Antonella D’Agostino

Semi-interpenetrated Hydrogels Composed of PVA and Hyaluronan or Chondroitin Sulphate: Chemico-Physical and Biological Characterization

Physical hydrogels based on poly (vinyl alcohol) (PVA) were synthesized in the presence of natural polysaccharides, either hyaluronan or Chondroitin sulphate, in order to obtain versatile biomaterials with enhanced performances. The physical network is obtained by the freeze thawing technique, a simple method widely used for structuring PVA blends. The chemico-physical characterization of resulting materials consisted of swelling studies and mechanical analysis. Furthermore the release of embedded polysaccharides from the network was evaluated to improve understanding of the strength of hydrogen bonding between the different polymeric chains, and the effect of the sulphate groups on the interaction promoting network formation and stability. Biological response in terms of cytotoxicity, adhesion and cell vitality of murine fibroblast and human keratinocytes showed that the addition of glycosaminoglycans to a PVA polymer leads to a biomaterial with potential applications in biomedical fields.

Properties of Newly-Synthesized Cationic Semi-Interpenetrating Hydrogels Containing Either Hyaluronan or Chondroitin Sulfate in a Methacrylic Matrix

Extracellular matrix components such as hyaluronan (HA) and chondroitin sulfate (CS) were combined with a synthetic matrix of p(HEMA-co-METAC) (poly(2-hydroxyethylmethacrylate-co-2-methacryloxyethyltrimethylammonium)) at 1% and 2% w/w concentration following a previously developed procedure. The resulting semi-interpenetrating hydrogels were able to extensively swell in water incrementing their dry weight up to 13 fold depending on the glycosamminoglycan content and nature. When swollen in physiological solution, materials water uptake significantly decreased, and the differences in swelling capability became negligible. In physiological conditions, HA was released from the materials up to 38%w/w while CS was found almost fully retained. Materials were not cytotoxic and a biological evaluation, performed using 3T3 fibroblasts and an original time lapse videomicroscopy station, revealed their appropriateness for cell adhesion and proliferation. Slight differences observed in the morphology of adherent cells suggested a better performance of CS containing hydrogels.

Short stature and azoospermia in a patient with Y chromosome long arm deletion.

We report on a 42-year old male with short stature, azoospermia and a wide deletion of long arm of Y chromosome. On physical examination, the patient showed height of 149 cm (<1° centile) and reduced volume (3 ml) and consistency of the testes. On hormonal evaluation, he showed increased serum gonadotropins and normal serum testosterone levels though its HCG stimulated levels were limited. Serum thyroid hormones were normal. Serum GH levels in baseline evaluation as well as after GHRH and GHRH + pyridostigmine administration were normal. Serum IGF I levels were lower than normal in baseline evaluation whereas its response to the GH administration was in the normal range. The bilateral testicular biopsy showed tubular atrophy, hyalinosis, interstitial sclerosis and a histological picture of a Sertoli cell only syndrome. Moreover the patient showed arthropathy, otopathy, small chin, small mouth and truncal obesity. On genetic evaluation, the patient showed a 46, X, delY(pter→q11.1:) karyotype and loss of several DNA loci on Yq. In fact he preserved short arm SRY, centromeric DYZ3 and more proximal euchromatic region Yq loci, including DYS270, DYS271, DYS272, DYS11, DYS273, DYS274, DYS148, DYS275, and missed more distal DNA loci from DYS246 to DYZ2. These results disclosed a wide Y long arm deletion, including all hypothized Yq azoospermia loci (except for AZFa and probably for one of the RBM genes, which lie proximally to the deletion) and possibly the Y-specific growth control region (GCY), mapped between DYS11 and DYS246 loci. This deletion is responsible for the complete azoospermia of the patient and probably also for his short stature, even if other factors could be implicated in the statural impairment. It further possibly allowed to relate the GCY gene(s) to the control of GH or IGF-I receptor or post-receptor pathway, being the alteration of this gene(s) consistent with the hormonal pattern of the patient

Development of nanocomposite based on hydroxyethylmethacrylate and functionalized fumed silica: mechanical, chemico–physical and biological characterization

In this research work organic/inorganic nano composites were synthesized from poly-2-hydroxyethylmethacrylate and properly modified silica nanoparticles by in situ polymerization. In particular, fumed nanosilica was functionalized with methacryloylpropyltrimetoxy silane (MPTMS) in order to obtain a more homogeneous, reliable and mechanically performing nano composite. For comparison, nano composites with non functionalised silica were also prepared. Scanning electron microscopy was performed in order to visualize the effects of functionalization on the mode and state of dispersion. This analysis demonstrated that MPTMS grafted onto silica surface acts as an effective coupling agent and assures a good dispersion and distribution of nanoparticles as well as a strong nano particle/matrix interfacial adhesion. As a result of strong interactions occurring between phases, a pronounced increase of the glass transition temperature and mechanical parameters were recorded. Finally, these novel nano composites were seeded with murine fibroblast and human mesenchymal stem cells, and observed in time-lapse experiments proving an effective biological response.

