Continuous multilayered composite hydrogel as osteochondral substitute.

Cartilage is a highly organized avascular soft tissue that assembles from nano-to macro-scale to produce a complex structural network. To mimic cartilage tissue, we developed a stable multilayered composite material, characterized by a tailored gradient of mechanical properties. The optimized procedure implies chemical crosslinking of each layer directly onto the previous one and ensures a drastic reduction of the material discontinuities and brittleness. The multilayered composite was characterized by infrared spectroscopy, differential scanning calorimetry, thermogravimetry, and scanning electron microscopy in order to compare its physico-chemical characteristics with those of cartilage tissue. The rheological behavior of the multilayered composite was similar to that of human cartilage. Finally its cytocompatibility toward chondrocytes and osteoblasts was evaluated.

Synthesis of esters of androgens with unsaturated fatty acids for androgen requiring therapy.

Androgens' metabolism and activity are gaining a more and more important role in human physiology particularly referring to aging and to neurodegenerative diseases. Androgen treatment is often required for long-lasting disorders. In order to improve their duration and effects, androgens can be administered as esters of carboxylic acids. The novelty of our research is the use of esters of androgens with specific unsaturated fatty acids, in order to reduce possible side effects particularly related to chronic pathologies with altered lipid homeostasis such as X-linked adrenoleukodystrophy and cardiovascular disorders. Thus the esters of the main androgenic substances testosterone, dihydrotestosterone (DHT) and their metabolite 5α-androstan-3α,17ß-diol were chemically obtained by coupling with different unsaturated fatty acids. To this aim, fatty acids with various degree of unsaturation and belonging to different series were selected. Specifically, oleic acid (18:1, n-9), linoleic acid (18:2, n-6), and the n-3 fatty acids, α-linolenic acid (18:3), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6) were used obtaining corresponding esters with acceptable yields and good degree of purity. All the synthesized compounds were tested for their cytotoxic activities in mouse NIH3T3 and human astrocyte cell lines. The esters demonstrated good tolerability and no in vitro cytotoxic effect in both cell cultures. After these promising preliminary results, the esters will be suitable for in vivo studies in order to ascertain their pharmacokinetic characteristics and their biological effects.

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.

Heterotypic interaction of fibroblasts and endothelial cells on restricted area.

The polysaccharide hyaluronic acid (Hyal) was photoimmobilized on glass surfaces to obtain a pattern with squares and rectangles of different dimensions and chemistry. The microstructured surfaces were characterized by Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Attenuated Total Reflection Infrared Spectroscopy (ATR FT-IR), and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). Surface analysis revealed the presence of a pattern consisting of alternating glass and Hyal microstructures whose dimensions decreased from the center to the edge of the sample. The behavior of Human Coronary Artery Endothelial Cells (HCAEC) and human tumoral dermal fibroblasts (C54) was studied on these micropatterned surfaces. Neither HCAEC nor C54 adhered to the immobilized Hyal but both adapted their shape to the different sizes of the glass squares and rectangles. The number of adherent HCAEC and C54 depended on the dimensions of both the glass domains and the nuclei of the cells. Co-cultured C54 on HCAEC patterned surfaces showed a heterotypic cell-cell interaction in the same chemical and topographic domain for the first time. In comparison to other techniques for patterning two different cell types, our approach was non cytotoxic and allowed arbitrary geometric patterns to form on different biocompatible substrata.

Role of fibrinogen conformation in platelet activation.

Platelet adhesion and activation induced by fibrinogen (Fbg) coating on polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) were analyzed. Hyal or HyalS was coated and grafted on the glass substrate using a photolithographic method. The Fbg coating was achieved by two different routes: the immobilization of Fbg by means of covalent bond to the polysaccharide layers and the mere adsorption of Fbg to Hyal and HyalS surfaces. Platelet adhesion and activation to the surfaces were evaluated using, respectively, scanning electron microscopy (SEM) and quantifying the release of Platelet Factor 4 by ELISA. The method used for the coating of the surfaces with the Fbg influenced the platelet response. In fact, platelet adhesion and activation took place on surfaces covered by bound Fbg but not on those containing adsorbed Fbg. To explain this difference, the molecular mechanism involved in the Fbg--platelet interaction was investigated blocking platelet membrane receptors by monoclonal antibodies. Because the interaction between Fbg and the GPIIb/IIIa platelet membrane receptor was the only molecular pathway involved, Fbg conformation after the interaction (adsorption or binding) with the Hyal and the HyalS chains and the role of serum proteins adsorbed on the Fbg containing surfaces were accurately analyzed. Both adsorbed and bound Fbg prevented the adsorption of further serum proteins; consequently, a direct interaction between Fbg and platelets was supposed and the different platelet behavior was ascribed to the different conformational changes that occurred after the adsorption and the chemical binding of the Fbg to the Hyal and HyalS surfaces.

