**2. Materials and methods**

#### **2.1. Preparation of polymer samples**

Preparation of PU/HPC samples was performed according to Fig. 1 as previously reported (Macocinschi et. al. 2009; Vlad et. al, 2010).

**Figure 1.** Scheme of chemical structure and synthesis way of PUs/HPC

Briefly, isocyanate terminated urethane prepolymers were first synthesized by the polyaddition reactions between 4',4'-diphenylmethane diisocyanate (MDI) and macrodiols in N,N-dimethylformamide (DMF) as solvent. Poly(ethylene adipate)diol (PEGA, Mn = 2000 g/mol), polytetrahydrofuran (PTHF, Mn = 2000 g/mol) or poly(propylene)glycol (PPG, Mn = 2000 g/mol) were used as macrodiols. The urethane prepolymers were treated in a subsequent step with ethylene glycol (EG) as chain extender. Finally, HPC (average weight molecular weight Mw = 95 000 g/mol) was added to PU solutions to obtain the following compositions for all PU/HPC samples: macrodiol/MDI/EG/HPC = 52.24 /36.57/7.27/3.92 (weight ratios). As the molar ratio between isocyanate groups in MDI and the sum of hydroxylic groups in macrodiol and EG was 1.02, the excess of isocyanate groups linked to PU prepolymers were available to bind a part of HPC chains. Membranes with about 1 mm thickness were prepared by pouring PU/HPC DMF solutions in distilled water, at 40 oC. The formed films were then dried under vacuum for several days and kept in distilled water for solvent removing.

To half of PUs with PEGA macrodiol in the soft segment no HPC was added to obtain PU-PEGA reference sample. HPC containing samples based on PEGA, PTHF and PPG macrodiols were codified as PU-PEGA/HPC; PU-PTHF/HPC and PU-PPG/HPC, respectively**.** 

#### **2.2. ζ potential determination**

ζ potential of the PU membranes was measured by streaming potential method using a commercial electrokinetic analyzer SurPASS, (Anton Paar GmbH, Graz, Austria). For each sample, ζ potential has been measured in 0.1 M NaCl solution at physiological 7.4 pH value, a 300 mbar electrolyte pressure and a 80 ml/min flow rate. For statistical reasons, four streaming potentials were measured. The mean value of these data was used for potential calculation by Fairbrother–Mastin equation, considering also the effect of surface conductivity (Luxbacher, 2006)

#### **2.3. Wettability**

202 Polyurethane

Valenta & Auner, 2004). In previous studies we found that when added to PU structure, HPC improves hydrophilicity and mechanical properties of PUs by increasing the elasticity

Considering the reviewed concept of biocompatibility as "the ability to exist in contact with tissues of the human body without causing an unacceptable degree of harm to the body" (Williams, 2008), our interdisciplinary work was focused on the synthesis of PU-based materials with improved ability to long-time functional integration. PU/HPC membranes were prepared by blending method. HPC was chosen due to its physical-chemical properties, its demonstrated biocompatibility and accessibility. The aim of the chapter is to highlight the most important criteria, able to predict the behaviour of material-tissue interfaces and the long-term material-tissue integration, in order to select most suitable compositions and morphologies for specific medical application. Thus, surface zeta (ζ) potential, wettability (as contact angle measurement and water uptake), pH modification after long time hydration and autoclaving, protein adsorption at protein physiological concentration and some relevant elements of bulk and surface morphology are treated as screening criteria for suitable membrane choice in the first part of the chapter. Biological performance evaluation, such as oxidative stress action, thrombogenicity and *in vivo*

Preparation of PU/HPC samples was performed according to Fig. 1 as previously reported

of the resulted materials (Macocinschi et. al., 2009).

behaviour of PU/HPC membranes are further discussed.

**Figure 1.** Scheme of chemical structure and synthesis way of PUs/HPC

**2. Materials and methods** 

**2.1. Preparation of polymer samples** 

(Macocinschi et. al. 2009; Vlad et. al, 2010).

*Wettability* of the PU membranes was determined by measuring the surface contact angle and water uptake. For surface contact angle, uniform drops of the tested liquid (doubledistilled water) with a volume of 2 μl were deposited on the film surface and the contact angles were measured after 30 s, using a video-based optical contact angle measuring device equipped with a Hamilton syringe in a temperature-controlled environmental chamber. All measurements were performed at room temperature of 25 C. Repeated measurements of a given contact angle were all within the range of ± 3 degrees. *Water uptake* was calculated as the ratio between fully hydrated and dried sample weights.

