Antonietta Stellavato

Biotechnological Chondroitin a Novel Glycosamminoglycan with Remarkable Biological Function on Human Primary Chondrocytes

Cartilage tissue engineering, with in vitro expansion of autologus chondrocytes, is a promising technique for tissue regeneration and is a new potential strategy to prevent and/or treat cartilage damage (e.g., osteoarthritis). The aim of this study was (i) to investigate and compare the effects of new biotechnological chondroitin (BC) and a commercial extractive chondroitin sulfate (CS) on human chondrocytes in vitro culture; (ii) to evaluate the anti‐inflammatory effects of the innovative BC compared to extractive CS. A chondrogenic cell population was isolated from human nasoseptal cartilage and in vitro cultures were studied through time‐lapse video microscopy (TLVM), immunohistochemical staining and cytometry. In order to investigate the effect of BC and CS on phenotype maintainance, chondrogenic gene expression of aggrecan (AGN), of the transcriptor factor SOX9, of the types I and II collagen (COL1A1 and COL1A2), were quantified through transcriptional and protein evaluation at increasing cultivation time and passages. In addition to resemble the osteoarthritis‐like in vitro model, chondrocytes were treated with IL‐1β and the anti‐inflammatory activity of BC and CS was assessed using cytokines quantification by multiplex array. BC significantly enhances cell proliferation also preserving chondrocyte phenotype increasing type II collagen expression up to 10 days of treatment and reduces inflammatory response in IL‐1β treated chondrocytes respect to CS treated cells. Our results, taken together, suggest that this new BC is of foremost importance in translational medicine because it can be applied in novel scaffolds and pharmaceutical preparations aiming at cartilage pathology treatments such as the osteoarthritis. J. Cell. Biochem. 117: 2158–2169, 2016.

Mancozeb, a fungicide routinely used in agriculture, worsens nonalcoholic fatty liver disease in the human HepG2 cell model

Mancozeb, a manganese/zinc ethylene-bis-dithiocarbamate, is a fungicide routinely used in pest control programs. However, it has been found to have deleterious effects on human health and on the environment. Indeed, its massive use has raised the issue of possible health risks for agrarian communities; the molecule can also reach human cells via the food chain and alter metabolism, endocrine activity and cell survival. In particular, mancozeb induces many toxic effects on hepatic cell metabolism. For this reason, we investigated its effect in an in vitro model of hepatic damage, namely fatty acid-induced nonalcoholic fatty liver disease in the HepG2 cell line. We found that the hepatic toxicity of the fungicide exacerbated fatty acid-induced steatosis, as manifested by an increase in intracellular lipid droplet accumulation. Furthermore, mancozeb altered cell metabolism and induced cell death through upregulation of lactate dehydrogenase and cytochrome c, respectively, in dose-dependent manners. Therefore, mancozeb may play an important role in the pathogenesis and progression of chronic disease in humans and represents a danger for human health in high doses.

Hybrid Complexes of High and Low Molecular Weight Hyaluronans Highly Enhance HASCs Differentiation: Implication for Facial Bioremodelling

Background/Aims: Adipose-derived Stem Cells (ASCs) are used in Regenerative Medicine, including fat grafting, recovery from local tissue ischemia and scar remodeling. The aim of this study was to evaluate hyaluronan based gel effects on ASCs differentiation and proliferation. Methods: Comparative analyses using high (H) and low (L) molecular weight hyaluronans (HA), hyaluronan hybrid cooperative complexes (HCCs), and high and medium cross-linked hyaluronan based dermal fillers were performed. Human ASCs were characterized by flow cytometry using CD90, CD34, CD105, CD29, CD31, CD45 and CD14 markers. Then, cells were treated for 7, 14 and 21 days with hyaluronans. Adipogenic differentiation was evaluated using Oil red-O staining and expression of leptin, PPAR-γ, LPL and adiponectin using qRT-PCR. Adiponectin was analyzed by immunofluorescence, PPAR-γ and adiponectin were analyzed using western blotting. ELISA assays for adiponectin and leptin were performed. Results: HCCs highly affected ASCs differentiation by up-regulating adipogenic genes and related proteins, that were also secreted in the culture medium. H-HA and L-HA induced a lower level of ASCs differentiation. Conclusion: HCCs-based formulations clearly enhance adipogenic differentiation and proliferation, when compared with linear HA and cross-linked hyaluronans. Injection of HCCs in subdermal fat compartment may recruit and differentiate stem cells in adipocytes, and considerably improving fat tissue renewal.

