**2. Methods**

#### **2.1. Production of sugar-modified chitosan films**

Chitosan solution was prepared as described in [13]. Briefly, four grams of chitosan powder (Chitosan 95/50 HMC+, Germany) were dissolved in a 1% (w/v) acetic acid aqueous solution until complete dissolution. Dibasic sodium phosphate (7.5 mM), sodium dihydrogen phos‐ phate (22 mM), potassium dihydrogen phosphate (1.5 mM), sodium chloride (125 mM) and‐ potassium chloride (2mM) were then sequentially added. The solution was filtered under vacuum using a 0.8 μM filter. Finally, D-(+) raffinose pentahydrate (290 mM) or D-(+) su‐ crose (290 mM) were added to the solution and allowed to dissolve for 2 hours under gentle stirring. About one mL of this solution was poured into a circular mould (1 cm diameter) and dried at 45 °C for 45 minutes in a ventilated oven. The obtained dry film was placed in a 5% (w/v) KOH aqueous solution for 12 hours then, washed in distilled water until neutrality of the wash water.

#### **2.2. Wettability**

Among them, chitosan (CS) a biodegradable [6], non-amphiphilic polymer of D-glucosamine obtained by partial de-acetylation of chitin [3], has shown interesting properties including bio-

Kind *et al*. [7] reported that it promotes plasma protein adsorption, platelet adhesion and ac‐ tivation, and thrombus development [8]. The positive charged CS surface induces a great degree of platelet adhesion. In fact, the Food and Drug Administration approved its use as haemostatic dressing for reducing haemorrhage [9-11]. Furthermore, it has been reported that the negatively charged-modified surface of CS prolongs clot formation after re-calcifica‐

Up to now, few data, often conflicting, on the haemocompatibility of negative charged-

In a previous work [13] we developed novel CS hydrogel prepared in the presence of phos‐ phate salts and relatively high amount of disaccharides such as D-(+)raffinose or D-(+)sac‐ charose and investigated the physico-chemical characteristics as well as the cytocompatibility of films obtained with this hydrogel. These sugars were not retained in the final structure of the film but were able to act as viscosity modifiers during the solidifica‐ tion/gelation process. The interference of salts and disaccharides resulted in smooth, amor‐ phous film with improved hydrophilicity and cytocompatibility compared to CS films produced with the same procedure but in low viscosity milieu. Differentiated human cells showed a great affinity for these sugar-modified chitosan (smCS) films, thus suggesting

The aim of the present study was to investigate qualities and aspects of the haemocompati‐ bility (platelet activation, haemolysis and activation of coagulation cascade) of smCS films produced according to Bettini*et al.* [13]. Moreover, the cytotoxicity of fragmented smCS was

These films were compared to materials able to activate platelets and induce thrombus for‐ mation such as plastic (standard polystyrene for cell culture) and glass (cover slips) as well

Chitosan solution was prepared as described in [13]. Briefly, four grams of chitosan powder (Chitosan 95/50 HMC+, Germany) were dissolved in a 1% (w/v) acetic acid aqueous solution until complete dissolution. Dibasic sodium phosphate (7.5 mM), sodium dihydrogen phos‐ phate (22 mM), potassium dihydrogen phosphate (1.5 mM), sodium chloride (125 mM) and‐ potassium chloride (2mM) were then sequentially added. The solution was filtered under vacuum using a 0.8 μM filter. Finally, D-(+) raffinose pentahydrate (290 mM) or D-(+) su‐ crose (290 mM) were added to the solution and allowed to dissolve for 2 hours under gentle

their candidature as promising biomaterial for tissue regeneration and repair.

mimetism due to the similarity of is structure with that of glycosaminoglycanes [4].

tion of plasma [12].

**2. Methods**

modified surfaces of CS films are available[11,12].

158 Advances in Biomaterials Science and Biomedical Applications

investigated in view of its bio-resorbability.

as a material able to trigger cell death such as latex.

**2.1. Production of sugar-modified chitosan films**

Contact angle measurements were performed at room temperature with a goniometer (AB Lorentzen & Wettre, Germany) on the surface of smC film in comparison to glass cover slip and plastic (standard polystyrene culture plates) to evaluate the wettability of the surface. Briefly, a drop (4 μL) of human serum was placed on the surface of the specimen. Images of the serum drop were recorded within 10 seconds of deposition by means of a digital camera (FinPix S602 Zoom, Fuji film, Japan). Digital pictures were analysed by ImageJ 1.43v soft‐ ware (NIH, USA) for angle determination. At least five measurements, taken at different po‐ sitions on each specimen, were carried out on both left and right side of the drop and averaged.

#### **2.3. Atomic force microscopy**

Atomic force microscopy (AFM) images of the films were analysed by AFM Nanoscope IIIA (Digital Instruments Inc., USA). Point probe silicon cantilever tip was used in contacting mode by the accompanying software to determine the surface roughness of investigated surfaces. The roughness parameters of each sample was evaluated on three scanned areas of 10μm x 10μm each.

