Phytochemical compound and non-cytotoxicity effect of sting bee and stingless bee honey against normal human gingival cell lines

Objective: Both honeybees (Apis spp.) and stingless bees (Trigona spp.) produce honeys which normally taken orally, have high nutritional and therapeutics value. Until recently, phytochemical comparison of both honey is still scarce and elucidating cytotoxicity effects on human gingival fibroblast cells (HGF) in oral cavity is of interest. Materials and Methods: Kelulut honey (KH), acquired from the stingless bees and acacia honey (AH) from the sting bees honey samples were underwent GC-MS analysis to ascertain their composition. HGF were exposed to various concentrations of KH and AH from the lowest 0.015% to the highest 5% by MTT assay for 24h, 48h and 72h. Results: GC-MS analysis determined various beneficial compounds such as flavonoids, furans, pyrans, levoglucosan and hydroxymethylfurfural from both of honey samples. MTT assay showed that the HGF cells demonstrated good viability up to percentages (v/v) as high as almost 2% in both honeys. The IC50 values for both honey for all time frames fall at above 2%. Conclusion: Both honey showed good survivability of HGF cells up to 2% of concentration. Bangladesh Journal of Medical Science Vol. 21(1) 2022 Page : 158-164

In Vitro Antimicrobial and Cytotoxicity Activities of Some Medicinal Plant Extracts against Oral Microbial Pathogens

Introduction: Medicinal plants have long been of great interest to scientists in the search for the best treatment of diseases, especially the infectious diseases. In recent years, the use of herbal medicines has become more well-known because of their antimicrobial, antifungal, anti-cancer and less side effects. Aim: The aim of this study was to investigate the antimicrobial and antifungal effects of Urtica dioica, Equisetum arvense, and Punica Granatum peel extracts on two common oral microorganisms, Streptococcus mutans and Candida albicans. Materials and methods: The study investigated the hydro-alcoholic extract of the plants. The antimicrobial activity of the extracts was evaluated using the method of measuring the inhibition of microorganisms, and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined using different concentrations of the extracts and also biofilm assay and SEM were determined. Also cell viability was assessed by MTT assay on human gingival fibroblast cells. Results: The lowest MIC against S. mutants and C. albicans was related to the hydro-alcoholic extract of U. dioica. There was a significant reduction in the microbial biofilms by all three extracts. Among them, U. dioica could decrease the biofilms of S. mutans and C. albicans more than other extracts. In addition, the best results for growth inhibition zone were the hydro-alcoholic extracts of E. arvense and U. dioica with 35 and 30 mm growth zone, respectively. The results of SEM showed that P. granatum peel, U. dioica and E. arvense could destroy microbial biofilms without exerting any cytotoxic effects on HGF cell. Conclusions: The results of the study suggest that U. dioica, E. arvense, and P. Granatum peel extracts can be used as mouthwash with the least significant difference with routine mouthwashes. Also, the plant-based mouthwashes may be more suitable substitutes for chemical types in the future.

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

SUMMARY 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.

Cytotoxic Tumour-Selective 1,5-Diaryl-3-Oxo-1,4-Pentadienes Mounted on a Piperidine Ring

A series of 3,5-bis(benzylidene)-4-piperidones 2a-u were prepared as candidate cytotoxic agents. In general, the compounds are highly toxic to human gingival carcinoma (Ca9-22), human squamous carcinoma-2 (HSC-2) and human squamous carcinoma-4 (HSC-4) neoplasms, but less so towards non-malignant human gingival fibroblast (HGF), human periodontal ligament fibroblast (HPLF) and human pulp cells (HPC), thereby demonstrating tumour-selective toxicity. A further study revealed that most of the compounds in series 2 were more toxic to the human Colo-205 adenocarcinoma cell line (Colo-205), human HT29 colorectal adenocarcinoma cells (HT-29) and human CEM lymphoid cells (CEM) neoplasms than towards non-malignant human foreskin Hs27 fibroblast line (Hs27) cells. The potency of the cytotoxins towards the six malignant cell lines increased as the sigma and sigma star values of the aryl substituents rose. Attempts to condense various aryl aldehydes with 2,2,6,6-tetramethyl-4-piperidone led to the isolation of some 1,5-diaryl-1,4-pentadien-3-ones. The highest specificity for oral cancer cells was displayed by 2e and 2r. In the case of 2r, its selective toxicity exceeded that of doxorubicin and melphalan. The enones 2k, m, o have the highest SI values towards colon cancer and leukemic cells. Both 2e,r inhibited mitosis and increased the subG1 population (with a transient increase in G2/M phase cells). Slight activation of caspase-3, based on the cleavage of poly(ADP-ribose)polymerase (PARP) and procaspase 3, was detected.

