**5. Results**

**4. Platelet morphology**

162 Advances in Biomaterials Science and Biomedical Applications

bridge Scientific Instruments, UK).

**4.2. Complement activation assay**

**4.3. Statistical analysis**

tained values were expressed as mean of two measurements.

of complement activity (10% of plasma + 90% saline).

**4.1. Platelet aggregation**

The platelets-coated testing surfaces were fixed with freshly prepared 2.5% glutaraldehyde for 20 minutes. After washing with PBS, the samples were dehydrated in a graded-ethanol series (50, 70, 90, and 100% v/v) for 15 minutes each and allowed to dry at room tempera‐ ture. The platelet-attached surfaces were carbon sputter coated under vacuum to a thickness of 100–200 Å and examined at 10 kV using a Cambridge StereoScan 200 microscope (Cam‐

The blood samples were collected in tubes containing PPACK (D-phenylalanyl-L-prolyl-Larginine chloromethyl ketone) as anticoagulant. Platelet aggregation was measured by means of light transmission aggregometry using Born's turbidimetric procedure and the PPACK-4 Platelet Aggregation Chromogenic Kinetic System (Helena Laboratories, USA). Briefly, 250 μL of PRP were incubated with specimen surfaces for 10 (baseline) and 60 mi‐ nutes. Thereafter,the PRP were placed in a cuvette containing a metal stir bar in the absence or in the presence (positive control) of the pro-aggregation agent, adenosine diphosphate (ADP) 20 μM. Upon the addition of ADP the platelets started to aggregate thus increasing light transmission through the sample. The degree of platelet aggregation was expressed as the maximum percentage change in light transmission from PPP used as baseline. The ob‐

The test, based on Complement Reagents Kit (Siemens Healthcare Diagnostic, Germany) was performed on BCT Siemens coagulometer (Siemens, Germany). The test focused on the ability of the complement system to lyse a standard suspension of sheep erythrocytes, sensi‐ tized with a rabbit anti-serum against sheep erythrocytes. Briefly, 1 mL of fresh blood sam‐ ples previously incubated for 1 hour with different substrates were incubated with sensitized erythrocytes to investigate the complement activation. Diminished levels of indi‐ vidual components (e.g. due to prior activation by a foreign surface) result in a prolongation of the time taken for lyses. The time necessary for the lyses of a defined amount of erythro‐ cytes is used as basis for determining the complement activity [14,15]. The results were eval‐ uated using a reference curve prepared by a serial dilution of standard plasma with isotonic saline to give 100% of complement activity, 75% (75% of plasma + 25% saline) down to 10%

Data were expressed as means ± standard deviation (SD). Where not differently stated, measurements were conducted at least in triplicate. Chi-square test or Student's t-test on un‐ paired data was used to assess the statistical significance of the difference between the re‐ sults obtained from the tested specimens (Kaleida-Graph, Synergy Software, USA).

Statistical significance was assumed at a confidence level of 95% (p < 0.05).

#### **5.1. Physico-chemical characterisation**

As already stated, the sugar added to the chitosan solution during the preparation of the smCS film was not retained in the final structure of the film.This assumption was mainly based on FT-IR spectra analysis for the identification of the absorption bands relevant to vi‐ bration of functional groups of chitosan [13]. The addition of phosphate salts and D-(+) raffi‐ nose to the chitosan solution used for film preparation led to non dramatic modifications in the IR spectrum of chitosan. The observation of the 1700-1500 cm-1 region evidenced that the amide I band (C=O in amide group) wavenumber was lower than the value for chitosan powder (1664 cm-1) for all the prepared films and particularly for those prepared from a sol‐ ution that did not contain the sugar [13]. This was interpreted as the result of a lower mobili‐ ty of the C=O group in the film due to its involvement in the week bound formation in the solid structure. The incorporation of phosphate salts and significant amount of sugar in the chitosan solution used for film preparation reduced this effect. On the other hand, the amino group band of films prepared from a solution that did not contain the sugar was at a lower wavenumber (1588 cm-1) than from chitosan powder (1592 cm-1), while it was practically un‐ changed in film prepared from the sugar containing solution (1590 cm-1).

D-(+)raffinose FT-IR spectrum evidenced characteristics bands at 2936 and 1649 cm-1. Inter‐ estingly, no trace of this bands was found in the FT-IR spectra of the chitosan film prepared from solutions containing D-(+)raffinose. Similarly, no trace of the characteristic series of peaks between 2994 and 2914 cm-1 of the sucrose powder was found in the spectrum of the film prepared from a solution containing a high amount of sucrose [13].

These observations allowed to conclude that the excipients added to chitosan in the film forming solutions though not retained in the solid film, interact or interfere with chitosan chains during the film formation likely acting as viscosity modifiers during the solidifica‐ tion/gelation process.

