periodontal tissue homeostasis through limiting inflammation and fostering an oral cavity microenvironment conducive of symbiotic microbiota associated with health. This proof of concept study provides key evidence for translational studies to improve overall health.

In Vitro Biocompatibility of CPP-ACP and Fluoride-containing Desensitizers on Human Gingival Cells.

OBJECTIVES: To analyze the biocompatibility of different desensitizers containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and fluoride in their composition: MI Varnish (MV), Clinpro White Varnish (3M Oral Care), Profluorid Varnish (VOCO), Duraphat (Colgate) and Embrace Varnish (Pulpdent) on human gingival fibroblast cells (hGF). METHODS AND MATERIALS: Human gingival fibroblast (hGF) cells were exposed to several desensitizer extracts at different concentrations (0.1%, 1%, and 4% eluates). Then, in vitro biocompatibility was studied by analyzing the IC50 value, cell proliferation (MTT assay and cell cycle), cell migration (wound healing assay), cell morphology and F-actin content (immunocytofluorescence), and induction of apoptosis/necrosis (flow cytometry). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test. RESULTS: The lowest cell viability and IC50 were observed in all concentrations of Embrace Varnish-treated hGFs (p<0.001), whereas the highest were exhibited by those treated with Clinpro White Varnish. Similar effects were evidenced when induction of apoptosis/necrosis and cell migration assays were assessed. Finally, MI Varnish, Profluorid Varnish, Duraphat, and Embrace Varnish extracts showed lower numbers of attached cells, some of them with an unusual fibroblastic morphology when cultured with 4% concentration of the varnishes, while Clinpro White Varnish exhibited a similar number of cells with an evident actin cytoskeleton compared to the control group. CONCLUSIONS: The results obtained in this study indicate that hGFs show better in vitro biocompatibility after exposure to Clinpro White Varnish, even at the highest concentration employed, making it the most eligible for topical applications. In contrast, Embrace Varnish exhibited a high cytotoxicity towards hGFs that could potentially delay the healing process and regeneration of the oral mucosa, although more studies are needed to confirm this hypothesis.

Effect of nano zinc oxide on proliferation and toxicity of human gingival cells.

BACKGROUND: Periodontal dressing is used to cover the gum surface and protect the wound after periodontal surgery. Nanomaterials have been widely applied in dentistry in recent years. Zinc oxide (ZnO) is one of the main components of periodontal dressing. AIM: This study aims to explore the toxicity ZnO nanoparticles (ZnO NPs) causes to human gingival fibroblast cells (HGF-1) and its effect on cell proliferation. METHODS: First, we identified and analyzed HGF-1, including cell morphology, growth curve, and immunohistochemistry staining. Then, we treated HGF-1 with ZnO NP. Cell viability, the integrity of the cell membrane, oxidative damage, and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release assay, fluorescent probe, and flow cytometry. Furthermore, the expression of murine double minute 2 (MDM2) and p53 was determined by quantitative real-time polymerase chain reaction (qPCR) and Western blotting. We finally overexpressed MDM2 in HGF-1 to verify the relationship between MDM2 and cell proliferation. RESULTS: Our research indicated ZnO NPs did not affect cell proliferation at low concentrations. However, high-concentration ZnO NP inhibited cell proliferation, destroyed the integrity of cell membranes, and induced oxidative stress and apoptosis. In addition, high concentration of ZnO NPs inhibited the proliferation of HGF-1 by regulating the expression of MDM2 and p53. CONCLUSION: High concentration of ZnO NP caused toxicity to HGF-1 cells and inhibited cell proliferation by regulating MDM2 and p53 expression.

Influence of Nano, Micro, and Macro Topography of Dental Implant Surfaces on Human Gingival Fibroblasts.

Current research on dental implants has mainly focused on the influence of surface roughness on the rate of osseointegration, while studies on the development of surfaces to also improve the interaction of peri-implant soft tissues are lacking. To this end, the first purpose of this study was to evaluate the response of human gingival fibroblasts (hGDFs) to titanium implant discs (Implacil De Bortoli, Brazil) having different micro and nano-topography: machined (Ti-M) versus sandblasted/double-etched (Ti-S). The secondary aim was to investigate the effect of the macrogeometry of the discs on cells: linear-like (Ti-L) versus wave-like (Ti-W) surfaces. The atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis showed that the Ti-S surfaces were characterized by a significantly higher micro and nano roughness and showed the 3D macrotopography of Ti-L and Ti-W surfaces. For in vitro analyses, the hGDFs were seeded into titanium discs and analyzed at 1, 3, and 5 days for adhesion and morphology (SEM) viability and proliferation (Cck-8 and MTT assays). The results showed that all tested surfaces were not cytotoxic for the hGDFs, rather the nano-micro and macro topography favored their proliferation in a time-dependent manner. Especially, at 3 and 5 days, the number of cells on Ti-L was higher than on other surfaces, including Ti-W surfaces. In conclusion, although further studies are needed, our in vitro data proved that the use of implant discs with Ti-S surfaces promotes the adhesion and proliferation of gingival fibroblasts, suggesting their use for in vivo applications.

