Stefania Lamponi

Hyaluronic acid hydrogel in the treatment of osteoarthritis

In order to overcome the problem of rapid clearance of the polysaccharide hyaluronic acid (Hyal) in the treatment of osteoarthritis (OA), a 50% cross-linked Hyal hydrogel (Hyal 50%) was synthesised. The 50% refers to the amount of COOH groups of the polysaccharide involved in the cross-linking reaction. i.e. 50% of the total amount. The rheological behaviour of the Hyal 50% hydrogel, and in particular the possibility to inject it through a needle, was studied. The results obtained demonstrated that the hydrogel injected through the needle still behaved like a gel, although it showed a reduction of the dynamic moduli. The most appropriate sterilisation technique for this kind of hydrogel was also evaluated. Liophilised Hyal 50% samples were sterilised by steam, Ethylene Oxide (EtO) and gamma-rays. EtO and gamma-rays did not modify the characteristics of the hydrogel in terms of swellability and morphology. Lastly, the in vivo effect of Hyal 50% hydrogel in the treatment of chondral defect in rabbit knee was also studied. The results obtained showed the Hyal 50% injections improved chondrocytes density and matrix appearance. Furthermore, the permanence in situ of the hydrogel was longer than that of the linear Hyal.

Biological Performance of Materials

The biomaterial science, the study of the application of materials to biological and biomedical problems is a field characterised by medical needs, basic research, advanced technological development, industrial involvement, ethical considerations and regulations. The biological performance of materials largely depends on their bulk and surface properties.

Blood-interaction performance of differently sulphated hyaluronic acids

Seven differently sulphated hyaluronic acid derivatives, having a general formula HyalSx where x can be 1, 2, 2.5, 3, 3.5, 3.8, 4, were synthetized. Coagulation tests i.e. whole blood clotting time and thrombin time were performed on these compounds and significant prolongations were observed from HyalS2.5 up to HyalS4. All that means the heparin like activity increases by increasing the sulphation degree of hyaluronic acid. The interaction of each of them with thrombin and FXa was studied in order to understand the mechanism of coagulation inactivation and the role of the sulphate position in the disaccharide unit to favour the protease inhibiting reaction. The bioactivity of HyalSx in terms of FXa and thrombin inactivation increases increasing with sulphation degree but the FXa inactivation seems to be mediated by ATIII, while the aspecific electrostatic interaction seems to play an important role in the inactivation of thrombin. Also the interaction with human serum albumin was studied by ATR/FT-IR technique and no changes of protein conformation was observed, as occurs in the case of heparin.

Micropatterned surfaces for the control of endothelial cell behaviour.

Micropatterned materials were synthesised by photoimmobilising the sulphated hyaluronic acid, adequately functionalised with a photoreactive moiety, on glass substrates. Four different patterns (10, 25, 50 and 100 microns) were obtained. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask was well reproduced on the sample surface. Analysis of endothelial cell behaviour on these micropatterned materials was performed in terms of adhesion, locomotion and orientation. Decreasing the stripe dimensions a more fusiform shape of the adhered endothelial cells was observed. At the same time the cell locomotion and orientation were increased. Furthermore, a photoimmobilisation of stripes of HyalS (10 and 100 microns) was performed on a continuous HyalS layer, in turn immobilised on glass substrate. Being excluded a different chemistry between the stripe and the substrate, the influence of topography on the behaviour of endothelia cells was thus envisaged.

Immobilization of Heparin and Highly-Sulphated Hyaluronic Acid onto Plasma-Treated Polyethylene

Heparin and highly-sulphated hyaluronic acid have been successfully immobilized onto plasma-processed polyethylene via a diamine polyethyleneglycol (PEG) spacer molecule. Two different plasma-processes have been utilized, i.e. a treatment and a deposition process, for providing polyethylene surface with the COOH groups necessary for the immobilization reactions. XPS integrated with derivatization procedures, ATR-FTIR and Water Contact Angle measurements have been carried out for characterizing each modification step: 1) the plasma-process, 2) the immobilization of the spacer molecule and 3) the immobilization of the biomolecules. The thrombin time of the modified surfaces has been measured, and their platelet activation characteristics evaluated. The results indicate a certain nonthrombogenic character of the biomolecule-immobilized polyethylene samples.

