**6. Discussion**

served at baseline (10 min) and after 1 hour of incubation with the substrates. smCS induced slightly higher aggregation of platelets (5-6%) compared to plastic (2.5%) or glass (less than 2%). However, these differences have to be considered with caution, as the coefficient of var‐

**Figure 4.** Fluorescence microscopy (100X)images of human platelets immunodecorated with CD62P (p-Selectin). Ar‐

Platelet activation was studied by the membrane expression of P-Selectin using the CD62P antibody. The expression of P-Selectin was evident on platelets adherent to plastic and glass

On glass and plastic (see arrows) the analysis of morphology showed several fully spread platelets expressing pseudopodia with the occurrence of focal clumps. This was also evident when platelets were examined by Scanning Electron Microscopy, SEM (Figure 5B and 5C).

SmCS film (Figure 5A) induced very limited morphological changes over the 90 minutes of

contact: platelets remained mostly discoid without the occurrence of pseudopodia.

surfaces and was negligible on platelets settled on smCS films (Figure 4).

rows indicate the presence of pseudopodia.

**5.11. Platelet activation assay**

iation estimated with human plasma in the absence of ADP was around 10%.

168 Advances in Biomaterials Science and Biomedical Applications

For blood-contact applications, haemocompatibility is largely determined by specific inter‐ actions with blood and its components [16]. Many, if not all, blood-contacting biomaterials are able to cause different undesired host responses like thrombosis, inflammatory reactions and infections.

The coagulation system and platelets are the main factors for thrombus formation on bioma‐ terials and represent a major unmet problem in the design of vascular implants and bloodhandling systems [17].

It is known that the endothelium is an active organ that maintains vessels integrity and pre‐ vent thrombosis and intimal hyperplasia [18,19]. Hence, biomaterials able to promote *in situ* endothelialisation of implants would be highly desirable.

Studies involving in vitro endothelialisation of grafts with cultured endothelial cells prior to implantation have shown that a confluent endothelium is able to prevent trombogenic complications and improves long-term patency [20,21]. Thus, taking into account that the endothelialisation of the blood-contacting polymeric materials is an important pre-requi‐ site for the success of the synthetic vascular grafts [22] we firstly investigated the ability of endothelial cells to adhere and proliferate on smCS film. The results obtained, in agreement with those shown in our previous paper [13], showed only little variation among the surfaces tested (glass, plastic and smCS). However, from the cell proliferation and morphology it was very difficult to discriminate difference in cytophilicity among the surfaces tested. Furthermore, the presence of fragmented smCS did not induce any decrement in the total number of endothelial cells compared to latex that, on the contra‐ ry, strongly affected cell survival.

The high hydrophilicity of smCS, indicated by the low contact angle, could ease the interac‐ tion with the bipolar extra-cellular matrix proteins such as fibronectin and vitronectin. Fur‐ thermore, the reduced cationic nature, due to a water shell does not allow anionic proteins such as collagen and fibronectin to dissociate from CS surface in a physiological environ‐ ment. This aspect is in agreement with the conclusion of [23] who reported that a hydrophil‐ ic surface is good for anti-non-specific protein adsorption. It was recently reported that the affinity for water of the cell-material interface seems to be a chief parameter in controlling cell adhesion, migration and differentiation [24].

Stevens and George [25] recognized that cells are sensitive to microscale patterns of chemis‐ try and topography, and Dalby [26] noted that cell behaviour is directly influenced by the surface structures such as grooves, pits, or ridges.

In this paper AFM images of smCS films evidenced a topographically patterned surface. In the light of the above reported literature, this observation can be used to speculate about the enhanced adhesion and proliferation of vascular cells compared to conventional, CS films previously observed in [13].

Surface properties such as wettability, surface topography and charge are known to affect endothelial cells attachment and growth [8]likely by altering the rate of the amount of adsor‐ bed proteins and their conformational changes [27,28]. The effect of surface materials on er‐ ythrocyte aggregation and platelet adhesion/activation becomes a chief parameter in haemocompatibility studies.

Several years ago Malette and co-workers [29] ascribed the pro-coagulation properties of chitosan to the negative charged surface of erythrocytes, while [30]showed that chitosan may induce the adhesion of erythrocytes.

In the present study, the surface of smCS films induced only a limited erythrocytes agglom‐ eration, thus indicating that smCS surface neither captures erythrocytes nor forms a threedimensional network structure with these cells.

The lack of erythrocyte aggregation may be likely due to a polymer chains rearrangement that masks the cationic nature of chitosan surface. Such a rearrangement can be ascribed to the larger amount of water in smCS films as described in [13].

One of the most important findings of this work is the observed difference in platelets mor‐ phology seeded on smCS in comparison with glass or plastic. On the latter surfaces platelets appeared flat with interconnecting pseudopodia coupled to strong P-Selectin membrane ex‐ pression.On the contrary, the platelets on smCS films were discoidal, and neither pseudopo‐ dia formation nor a P-Selectin membrane translocation was observed.