PDRN, a natural bioactive compound, blunts inflammation and positively reprograms healing genes in an "in vitro" model of oral mucositis.

Oral mucositis is a side effect hard to treat following high dose chemotherapy or radiotherapy. Adenosine A2A receptor stimulation blocks NF-κB and boosts the Wnt/ß-catenin signaling, thus blunting inflammation and triggering growth factor codifying genes. Polydeoxyribonucleotide (PDRN) is a registered drug that activates the A2A receptor. Therefore, the aim of this study was to evaluate PDRN effects in an "in vitro" model of oral mucositis induced by prompting an inflammatory phenotype in human gingival fibroblasts (GF) and human oral mucosal epithelial cells (EC). GF and EC were stimulated with LPS (2 µg/ml) alone or in combination with i) PDRN (100 µg/ml); ii) PDRN plus ZM241385 (1 µM) as an A2AR antagonist; iii) CGS21680 (1 µM) as an A2AR agonist. LPS boosted NF-κB, TNF-α and IL-6 expression, decreased IL-10 levels and downregulated both Wnt/ß-catenin, VEGF and EGF expression. PDRN reverted the LPS-induced phenotype as well as CGS21680. Co-incubation with ZM241385 abolished PDRN effects, thus confirming A2A receptor involvement in PDRN mechanism of action. These results suggest that PDRN efficacy may be due to a "dual mode" of action: NF-κB inhibition and Wnt/ß-catenin signaling activation. However, these interesting findings need to be confirmed by animal and clinical studies.

Evaluation of Wound Healing Activity of Salvianolic Acid B on In Vitro Experimental Model.

Despite a wide range of bactericides and antiseptics, the treatment of chronic or complicated wounds is still a major challenge for modern medicine. Topical medications are the most sought-after new agents for use as treatment. The therapeutic concentration of their active substances is easy to achieve with the lowest possible burden on the patient's body. This study assesses the effect of salvianolic acid B (Sal B) on the proliferation, migration, and production of collagen type III by fibroblasts, which are the most important processes in wound healing. The study was conducted on human gingival fibroblasts obtained from primary cell culture. The results showed that Sal B at a dose of 75 µg/mL increases the cell viability with significant stimulation of the cell migration as demonstrated in the wound healing assay, as well as an increase in the expression of collagen type III, which has great importance in the initial stages of wound scarring. The results obtained in the conducted studies and previous scientific reports on the antibacterial properties and low toxicity of Sal B indicate its high potential in wound healing.

The role of nuclear receptor 4A1 (NR4A1) in drug-induced gingival overgrowth.

Drug-induced gingival overgrowth (DIGO) is a side effect of cyclosporine A (CsA), nifedipine (NIF), and phenytoin (PHT). Nuclear receptor 4A1 (NR4A1) plays a role in fibrosis in multiple organs. However, the relationship between NR4A1 and DIGO remains unclear. We herein investigated the involvement of NR4A1 in DIGO. In the DIGO mouse model, CsA inhibited the up-regulation of Nr4a1 expression induced by periodontal disease (PD) in gingival tissue, but not that of Col1a1 and Pai1. We detected gingival overgrowth (GO) in Nr4a1 knock out (KO) mice with PD. A NR4A1 agonist inhibited the development of GO in DIGO model mice. TGF-ß increased Col1a1 and Pai1 expression levels in KO mouse gingival fibroblasts (mGF) than in wild-type mice, while the overexpression of NR4A1 in KO mGF suppressed the levels. NR4A1 expression levels in gingival tissue were significantly lower in DIGO patients than in PD patients. We also investigated the relationship between nuclear factor of activated T cells (NFAT) and NR4A1. NFATc3 siRNA suppressed the TGF-ß-induced up-regulation of NR4A1 mRNA expression in human gingival fibroblasts (hGF). CsA suppressed the TGF-ß-induced translocation of NFATc3 into the nuclei of hGF. Furthermore, NIF and PHT also decreased NR4A1 mRNA expression levels and suppressed the translocation of NFATc3 in hGF. We confirmed that CsA, NIF, and PHT reduced cytosolic calcium levels increased by TGF-ß, while CaCl2 enhanced the TGF-ß-up-regulated NR4A1 expression. We propose that the suppression of the calcium-NFATc3-NR4A1 cascade by these three drugs plays a role in the development of DIGO.