Fetal Fascial Reinforcement Development: From “a White Tablet” to a Sculpted Precise Organization by Movement

Simple Summary Nowadays, the number of studies concerning fasciae is increasing, but few studies focus on fetal fasciae development and there is no study on the retinacula. The latter are fascial reinforcements with a crucial role in proprioception and coordination. We aimed to identify their structural organization by qualitative and quantitative assessments, to establish their role in myofascial development, highlighting their appearance and organization. Our data strongly suggest that the movement models the fascial reinforcements, structuring the fascial system, particularly at the end of the pregnancy. Fasciae have received much attention in recent years due to their important role in proprioception and muscular force transmission, but few studies have focused on fetal fasciae development and there is no study on the retinacula. The latter are fascial reinforcements that play a key role in proprioception and motor coordination. Furthermore, it is still unclear if they are genetically determined or if they are defined by movements, and if they are present during gestation or if they appear only later in the childhood. We aim to identify their structural organization by qualitative and quantitative assessments to establish their role the myofascial development, highlighting their appearance and organization. Samples from the wrist retinacula, posterior forearm, ankle retinacula, anterior leg, iliotibial tract and anterior thigh of six fetus body donors (from 24th to 40th week of gestation) and histological sections were obtained and a gross anatomy dissection was performed. Sections were stained with hematoxylin-eosin to observe their overall structure and measure their thicknesses. Using Weigert Van Gieson, Alcian blue and immunostaining to detect Hyaluronic Acid Binding Protein (HABP), Collagens I and III (Col I and III) were realized to assess the presence of elastic fibers and hyaluronan. This study confirms that the deep fasciae initially do not have organized layers and it is not possible to highlight any reinforcement. The fascial development is different according to the various area: while the deep fascia and the iliotibial tract is already evident by the 27th week, the retinacula begin to be defined only at the end of pregnancy, and their complete maturation will probably be reached only after birth. These findings suggest that the movement models the retinacula, structuring the fascial system, in particular at the end of pregnancy and in the first months of life. The fasciae can be imagined, initially, as white tablets composed of few elastic fibers, abundant collagens and HA, on which various forces, u movements, loads and gravity, write their history

The Crural Interosseous Membrane Re-visited: a Histological and Microscopic Study

The aim of this study was to characterize the microscopic structure and sensory nerve endings of the crural interosseous membrane (IM). 13 IMs from 7 cadavers were used to analyze the organization of the collagen fibers, IM’s thickness, distribution of elastic fibers and nerve elements. The IM is mainly a two-layer collagen fascicle structure with the collagen fibers of adjacent layers orientated along different directions, forming angles of 30.5 +/- 1.7° at proximal and 26.6 +/- 2.1° at distal part (P>0.05). The percentage of elastic fibers between the two layers and inside the collagen fascicle layer is 10.1 +/- 0.5% and 2.2 +/- 0.1% (P<0.001). The IM’s thickness at proximal, middle, and distal parts is 268.5 +/- 18.6μm; 293.2 +/- 12.5μm; 365.3 +/- 19.3 μm, respectively (Proximal vs Distal: P<0.001; Middle vs Distal: P<0.05). Nerve elements were present and located both inside and on the surface of the IM, whereas the mechanoreceptors are mainly located on the surface of the IM. Free nerve endings (33.3 +/- 5.0/cm2) and Ruffini corpuscles (3.4 +/- 0.6/cm2) were the predominant sensory elements, while Pacinian corpuscles (1.3 +/- 0.7/cm2) were rarely found. The type of mechanoreceptors found suggests that the IM may play a role in proprioception.https://doi.org/10.4081/ejtm.2019.834

Fasciacytes: specialized fibroblast-like cells that secrete the hyaluronan-rich matrix in fascial tissue

