Acute and chronic cannabidiol treatment: In vitro toxicological aspects on human oral cells.

Cannabidiol is gaining increasing interest for its potential anti-inflammatory, immunomodulatory, and antineoplastic effects. The purpose of this study is to investigate the biological effects of acute and chronic CBD administration on gingival fibroblasts and oral keratinocytes. Viability, morphology, migration, apoptosis and cell cycle, and expression of related genes (p53, BCL2, p21, and BAX) and of endocannabinoid system receptors (CB1, CB2 and GPR55) with real-time PCR and DNA damage with phospho-γ-H2AX immunofluorescence detection were analyzed. Concentrations between 100 µM and 0.001 µM were used: 50 µM (toxic dose), 25 µM (viability promoter), and 1 µM (nontoxic), were selected for subsequent chronic analysis. Acute treatment reveals significant effects than chronic, in particular in fibroblasts: concentrations ≥50 µM are highly cytotoxic, with increased apoptosis and reduced migration. Cell death correlates with increased p53 and BAX, followed by arrest in G0/G1 phase, with elevated p21 levels, suggesting a time- and dose-dependent damage. An increase in H2AX phosphorylation was observed with 25 µM and 50 µM, while 1 µM was biocompatible. Keratinocytes showed less cytotoxic effect than fibroblasts. Induced cell damage was dose- and time-related, with less damage after chronic treatment. Further investigations are needed with longer time frames to evaluate CBD dose- and time-dependent effects to identify an effective therapeutic dose.

In Vitro effects of alternative smoking devices on oral cells: Electronic cigarette and heated tobacco product versus tobacco smoke.

OBJECTIVE: To investigate the biological effects of electronic cigarette (e-cigarette) and heated tobacco product extracts respect to tobacco smoke extract on human gingival fibroblasts and human oral keratinocytes analysing cell viability, morphology, migration, apoptosis, cell cycle and epithelial-mesenchymal transition (EMT). DESIGN: Human gingival fibroblasts and human oral keratinocytes viability was analysed by MTT assay, cell morphology using scanning electron microscope (SEM) and cell migration by Scratch assay, a method that mimics the cell migration during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry and the related-gene expression of TP53, BCL2, CDKN2A and CDKN1A was indagated using real-time polymerase chain reaction. EMT process was analysed through expression of specific markers: CDH1, SNAI2, TWIST1, MMP2, FN1 and VIM. All investigations were evaluated after 24 h an in vitro exposure. RESULTS: Undiluted tobacco smoke extract induced significant inhibition of cell viability and cell migration, caused morphological alterations and induced an increase in cell death. No alterations or damage were observed after treatment with e-cigarette extracts. Heated tobacco product extract induced proliferation as highlighted by an increase of cell viability, cell migration and alterations of cycle analysis. CONCLUSIONS: Comparing the different cigarette extracts, tobacco smoke turns out to be the most harmful, e-cigarette did not determine morphological and functional alterations and heated tobacco product must be carefully investigated for its possible clinical effects on oral cell populations.

Use of the Er:YAG Laser in Conservative Dentistry: Evaluation of the Microbial Population in Carious Lesions.

The aim of this study is to investigate the Erbium:Yttrio-Aluminum-Granate (Er:YAG) laser photothermal and mechanical effects on cariogenic species concentration and on the microbial load composition of therapeutic cavities, in order to evaluate the possible micro-organisms reduction and make a comparison with manual and rotating conventional therapy (CT). A clinical trial was designed, including adults with active deep carious lesions on permanent teeth who were divided into two groups, i.e., control group and intervention group treated with CT and Er:YAG therapy, respectively. Before and after any conservative treatment, two oral samples were collected using a small sterile microbrush scrubbed within the base of the dentinal cavity tissue. The percentage of reduction and the colony-forming units (CFUs) count after Er:YAG and conventional treatments were compared for total microorganisms, including Candida spp., Streptococcus spp., and Lactobacillus spp. The microbial reduction varied from 90.2% to 100% and was significantly observed for total microorganisms and Streptococcus spp. (p < 0.05). The Er:YAG laser shows the potential for clinical applications, especially with paediatric and complicated patients, thanks to its minimally invasive properties and its effect on the reduction of microbial load.

Heat-not-burn tobacco (IQOS), oral fibroblasts and keratinocytes: cytotoxicity, morphological analysis, apoptosis and cellular cycle. An in vitro study.

OBJECTIVES: The aim of this work is to investigate the biological effects of IQOS smoking on human gingival fibroblasts and human keratinocytes analysing cell viability, morphology, migration, apoptosis and cell cycle. BACKGROUND: Electronic cigarettes and tobacco heating systems have been marketed to reduce smoking damages caused by combustion. METHODS: Human gingival fibroblasts and human keratinocytes viability was determined by a colorimetric assay measuring mitochondrial dehydrogenase activity (MTT assay); after an in vitro exposure of 24 h, cell morphology was analysed with scanning electron microscope and cell migration was tested by Scratch assay, a method to mimic the migration of the cells during wound healing in vivo. Apoptosis and cell cycle were analysed with flow cytometry, and the expression of related genes (p53, Bcl2, p16 and p21) was indagated using real-time polymerase chain reaction. RESULTS: IQOS extracts increased both cell viability (23%-41% with fibroblasts and 30%-79% with keratinocytes) and migration. No morphological alterations were observed. IQOS extracts did not induced an increase in cell death, but rose the number of S- and G2/M-phase cells. IQOS extracts also significantly increased p53 expression by fibroblasts (undiluted and 6.25% dilution, 2- and 3.6-fold higher, respectively) and reduced both Bcl2 (about two- and fivefold, respectively) and p21 expressions (about twofold with both extracts), while on keratinocytes both undiluted and 6.25% dilution extracts increased Bcl2 expression (about four- and threefold higher, respectively) and reduced p53 expression (about two- and fivefold, respectively). CONCLUSION: IQOS smoke seemed to induce proliferation as highlighted by a viability assay, and migration and cell cycle analysis. The increased cell proliferation induced by IQOS devices must be carefully investigated for its possible clinical effects on oral cell populations.

