Blood vessel formation after soft-tissue implantation of hyaluronan-based hydrogel supplemented with copper ions.

The possibility of ameliorating bone healing of implanted bone allografts is a field of great interest. Early vascular invasion is a key factor in bone allograft incorporation. It is well known that copper ions (Cu2+) show a proangiogenic action favouring the development of new vessels. In this work a hyaluronan based 50% hydrogel (Hyal-50%) was enriched with (Cu2+) and its proangiogenic activity was evaluated. Fifteen Sprague Dawley female rats were submitted to the subcutaneous implantation of Hyal-50%, freeze-dried bone allografts, Hyal-50%-Cu2+, freeze-dried bone allografts plus Hyal-50% and freeze-dried bone allografts plus Hyal-50%-Cu2+. One month later, histomorphometric analysis evidenced the presence of a fibrous-reactive capsule around all specimens showing significant differences among groups (p<0.0005). The highest thickness of the fibrous capsule was found around the freeze-dried bone implants (p<0.05); as well as the Hyal-50%-Cu2+ plus freeze-dried bone (15.2%, p<0.05) and Hyal 50% plus freeze-dried bone (21.4%, p<0.0005) implants showed a significant higher thickness compared with Hyal 50% and Hyal-50%-Cu2+. Statistical analysis showed a significant (p<0.01) higher vascular density in Hyal- 50%-Cu2+ and Hyal-50%-Cu2+ plus freeze-dried bone group when compared to other groups. The present preliminary results suggest the advantages offered by the combined use of a well-known biocompatible and tissue healing promoting material (Hyal-50%) and a new technique that consists of stimulating tissue vascularization using Cu2+ and that bone allograft incorporation may benefit from this technology.

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.

Conformation of human plasma proteins at polymer surfaces: the effectiveness of surface heparinization.

Studies were made on the adsorption of two human plasma proteins, albumin (HSA) and fibrinogen (HFg), onto three different polymeric surfaces: commercial pellethane 2363-80AE (PU); pellethane crosslinked with a poly(amido-amine) (PUPA); and heparinized PUPA, using in situ ATR/FTIR spectroscopy (attenuated total reflection Fourier transform infrared spectroscopy). Conformational changes were found to occur on the two proteins upon adsorption onto bare PU and PUPA, and the protein unfolding on bare PU was also found to be time dependent. On the contrary, the two proteins do not change conformation when they are adsorbed onto the heparinized surface, emphasising the effectiveness of surface heparinization.

Physiochemical characterization and coating of polyurethane with a new heparin-adsorbing material.

The coating of polyurethane devices was obtained by using a solution of a new heparin-adsorbing material, PUPA. The structure of the coated material was investigated at different depths by FTIR techniques. The bond with heparin is electrostatic, as revealed by subtracting the spectrum of the native PUPA from that of the heparinized sample.

Biocompatibility and enzymatic degradation studies on sulphated hyaluronic acid derivatives.

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.

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 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.

Chemical and biological evaluation of heparinized poly(amido-amine) grafted polyurethane.

By a simple process poly(amido-amine) chains have been grafted onto the surface of polyurethane. The poly(amido-amine) was found to be able to complex heparin by electrostatic interaction. Heparin can be released only at pH greater than 10 with NaOH solution. The heparin adsorbing capacity of the material was biologically tested, and the anticoagulant activity of the heparinized polyurethane was demonstrated.

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.

Heparinizable materials (IV). Surface-grafting on poly(ethylene terephthalate) of heparin-complexing poly(amido-amine) chains.

By a chemical process poly(amido-amine) chains have been grafted on the surface of poly(ethylene terephthalate) (Dacron) devices. After treatment, it was shown that the devices could adsorb significant amounts of heparin. Most of the adsorbed heparin can be recovered only by eluting at pH greater than 10 with NaOH solution.

In vitro biocompatibility evaluation of a heparinizable material (PUPA), based on polyurethane and poly(amido-amine) components.

The biocompatibility of a new heparinizable material based on polyurethane and poly(amido-amine) (PUPA) was evaluated both in the heparinized and non-heparinized forms. The quantity of heparin present on the material was measured using radiolabelled heparin and biological tests. Heparin release in plasma from heparinized PUPA was investigated using in vitro methods. The behaviour of PUPA towards cellular and plasmatic blood components was studied. The influence of sterilization on the cytocompatibility response of both heparinized and non-heparinized PUPA was investigated; gamma-rays were found to be a suitable method of sterilization as no toxic response was noticed.

A material (PUPA) presenting both the properties of polyurethanes and the capacity of adsorbing a high quantity of heparin.

Polyurethanes are widely used for biomedical applications, but there is still a constant search for improved blood-compatible materials. We studied a material (PUPA) obtained by the interconnection between a poly(amido-amine), N2LL, capable of forming stable complexes with heparin and a commercial polyurethane, Pellethane 2363-80AE, using hexamethylenediisocyanate as the crosslinking agent. The amount of absorbed heparin (evaluated by biological tests) was generally much higher than that found on the poly(amido-amine) surface-grafted polyurethane.