Hyaluronan Hybrid Cooperative Complexes as a Novel Frontier for Cellular Bioprocesses Re-Activation.

Hyaluronic Acid (HA)-based dermal formulations have rapidly gained a large consensus in aesthetic medicine and dermatology. HA, highly expressed in the Extracellular Matrix (ECM), acts as an activator of biological cascades, stimulating cell migration and proliferation, and operating as a regulator of the skin immune surveillance, through specific interactions with its receptors. HA may be used in topical formulations, as dermal inducer, for wound healing. Moreover, intradermal HA formulations (injectable HA) provide an attractive tool to counteract skin aging (e.g., facial wrinkles, dryness, and loss of elasticity) and restore normal dermal functions, through simple and minimally invasive procedures. Biological activity of a commercially available hyaluronic acid, Profhilo®, based on NAHYCO™ technology, was compared to H-HA or L-HA alone. The formation of hybrid cooperative complexes was confirmed by the sudden drop in η0 values in the rheological measurements. Besides, hybrid cooperative complexes proved stable to hyaluronidase (BTH) digestion. Using in vitro assays, based on keratinocytes, fibroblasts cells and on the Phenion® Full Thickness Skin Model 3D, hybrid cooperative complexes were compared to H-HA, widely used in biorevitalization procedures, and to L-HA, recently proposed as the most active fraction modulating the inflammatory response. Quantitative real-time PCR analyses were accomplished for the transcript quantification of collagens and elastin. Finally immunofluorescence staining permitted to evaluate the complete biosynthesis of all the molecules investigated. An increase in the expression levels of type I and type III collagen in fibroblasts and type IV and VII collagen in keratinocytes were found with the hybrid cooperative complexes, compared to untreated cells (CTR) and to the H-HA and L-HA treatments. The increase in elastin expression found in both cellular model and in the Phenion® Full Thickness Skin Model 3D also at longer time (up to 7 days), supports the clinically observed improvement of skin elasticity. The biomarkers analyzed suggest an increase of tissue remodeling in the presence of Profhilo®, probably due to the long lasting release and the concurrent action of the two HA components.

A time-lapse approach to examine chromium and nickel effects on wound healing in vitro.

Chromium and nickel cause allergic contact dermatitis, a common biological skin response to sensitizing agents. This study used a conventional in vitro wounding model to study the impact of sensitizing agents on the innate immune response of human keratinocytes. Experiments were designed to evaluate the involvement of specific Toll-like receptors and metalloproteinases as effectors molecules downstream, at a molecular level. Further, keratinocytes were co-cultured with monocytes (THP-1 cells) to reproduce an inductive stimulus on monocytes made by metals. Human keratinocytes (HaCat) were grown on plates covered with collagen type I, chemically treated, and then mechanically injured with a sterile pipette tip. Restoration of the monolayer integrity was monitored by time-lapse video microscopy. Effector gene expression was evaluated by real-time PCR. The presence of chromium significantly dropped the rate of wound closure, while nickel-induced hyper-proliferation ended in an acceleration of the healing process, an event that does not occur in vivo. This latter outcome led to considering nickel as an unsuitable example for use in the experimental model. Focusing solely on the chromium aspect of this study, RNA profiles of selected molecular markers were generated to ascertain if the detrimental stimulus from chromium was eliminated or persisted both in keratinocytes alone and/or during co-cultures of keratinocytes and monocytes. Monocytes accelerated the process of wound repair. This in vitro experimental model highlighted the involvement of innate immunity in response to chromium and might be useful for test molecules of therapeutic interest for the treatment of skin lesions. However, the experience with nickel reveals that there are limitations to the utility of this wound model system after all.

