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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.

Sulphated hyaluronic acids having a sulphation degree of 3.5 per disaccharide unit, HyalS3.5, were prepared with different molecular weights corresponding to 21 x 10(3), 320 x 10(3) and 3500 x 10(3). The thrombin inhibition in plasma and in the presence of purified molecules, i.e. fibrinogen, antithrombin III (AT III) and heparin cofactor II (HC II), were studied for the three different MW compounds at different concentrations. The thrombin time in plasma depended on the length of the chain, and the two lower MW HyalS3.5 inhibited thrombin both by direct aspecific interaction and via HC II, whereas the activity of the highest MW compound was mainly related to the electrostatic interaction with HC II. The inactivation of FXa serine protease was only attributed to HyalS3.5-AT III complex.

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.

The biocompatibility and susceptibility to enzymatic degradation of new heparin-like polysaccharides obtained by hyaluronic acid sulphation (HyalSx) were evaluated. In particular, HyalSx cytotoxicity and cytocompatibility were assessed by the direct contact method using fibroblasts L929 and human endothelial cells. The results showed that hyaluronic acid derivatives are devoid of any cytotoxic effects on mouse fibroblasts and they are cytocompatible. The haemolysis test showed that the sulphated polysaccharides are not haemolytic. HyalSx susceptibility to enzymatic degradation was tested in the presence of both hyaluronidase and chondroitinase ABC. It was demonstrated that the introduction of sulphate groups along the hyaluronic acid chain makes the macromolecules resistant to enzymatic digestion.