This finding could be attributed to a new conformation of the adsorbed plasma proteins on glass or plastic that could have facilitated platelet aggregation. Indeed, it is well known that the surface topography can induce a spatial reorganization of adsorbed proteins as well as how this phenomenon occurs [31]. In contrast, when the adsorbed proteins maintain their native state, they do not support platelet adhesion and aggregation [32].

The absence of platelet activation on smCS surfaces suggests this outcome.

complications and improves long-term patency [20,21]. Thus, taking into account that the endothelialisation of the blood-contacting polymeric materials is an important pre-requi‐ site for the success of the synthetic vascular grafts [22] we firstly investigated the ability of endothelial cells to adhere and proliferate on smCS film. The results obtained, in agreement with those shown in our previous paper [13], showed only little variation among the surfaces tested (glass, plastic and smCS). However, from the cell proliferation and morphology it was very difficult to discriminate difference in cytophilicity among the surfaces tested. Furthermore, the presence of fragmented smCS did not induce any decrement in the total number of endothelial cells compared to latex that, on the contra‐

The high hydrophilicity of smCS, indicated by the low contact angle, could ease the interac‐ tion with the bipolar extra-cellular matrix proteins such as fibronectin and vitronectin. Fur‐ thermore, the reduced cationic nature, due to a water shell does not allow anionic proteins such as collagen and fibronectin to dissociate from CS surface in a physiological environ‐ ment. This aspect is in agreement with the conclusion of [23] who reported that a hydrophil‐ ic surface is good for anti-non-specific protein adsorption. It was recently reported that the affinity for water of the cell-material interface seems to be a chief parameter in controlling

Stevens and George [25] recognized that cells are sensitive to microscale patterns of chemis‐ try and topography, and Dalby [26] noted that cell behaviour is directly influenced by the

In this paper AFM images of smCS films evidenced a topographically patterned surface. In the light of the above reported literature, this observation can be used to speculate about the enhanced adhesion and proliferation of vascular cells compared to conventional, CS films

Surface properties such as wettability, surface topography and charge are known to affect endothelial cells attachment and growth [8]likely by altering the rate of the amount of adsor‐ bed proteins and their conformational changes [27,28]. The effect of surface materials on er‐ ythrocyte aggregation and platelet adhesion/activation becomes a chief parameter in

Several years ago Malette and co-workers [29] ascribed the pro-coagulation properties of chitosan to the negative charged surface of erythrocytes, while [30]showed that chitosan

In the present study, the surface of smCS films induced only a limited erythrocytes agglom‐ eration, thus indicating that smCS surface neither captures erythrocytes nor forms a three-

The lack of erythrocyte aggregation may be likely due to a polymer chains rearrangement that masks the cationic nature of chitosan surface. Such a rearrangement can be ascribed to

ry, strongly affected cell survival.

170 Advances in Biomaterials Science and Biomedical Applications

cell adhesion, migration and differentiation [24].

surface structures such as grooves, pits, or ridges.

previously observed in [13].

haemocompatibility studies.

may induce the adhesion of erythrocytes.

dimensional network structure with these cells.

the larger amount of water in smCS films as described in [13].

As far as the surface morphology is concerned, it has been reported that platelets adhere in similar manner on smooth and rough surfaces when tested under static conditions [33]. Sim‐ ilarly Ward et al. [34] concluded that it is not the roughness *per se* which affects the platelet adhesion.

One decade ago, Suzuki and Minami [35,36] showed that Chitosan depleted complement proteins from plasma, suggesting that chitosan activates complement. A greater depletion of complement activity was seen for a highly de-acetylated form of chitosan [36]. It is however, important to note that the results obtained about the complement activation were based on binding and depletion assays. This complement depletion can equally be explained by as‐ suming a tight binding to the chitosan surface without activation [37].

The results presented here indicate that although large amounts of serum were deposited on smCS surface no activation of the complement system occurred, suggesting that the comple‐ ment is not directly activated by the smCS surface in the process of blood coagulation.

Haemolysis testing of biomaterials has been advocated for, and used in, standard biological safety testing of materials for more than 30 years. The results of test for haemolysis should be considered with care even if they represent the only recommended test for some medical devices as stated in Part 4 of ISO 10993 guideline.

Different papers have reported that chitosan promotes surface-induced haemolysis likely through an electrostatic interactions [38]. In the present work, in the presence of whole blood smCS triggered less than 5% of haemolysis that, along with the low erythrocyte adhe‐ sion, indicates a wide safety margin in blood contacting applications and suitability for vas‐ cular implants.

In the process of haemostasis, the activation of platelet adhesion and aggregation could rep‐ resent an initial and critical step. Here we showed that the surface of smCS films does not interfere with coagulation mechanism and supportswell endothelial cell adhesion and pro‐ liferation even if [39] reported that the haemostatic mechanism of chitosan may be inde‐ pendent of the classical coagulation cascade.