#### **2.4. Procurement and processing of blood perfusates**

This procedure was conducted in accordance with the tenets of the Declaration of Helsinki. Following the indication of Italian DLgs no.196/03 (Codex on Privacy) in order to guarantee the respect of the privacy of the patients and the confidentiality of the donors' information. Blood (3.5-4 mL/test) was drawn by venipuncture from four healthy volunteers and added with tri-sodium citrate (0.109 M, 3.2% final concentration) in a 9:1 volumetric ratio to pre‐ vent coagulation. Whole blood was used for the haemolysis and thrombus formation tests.

Platelet-rich plasma (PRP) was obtained by centrifugation (400xg for 10 minutes, at room temperature) while platelet-poor plasma (PPP, platelets less than 10.000/μL) by centrifuga‐ tion at 2000xg for 20 minutes at room temperature.

Coagulation- and factor XII-assays were performed with platelet-poor plasma isolated from whole blood. For platelet function studies, PRP was volume adjusted with PPP to obtain a final physiologic stock platelet count of 3 105 platelets μL-1.

#### **2.5. Cell proliferation**

Human endothelial cells derived from foetal umbilical vessels (HUVEC) were provided by the American Type Culture Collection (Rockville, MD, USA). Cell monolayer were cultured in complete medium (D-MEM containing antibiotics and 10% foetal calf serum) supplement‐ ed with 50 μg mL-1 of endothelial cell growth factor (Sigma-Aldrich, USA) and kept in a in‐ cubator at 37 °C in a water-saturated atmosphere with 5% CO2. Endothelial cells were seeded onto smCS films as well as on tissue culture plates (TCPS, Corning, USA) or glasses (20x20 mm, ForLab, Carlo Erba, Italy,) at a density of 1–2.5 104 cells cm-2 in 24-well plates. After 1, 3 and 7 days, the monolayer was rinsed twice with phosphate buffer solution, PBS, and cells detached from the substrate by 0.02% trypsin in PBS. The number of adherent cells was then, counted with a Burkerhaemocytometer.

#### **2.6. Cell morphology**

For morphological characterization, endothelial cells cultured on smCS films were examined by contrast-phase microscopy. After 7 days, the cell monolayer adherent to the film was gently washed with PBS three times. Then, the film was fixed with 2.5% glutaraldehyde in PBS for 1 h at 4 °C. After thorough washing with PBS, the cells were dehydrated through graded alcohol series and positioned under the microscope (Zeiss AxioPhot, Germany) for observation and image recording (Zeiss AxioCam, Germany).

#### **2.7. Cytotoxicity test**

Endothelial cells were grown until confluence. The smCS films was cut in small pieces (0.5x0.5 mm) and placed in direct contact with the cell layer for 72 hours. Cells were detached and the resulting suspension was counted in a Burkerhaemocytometer after proper dilution.

Duplicate cell counts on each suspension from 3 culture wells were performed for each sub‐ strate investigated. Not less than 50 cells were scored for each counting. Counts from tripli‐ cate seeding differed by not more than 10% among replications throughout the experiments.

#### **2.8. Haemolysis assay**

Two positive controls, copper and deionised water, and a negative control, glass cover slip, were used in this study, SmCS films were dried and washed three times with PBS and then sterilized by soaking in 75% (v/v) ethanol for 15 minutes. Then, washed 5 times in sterile PBS and kept in the same buffer until use. Thereafter, the samples were put in vacutainers contain‐ ing sodium citrate (0.109 M, 3.2% w/v final concentration) (Greiner Bio-One International AG, Austria) in which 3.5 mL of healthy volunteers blood was finally collected. The substrates were incubated with blood at 37 °C, with gentle shaking twice every 30 minutes. After 3 hours, 1.5 mL of each vacutainer was centrifuged at 740xg for 10 minutes at room temperature. The ob‐ tained pellet was re-centrifuged at 3000g for 15 minutes at room temperature. The haemolyses was quantified on a ADVIA 2120 system (Siemens-Bayer, Germany) using a colorimetric assay.

#### **2.9. Coagulation assays**

Human whole blood (3.5 mL) from a healthy volunteer was collected and mixed with an aqueous solution containing sodium citrate, then the human whole blood was centrifuged at 1500g for 15 min at room temperature to separate the blood corpuscles, and the resulting PPP was used to study the coagulating ability of the CS film. All tests were performed on *IL Coagulation and ELECTRATM* system (Instrumentation Laboratories, USA). The level of Pro‐ thrombin Time (PT), activated Partial Thromboplastin Time (aPTT) and Thrombin Time (TT) were determined by using three different kits (Instrumention Laboratory USA): HemosIL TM RecombiPlasTin 2G is a high sensitivity thromboplastin reagent based on recombinant hu‐ man tissue factor (RTF) for the quantitative determination in human citrate plasma of Pro‐ thrombin Time (PT); HemosIL TM SynthASil is a high synthetic phospholipids reagent for the *in vitro* determination of APTT (Activated Partial Thromboplastin Time). After incubation at 37 °C for an optimized period of time, calcium is added to trigger the coagulation process and the time required for clot formation is determined; HemosIL TM Thrombin Time was used for the determination of TT in human citrated plasma.