The presence of hyaluronan (HA) in samples of fascia lata were determined in eight volunteers undergoing elective surgical procedures at the Orthopedic Clinic of Padova University. The methods used were: Alcian Blue staining with differential concentrations of the electrolyte MgCl2, immunohistochemistry with anti-HABP (HA-binding protein) and transmission electron microscopy (TEM). In additional biochemical studies, we extracted and quantified HA in fascial tissue using an HA assay (Biocolor). The results demonstrated the rich presence of HA within fascia. HA forms thin layers throughout the various fascial fibrous layers. The quantification assay documented a mean value of 40 µg HA/g in fascia. Histological and TEM analyses demonstrated the presence of two different types of stromal cells within fasciae: apparent fibroblasts and some modified fibroblast-like cells with specialized functions of HA synthesis and secretion. We termed these cells “fasciacytes”. They may represent a new class of cells not previously recognized. This study confirmed that copious levels of HA occur within fascia, and provide quantification for the first time. In future studies, it will be important to compare these results in tissue from patients with myofascial pain and with rheumatic diseases

Distribution of cannabinoid receptor 1 (CB1) and 2 (CB2) in subcutaneous tissue and fasciae

The endocannabinoid system is constituted by the endocannabinoid receptors (CB1 and CB2), by the endocannabinoids and the machinery for their biosynthesis and metabolism. Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system but recently they are discovered at epidermis and dermis1. The endocannabinoid system has been involved in different physiological processes, in particular many works in animal models have discovered an antinociceptive activities in inflammatory state and chronic inflammatory disease. Those findings suggest the possibility that the endocannabinoid system interacts with different cells so it will be interesting to provide a description of endocannabinoid receptors distribution. Immunohistochemical and molecular investigation for CB1 and CB2 localization was carried out in human skin and subcutaneous tissue and direct analysis on fibroblasts isolated from deep fascia2 and subcutaneous tissue of human and rat samples. The majority of endocannabinoid receptors were found in the keratinocytes of skin and mast cells close to subcutaneous adipose tissue, whilst in the deep fascia the presence is scarse. The CB2 receptors were more frequently highlighted respect to CB1 receptors. The abundant distribution of cannabinoid receptors on skin, mast cells and subcutaneous tissue provides implications for an anti-inflammatory, and this suggests more studies to evaluate the therapeutic potential of endocannabinoids

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p &lt; 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p &lt; 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p &lt; 0.0001) or urgent (20.4% vs. 38.5%; p &lt; 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p &lt; 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

Studi di tossicità di nanomateriali e valutazione dei meccanismi d'azione in sistemi cellulari eucariotici