#### **5.2. Wettability**

Contact angle measurements were performed by using serum droplets on plastic surface and on smCS film. As expected, plastic showed the least wettable surfaces with significantly higher contact angle (50° ± 6.3) compared to smCS film (15° ± 0.1) (Chi Square P< 0.001), thus confirming the high hydrophilicity of smCS [13].

The hydrophilicity of the sm CS film was also investigated by measuring the swelling index in water at the equilibrium according to the following equation:

$$S\_{\rm av} = \frac{\mathcal{W}\_s - \mathcal{W}\_d}{\mathcal{W}\_s} \text{x100} \tag{1}$$

where Ws and Wd represent the weight of the fully hydrated and the dry film respectively. The smCS film afforded a degree of swelling at the equilibrium more than 3 order of magni‐ tude (1285%) higher than that of the dry film. These data confirmed the very high hydrophi‐ licity of the films obtained by adding a sugar to the solution used for the film preparation.

#### **5.3. Roughness (AFM measurements)**

The AFM analysis (Figure 1) revealed that the plastic specimen exhibited rather low surface roughness (average= 28 nm) in contrast to smCS film that showed a roughness approximate‐ ly 1.7-fold higher, around 50 nm. It is interesting to note the almost regular appearance of groove and pits in smCS compared with plastic surface.

**Figure 1.** AFM 3D image of (A) standard colture plastic dish (plastic) and (B) smCS film surfaces.

#### **5.4. Adhesion and proliferation assay of endothelial cells**

As shown in Figures 2 endothelial cells attached (A), extended and proliferated (B) very well on all surfaces tested. Cell attachment (panel A, C) and proliferation (panel B) on smCS films were comparable to control cells grown on standard tissue culture surface (plastic). Con‐ trast-phase microscopy showed that cells were well attached to the different surfaces and closely packed maintaining their original shapes. Moreover, endothelial cells did not evi‐ dence any morphological indication of cell death 72 hours after seeding (panel D). The counts of cells showed little variation for the three surfaces used. In the case of plastic sur‐ face (control) the growth of HUVEC reached the values of 21284 ± 650 cm-2, while in the case of smCS reached a lower value of 19805 ± 305 cm-2 similarly to that obtained on glass surface (19543 ± 1050 cm-2).

#### **5.5. In vitro cytotoxicity**

Data relevant to cell growth in the presence of small pieces of smCS film or latex (positive control) are reported in Table 1.

where Ws and Wd represent the weight of the fully hydrated and the dry film respectively. The smCS film afforded a degree of swelling at the equilibrium more than 3 order of magni‐ tude (1285%) higher than that of the dry film. These data confirmed the very high hydrophi‐ licity of the films obtained by adding a sugar to the solution used for the film preparation.

The AFM analysis (Figure 1) revealed that the plastic specimen exhibited rather low surface roughness (average= 28 nm) in contrast to smCS film that showed a roughness approximate‐ ly 1.7-fold higher, around 50 nm. It is interesting to note the almost regular appearance of

**5.3. Roughness (AFM measurements)**

164 Advances in Biomaterials Science and Biomedical Applications

groove and pits in smCS compared with plastic surface.

**Figure 1.** AFM 3D image of (A) standard colture plastic dish (plastic) and (B) smCS film surfaces.

As shown in Figures 2 endothelial cells attached (A), extended and proliferated (B) very well on all surfaces tested. Cell attachment (panel A, C) and proliferation (panel B) on smCS films were comparable to control cells grown on standard tissue culture surface (plastic). Con‐ trast-phase microscopy showed that cells were well attached to the different surfaces and closely packed maintaining their original shapes. Moreover, endothelial cells did not evi‐ dence any morphological indication of cell death 72 hours after seeding (panel D). The counts of cells showed little variation for the three surfaces used. In the case of plastic sur‐ face (control) the growth of HUVEC reached the values of 21284 ± 650 cm-2, while in the case of smCS reached a lower value of 19805 ± 305 cm-2 similarly to that obtained on glass surface

Data relevant to cell growth in the presence of small pieces of smCS film or latex (positive

**5.4. Adhesion and proliferation assay of endothelial cells**

(19543 ± 1050 cm-2).

**5.5. In vitro cytotoxicity**

control) are reported in Table 1.

**Figure 2.** A) Percentage of cells adhered after 24 hoursand (B) proliferation assay of endothelial cells on the different surfaces tested. Pictures taken at the optical microscope, in phase contrast (40x), showing the morphology of endo‐ thelial cells 8 hours (C) and 72 hours (D) after seeding on smCS film.


**Table 1.** Number of endothelial cells attached to the different substrates in the presence of latex or smCS film fragments.

The initial plating corresponds to the number of cells attached to the substrate 6 hours after their inoculation into the well. The measured plating efficiency was around 95%. When smCS fragments were present in the colture medium, a progressive increase of cell numbers was observed, while in the presence of latex a progressive detachment was noticed with al‐ most all plated cells detached from the substratum after 72 hours.