Hyaluronan hydrogels with a low degree of modification as scaffolds for cartilage engineering

In the field of cartilage engineering, continuing efforts have focused on fabricating scaffolds that favor maintenance of the chondrocytic phenotype and matrix formation, in addition to providing a permeable, hydrated, microporous structure and mechanical support. The potential of hyaluronan-based hydrogels has been well established, but the ideal matrix remains to be developed. This study describes the development of hyaluronan sponges-based scaffolds obtained by lysine methyl-ester crosslinking. The reaction conditions are optimized with minimal chemical modifications to obtain materials that closely resemble elements in physiological cellular environments. Three hydrogels with different amounts of crosslinkers were produced that show morphological, water-uptake, mechanical, and stability properties comparable or superior to those of currently available hyaluronan-scaffolds, but with significantly fewer hyaluronan modifications. Primary human chondrocytes cultured with the most promising hydrogel were viable and maintained lineage identity for 3 weeks. They also secreted cartilage-specific matrix proteins. These scaffolds represent promising candidates for cartilage engineering.

Myclobutanil worsens nonalcoholic fatty liver disease: An in vitro study of toxicity and apoptosis on HepG2 cells

Myclobutanil is a conazole class fungicide widely used as an agrichemical. It is approved for use on fruit, vegetables and seed commodities in the EU and elsewhere to control fungi such as Ascomycetes, Fungi Imperfecti and, Basidiomycetes. Its widespread use has raised the issue of possible health risks for agrarian communities and the general population, which can be exposed to residues present in food and drinking water. The toxicities identified include adverse effects on liver and kidney and on the development of male reproductive organs. Since the liver is the first-line organ in the defense against xenobiotics, toxic effects on hepatic metabolism cause degeneration, necrosis, and tissue hypertrophy. Therefore, we investigated myclobutanils effects on the human liver cell line HepG2. We found that myclobutanil increases the amount of fatty acids in these hepatic cells, as evaluated with Oil Red O staining, and progressively reduces cell viability from 1ppm to 500ppm. Analysis of biomarkers such as Bcl-xL/Bak and Mcl-1/Bak confirmed activation of cell death pathways at low doses. Therefore, myclobutanil may play an important role in the pathogenesis and progression of chronic hepatocellular diseases in humans.

Effects of Pyriproxyfen on Viability and Increase of Intracellular Lipids inHepG2 Cell Line

Abstract Introduction: Pyriproxyfen, (2-[1-methyl-2-(4-phenoxyphenoxy) ethoxy] pyridine) (PPF) is an insecticidal used in household, agricultural, and horticultural applications to control many insect species. We tested its hepatic toxicity in hepatoma HepG2 cell line, we also evaluate if PPF could induce nonalcoholic fatty liver disease. Materials and methods: The hepatoma HepG2 cell line was exposed for 24-48 hrs with serum-free DMEM to the active principles at different concentrations. The cell viability was assessed by measuring reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). For the evaluation of in vitro steatosis, the cells were rinsed with cold phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde. Images of cell were captured using an optic microscope and stained lipid droplets were then extracted with isopropanol (60%) for quantification by measuring its absorbance at 510 nm. Results: The MTT-test showed that PPF is cytotoxic at all concentrations tested both at 24 h and 48 h. Cell viability is below 50% for concentrations 1-10 ppm while the viability is less than 10% for the concentration 100 ppm. PPF induces the increasing intracellular lipids from 1 ppm concentration. The maximum effect is observed at 100 ppm. Discussion: In our in vitro study we found a loss of cell viability of about 50% for concentrations from 1-10 ppm by the MTT-Test that measures mitochondrial enzyme activity. Because the mitochondrial enzyme activity affected major changes at the starting/beginning of the apoptotic this condition suggested that PPF is strongly cytotoxic to human hepatocytes in the presented assays. Already at 1 ppm concentration PPF induces the increasing intracellular lipids, in HepG2 in vitro culture.