Nanoparticles (NPs) are particulate structures of various shapes and different composition with size ranging 1 and 100 nm. They are divided in NPs of natural origin (produced by combustion as in volcanoes), NPs of anthropogenic origin (produced by diesel engines or industrial incinerators) and artificial NPs (obtained through nanotechnology). These structures possess unique and innovative physical and chemical properties, dependent on their nanoscale dimensions and especially on the high ratio surface area/volume, that give to the NPs a new chemical reactivity and new optical, magnetic, catalytic and electrochemical properties (Sanvincens et al., 2008). In the last decades, these characteristics have made the NPs of considerable interest in technological development and wide used in medicine and diagnostics (Sanvincens et al., 2008), in biotechnology (Abu-Salah et al., 2010; Karn et al., 2009) and in cosmetics, food and materials (Liu et al., 2009). However, the increasing exposure to nanoscale particles requires studies that characterize the properties and potential cytotoxic effects. Although they are applied in a vast number of fields that seem to be destined to increase, their behavior inside the cell remains undetermined. It seems that NPs may lead to in vitro alteration of gene expression and cell death and that they are able to induce DNA damage both directly and indirectly, causing oxidative stress and inflammatory responses (Singh et al., 2009). Although many suppositions have been made about the possible harmful effects of nanoparticles on the body, it is not clear yet what is the exact mechanism by which these nanostructures interact with cells and subcellular structures. My Ph.D. work is part of the project funded by ECSIN (European Centre for the Sustainable Impact of Nanotechnology) that aims to assess the toxicity induced by nanostructures produced at industrial scale. In particular, a research was carried out on in vitro cytotoxicity of commercial Ludox® nanoparticles (a trademark product of W. R. Grace & Co) in human cell systems. These colloidal amorphous silica NPs are widely used in various industrial fields, such as in the production of printer's inks and paints, in textile industry, and in food industry for the fining of drinks. In particular the formulations that have been used are AS30 and SM30, 20 and 7 nm in diameter respectively. First of all, the two aqueous solutions of nanoparticles were characterized in collaboration with the Department of Chemistry, University of Padua, by measuring the ζ,potential, an indicator of the stability of colloidal suspension, by analyzing the form and the size with the transmission electron microscope, and finally with the analysis of the diameter by dynamic light scattering. It was also investigated the interaction of nanoparticles with the components of cell culture medium and serum proteins: studies of spectroscopy and analysis by dynamic light scattering have shown that even at low concentrations (0.01 mg/ml), Ludox® NPs aggregate in presence of even small percentages of serum (3%). The interaction with serum proteins resulting in large aggregates takes place immediately after preparing the solution of NPs in medium with serum and increases with incubation time. This phenomenon does not occur when the NPs are retained in aqueous suspension or in culture medium without serum. Because of the many toxicological studies conducted on cultured lung fibroblasts (Mroz et al., 2007; Foldbjerg et al., 2010) and the high risk of exposure to nanoparticles in the lungs (Gwinn et al., 2006; Nel et al., 2006), I selected a human cell line, CCD-34 Lu, derived from neonatal lung fibroblasts, and two human tumoral cell lines, A549 from a lung cancer and fibrosarcoma's cells HT-1080, were selected for this work. Initially the exposure's effects to various concentrations of Ludox® nanoparticles on cell viability were tested using the MTS colorimetric assay and the clonogenic assay. Cell viability was measured by incubating the cells in culture medium supplemented with 3% of serum for different times (24, 48 and 72 h) and for only 2 h in absence of serum to avoid the aggregation phenomena. The results showed that the two tested silica NPs give a dose and time-dependent toxicity in all the three cell lines. In addition, it was found that NPs with smaller diameter and greater surface air (Ludox SM30®) have generally a higher cytotoxic activity in agreement with literature studies (Lin et al., 2006; Napierska et al., 2009). Probably the smaller NPs can more easily penetrate the membranes and, at the same weight, they are also administered to the cells in a bigger amount than the AS30 NPs. The results of cell viability tests were compared by treating the cells in presence or in absence of serum in culture medium for a short time (2 h): both the clonogenic and the MTS assays showed that cells have a higher viability when the treatment occurs in medium with 3% of serum. I hypothesize that the NPs, forming reversible and unstable aggregates with serum proteins, are less toxic, probably because they are unable to penetrate the cell membrane because of their larger size. Finally, the normal cell line CCD-34 Lu was more sensitive to treatment with NPs than the two tumor cell lines, which show a significant decrease in cell viability only at doses that resulted almost lethal to normal cells (~0.02 mg/ml). Given the many