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.

Optimization of hyaluronan-based eye drop formulations.

Hyaluronan (HA) is frequently incorporated in eye drops to extend the pre-corneal residence time, due to its viscosifying and mucoadhesive properties. Hydrodynamic and rheological evaluations of commercial products are first accomplished revealing molecular weights varying from about 360 to about 1200kDa and viscosity values in the range 3.7-24.2mPa s. The latter suggest that most products could be optimized towards resistance to drainage from the ocular surface. Then, a study aiming to maximize the viscosity and mucoadhesiveness of HA-based preparations is performed. The effect of polymer chain length and concentration is investigated. For the whole range of molecular weights encountered in commercial products, the concentration maximizing performance is identified. Such concentration varies from 0.3 (wt%) for a 1100kDa HA up to 1.0 (wt%) for a 250kDa HA, which is 3-fold higher than the highest concentration on the market. The viscosity and mucoadhesion profiles of optimized formulations are superior than commercial products, especially under conditions simulating in vivo blinking. Thus longer retention on the corneal epithelium can be predicted. An enhanced capacity to protect corneal porcine epithelial cells from dehydration is also demonstrated in vitro. Overall, the results predict formulations with improved efficacy.

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.

Nanoparticles for the delivery of zoledronic acid to prostate cancer cells: A comparative analysis through time lapse video-microscopy technique

Time-lapse live cell imaging is a powerful tool for studying the responses of cells to drugs. Zoledronic acid (ZOL) is the most potent aminobiphosphonate able to induce cell growth inhibition at very low concentrations. The lack of clear evidence of ZOL-induced anti-cancer effects is likely due to its unfavorable pharmacokinetic profile. The use of nanotechnology-based formulations allows overcoming these limitations in ZOL pharmaco-distribution. Recently, stealth liposomes (LIPOs) and new self-assembly PEGylated nanoparticles (NPs) encapsulating ZOL were developed. Both the delivery systems showed promising anticancer activity in vitro and in vivo. In this work, we investigated the cytostatic effect of these novel formulations (LIPOs and NPs) compared with free ZOL on 2 different prostate cancer cell lines, PC 3 and DU 145 and on prostate epithelial primary cells EPN using time lapse video-microscopy (TLVM). In PC3 cells, free ZOL showed a significant anti-proliferative effect but this effect was lower than that induced by LIPOs and NPs encapsulating ZOL; moreover, LIPO-ZOL was more potent in inducing growth inhibition than NP-ZOL. On the other hand, LIPO-ZOL slightly enhanced the free ZOL activity on growth inhibition of DU 145, while the anti-proliferative effect of NP-ZOL was not statistically relevant. These novel formulations did not induce anti-proliferative effects on EPN cells. Finally, we evaluated cytotoxic effects on DU145 where, LIPO-ZOL induced the highest cytotoxicity compared with NP-ZOL and free ZOL. In conclusion, ZOL can be transformed in a powerful anticancer agent, if administered with nanotechnology-based formulations without damaging the healthy tissues.

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.

Protective effect of extractive and biotechnological chondroitin in insulin amyloid and advanced glycation end product-induced toxicity: IANNUZZI et al.

Glycosaminoglycans are extracellular matrix components related to several biological functions and diseases. Chondroitin sulfate is a sulphated glycosaminoglycan synthesized as part of proteoglycan molecules. They are frequently associated with amyloid deposits and possess an active role in amyloid fibril formation. Recently, a neuroprotective effect of extracellular matrix components against amyloid toxicity and oxidative stress has been reported. Advanced glycation end products (AGEs), the end products of the glycation reaction, have been linked to amyloid‐based neurodegenerative disease as associated with oxidative stress and inflammation. In this study we have analyzed the effect of chondroitin sulfate isolated from different species, in comparison with a new biotechnological unsulfated chondroitin, in the amyloid aggregation process of insulin, as well as the ability to prevent the formation of AGEs and related toxicity. The results have showed a determining role of chondroitin sulfate groups in modulating insulin amyloid aggregation. In addition, both sulfated and unsulfated chondroitins have shown protective properties against amyloid and AGEs‐induced toxicity. These data are very relevant as a protective effect of these glycosaminoglycans in the AGE‐induced toxicity was never observed before. Moreover, considering the issues related to the purity and safety of chondroitin from natural sources, this study suggests a new potential application for the biotechnological chondroitin.