Anti-inflammatory properties of superoxide dismutase modified with carboxymetil-cellulose polymer and hydrogel.

Superoxide dismutase (SOD) was chemically bound to carboxymethyl-cellulose (CMC) polymer.Furthermore, SOD was also trapped into two hydrogels of CMC with 50% and 90% crosslinking degree. The ability of the two SOD-CMC hydrogels to capture SOD and their release kinetics were investigated. ATR FT-IR spectrometry was used to study the conformation of SOD interacting with both CMC polymer and hydrogels. The effect of SOD-CMC polymer conjugate and SOD-CMC hydrogel systems upon human fibroblasts was studied in vitro measuring the cell proliferation inhibition index and evaluating cell morphology. Using the xanthine oxidase-nitroblue tetrazolium assay, the specific activity of bound SOD to CMC polymer or trapped into hydrogels was evaluated. The specific activity of the enzyme was higher in SOD-CMC hydrogels than in SOD-CMC polymer conjugates.

Immobilizing Cu,Zn-superoxide dismutase in hydrogels of carboxymethylcellulose improves its stability and wound healing properties.

Hydrogels of carboxymethylcellulose (CMC) with 50 and 90% cross-linking degree (CMC50% and CMC90%, respectively) were prepared and loaded with bovine erythrocyte Cu,Zn-superoxide dismutase (SOD) to obtain two drug delivery systems: SOD-CMC50% and SOD-CMC90%. Resistance of native SOD to inactivation by H2O2 and the effect of applying SOD-CMC hydrogels to open wounds of rats' back skin were examined and compared to that of SOD trapped into CMC50% and CMC90% hydrogels. Also, the effect of CMC50% and SOD-CMC90% on human fibroblasts proliferation was evaluated at different times. It was found that SOD in the hydrogel was more resistant to H2O2 inactivation than the native enzyme and at the same time it reduced the time necessary for wound healing. Furthermore, the highest cell proliferation value was found for the CMC50% hydrogels, which had a three-dimensional structure suitable for gas and nutrient exchanges and improving cell life conditions.

Hyaluronan and sulphated hyaluronan micropatterns: effect of chemical and topographic cues on lymphatic endothelial cell alignment and proliferation.

While tissue engineered blood vessels have entered surgical practice, the construction of artificial lymphatic vessels has never been attempted due to the small dimensions and fragility of lymphatic vessels. A possible alternative would be to obtain a new growth of interrupted lymphatic vessels. We have previously reported that lymphatic endothelial cells align when cultured on striped micropatterns of hyaluronan (Hyal) and aminosilanized glass. We here report a comparative study in which lymphatic endothelial cells have been plated on micropatterns with stripes of different width and height obtained by the photoimmobilization of Hyal and its sulphated derivative (HyalS) on aminosilanized glass to verify whether their response correlated with surface-chemistry andlor topography. On Hyal micropatterns, cells adhered to aminosilanized glass, avoiding Hyal stripes and molding their shape in accordance to the micropattern topography. Stress fibers, integrins and focal adhesion kinase organized accordingly. HyalS micropatterns with the same topography were unable to guide cell response, cells randomly adhered to HyalS and glass stripes, and polarization was attained only by increasing stripe height. These data indicate that surface chemistry is the main cue responsible for lymphatic endothelial cell guidance. When surface chemistry of stripes promotes cell adhesion as well as that of the substrate, topographical parameters become prevalent. Micropatterns with defined chemical and topographical properties may contribute to the design of new platforms for controlled cell growth in tissue engineering of lymphatic vessels.

Fibroblast cell behavior on bound and adsorbed fibronectin onto hyaluronan and sulfated hyaluronan substrates.

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.

Micropatterned hyaluronan surfaces promote lymphatic endothelial cell alignment and orient their growth.

The implant of a biocompatible device capable of guiding lymphatic vessel regeneration in patients who underwent removal of lymph nodes might contribute to restoring an efficient lymphatic drainage and help to prevent the occurrence of lymphedema. The aim of this study was to evaluate whether a microstructured surface could provide a guidance for the growth of cultured lymphatic endothelial cells. The presence of microstructures on a surface permits the control of cell adhesion, migration, proliferation, and differentiation. We report here that lymphatic endothelial cells align on microstructures of alternating hyaluronan and aminosylanized glass stripes obtained by photoimmobilization. Cells consistently spread and proliferate only on aminosylanized glass. They orient parallel to the longitudinal axis of the stripe. A pattern of alternating stripes of aminosylanized glass uniformly covered by elongated cells and of hyaluronan devoid of cells eventuallyforms. The presence of alpha(v)-integrins along cell borders of cells in search of contact with each other and at the leading edge of migrating cells, sites where new focal adhesions are presumably formed, indicates that integrin-mediated adhesion to the substrate guides cell migration along the microstructure. Micropatterned surfaces of hyaluronan thus proved to adequately orient the growth of cells allowing the regeneration of lymphatic endothelium in the desired direction.