#### **2.10. Erythrocytes adhesion assay**

in complete medium (D-MEM containing antibiotics and 10% foetal calf serum) supplement‐ ed with 50 μg mL-1 of endothelial cell growth factor (Sigma-Aldrich, USA) and kept in a in‐ cubator at 37 °C in a water-saturated atmosphere with 5% CO2. Endothelial cells were seeded onto smCS films as well as on tissue culture plates (TCPS, Corning, USA) or glasses

After 1, 3 and 7 days, the monolayer was rinsed twice with phosphate buffer solution, PBS, and cells detached from the substrate by 0.02% trypsin in PBS. The number of adherent cells

For morphological characterization, endothelial cells cultured on smCS films were examined by contrast-phase microscopy. After 7 days, the cell monolayer adherent to the film was gently washed with PBS three times. Then, the film was fixed with 2.5% glutaraldehyde in PBS for 1 h at 4 °C. After thorough washing with PBS, the cells were dehydrated through graded alcohol series and positioned under the microscope (Zeiss AxioPhot, Germany) for

Endothelial cells were grown until confluence. The smCS films was cut in small pieces (0.5x0.5 mm) and placed in direct contact with the cell layer for 72 hours. Cells were detached and the

Duplicate cell counts on each suspension from 3 culture wells were performed for each sub‐ strate investigated. Not less than 50 cells were scored for each counting. Counts from tripli‐ cate seeding differed by not more than 10% among replications throughout the experiments.

Two positive controls, copper and deionised water, and a negative control, glass cover slip, were used in this study, SmCS films were dried and washed three times with PBS and then sterilized by soaking in 75% (v/v) ethanol for 15 minutes. Then, washed 5 times in sterile PBS and kept in the same buffer until use. Thereafter, the samples were put in vacutainers contain‐ ing sodium citrate (0.109 M, 3.2% w/v final concentration) (Greiner Bio-One International AG, Austria) in which 3.5 mL of healthy volunteers blood was finally collected. The substrates were incubated with blood at 37 °C, with gentle shaking twice every 30 minutes. After 3 hours, 1.5 mL of each vacutainer was centrifuged at 740xg for 10 minutes at room temperature. The ob‐ tained pellet was re-centrifuged at 3000g for 15 minutes at room temperature. The haemolyses was quantified on a ADVIA 2120 system (Siemens-Bayer, Germany) using a colorimetric assay.

Human whole blood (3.5 mL) from a healthy volunteer was collected and mixed with an aqueous solution containing sodium citrate, then the human whole blood was centrifuged at 1500g for 15 min at room temperature to separate the blood corpuscles, and the resulting

resulting suspension was counted in a Burkerhaemocytometer after proper dilution.

cells cm-2 in 24-well plates.

(20x20 mm, ForLab, Carlo Erba, Italy,) at a density of 1–2.5 104

observation and image recording (Zeiss AxioCam, Germany).

was then, counted with a Burkerhaemocytometer.

160 Advances in Biomaterials Science and Biomedical Applications

**2.6. Cell morphology**

**2.7. Cytotoxicity test**

**2.8. Haemolysis assay**

**2.9. Coagulation assays**

SmCS films (10x10 mm) were equilibrated in PBS for 1 hour at 37°C. A washed-erythrocytes stock suspension containing 3 105 mL-1 was poured on plastic and smCS film surfaces and incubated for 30 minutes. The incubation volume was kept low (100 μL) to (a) minimize the floating population of erythrocytes and (b) maintain the total erythrocytes count at a level such as to prevent saturation-levels of adhesion and (c) to prevent other still suspended er‐ ythrocytes from contacting the surface. After that, the specimens were rinsed with PBS, fixed with glutaraldheyde and detached from surface with 1% sodium dodecyl sulphate, SDS. Ten microliters of recovered erythrocytes suspension were counted with a Burkerhae‐ mocytometer.

#### **2.11. Platelet adhesion assay**

SmCS films, plastic and cover slips glass were sterilized with 75% (v/v) ethanol solution. Then air dried under a laminar-flow hood and rehydrated with 1 mL of sterile PBS for 1 hour. The surfaces were overlaid with 300 μL PRP at 37 °C for 2 hours. Then, the films were washed three times in PBS with mild shaking to remove non- or poorly-adherent platelets. After that, the specimens were rinsed with PBS, fixed with glutaraldheyde and cells detach‐ ed with 1% SDS. Ten microliters of recovered platelets suspension were counted with a Bur‐ kerhaemocytometer.