In vitro assessment of nutraceutical compounds and novel nutraceutical formulations in a liver-steatosis-based model

BackgroundSteatosis is a chronic liver disease that depends on the accumulation of intracellular fatty acids. Currently, no drug treatment has been licensed for steatosis; thus, only nutritional guidelines are indicated to reduce its progression. The aim of this study is to combine different nutraceutical compounds in order to evaluate their synergistic effects on a steatosis in vitro model compared to their separate use. In particular, three different formulations based on silymarin, curcumin, vitamin E, docosahexaenoic acid (DHA), choline, and phosphatidylcholine were assayed.MethodsHuman hepatocellular carcinoma cells (HepG2 cell line) were treated with a mixture of fatty acids in order to induce an in vitro model of steatosic cells, and then the amount of intracellular fat was evaluated by Oil Red O staining. The peroxisome proliferator-activated receptors α and γ (PPARα and γ) expression, closely correlated to lipid metabolism, was evaluated. The efficiency of these receptors was evaluated through the study of LPL mRNA expression, a marker involved in the lipolysis mechanism. Superoxide dismutase (SOD-2) and malondialdehydes (MDA) in lipid peroxidation were assayed as specific biomarkers of oxidative stress. In addition, experiments were performed using human liver cells stressed to obtain a steatosis model. In particular, the content of the intracellular fat was assayed using Oil Red O staining, the activation of PPARα and γ was evaluated through western blotting analyses, and the LPL mRNA expression level was analyzed through qRT-PCR.ResultsAll formulations proved effective on lipid content reduction of about 35%. The oxidative stress damage was reduced by all the substances separately and even more efficiently by the same in formulation (i.e. Formulation 1 and Formulation 3, which reduced the SOD-2 expression and induced the PPARs activation). Lipid peroxidation, was reduced about 2 fold by foormulation2 and up to 5 fold by the others compared to the cells pretreated with H2O2.Formulation 1, was more effective on PPARγ expression (2.5 fold increase) respect to the other compounds on FA treated hepathocytes. Beside, LPL was activated also by Formulation 3 and resulted in a 5 to 9 fold-increase respect to FA treated control.ConclusionsOur results proved that the formulations tested could be considered suitable support to face steatosis disease beside the mandatory dietetic regimen.

Positive Effects against UV-A Induced Damage and Oxidative Stress on an In Vitro Cell Model Using a Hyaluronic Acid Based Formulation Containing Amino Acids, Vitamins, and Minerals

Ultraviolet (UV) radiations are responsible for skin photoaging inducing alteration of the molecular and cellular pathways resulting in dryness and reduction of skin elasticity. In this study, we investigated, in vitro, the antiaging and antioxidant effects of hyaluronan formulations based hydrogel. Skinkò E, an intradermic formulation composed of hyaluronic acid (HA), minerals, amino acids, and vitamins, was compared with the sole HA of the same size. For this purpose, HaCaT cells were subjected to UV-A radiations and H2O2 exposure and then treated with growth medium (CTR) combined with M-HA or Skinkò E to evaluate their protective ability against stressful conditions. Cells reparation was evaluated using a scratch in vitro model and Time-Lapse Video Microscopy. A significant protective effect for Skinkò E was shown with respect to M-HA. In addition, Skinkò E increased cell reparation. Therefore, NF-kB, SOD-2, and HO-1 were significantly reduced at the transcriptional and protein level. Interestingly, γ-H2AX and protein damage assay confirmed the protection by hyaluronans tested against oxidative stress. G6pdΔ ES cell line, highly susceptible to oxidative stress, was used as a further cellular model to assess the antioxidant effect of Skinkò E. Western blotting analyses showed that the treatment with this new formulation exerts marked antioxidant action in cells exposed to UV-A and H2O2. Thus, the protective and reparative properties of Skinkò E make it an interesting tool to treat skin aging.