Vincenzo Rocchetti

Chronic exposure to cigarette smoke increases matrix metalloproteinases and Filaggrin mRNA expression in oral keratinocytes: Role of nicotine stimulation

The vegetal alkaloid nicotine has been proved to modify the expression of many keratinocyte markers. In this study, the basal expression of MMP-2, MMP-9, MMP-28, and Filaggrin has been evaluated in oral keratinocytes, in order to collect information about the ability of cigarette smoke to modify the basal expression pattern of these key enzymes in the absence of evident clinical signs in the oral epithelium. MMP-2, MMP-9, MMP-28, and Filaggrin basal expression was investigated by RT-PCR in oral keratinocytes derived from smokers (n=11), non-smokers (n=11), and ex-smokers (n=6) healthy volunteers. Moreover keratinocytes from non-smokers volunteers were stimulated in vitro by a single dose administration of nicotine (10 μM) in order to estimate the effect of nicotinic receptors activation on the basal expression of the studied markers. RT-PCR analysis showed that all the markers studied were overexpressed in keratinocytes from smoker donors compared to control keratinocytes, while a single dose of nicotine was able to induce only Filaggrin expression in keratinocytes from non-smoking donors. Markers expression in ex-smoker donors was similar to that observed in normal non-smoker donors. These data indicate for the first time that cigarette smoking affects basal expression of some important markers in oral mucosa keratinocytes in vivo in the absence of clinical signs and that smoke quitting restores basal expression levels of these markers.

Nicotine modulates gelatinase B (MMP-9) and epilysin (MMP-28) expression in reconstituted human oral epithelium.

Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn-dependent enzyme involved in cell migration. Among them, gelatinase B (MMP-9) and epilysin (MMP-28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1-50 μM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP-28 expression has been analyzed in epithelium sections using an anti-MMP-28 antibody, whereas MMP-9 presence and activity has been measured in cell-conditioned medium analyzed by gelatine zymography. The expression of MMP-9 was reduced by Nic in a dose-dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP-28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP-9 and MMP-28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.

Alveolar bone regeneration in post-extraction socket: A review of materials to postpone dental implant

Tooth extraction usually involves alveolar bone loss and reduction in height and width of the remaining alveolar socket, owing to the physiological bone resorption. This occurrence may perform an inadequate bone profile, that make difficult orthodontic applications, compromising the functional and aesthetic restoration of dental implants. The present review will provide an update on the biological and clinical profile of materials currently in use and those under investigation, in the recovering of bone margins of edentulous sockets.

Near-infrared laser increases MDPC-23 odontoblast-like cells proliferation by activating redox sensitive pathways.

Near infrared laser is known to induce biostimulatory effects, resulting in cell proliferation enhancement. Although such positive effect is widely exploited in various clinical applications, molecular mechanisms involved are still poorly understood. The aim of the study was to investigate the ability of laser stimulation to increase cell proliferation through an early activation of three redox sensitive pathways, namely Nrf-2, NF-κB and ERK in a rat odontoblast-like cell line (MDPC-23 cells). MDPC-23 cells were irradiated with different energy settings (0-50J, corresponding to 0-32.47J/cm2) and cell proliferation was evaluated by cell counting. Nrf-2, NF-κB and ERK signaling pathways activation was investigated through Western blot analysis. Our results show that a single 25J laser stimulation is able to increase cell proliferation and that this effect could be increased by repeating the stimulation twice with a time lapse of 24h. Western blot experiments demonstrated that laser stimulation is able to induce an early activation response in intracellular signaling, with an overlapping time pattern between the three considered pathways. Results discussed in this paper reveal a complex mechanism underlying near-infrared induced increase in pre-odontoblasts proliferation, involving three survival pathways that can act both separately or through reciprocal crosstalk. In particular, data presented suggest an important role for ERK pathway that could act directly by stimulating cell proliferation but can also induce both Nrf-2 and NF-κB activation, acting as a critical cellular checkpoint in response to imbalanced redox state generated by a laser induced increase in ROS production.

Low concentrations of neutrophil extracellular traps induce proliferation in human keratinocytes via NF-kB activation

INTRODUCTION Granulocytes play a pivotal role in innate immune response, as pathogen invasion activates neutrophils, a subclass of granulocytes, inducing the production of neutrophil extracellular traps (NETs). In this study, it has been evaluated how NETs could affect human keratinocytes (HaCaT cells) behaviour. MATERIALS AND METHODS HaCaT cells were treated with increasing NETs concentrations (0.01-200ng/ml) and the effect on cell proliferation was evaluated by MTT assay. Inhibition studies were performed by pre-treating cells with dexamethasone, chloropromazine or amiloride. NF-kB pathway activation was evaluated by western blot. RESULTS HaCaT cells stimulation with increasing concentrations of NETs (0.01-50ng/ml) for 48h resulted in a modulation of cell proliferation with a maximum increase corresponding to 0.5-1ng/ml stimulation. NETs low concentrations not only increased cell proliferation, but were also able to induce a faster wound closure in an in vitro scratch assay. NETs scaffold, composed by histone proteins and DNA, is recognized by Toll Like Receptor 9 (TLR 9) that, in turn, activates the NF-kB pathway. In fact, NETs induced proliferation was inhibited by chloropromazine (1nM), that blocks chlatrin vesicles formation, and by amiloride (50nM) that inhibits macropinocytosis. Moreover, dexamethasone, an inhibitor of NF-kB, was able to abolish the NETs effect. DISCUSSION This study thus demonstrates that low NETs concentrations undergo internalization finally resulting in a quick NF-kB pathway activation and HaCaT cells proliferation increase, suggesting a close relationship between first immune response and wound healing onset.

Charged polyhedral oligomeric silsesquioxanes trigger in vitro METosis via both oxidative stress and autophagy

Aims: Monocytes/macrophages are essential in innate immune response against pathogens also because their ability to release extracellular traps named METs (monocytes/macrophages extracellular traps). These structures are composed of DNA fibers decorated with nuclear and cytoplasmic proteins and their production process is called METosis. In this study attention has been focused on the ability of differently charged molecular systems (polyhedral oligomeric silsesquioxanes, POSS positively or negatively charged) to induce METosis. Main methods: METs formation was induced by lipopolysaccharide (250 &mgr;g/ml, positive control) and POSS positive and negative (0.05–1 mg/ml) treatment. METs were visualized and quantified by confocal microscopy using Sytox green staining. Oxidative stress, autophagy, as well as endocytosis involvement in the POSS induced METosis was evaluated. Key findings: Results obtained indicate a POSS positive or negative dose dependent ability in inducing MET release independently to their charge and that this phenomenon is a consequence of POSS +/− internalization. Moreover, studies using many reactive oxidative species (ROS) blockers and autophagy inhibitor showed a strong reduction in POSS induced METosis indicating their involvement. Significance: POSS +/− induce extracellular traps production in human monocytes/macrophages by oxidative and autophagic pathway.

Correction to: Near infrared laser irradiation induces NETosis via oxidative stress and autophagy

The published online version contains mistake on the author names. The first names and family names were interchanged. Corrected names are shown in the author group section above.