Heparinizable materials (III). Heparin retention power of a poly(amido-amine) either as crosslinked resin, or surface-grafted on PVC.

The retentive power of a poly(amido-amine), in the form of a highly hydrophilic crosslinked resin, has been evaluated at different pH's. The resin releases heparin quantitatively in a very narrow pH range (10.8-11.4). This poly(amido-amine) has also been grafted on PVC tubes, and the heparin-adsorbing capacity of the materials so obtained has been tested biologically. In this case heparin is only released at pH greater than 10 so confirming the strong interaction between our polymer and heparin.

Preparation and ESCA characterization of poly(vinyl chloride) surface-grafted with heparin-complexing poly(amido amine) chains.

By a simple process poly(amido-amine) chains have been grafted on the surface of poly(vinyl chloride). Grafted poly(vinyl chloride) is able to adsorb heparin, thus providing potentially non-thrombogenic surfaces. The grafting of poly(amido-amine), and the heparin adsorption have been studied by ESCA. It has been found that the total amount of grafted poly(amido-amine) depends on the molecular weight of the poly(amido-amine) used in the grafting reaction, but the amount of heparin adsorbed on the grafted material is relatively independent of the length of the poly(amino-amine) grafted chains.

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.

Synthesis and physicochemical characterization of a new material (PUPA) based on polyurethane and poly(amido-amine) components capable of strongly adsorbing quantities of heparin.

The synthesis of new materials (PUPAs) based on a commercial polyurethane and a heparin-complexing polymer, poly(amido-amine), was studied. PUPAs are capable of adsorbing heparin because the basic nitrogens of poly(amido-amine), once protonated, interact with the negative charges carried by the heparin molecule. Six different samples of PUPA were synthesized having a varied ratio of the components. The quantity of basic nitrogen on the surface and the bound heparin for each sample was determined. Two different kinds of heparin are present on a PUPA surface: one is strongly bound but can be detached by 0.1 M NaOH solution, the other is physically adsorbed and is slowly released by a stream of saline solution. A relationship between the quantity of strongly bound heparin and basic nitrogen was found. SEM and FTIR-ATR analysis were performed on all the PUPA samples. The mechanical characteristics change according to chemical composition.

Endothelization and adherence of leucocytes to nanostructured surfaces.

We analyse the leucocyte and endothelial cell response to polybromostyrene-polystyrene (PS/PBrS) and the poly-n-butylmethacrylate-polystyrene (PnBMA/PS) systems, both in flat form or nanostructured surfaces consisting of nanohills with increasing hill height (13-95nm). Experiments were carried out first with blood leucocytes alone, endothelial cells (of three different types) alone, and finally, using blood cells and endothelized nanosurfaces. Blocking monoclonal antibodies specific for CD11, CD29, CD31, CD54, CD166 were used to analyse whether and to what extent adhesion molecules could be involved in the adherence of both blood leucocytes and endothelial cells to different nanosurfaces. Expression of CD29 (beta-1 integrin), CD54 (ICAM-1) and CD166 (ALCAM) on blood leucocytes was dependent on the hill height, being most prominent with 13nm (PS/PBrS) and 45nm hill (PnBMA/PS) nanosurfaces. Adherence of a human microvascular endothelial cell line and umbilical primary endothelial cells was also related to hill height, being most prominent with 13nm hill height. An indirect correlation was observed between the extent of endothelization and the degree of leucocyte adherence. In cases of low to medium extent of endothelization, the adherence of monocytes and granulocytes was mediated by the expression of CD166, CD29 and CD11a (alpha-L integrin), CD29, CD31 (PECAM-1), respectively. Scanning electron microscopy studies showed the predominant emission of pseudopodia at the holes of the surfaces and the focal contacts with the nanosurfaces. Our studies emphasize the relevance of testing functional properties in co-culture experiments in the development and optimization of nanosurfaces for biomedical application.

Heparin adsorbing capacities at physiological pH of three poly(amido-amine) resins, and of poly(amido-amine)-surface-grafted glass microspheres.

Three poly(amido-amine)s of similar structure in the form of highly hydrophilic crosslinked resins, have been prepared, and tested for their heparin-adsorbing capacity at physiological pH. They showed different capacities, and their capacities were related to their basicities. One of the same polymers was grafted on the surface of glass microspheres. After treatment, it was shown that the microspheres could adsorb significant amounts of heparin. In all cases most of the adsorbed heparin was hardly eluted with saline, plasma, or blood, but could be recovered by eluting with 0.1 M NaOH. The resins were found to have some haemolytic properties, but no haemolysis was observed with the grafted microspheres.

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.

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.