#### **2.4. Material extraction in a simulated biological microenvironment**

Material extraction in a simulated biological microenvironment was done for long period of time (over 2 months) in Hank's Balanced Salt Solution (HBSS) without Ca2+ and Mg2+, with

glucose, and phenol red as pH indicator. For extraction experiments, 0.2 g of each membrane, cut in very small pieces (see Fig. 2), were incubated in 2 ml of HBSS solution at 37oC. pH variation was monitored daily, based on phenol red indicator colour and measured after 1, 2, 3, 30 and 60 days of incubation using Mettler Toledo SevenGo SG2ELK pH-meter.

Biocompatibility and Biological Performance

of the Improved Polyurethane Membranes for Medical Applications 205

A blank- Astandard (2)

TASmMol/L=FactorΔAblank-ΔAsample (3)

spectrophotometer mentioned in the previous section. By adding blood plasma containing antioxidants a suppression of this colour to a degree which is proportional to their concentration is observed. Control serum ("standard" provided by the determination kit) was used for data validation. TAS values were calculated based on the measured absorbance in the standard, blood plasma sample and blank (buffer provided by the kit) before and after H2O2 adding. The absorbance differences (ΔA) between measurement before and after H2O2 adding for standard, sample or blank solutions were used for

concentrationof standard Factor

Haemocompatibility of membrane surface was evaluated by haemolysis and coagulation tests. All tests were performed on well swollen PU samples in PBS. *Haemolysis* was determined using 0.25 ml of blood (human blood from healthy voluntary donors, collected on 3.8 % sodium citrate solution as anticoagulant in 9:1 v/v ratio) that was incubated with 1 cm2 surface area PU samples for 30 min at 37 oC. Haemoglobin released from lysed erythrocytes was measured by spectrophotometric method at λ = 545. *Prothrombin time* was measured after 1 hour incubation of polymer sample in blood plasma. Standard laboratory method was applied using PT kit (Biodevice, Italy) and ACL 100 coagulometer. Blood

*Platelet adhesion* on material surface was determined based on number of platelet counted in 0.1 ml platelet rich blood plasma (PRP), before and after membrane (0.5 cm x 0.5 cm) incubation for 1 hour at 37 oC. PRP was obtained by blood centrifugation at 400 G for 20 min. Improved Neubauer haemocytometer was used for platelet counting. *Clot weight test* was performed by adding 0.2 ml of human blood upon well swollen samples with 1cm2 surface area. The thrombus formation was started by adding 0.05 ml CaCl2 solution (0.025 mol/l). Each formed thrombus was weighed and compared with control. Collagen film was used as positive pro-coagulant control and normal blood plasma without polymer sample as

Subcutaneous implantation experiment was performed on Wistar 200 g weight male rats. Testing protocol was designed according to ISO 10993-2 (Animal Welfare Requirements) and the guidelines of Council for International Organizations of Medical Sciences (CIOMS). The pieces of autoclaved purified or unpurified membranes (0.5 x 0.5 cm size) were implanted under both sites (right and left) of dorso-lateral skin. Material purification was performed by immersion in sterile distilled water for 1 week and equilibration in

calculation of TAS concentration according to relations 2 and 3:

plasma was obtained by blood centrifugation at 1000 G for 10 min.

**2.8. Haemocompatibility testing** 

negative control.

**2.9.** *In vivo* **biocompatibility** 

#### **2.5. Scanning Electron Microscopy (SEM)**

SEM analysis of PU/HPC membrane cross-sections was performed using a VEGA TESCAN microscope, in high vacuum mode, at an acceleration voltage of 30 kV.

#### **2.6. Protein adsorption**

Amount of protein adsorption on membrane surfaces was measured in three different conditions: (a) on individual protein solutions of fibrinogen (FB) at 3 mg/ml (95% clotable from Sigma-Aldrich) and serum albumin (SA) at 45 mg/ml (bovine SA (BSA) from Sigma-Aldrich); (b) FB and BSA mixed solutions of physiological concentrations (3 mg/ml for BSA and 45 mg/ml for FB); (c) complex protein conditions (platelet poor blood plasma (PPP)). Prior adsorption experiment, the PU/HPC films were brought to equilibrium with phosphate buffer saline (PBS) up to reaching maximum hydration, for about 72 h. Briefly, PU/HPC hydrated membranes with 0.5 cm x 0.5 cm surface area were covered with 0.25 ml of one of the protein solutions or with blood plasma and kept at 37 oC for 30 min. FB and BSA concentration in incubated medium was determined before and after incubation. A turbidimetric method based on the formation of an insoluble complex with Na2SO4 was used for FB determination. The method based on antigen–antibody reaction was performed for SA measuring, using a Dialab kit, Austria. FB and SA reaction products were assessed on a Piccos 05 UV–VIS spectrophotometer at λ = 530 nm for FB and λ = 340 nm for SA. The adsorbed amount of proteins was calculated with the following relation:

$$\text{Adsorbed protein} \left(\text{mg/cm}^2\right) = \frac{\left(\text{Co} - \text{Ce}\right) \cdot V}{S} \tag{1}$$

where *Co* and *Ce* are the initial and post-incubation concentrations of protein solution (mg/ml), *V* is the incubated volume of the protein solution (ml) and *S* is the surface of the incubated PU/HPC sample