These results indicate that smCS film were not cytotoxic while latex, as expected, was found to markedly affect endothelial cell survival.

#### **5.6. Haemolysis assay**

Haemolysis of red blood cells was used to evaluate the membrane damaging potential of the surface of the smCS film. Two positive controls, distilled water and Copper, and one nega‐ tive control, glass, were used.


**Table 2.** Percentage of haemolysis measured on different substrates. The tested surfaces were incubated with whole blood for 1 hour. Distilled water was used as positive control.

As shown in Table 2, distilled water resulted in about 100% haemolysis, while Copper led to 7% haemolysis. Glass and smCS film caused negligible haemolysis (within the experimental error) indicating very low membrane damaging properties of smCS material.

#### **5.7. Blood coagulation assay**

The effects of the biomaterial on coagulation process were tested by means of the (aPTT), the (PT) and the (TT) selected as reliable measurements of the capacity of blood to coagulate through the intrinsic, extrinsic and common coagulation mechanisms, respectively. As shown in Figure 3 the values obtained for PT, TT and aPTT were similar to those observed for human plasma, thus indicating that all materials tested, including smCS, did not affect coagulation pathways.


**Table 3.** Erythrocyte lyses time determined by plastic and glass surfaces in comparison with smCS film.

#### **5.8. Complement activation assay**

The erythrocyte lyses time observed and reported in Table 3 shows no significant difference among the material studied and the control. The data presented demonstrate that smCS is a nonreactive biomaterial that does not directly activate complement.

**Figure 3.** Effect of the different surfaces on coagulation time tested by means of the (aPTT), the (PT) and the (TT).

#### **5.9. Erythrocytes and platelets adhesion assay**

**5.6. Haemolysis assay**

tive control, glass, were used.

166 Advances in Biomaterials Science and Biomedical Applications

**5.7. Blood coagulation assay**

coagulation pathways.

**5.8. Complement activation assay**

blood for 1 hour. Distilled water was used as positive control.

Haemolysis of red blood cells was used to evaluate the membrane damaging potential of the surface of the smCS film. Two positive controls, distilled water and Copper, and one nega‐

**Substrate Haemolysis (%)**

**Table 2.** Percentage of haemolysis measured on different substrates. The tested surfaces were incubated with whole

As shown in Table 2, distilled water resulted in about 100% haemolysis, while Copper led to 7% haemolysis. Glass and smCS film caused negligible haemolysis (within the experimental

The effects of the biomaterial on coagulation process were tested by means of the (aPTT), the (PT) and the (TT) selected as reliable measurements of the capacity of blood to coagulate through the intrinsic, extrinsic and common coagulation mechanisms, respectively. As shown in Figure 3 the values obtained for PT, TT and aPTT were similar to those observed for human plasma, thus indicating that all materials tested, including smCS, did not affect

> Plasma (*control*) 35.4 Glass 38.2 Plastic 35.7 SmCS film 36.3

The erythrocyte lyses time observed and reported in Table 3 shows no significant difference among the material studied and the control. The data presented demonstrate that smCS is a

**Table 3.** Erythrocyte lyses time determined by plastic and glass surfaces in comparison with smCS film.

nonreactive biomaterial that does not directly activate complement.

**Substrates Erythrocyte lyses time (seconds)**

error) indicating very low membrane damaging properties of smCS material.

Distilled Water 97 (± 5) Copper 7 (± 2) Plastic 5 (± 1) Glass 2 (± 1) smCS 1 (±3)

> In Table 4 the number of cells detached with SDS from the different surfaces after adhesion test is reported. The smCS film presented a lower overall erythrocyte and platelet adhesion in comparison to plastic surface.


**Table 4.** Numbers of erythrocytes and platelets adhered to the studied surfaces

The test showed a high significant difference in the number of adhered erythrocytes on ma‐ terials studied (p<0.0001): the erythrocyte adhesion on smCS film was about 5 fold less than the adhesion on plastic surface. A similar behaviour was observed for platelets. In fact, the platelets recovered from plastic and glass surfaces ranged from 2 to 3 fold more than plate‐ lets recovered from smCS film surface.

#### **5.10. Platelet aggregation**

This test was performed in order to investigate the ability of plastic, smCS film and glass surfaces to induce platelet aggregation. The presence of ADP (adenosine-diphosphate, a pro-aggregation agent) determined a normal profile of platelets aggregation (range 90-95% after 5 minutes of incubation). Subsequently, the influence of materials on platelets aggrega‐ tion in the absence of ADP was studied. There were no differences between materials ob‐ 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‐ iation estimated with human plasma in the absence of ADP was around 10%.

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

#### **5.11. Platelet activation assay**

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

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.