clinical and experimental evidences that nanoparticles can damage cells and cause toxic effects (Nel et al., 2006), the production of reactive oxygen species (ROS) by cells after incubation with Ludox® AS30 and SM30 was analyzed. In agreement with the results of viability tests, it was observed that the normal line CCD-34 Lu produces high levels of ROS at concentrations of NPs at which the tumor cell lines were unaffected (~0.03mg/ml). For all the three cell lines, however, it was found a dose-dependent production of ROS after 2 h of incubation in culture medium without serum. Previous data have shown that the formation and accumulation of reactive oxygen species may cause serious damages to cells and are able to induce double breaks to DNA (Mroz et al., 2007; Mroz et al., 2008). For this reason, the induction of DSBs (double strand breaks) has been analyzed by the presence of foci of the phopshorylated form of the histone H2AX (γH2AX) in the nucleus of treated cells. The phosphorylation of this histone is necessary for the signalling of the damage and the consequent recruitment of proteins of DNA repair in the breaking point. The histone H2AX phosphorylation is not exclusively induced by the presence of DNA double strand breaks, but it is highly correlated to them, as demonstrated by several studies of induction of oxidative stress and exposure to ionizing radiation (Hamer et al., 2003). The results obtained in this work have revealed that only the fibrosarcoma human cell line HT-1080 was positive for the presence of foci after treatment with Ludox® nanoparticles AS30 and SM30, at concentrations of 0.02-0.04-0.06 mg/ml, whereas CCD-34 Lu and A549 cells were negative for all times and doses of treatment analysed. To assess whether the treatment with Ludox® NPs induces cell death by apoptosis, the cells were analysed by means of fluorescence microscopy after staining with the nuclear dye DAPI to detect the nuclear morphology and the presence of apoptotic bodies. An increase of apoptotic index was found following the treatment with nanoparticles, especially those with a smaller diameter (SM30, 0.04 mg/ml), and mainly in tumour cell line HT-1080. CCD-34 Lu cells are proved negative instead, in agreement with data showing that the normal fibroblasts does not meet to apoptosis but present different ways of response to cytotoxic activity of various agents. These data were then confirmed in the two cancer lines through the fluorimetric assay of caspase-3 activation, a cistein-protease involved in the initial stages of apoptosis. Finally to assess the possible genotoxic effects caused by incubation with Ludox® NPs, the gene expression alteration is assessed through Agilent®'s kit "Whole Human Genome Oligo Microarray". With DNA microarray technology it is possible to measure the expression levels of thousands of genes simultaneously, using the basic principles of hybridization of nucleic acids. A typical microarray experiment is divided into four distinct phases: 1) marking of the sample; 2) hybridization on a solid support; 3) image acquisition; 4) extraction of raw data and statistical analysis of measured values. The signal intensity detected in each spot of the array is ultimately an indirect measure of the concentration of that target (in this case mRNA) in the cell. Through the microarray analysis it is possible therefore to understand not only which genes are expressed in the examined conditions, but also if their expression is altered compared to the control sample (Kronick, 2004). The preliminary results achieved in this work relate to the A549 cell line incubated with Ludox® nanoparticles AS30 and SM30 at a concentration of 0.02 mg/ml, with a treatment of 2 h without serum, followed by a recovery in complete medium for 3 h (for SM30 and AS30 NPs) or 22 h (only for AS30 NPs). The number of genes whose expression is significantly altered compared to the control is higher in the sample treated with SM30 NPs (354 genes) compared to the sample treated with AS30 NPs (222 at 3 h after the end of treatment and 118 at 22 h). In both cases a greater number of altered genes are over-expressed in relation to those under-expressed when compared with the control sample. Furthermore, the level of gene expression is more altered when the analysis is conducted after 3 h of incubation in normal medium compared to 22 h. Investigating the expression levels of the main altered genes and the cellular pathways in which they are involved, it was observed that the main altered pathways are cell cycle control, the ways regulated by p53, the signaling pathway of the MAPK and the organization of the cell cytoskeleton. Although the study of gene expression profiles has revealed an alteration of the expression of genes in the cell cycle after treatment with Ludox® NPs, cell cycle analysis by flow cytometry in all the three cell lines examined did not bring out any change due to the NPs in any of the treatment conditions studied. The results obtained during my Ph.D. thesis constitute a preliminary study conducted in vitro on human cells about the cytotoxic effects caused by Ludox® silica nanoparticles, which are a model of commercial nanoparticles. In recent decades the NPs have found an increasingly wide use in various fields, from construction to textiles and food, imposing at the same time the need to provide exhaustive information for an assessment of the impact of nanomaterials