**3.1.** P**rimary screening criteria for the appropriate selection of PU/HPC membranes for medical usage**

Biocompatibility of PUs, seen in terms of specific application, is a result of a "bioappropriate" expression of surface and bulk properties achieved by synthesis and scaffold fabrication methods. Thus, surface ζ potential and surface wettability are important characteristics responsible for specific tissue-material interaction mechanisms, starting with protein adsorption that can be influenced in turn by specific physiological/pathological tissue environment.

### *3.1.1. Surface ζ potential and wettability*

Surface charge plays an important and active role in tissue-material interaction and must be considered in accordance with the targeted application. The importance of surface charge on cell adhesion, biofilm formation or thrombogenesis was demonstrated (Cai et. al., 2006; Colman & Schmaier, 1997; Kang et. al., 2006; Khorasani et. al., 2006). These phenomena are a consequence of adsorptive behavior of proteins on charged surface rather than the effect of electrostatic interactions with cells (Keselowsky et. al., 2003; Wilson et. al., 2005). Many data refer to the effect of surface charge on biological phenomena (Jelinek et. al., 2010; Kang et. al., 2006). However, there are not many data reporting surface charge and its clear relevance for biocompatibility of PU-based membranes. Moreover, it is difficult to estimate the electrokinetic properties of such surfaces, mainly due to the complexity of the chemical composition but also due to membrane variable porosity and swelling behavior that can influence surface charge values (Yaroshchuk & Luxbacher, 2010). ). Surface ζ potential of material is a property that reflects surface charge. Some reported data have shown that poly(ether-urethane)s exhibit a very negative (– 25 mV) ζ potential, while poly(esterurethane)s are less negative (- 12 mV). Contradictory data were published on the beneficial effect of positively (Khorasani et. al., 2006) or negatively charged surfaces (Sanders et. al., 2005) on cells attachment and proliferation.

Thus, this section is aimed to predict the influence of surface potential and wettability on the biocompatibility and biological performances of PU-based samples. Table 1 shows hydrophilic/hydrophobic properties and ζ potential of examined PU-based samples.

#### Biocompatibility and Biological Performance of the Improved Polyurethane Membranes for Medical Applications 207


**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 the PU samples

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-Askargorta et. al., 2003).

#### *3.1.2. Extraction microenvironment*

206 Polyurethane

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

**3.1.** P**rimary screening criteria for the appropriate selection of PU/HPC** 

Biocompatibility of PUs, seen in terms of specific application, is a result of a "bioappropriate" expression of surface and bulk properties achieved by synthesis and scaffold fabrication methods. Thus, surface ζ potential and surface wettability are important characteristics responsible for specific tissue-material interaction mechanisms, starting with protein adsorption that can be influenced in turn by specific physiological/pathological

Surface charge plays an important and active role in tissue-material interaction and must be considered in accordance with the targeted application. The importance of surface charge on cell adhesion, biofilm formation or thrombogenesis was demonstrated (Cai et. al., 2006; Colman & Schmaier, 1997; Kang et. al., 2006; Khorasani et. al., 2006). These phenomena are a consequence of adsorptive behavior of proteins on charged surface rather than the effect of electrostatic interactions with cells (Keselowsky et. al., 2003; Wilson et. al., 2005). Many data refer to the effect of surface charge on biological phenomena (Jelinek et. al., 2010; Kang et. al., 2006). However, there are not many data reporting surface charge and its clear relevance for biocompatibility of PU-based membranes. Moreover, it is difficult to estimate the electrokinetic properties of such surfaces, mainly due to the complexity of the chemical composition but also due to membrane variable porosity and swelling behavior that can influence surface charge values (Yaroshchuk & Luxbacher, 2010). ). Surface ζ potential of material is a property that reflects surface charge. Some reported data have shown that poly(ether-urethane)s exhibit a very negative (– 25 mV) ζ potential, while poly(esterurethane)s are less negative (- 12 mV). Contradictory data were published on the beneficial effect of positively (Khorasani et. al., 2006) or negatively charged surfaces (Sanders et. al.,

Thus, this section is aimed to predict the influence of surface potential and wettability on the biocompatibility and biological performances of PU-based samples. Table 1 shows

hydrophilic/hydrophobic properties and ζ potential of examined PU-based samples.

examination and Masson's trichrome for collagen fibres.

**3. Results and discussions** 

**membranes for medical usage** 

*3.1.1. Surface ζ potential and wettability* 

2005) on cells attachment and proliferation.

tissue environment.