#### **2.7. Total Antioxidant Status (TAS)**

TAS was measured in blood plasma obtained by human blood centrifugation at 1000 G for 20 min. PU samples were incubated in blood plasma for 1, 2 and 3 days at 37 oC and mild orbital shacking. The TAS measurement was made by standard protocol provided by Randox TAS kit. Thus, 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] (ABTS)® was incubated with a peroxidase (metmyoglobine) and H2O2 to produce the ABTS®+ radical cations having a stable blue-green colour that was measured at 600 nm on a spectrophotometer mentioned in the previous section. By adding blood plasma containing antioxidants a suppression of this colour to a degree which is proportional to their concentration is observed. Control serum ("standard" provided by the determination kit) was used for data validation. TAS values were calculated based on the measured absorbance in the standard, blood plasma sample and blank (buffer provided by the kit) before and after H2O2 adding. The absorbance differences (ΔA) between measurement before and after H2O2 adding for standard, sample or blank solutions were used for calculation of TAS concentration according to relations 2 and 3:

$$\text{Factor} = \frac{\text{concentration of standard}}{\text{AA blank} \cdot \text{AA standard}} \tag{2}$$

$$\text{TASmMol/L} = \text{Factor} \cdot \Delta \text{A blank} \cdot \Delta \text{A sample} \tag{3}$$

#### **2.8. Haemocompatibility testing**

204 Polyurethane

pH-meter.

**2.6. Protein adsorption** 

incubated PU/HPC sample

**2.7. Total Antioxidant Status (TAS)** 

**2.5. Scanning Electron Microscopy (SEM)** 

glucose, and phenol red as pH indicator. For extraction experiments, 0.2 g of each membrane, cut in very small pieces (see Fig. 2), were incubated in 2 ml of HBSS solution at 37oC. pH variation was monitored daily, based on phenol red indicator colour and measured after 1, 2, 3, 30 and 60 days of incubation using Mettler Toledo SevenGo SG2ELK

SEM analysis of PU/HPC membrane cross-sections was performed using a VEGA TESCAN

Amount of protein adsorption on membrane surfaces was measured in three different conditions: (a) on individual protein solutions of fibrinogen (FB) at 3 mg/ml (95% clotable from Sigma-Aldrich) and serum albumin (SA) at 45 mg/ml (bovine SA (BSA) from Sigma-Aldrich); (b) FB and BSA mixed solutions of physiological concentrations (3 mg/ml for BSA and 45 mg/ml for FB); (c) complex protein conditions (platelet poor blood plasma (PPP)). Prior adsorption experiment, the PU/HPC films were brought to equilibrium with phosphate buffer saline (PBS) up to reaching maximum hydration, for about 72 h. Briefly, PU/HPC hydrated membranes with 0.5 cm x 0.5 cm surface area were covered with 0.25 ml of one of the protein solutions or with blood plasma and kept at 37 oC for 30 min. FB and BSA concentration in incubated medium was determined before and after incubation. A turbidimetric method based on the formation of an insoluble complex with Na2SO4 was used for FB determination. The method based on antigen–antibody reaction was performed for SA measuring, using a Dialab kit, Austria. FB and SA reaction products were assessed on a Piccos 05 UV–VIS spectrophotometer at λ = 530 nm for FB and λ = 340 nm for SA. The

<sup>2</sup> ( ) Adsorbedproetin(mg/cm ) *Co Ce V*

where *Co* and *Ce* are the initial and post-incubation concentrations of protein solution (mg/ml), *V* is the incubated volume of the protein solution (ml) and *S* is the surface of the

TAS was measured in blood plasma obtained by human blood centrifugation at 1000 G for 20 min. PU samples were incubated in blood plasma for 1, 2 and 3 days at 37 oC and mild orbital shacking. The TAS measurement was made by standard protocol provided by Randox TAS kit. Thus, 2,2'-azino-di-[3-ethylbenzthiazoline sulphonate] (ABTS)® was incubated with a peroxidase (metmyoglobine) and H2O2 to produce the ABTS®+ radical cations having a stable blue-green colour that was measured at 600 nm on a

*S*

(1)

microscope, in high vacuum mode, at an acceleration voltage of 30 kV.

adsorbed amount of proteins was calculated with the following relation:

Haemocompatibility of membrane surface was evaluated by haemolysis and coagulation tests. All tests were performed on well swollen PU samples in PBS. *Haemolysis* was determined using 0.25 ml of blood (human blood from healthy voluntary donors, collected on 3.8 % sodium citrate solution as anticoagulant in 9:1 v/v ratio) that was incubated with 1 cm2 surface area PU samples for 30 min at 37 oC. Haemoglobin released from lysed erythrocytes was measured by spectrophotometric method at λ = 545. *Prothrombin time* was measured after 1 hour incubation of polymer sample in blood plasma. Standard laboratory method was applied using PT kit (Biodevice, Italy) and ACL 100 coagulometer. Blood plasma was obtained by blood centrifugation at 1000 G for 10 min.