on human health and the consequent regulation of their use.Le nanoparticelle (NP) sono strutture particolate, di varia forma e di diversa composizione, con dimensioni comprese tra 1 e 100 nm. Si distinguono in NP di origine naturale (prodotte da combustioni come nei vulcani), NP di origine antropogenica (prodotte da motori diesel o inceneritori industriali) e NP artificiali (ottenute attraverso le nanotecnologie). Queste strutture possiedono proprietà  fisico-chimiche innovative uniche, dipendenti dalle loro dimensioni nanometriche e soprattutto dall'elevato rapporto area superficiale/volume, che conferiscono una nuova reattività  chimica e nuove proprietà  ottiche, magnetiche, catalitiche ed elettrochimiche (Sanvincens et al., 2008). Queste caratteristiche hanno reso le NP negli ultimi decenni di notevole interesse nello sviluppo tecnologico e di largo impiego in campo medico-diagnostico (Sanvincens et al., 2008), in campo biotecnologico (Abu-Salah et al., 2010; Karn et al., 2009) e nell'industria cosmetica, alimentare e dei materiali (Liu et al., 2009). Tuttavia, la crescente esposizione a particelle nanometriche necessita di studi che ne caratterizzino le proprietà e i potenziali effetti citotossici. Nonostante esse siano applicate in un numero così vasto di campi che sembra essere destinato ad aumentare, il loro comportamento all'interno della cellula resta ancora da accertare; sembra che possano indurre in vitro alterazione dell'espressione genica e morte cellulare e che siano in grado di causare danni al DNA sia in modo diretto che indiretto, inducendo stress ossidativi o risposte infiammatorie (Singh et al., 2009). Sebbene si siano fatte numerose ipotesi sui possibili effetti dannosi delle NP per l'organismo, tuttavia non è ancora chiaro quale sia l'esatto meccanismo con il quale queste nanostrutture interagiscano con le cellule e con le strutture subcellulari. Questo lavoro di Dottorato di Ricerca si colloca all'interno del progetto promosso e finanziato da ECSIN (European Centre for the Sustainable Impact of Nanotechnology) volto a valutare la tossicità  indotta da nanostrutture prodotte a livello industriale. In particolare è stata condotta un'indagine in vitro sulla citotossicità  di nanoparticelle commerciali Ludox® (prodotto a marchio registrato della W. R. Grace & Co) in sistemi cellulari umani. Queste NP colloidali di silice amorfa sono ampiamente utilizzate in vari campi industriali, ad esempio nella produzione di inchiostri per la stampa e vernici, nell'industria tessile e in quella alimentare per la chiarificazione di bevande. In particolare sono state utilizzate le formulazioni AS30 e SM30, rispettivamente di 20 e 7 nm di diametro. Prima di tutto le due soluzioni acquose di nanoparticelle sono state caratterizzate in collaborazione con il Dipartimento di Scienze Chimiche dell'Università  di Padova, attraverso la misura del potenziale ζ,, indicatore della stabilità  della sospensione colloidale, attraverso analisi al microscopio elettronico a trasmissione per visualizzarne forma e dimensione, e infine con la misura del diametro mediante analisi al dynamic light scattering. Inoltre si è indagato sulla possibile interazione delle nanoparticelle con le componenti del terreno di coltura cellulare e con le proteine del siero: studi di spettrofotometria e analisi al dynamic light scattering hanno dimostrato che anche a concentrazioni basse (0,01 mg/ml), le NP Ludox® aggregano in presenza anche di piccole percentuali di siero (3%). L'interazione con le proteine del siero con conseguente formazione di aggregati di dimensioni maggiori avviene subito dopo la preparazione della soluzione di NP in terreno con siero e aumenta con l'aumentare del tempo di incubazione. Questo fenomeno non si verifica quando le NP vengono mantenute in sospensione acquosa o in terreno di coltura privo di siero. Dati i numerosi studi di tossicità  condotti su colture di fibroblasti polmonari (Mroz et al., 2007; Foldbjerg et al., 2010) e l'alto rischio di esposizione alle nanoparticelle a livello polmonare (Gwinn et al., 2006; Nel et al., 2006), per questo lavoro sono state selezionate una linea cellulare umana, CCD-34 Lu, derivata da fibroblasti di polmone neonatali, e due linee umane tumorali: A549, di carcinoma polmonare, e HT-1080, di fibrosarcoma. Gli effetti dell'esposizione a varie concentrazioni di nanoparticelle Ludox® sulla vitalità  cellulare sono state innanzitutto analizzati mediante il saggio colorimetrico MTS e il saggio clonogenico, dopo incubazione a diversi tempi: 24, 48 e 72 h in terreno di coltura addizionato al 3% di siero. La vitalità  cellulare è stata misurata anche incubando le cellule per 2 h in assenza di siero, nella condizione in cui le NP non vanno incontro a fenomeni di aggregazione. I risultati ottenuti hanno evidenziato innanzitutto che le due NP di silice saggiate danno una tossicità  dose e tempo dipendente in tutte e tre le linee cellulari. Inoltre, è stato verificato che le NP di diametro inferiore e aerea superficiale maggiore (Ludox® SM30) possiedono generalmente una maggiore attività  citotossica in accordo con studi di letteratura (Lin et al., 2006; Napierska et al., 2009), probabilmente perché, essendo più piccole, possono penetrare più facilmente nelle membrane e inoltre, a parità  di peso, ne vengono somministrate alle cellule un numero maggiore rispetto alle NP AS30. Sono stati poi confrontati i risultati dei saggi di