Influence of cold atmospheric plasma on dental implant materials — an in vitro analysis

Background and objectives Alterations in the microenvironment of implant surfaces could influence the cellular crosstalk and adhesion patterns of dental implant materials. Cold plasma has been described to have an influence on cells, tissues, and biomaterials. Hence, the mechanisms of osseointegration may be altered by non-thermal plasma treatment depending on different chemical compositions and surface coatings of the biomaterial. The aim of the present study is to investigate the influence of cold atmospheric plasma (CAP) treatment on implant surfaces and its biological and physicochemical side effects. Materials and methods Dental implant discs from titanium and zirconia with different surface modifications were treated with CAP at various durations. Cell behavior and adhesion patterns of human gingival fibroblast (HGF-1) and osteoblast-like cells (MG-63) were examined using scanning electron microscopy and fluorescence microscopy. Surface chemical characterization was analyzed using energy-dispersive X-ray spectroscopy (EDS). Quantitative analysis of cell adhesion, proliferation, and extracellular matrix formation was conducted including real-time PCR. Results CAP did not affect the elemental composition of different dental implant materials. Additionally, markers for cell proliferation, extracellular matrix formation, and cell adhesion were differently regulated depending on the application time of CAP treatment in MG-63 cells and gingival fibroblasts. Conclusions CAP application is beneficial for dental implant materials to allow for faster proliferation and adhesion of cells from the surrounding tissue on both titanium and zirconia implant surfaces with different surface properties. Clinical relevance The healing capacity provided through CAP treatment could enhance osseointegration of dental implants and has the potential to serve as an effective treatment option in periimplantitis therapy.

Er:YAG laser irradiation enhances bacterial and lipopolysaccharide clearance and human gingival fibroblast adhesion on titanium discs

To investigate the effect of Er:YAG laser treatment on lipopolysaccharide (LPS) clearance and fibroblast adhesion on titanium disks. Grade IV titanium discs (n = 216) were used and allocated to 6 groups. Group 1 was the negative control without Porphyromonas gingivalis inoculation. Discs in Groups 2–6 were incubated with P. gingivalis to form a biofilm. Group 3 received 0.12% chlorhexidine irrigation and Group 4 received titanium curettage to remove the biofilm. Group 5 was treated with Er:YAG laser irradiation and Group 6 was treated with titanium curettage plus Er:YAG laser irradiation. The contact angle and surface roughness were measured after the various treatments. The surface microstructure and residual bacteria were examined using scanning electron microscopy and confocal laser scanning microscopy, respectively. Residual LPS was examined using a limulus amoebocyte lysate assay and human gingival fibroblast adhesion was quantified using fluorescent microscopy. Curettage plus Er:YAG laser irradiation was the most effective method for removing bacteria and LPS. No significant difference in the amount of fibroblast adhesion was found between the control and Group 6. Combined use of Er:YAG laser irradiation and curettage optimizes LPS clearance and fibroblast adhesion on titanium discs.

The Application of a Recombinant Antimicrobial Peptide of Thrombocidin-1 expressed in Pichia pastoris as a Novel Approach Against Some Oral Pathogenic Bacteria: An In-vitro Study

Objective: Oral infections and dental caries are considered serious health problems. Therefore, searching for new agents with antimicrobial properties seems to be crucial. This study aimed to evaluate the antimicrobial activity of the recombinant Thrombocidin-1 [TC-1] peptide on some oral pathogens. Also, the cytotoxicity of this peptide on human gingival fibroblast cells was investigated. Methods & Materials: In this study, Pichia pastoris was used for the expression of recombinant TC-1. The microbroth dilution method was used to determine the minimum inhibitory concentration [MIC] and minimum bacterial concentration [MBC]. It tested against four main oral pathogens; Streptococcus mutans, Streptococcus salivarius, Streptococcus oralis, and Enterococcus faecalis. Moreover, the cytotoxicity analysis was done on gingival fibroblast cells by the MTT method. The data were analyzed using a two-way analysis of variance [ANOVA] and Tukey’s HSD tests. Results: The most bactericidal effect of TC-1 was against S. salivarius, the highest bacteriostatic effect was against S. salivarius, and S. oralis had the lowest MIC value of 1.512 μg/ml. The Thrombocidin-1 peptide showed lower antibacterial properties against E. faecalis compared with CHX, unlike the stronger antimicrobial effect on examined streptococci. According to cytotoxicity examination, no concentration of TC-1 presented over 50% growth inhibition [IC50] of the fibroblasts cells. Conclusion: Based on antimicrobial tests and cytotoxicity results, the Thrombocidin-1 peptide may be useful as a safe antibacterial agent against some oral pathogens in dental materials.

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.

Titanium Substratum Roughness as a Determinant of Human Gingival Fibroblast Fibronectin and α-Smooth Muscle Actin Expression

The most appropriate surface treatment to enhance gingival connective tissue formation on the abutment of dental implants remains undefined, with healing associated with a scar-like response. We have previously shown that topographies with an arithmetic average of the absolute profile height deviations (Ra) = 4.0 induces an anti-fibrotic phenotype in human gingival fibroblasts (HGFs) by causing nascent adhesion formation. With bacterial colonization considerations, we hypothesized that a lower Ra could be identified that would alter adhesion stability and promote a matrix remodeling phenotype. Focal adhesions (FAs) area decreased with increasing roughness, although no differences in cell attachment or proliferation were observed. Alpha smooth muscle actin (α-SMA) protein levels were significantly reduced on Ra = 3.0 and 4.0 vs. 0.1 (p < 0.05), with incorporation of α-SMA into stress fibers most prominent on Ra = 0.1. Fibronectin protein levels were reduced on 3.0 and 4.0 vs. 0.1 (p < 0.05), and Ra = 1.5 and deeper significantly altered fibronectin deposition. Addition of exogenous TGF-β3 increased HGF adhesion size on 0.1 surfaces, but not on any other topography. We conclude that Ra = 1.5 is sufficient to reduce adhesion size and inhibit α-SMA incorporation into stress fibers in HGFs, but 3.0 is required in the presence of exogenous TGF-β3. Our findings have implications for inhibiting fibrotic tissue formation surrounding percutaneous devices such as dental implants.