*Platelet adhesion* on material surface was determined based on number of platelet counted in 0.1 ml platelet rich blood plasma (PRP), before and after membrane (0.5 cm x 0.5 cm) incubation for 1 hour at 37 oC. PRP was obtained by blood centrifugation at 400 G for 20 min. Improved Neubauer haemocytometer was used for platelet counting. *Clot weight test* was performed by adding 0.2 ml of human blood upon well swollen samples with 1cm2 surface area. The thrombus formation was started by adding 0.05 ml CaCl2 solution (0.025 mol/l). Each formed thrombus was weighed and compared with control. Collagen film was used as positive pro-coagulant control and normal blood plasma without polymer sample as negative control.

#### **2.9.** *In vivo* **biocompatibility**

Subcutaneous implantation experiment was performed on Wistar 200 g weight male rats. Testing protocol was designed according to ISO 10993-2 (Animal Welfare Requirements) and the guidelines of Council for International Organizations of Medical Sciences (CIOMS). The pieces of autoclaved purified or unpurified membranes (0.5 x 0.5 cm size) were implanted under both sites (right and left) of dorso-lateral skin. Material purification was performed by immersion in sterile distilled water for 1 week and equilibration in

physiological salted sterile solution for 24 hours before subcutaneous implantation. All surgical procedures were done under thiopental anaesthesia, using a dosage of 35 mg/kg body weight. Lots of six animals for each material were taken in each experiment. The period of 10 or 30 days was chosen for material examination. Explanted samples together with surrounding tissue were fixed in 10% formaldehyde solution embedded in paraffin wax, sliced in 15 μm pieces and stained using Hematoxylin – Eosin (HE) method for cell examination and Masson's trichrome for collagen fibres.

Biocompatibility and Biological Performance

of the Improved Polyurethane Membranes for Medical Applications 207

*First immersion Second immersion* **WU (%) ζ (mV)** 

**Contact angle**

θadv(o) θrec(o) H(%) θadv(o) θrec(o) H(%) PU-PEGA 85.3±1.1 54.3±0.6 36.3 51.0±0.5 54.1±0.6 5.6 141±10 - 4.31 PU-PEGA/HPC 84.8±1.1 44.2±0.5 47.9 52.6±0.5 43.7±0.5 16.9 140±4 + 3.14 PU-PTHF/HPC 77.4±1.1 42.9±0.5 44.5 31.6±0.4 42.3±0.4 25.2 167±3 + 0.78 PU-PPG/HPC 85.6±1.1 44.8±0.5 47.7 60.3±0.6 44.1±0.5 27.0 92±6 + 4.85 **Table 1.** Dynamic contact angle values (θ) in contact with water, hysteresis (H) resulted from advanced (*adv*) and receded (*rec*) contact angles, water uptake (WU) (Macoconschi et. al., 2009) and ζ potential of

As one can see from Table 1, PU-PEGA has a slightly negative ζ potential, probably due to the presence of carboxylic groups resulted by the hydrolysis of residual isocyanate groups during membrane precipitation in water. After blending with HPC, the residual isocyanated groups linked to PU prepolymer are reacted with the hydroxyl groups of HPC and all PU/HPC membranes showed a slightly positive surface. The most hydrophilic sample (PU-PTHF/HPC) exhibited the most neutral ζ potential. This observation is in accordance to other data that report dependence of surface charge on water swelling capacity (Aranberri-

The material biocompatibility can be appreciated through its effects on the physico-chemical properties of the physiological environment, especially on the pH. Thus, pH modification of HBSS buffer solutions after unsterilized and sterilized membranes incubation was measured. The results are shown in Figs 2 and 3 (1, PU-PEGA; 2- PU-PEGA/HPC; 3, PU-

**Figure 2.** pH variation of HBSS buffer in which unsterilized membranes were incubated: A, PU-PEGA;

**Material samples** 

the PU samples

Askargorta et. al., 2003).

*3.1.2. Extraction microenvironment* 

PTHF/HPC; 4, PU-PPG/HPC).

B, PUs/HPC; C, pH variation curves