vitalità  cellulare trattando le cellule in presenza di siero nel terreno di coltura o in assenza di siero per tempi brevi (2 h): sia il saggio clonogenico che il test MTS hanno messo in evidenza che le cellule hanno una vitalità  superiore quando il trattamento con le NP Ludox® avviene in terreno con il 3% di siero. Questo probabilmente è determinato dal fatto che le NP, formando aggregati reversibili ed instabili con le proteine del siero, risultano meno tossiche, probabilmente perché non sono in grado di penetrare nella membrana cellulare date le maggiori dimensioni. Infine, la linea cellulare normale CCD-34 Lu è risultata più sensibile al trattamento con le NP rispetto alle due linee tumorali, che mostrano un calo significativo della vitalità  cellulare solo a dosi di NP che risultano pressoché letali per la linea normale (~0,02 mg/ml). Date le numerose evidenze sperimentali e cliniche che le nanoparticelle possono causare danni a livello cellulare e avere effetti tossici (Nel et al., 2006), in questo lavoro è stata analizzata la produzione di specie reattive dell'ossigeno (ROS) da parte delle cellule, come conseguenza dell'incubazione con Ludox® AS30 e SM30. In accordo con i risultati dei test di vitalità , si è osservato che la linea normale CCD-34 Lu produce alti livelli di ROS a concentrazioni di NP a cui le linee cellulari tumorali risultano insensibili (~0,03 mg/ml). Per tutte e tre le linee cellulari prese in esame si è comunque riscontrata una produzione di ROS dose-dipendente dopo 2 h di incubazione in terreno di coltura in assenza di siero. Dati precedenti hanno dimostrato che la formazione e l'accumulo di specie reattive dell'ossigeno possono causare notevoli danni a livello cellulare e sono in grado di indurre doppie rotture a livello del DNA (Mroz et al., 2007; Mroz et al., 2008). Per questo motivo è stata analizzata tramite immunofluorescenza l'induzione di DSBs (double strand breaks) al DNA per mezzo di un marcatore di tale lesione, ovvero i foci dell'stone H2AX fosforilato. La fosforilazione di questa variante istonica è necessaria per la segnalazione del danno e il conseguente reclutamento di proteine di riparazione del DNA nel sito di rottura. La fosforilazione dell'istone H2AX non è esclusivamente indotta dalla presenza di doppie rotture al DNA, ma è altamente correlabile ad esse, come dimostrato da diversi studi di induzione di stress ossidativo e di esposizione a radiazioni ionizzanti (Hamer et al., 2003). I risultati ottenuti in questo lavoro hanno evidenziato che solo la linea cellulare di fibrosarcoma umano HT-1080 è risultata positiva per la presenza di foci di riparazione dopo trattamento con nanoparticelle Ludox® AS30 e SM30, alle concentrazioni di 0,02-0,04-0,06 mg/ml, mentre le cellule CCD-34 Lu e A549 sono risultate negative per tutti i tempi e le dosi di trattamento analizzate. Per valutare poi se il trattamento con NP Ludox® induce morte cellulare per apoptosi, tutte e tre le linee cellulari prese in esame sono state analizzate al microscopio a fluorescenza dopo fissazione con il colorante nucleare DAPI per evidenziare la morfologia nucleare e l'eventuale presenza di corpi apoptotici. Si è così potuto evidenziare un aumento dell'indice apoptotico in seguito al trattamento con nanoparticelle soprattutto per quelle di diametro inferiore (SM30, 0,04 mg/ml), principalmente nella linea cellulare tumorale HT-1080 e in misura minore anche nella linea cellulare A549. Le cellule CCD-34 Lu sono invece risultate negative a conferma di dati riportati in letteratura che dimostrano che questa linea di fibroblasti polmonari umani normali non va incontro ad apoptosi ma presenta differenti modalità  di risposta all'attività  citotossica di diversi agenti. Questi dati sono stati poi confermati nelle due linee tumorali tramite il saggio fluorimetrico di attivazione della caspasi 3, una cistein-proteasi coinvolta nelle fasi iniziali dell'apoptosi. Per valutare poi gli eventuali effetti genotossici causati dall'incubazione con NP Ludox® si è valutata l'alterazione dell'espressione genica tramite il kit della Agilent® 'Whole Human Genome Oligo Microarray'. Con la tecnologia dei microarray a DNA è possibile misurare il livello di espressione di migliaia di geni contemporaneamente, sfruttando i principi di base dell'ibridazione degli acidi nucleici. Un tipico esperimento di microarray si divide in quattro fasi distinte: 1) marcatura del campione; 2) ibridazione sul supporto solido; 3) acquisizione dell'immagine; 4) estrazione dei dati grezzi ed analisi statistica dei valori misurati. L'intensità del segnale rilevata in ogni spot dell'array è in definitiva una misura indiretta della concentrazione di quel target (in questo caso RNA messaggero) nella cellula. Tramite l'esperimento di microarray è possibile quindi capire non solo quali sono i geni espressi nelle condizioni esaminate, ma anche se la loro espressione è alterata rispetto al campione di controllo (Kronick, 2004). I risultati preliminari raggiunti in questo lavoro riguardano la linea cellulare A549 incubata con nanoparticelle Ludox® AS30 e SM30 alla concentrazione di 0,02 mg/ml, con un trattamento di 2 h senza siero, seguito poi da un ripristino in terreno completo di 3 h (per le NP AS30 e SM30) o di 22 h (solo per le NP AS30). Il numero di geni la cui espressione risulta significativamente alterata rispetto al controllo è più alto nel campione trattato con le NP SM30 (354 geni) r