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.

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.

Cu(II) and Zn(II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells.

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS3.5) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH)2L and ZnL, Zn(OH)2L (L means the disaccharide unit of the ligands) in aqueous solution at 37 degrees C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH)2HyalS3.5](4.5)- induced cell adhesion, while [ZnHyalS3.5](2.5)- and [Zn(OH)2HyalS3.5](4.5)- inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH)2HyalS3.5](4.5)- complex at 1 microM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.

Immobilisation of sulphated hyaluronan for improved biocompatibility.

Hyaluronan (Hyal) was modified by the insertion of sulphate to hydroxyl groups. A series of heparin-like compounds with controllable properties was obtained. The physicochemical and biological behaviours of all these sulphated hyaluronan acids (HyalSx) and their complexes with heavy metal ions (Cu2+ and Zn2+) were investigated. HyalS, derivatives showed a good anticoagulant activity and low platelet aggregation which increased with increasing degree of sulphation. Moreover HyalSx and their Cu2+ complexes were demonstrated to favour the growth of human endothelial cells. However, the utilisation of HyalSx as a material is hindered by its high solubility in physiological solution. Our approach to improve its stability was directed to the synthesis of new HyalSx-based hydrogels and on the preparation of new biocompatible polymeric surfaces obtained through covalent photoimmobilisation of HyalSx. The reaction of primary ovine chondrocytes and B10D2 endothelial cells was studied on both matrices in terms of cell number, F-actin and CD44 receptor immunostaining. Analysis of cell movement showed that the cells respond to HyalSx showing good adhesion and spreading. These results suggest that HyalSx containing materials could be used as biomaterials to aid cartilage repair and vessel endothelisation.

In vitro study of blood-contacting properties and effect on bacterial adhesion of a polymeric surface with immobilized heparin and sulphated hyaluronic acid.

The blood-contacting properties and the effect on bacterial adhesion of a material based on polyurethane and poly(amido-amine) (PUPA), both in its native form and with the anticoagulant molecules heparin or sulphated hyaluronic acid (HyalS3.5) electrostatically bonded to its surface, were evaluated and compared in vitro. The presence of the biological molecules on the surface was revealed by a dye test and ATR/FTIR analysis. Bound heparin was found to maintain its physiological action, in terms of thrombin inactivation, as well as did free heparin. Moreover, it reduced the degree of platelet adhesion. On the contrary, bound HyalS3.5 lost its anticoagulant activity, though it reduced platelet adhesion. The number of platelets on both modified surfaces was low. Their shape distribution, as determined by SEM, did not differ significantly on the two modified surfaces or with respect to the bare PUPA surface. HyalS3.5 and heparin also inhibited adhesion of Staphylococcus epidermidis to the material. A possible relationship between the platelet and bacterial adhesion is ascribed to the mediating role of plasma proteins.

The role of Fbg in platelet adhesion to polymeric materials in conditions of psychological stress.

The effect of psychological stress on platelet adhesion to five polymeric materials (polyurethane, polyurethane filled with BaSO4, polyethyleneterephthalate, silicone and low-density polyethylene) was studied. The platelets were obtained from non-stressed and stressed rabbits as platelet-rich plasma (PRP) and, once washed (Pw), were suspended in different media, i.e. in platelet poor plasma (Pw-PPP), in serum (Pw-S) and in Krebs-Ringer solution (Pw-KR). Scanning electron microscopy of platelet adhesion and morphology revealed differences in the platelet activating power of the various materials. The washing procedure and resuspension in PPP generally resulted in an increased number of adherent platelets, compared with the number of platelets adherent to the same material in PRP. However, platelets washed and suspended in Pw-KR or Pw-S showed the same shape distribution as in PRP. When platelets from stressed rabbits were used, there was very strong aggregation and activation of the platelets in both PRP and Pw-PPP, independent of the chemical nature and surface structure of the material. In contrast, in Pw-KR and Pw-S (in which Fbg is absent) a general picture of single, not very modified platelets was observed. Their number and shapes changed according to the nature of the different materials. On the whole, the present results confirm our original hypothesis of a key role of the psychological condition of the blood donor and strongly indicate Fbg as the determinant factor in the pattern of platelet adhesion.

The role of plasma proteins and stress in the assessment of hemocompatibility.