Bio-mechanical characterization of a CAD/CAM PMMA resin for digital removable prostheses.

OBJECTIVE: To compare the mechanical and biological features of a polymethylmethacrylate (PMMA) disc for CAD/CAM prostheses (test samples, TG) with a traditional resin (control samples, CG). METHODS: Mechanical analysis was performed using Dynamic Mechanical Analysis (DMA) and Brillouin's micro-spectroscopy. Human keratinocyte morphology and adhesion were analyzed by scanning electronic microscopy (SEM), cytotoxicity by the MTT assay, apoptosis by flow cytometry and p53, p21 and bcl2 gene expression by real time PCR. RESULTS: TG exhibited a higher elastic modulus than CG (range 5100-5500 ± 114.3 MPa vs 3000-3300 ± 99.97 MPa). The Brillouin frequency was found at ωB= (15.50 ± 0.05) GHz for TG and at ωB_1 = (15.50 ± 0.05) GHz and ωB_2 = (15.0 ± 0.1) GHz for CG where two peaks were always present independently of the sample point. SEM analysis revealed that keratinocytes on TG disks appeared to be flattened with lamellipodia. Keratinocytes on CG disks rose above the substrate with cytoplasmatic filaments. MTT viability data at 3 h and 24 h showed TG was significantly less cytotoxic than CG (p < 0.001). No significant differences emerged in apoptosis on CG and TG. Real-time PCR showed p53 expression increased after 3 h by about 9-fold in keratinocytes on TG (p < 0.001) and about 5-fold in those on CG (p < 0.001). High p53 expression persisted after 24 h on both disks. No significant variations were observed in p21 and bcl2 expression at any time-point. SIGNIFICANCE: PMMA resins, as used in CAD/CAM technology, displayed suitable biocompatible and mechanical properties for removable prostheses.

Morpho-functional effects of different universal dental adhesives on human gingival fibroblasts: an in vitro study.

To analyze the effects of four universal adhesives (Optibond Solo Plus-OB, Universal Bond-UB, Prime&Bond Active-PBA, FuturaBond M + -FB) on human gingival fibroblasts in terms of cytotoxicity, morphology and function. After in vitro exposure for up to 48 h, fibroblast viability was determined by the MTT assay determined, morphology by phase-contrast microscopy and migration by the scratch wound assay. Expression levels of IL1ß, IL6, IL8, IL10, TNFα and VEGF genes were assessed by RT-PCR and their protein production by Western blot analysis. Apoptosis and cell cycle were analyzed by flow cytometry. OB and UB induced early morphological changes on fibroblasts (3 h) with extended cell death at 24 h/48 h. Gene expression of collagen type I and fibronectin increased fivefold compared with controls, elastin disappeared and elastase increased threefold, indicating gingival tissue tended to become fibrotic. Only UB and OB increased gene expression of inflammatory markers: IL1ß at 3 and 48 h (up to about three times), IL6 and IL8 at 3 h (up to almost four times) which corresponded to the increase of the activated form NF-kB. All adhesives showed an effect on the functionality of fibroblasts with cytotoxic effect time and concentration dependent. Among all the OB and UB adhesives, they showed the greatest cell damage. The in-depth analysis of the effects of universal adhesives and possible functional effects represents an important information for the clinician towards choosing the most suitable adhesive system.

Biological effects of Cannabidiol on normal human healthy cell populations: Systematic review of the literature.

A systematic review was performed to evaluate the biological effects of Cannabidiol (CBD), one of the major components of Cannabis Sativa, on normal human healthy cell populations in terms of cell viability, proliferation, migration, apoptosis and inflammation. Inclusion criteria were: studies on cell lines and primary cell culture from healthy donors, CBD exposure as variable, no CBD exposure as control and published in English language. Quality assessment was based on ToxR tool, with a score of reliability ranging from 15 to 18.Following the PRISMA statement, three independent reviewers performed both a manual and an electronic search using MEDLINE via PubMed, Scopus, Web of Science and Cochrane. From a total of 9437eligible articles, 29 studies have been selected. The average quality assessment score was 16.48.Theresults showed heterogeneous CBD concentration exposure (0.01-50 µM or 0.1 nmol/mL-15 mg/mL). The definition of a threshold limit would allow the identification of specific effects on expected outcomes. From the data obtained CBD resulted to inhibit cell viability in a dose-dependent manner above 2 µM, while in oral cell populations the inhibitory concentration is higher than 10 µM. Moreover, it was observed a significantly inhibition of cell migration and proliferation. On the contrary, it was highlighted a stimulation of apoptosis only at high doses (from 10 µM).Finally, CBD produced an anti-inflammatory effect, with a reduction of the pro-inflammatory cytokine gene expression and secretion. CBD down-regulated ROS production, although at high concentrations (16 µM) increased ROS-related genes expression. The diffusion of CBD for therapeutic and recreational uses require a precise definition of its potential biological effects. A thorough knowledge of these aspects would allow a safe use of this substance without any possible side effects.