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

**Figure 2.** pH variation of HBSS buffer in which unsterilized membranes were incubated: A, PU-PEGA; B, PUs/HPC; C, pH variation curves

Biocompatibility and Biological Performance

of the Improved Polyurethane Membranes for Medical Applications 209

PU-PEGA sample showed important bulk microporosity, with isolated pores, while PU-PEGA/HPC presented smaller but interconnected pores allowing water diffusion and the

Thus one can conclude that PU/HPC membranes are slightly positively charged and they possess interconnected porous morphology influencing the wettability and floatability. They also showed a less pronounced influence on the biological media as compared to the pure

There are many data concerning mechanisms of protein adsorption on different surfaces (Gray, 2004; Scott & Elbert, 2007; Van Tassel, 2006; Wilson et. al., 2005). It was clearly demonstrated that proteins have amphoteric properties, being able to adsorb on both negatively and positively charged surfaces (Michelsen et. al., 2000; Van Tassel, 2006). The amount of adsorbed proteins is depending on their isoelectric points as well as on surface chemistry and hydrophilicity (Keselowsky et. al., 2003; Wertz & Santore, 2001). Hydrophobic surfaces manly interact with hydrophobic protein core that leads to the modification of the protein physiological conformation and its functionality. Opposite to hydrophobicity, superficial water maintains native protein conformation and specific

Many authors have reported protein adsorption behaviour on different surfaces using simulated solutions and highly sensible methods in which very low protein concentrations are detected. Thus, the adsorption of albumin solutions of different concentrations on pure silica or on silica modified with NH2 and CH3 terminated self-assembled monolayers (SAMs) (Noinville & Revault, 2006) and on silica-titanium surfaces (Kurrat et. al. 1997) was studied. Other authors reported the competitive adsorption of fibrinogen on mica (Gettens et. al., 2005; Tsapikouni & Missirlis, 2007). Surface adsorption of SA, FB, fibronectin (FN), immunoglobulins (IGs) and lysozyme were investigated to evaluate the surface biocompatibility (Bernsmann et. al., 2010; Pompe et. al., 2006; Rezwan et. al., 2005), each class of these proteins providing specific surface properties for targeted application. Thus, FN adsorption is relevant for the prediction of cell adhesion, lysozyme – for enzymatic degradability predisposition, IGs - for immune-specific interactions, while SA and FB

In order to estimate protein adsorption (retention) capacity of materials at blood or tissues contact, simulated physiological environment, close to normal blood conditions is required. For example, Bajpai, 2005 followed SA adsorption capacity of biomaterials at SA bulk concentration from 1 to 6 mg/ml, while Alves et. al., 2010 used mix protein conditions, considering physiological value for each protein. The mix protein adsorption conditions are considered to better reflect the complex interactions that occur between different proteins

PU/HPC membranes were previously demonstrated to possess good mechanical properties (elongation at break for dried/hydrated PU-PEGA/HPC = 71/84; for PU-PTHF/HPC = 72/159

functionality (Keselowsky et. al., 2003; Noinville & Revault, 2006).

adsorption have haemocompatibility predictive value.

decrease of the floatability.

**3.2. Protein adsorption** 

PU membrane.

(Latour, 2008).

**Figure 3.** pH variation of HBSS buffer in which autoclaved PU/HPC membranes were incubated: A, 24 h of incubation; B, 72 h of incubation; C, pH variation curves

As one can see from Figs. 2 and 3, a long-period of incubation of unsterilized and sterilized (by autoclaving at 121 oC and 1 atm) PU/HPC membranes in simulated biological fluid did not meaningfully modify the physiological range pH value of the incubation environment, while a pronounced decrease of the environment pH was observed for pure PU-PEGA sample (Fig. 2 A). Thus, one can say that HPC gives an important contribution to hydrolytic stability of urethane and ester bonds of PU chains.

For autoclaved samples, the variation of pH values of the environment for poly(etherurethane)s remains in the range of the physiological value, while PU-PEGA/HPC membrane induced a higher decrease of pH (Fig. 3), a normal result owing the higher thermal degradability of poly(ester-urethane)s (Guelcher, 2008).

Another property that was changed by modifying PU membranes with HPC was the floatability (see Fig. 2B). As the surface wettability and water uptake for PU-PEGA sample is similar to HPC modified one (see Table 1), the reason of these different behavior could reside in different morphologies, as seen from SEM images of membrane cross-sections (Fig. 4).

**Figure 4.** SEM image of PU-PEGA (A) and PU-PEGA/HPC (B) membrane cross-sections

PU-PEGA sample showed important bulk microporosity, with isolated pores, while PU-PEGA/HPC presented smaller but interconnected pores allowing water diffusion and the decrease of the floatability.

Thus one can conclude that PU/HPC membranes are slightly positively charged and they possess interconnected porous morphology influencing the wettability and floatability. They also showed a less pronounced influence on the biological media as compared to the pure PU membrane.