The physiological and psychological conditions of subjects supplying blood for hemocompatibility tests significantly affect the behavior of platelets in terms of both adhesion and activation. The responses of platelets to a standard biomaterial, polyethylene (PE), were examined with blood collected from male rabbits both in basal conditions and after stress. Different media were utilized. First, platelet-rich plasma (PRP) was used to obtain a PE response to contact with platelets. Then platelets drawn from PRP were isolated and washed with Krebs-Ringer solution. One aliquot was suspended in serum (Pw-S) where fibrinogen was absent, another aliquot in Krebs-Ringer solution (Pw-KR) (in order to avoid the influence of the plasma proteins on platelets), and a third aliquot in the original plasma from which the platelets were drawn (Pw-PPP) (in order to restore the initial condition of the plasma but with washed platelets). The analysis of platelet adhesion and morphology was performed by Scanning Electron Microscopy (SEM). Differences in platelet adhesion and morphology were observed with four different media in nonstressed animals, with Pw-PPP showing a higher number and Pw-S and PW-KR lower numbers. Platelet morphology indicated low levels of activation. The platelets drawn from stressed subjects could not be counted in either PRP or PPP medium because they were fully aggregated and adhered; in contrast, in Pw-KR and Pw-S, no significant differences were found with respect to nonstressed conditions, and there was little difference in platelet morphology. All of these factors underline the role of plasma proteins, in particular fibrinogen, in the stress-induced activation of platelets.

Effect of behavioral stress on platelet reactivity on polymeric surfaces.

It is well known that stressful stimuli change blood functions and platelet parameters are altered in humans and animals subjected to stress. We have examined the influences of behavioral stress on the morphological responses of platelets on a standard biomaterial, polyethylene (PE). Male rabbits were used. Blood was collected from the marginal vein of the ear 2 times per subject: the first sample was used as the baseline; 1 week later, the second was preceded in half of the subjects by 20 min of immobilization stress. In vitro adhesion of platelets on the PE was evaluated. The exposure of animals to stress induced a dramatic change in platelet morphology and functions on the PE: a higher degree of platelet adhesion, increased platelet spreading, and the appearance of pseudopodia. In the unstressed subjects there were no modifications of the platelets on the PE with respect to the baseline. The present experiment emphasizes for the first time the possible problems involved with the varying physiological conditions of patients before and after any biomaterial application surgery and of subjects who supply the blood for hemocompatibility tests performed on biomaterials. Therefore, in assessments of the performance of different biomaterials, the reactivity of blood factors in the patients should be considered and the test of blood compatibility should be performed with blood collected from donors in appropriate physiological conditions.

The influence of molecular weight on the biological activity of heparin like sulphated hyaluronic acids.

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.

Influence of Sulfation on Platelet Aggregation and Activation with Differentially Sulfated Hyaluronic Acids.

A number of sulfated hyaluronic acid derivatives (HyalS(2.5), HyalS(3), and HyalS(4)) were prepared by sulfation of the -OH groups present on hyaluronic acid and were generically termed HyalS(x). The anticoagulant properties of this series of compounds has previously been shown to be good in terms of their whole blood clotting inhibition and factor Xa and thrombin inactivation. The purpose of the present study was to investigate whether the use of these compounds would be beneficial to patients who would normally be given heparin, and to perform some preliminary investigations into their effects on platelets. The three compounds were thus studied by investigating their ability to inhibit von Willebrand factor-dependent platelet agglutination in comparison with unfractionated heparin. Agglutination was determined turbidometrically after the addition of ristocetin to stirred formaldehyde-fixed platelets and was demonstrated to be dependent on the presence of sulfate groups on the polysaccharide chain and correlated with the degree of HyalS(x) sulfation. Interactions possibly important in low shear environments were investigated by measuring the pharmacological action of the HyalS(x) on spontaneous platelet activation and aggregate formation by flow cytometry. The data indicate that platelet activation is not correlated with the number of sulfate or hydroxyl groups on HyalS(x), suggesting that activation occurs not via electrostatic interactions or H bonding, but via some other mechanism. A differentiation between low and high glycosaminoglycan sulfation densities is observed with respect to platelet aggregation, which is correlated with the number of sulfated groups per disaccharide unit. The ability of HyalS(x) to inhibit platelet aggregation induced by ADP and thrombin was measured by aggregometry. HyalS(4) resisted thrombin stimulation to a similar extent as heparin. All Hyal derivatives, however, were better at inhibiting ADP-induced aggregation than was heparin. We conclude, therefore, that clinical use of HyalS(x) in place of heparin may be beneficial because ristocetin-dependent agglutination, and therefore resistance to platelet aggregation in high shear environments, in addition to resistance to stimulation by ADP, has been shown to be superior to heparin. Spontaneous platelet activation and aggregation are induced at an overall low level, even at high HyalS(x) concentrations, and are comparable with that of heparin.