Sensitivity of the Fasciae to the Endocannabinoid System: Production of Hyaluronan-Rich Vesicles and Potential Peripheral Effects of Cannabinoids in Fascial Tissue

The demonstrated expression of endocannabinoid receptors in myofascial tissue suggested the role of fascia as a source and modulator of pain. Fibroblasts can modulate the production of the various components of the extracellular matrix, according to type of stimuli: physical, mechanical, hormonal, and pharmacological. In this work, fascial fibroblasts were isolated from small samples of human fascia lata of the thigh, collected from three volunteer patients (two men, one woman) during orthopedic surgery. This text demonstrates for the first time that the agonist of cannabinoid receptor 2, HU-308, can lead to in vitro production of hyaluronan-rich vesicles only 3&ndash;4 h after treatment, being rapidly released into the extracellular environment. We demonstrated that these vesicles are rich in hyaluronan after Alcian blue and Toluidine blue stainings, immunocytochemistry, and transmission electron microscopy. In addition, incubation with the antagonist AM630 blocked vesicles production by cells, confirming that release of hyaluronan is a cannabinoid-mediated effect. These results may show how fascial cells respond to the endocannabinoid system by regulating and remodeling the formation of the extracellular matrix. This is a first step in our understanding of how therapeutic applications of cannabinoids to treat pain may also have a peripheral effect, altering the biosynthesis of the extracellular matrix in fasciae and, consequently, remodeling the tissue and its properties

Nerve cells developmental processes and the dynamic role of cytokine signaling

The stunning diversity of neurons and glial cells makes possible the higher functions of the central nervous system (CNS), allowing the organism to sense, interpret and respond appropriately to the external environment. This cellular diversity derives from a single primary progenitor cell type initiating lineage leading to the formation of both differentiated neurons and glial cells. The processes governing the differentiation of the progenitor pool of cells into mature nerve cells will be here briefly reviewed. They involve morphological transformations, specialized modes of cell division, migration, and controlled cell death, and are regulated through cell-cell interactions and cues provided by the extracellular matrix, as well as by humoral factors from the cerebrospinal fluid and the blood system. In this respect, a quite large body of studies have been focused on cytokines, proteins representing the main signaling network that coordinates immune defense and the maintenance of homeostasis. At the same time, they are deeply involved in CNS development as regulatory factors. This dual role in the nervous system appears of particular relevance for CNS pathology, since cytokine dysregulation (occurring as a consequence of maternal infection, exposure to environmental factors or prenatal hypoxia) can profoundly impact on neurodevelopment and likely influence the response of the adult tissue during neuroinflammatory events