Using Liposomes as Carriers for Polyphenolic Compounds: The Case of Trans-Resveratrol

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol found in various plants, especially in the skin of red grapes. The effect of resveratrol on human health is the topic of numerous studies. In fact this molecule has shown anti-cancer, anti-inflammatory, blood-sugar-lowering ability and beneficial cardiovascular effects. However, for many polyphenol compounds of natural origin bioavailability is limited by low solubility in biological fluids, as well as by rapid metabolization in vivo. Therefore, appropriate carriers are required to obtain efficient therapeutics along with low administration doses. Liposomes are excellent candidates for drug delivery purposes, due to their biocompatibility, wide choice of physico-chemical properties and easy preparation. In this paper liposome formulations made by a saturated phosphatidyl-choline (DPPC) and cholesterol (or its positively charged derivative DC-CHOL) were chosen to optimize the loading of a rigid hydrophobic molecule such as resveratrol. Plain and resveratrol loaded liposomes were characterized for size, surface charge and structural details by complementary techniques, i.e. Dynamic Light Scattering (DLS), Zeta potential and Small Angle X-ray Scattering (SAXS). Nuclear and Electron Spin magnetic resonances (NMR and ESR, respectively) were also used to gain information at the molecular scale. The obtained results allowed to give an account of loaded liposomes in which resveratrol interacted with the bilayer, being more deeply inserted in cationic liposomes than in zwitterionic liposomes. Relevant properties such as the mean size and the presence of oligolamellar structures were influenced by the loading of RESV guest molecules. The toxicity of all these systems was tested on stabilized cell lines (mouse fibroblast NIH-3T3 and human astrocytes U373-MG), showing that cell viability was not affected by the administration of liposomial resveratrol.

The use of hyaluronan and its sulphated derivative patterned with micrometric scale on glass substrate in melanocyte cell behaviour

Surface microfabrication techniques were widely utilised for the spatial control of in vitro cell behaviour. A photo-immobilisation procedure was utilised to create micropatterned surfaces: four different stripe patterns (100, 50, 25 and 10 microm) of hyaluronan (Hyal) and its sulphated derivative (HyalS) on silanised glass substrate were obtained.The morphological analysis showed that the surface topography showed regular stripes of 100, 50, 25 and 10 microm wide and ranging from 300 nm up to 1 microm in thickness. They reproduced the exact photo-mask pattern: glass stripes alternating with polysaccharide ones. On the contrary, Hyal microstructures showed just a topographic pattern as the glass stripes appeared to be covered by a thin layer of the macromolecule by TOF-SIMS. Cell adhesion studies demonstrated that melanocytes adhered and oriented within the first 2h of culture on HyalS microdomains and not on Hyal microstructures where they spread on glass substrate around the patterned area. Double photo-immobilised samples characterised by a 100 microm stripe pattern of Hyal or HyalS on the top of a continuous layer of the two polysaccharides were also created in order to investigate the effect of the topography on cell behaviour. The obtained results demonstrated that melanocytes adhered on HyalS stripes while on the Hyal micropatterned surfaces they spread on silanised glass substrate around the structured area, resulting in the exclusion of the topographic pattern.

Novel polysaccharide hydrogels: characterization and properties

New chemical hydrogels, potentially suitable for biomedical applications, have been synthesized and characterized by 13C NMR, and FT-IR spectroscopy. The polysaccharide components of these hydrogels are hyaluronane, alginate and carboxymethylcellulose, while the novel cross-linking procedure consists of activating the carboxylate moieties by 2-chloro-1-methylpyridinium iodide CMPJ and using 1,3-diaminopropane as a chemical bridge. Varying the amount of CMPJ three series of hydrogels were obtained with different cross-linking degrees (5, 50, 100%). Their percentages were determined by 13C NMR and FT-IR analysis. The morphology of the gels was studied by scanning electron-microscopy and the pore sizes were determined in order to find a relationship with the swelling properties. Cell adhesion, using human hepatocytes, and platelet adhesion studies on the different series of cross-linked compounds allowed us to envisage their utilization as extracellular matrix materials and cardiovascular biomaterials. Copyright

New perspectives in cell communication: Bioelectromagnetic interactions

This paper explores physical signalling in biological communications, the so-called biophysical pathways, and especially the role of electromagnetic signalling in cell-cell interactions. The experiments were designed to evaluate whether different cell populations physically interfere when incubated in separate Petri dishes placed in close proximity. Two different cell populations, immortalized mouse fibroblasts (NIH3T3) and adult human microvascular endothelial cells (HMVECad) were selected and seeded in separate polystyrene Petri dishes. Dishes seeded with NIH3T3 were then placed on top of those seeded with HMVECad at distances of 4mm and 11mm. A black filter was placed between dishes containing the two cell populations in another experiment, to prevent transmission of electromagnetic radiation between the two. Cell number and morphology of NIH3T3 and endothelial cells were found to be modified in dishes without the black filter, suggesting that specific signals emitted by the cells were transmitted through the polystyrene wall, affecting cell proliferation rate and morphology, even though the cells were growing in separate dishes.

Disease modifying anti-Alzheimer’s drugs: inhibitors of human cholinesterases interfering with β-amyloid aggregation

We recently described multifunctional tools (2a–c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Aβ aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level.