Cell membrane-mimicking coating for blood-contacting polyurethanes

Please cite as: Cell membrane mimicking coating for blood-contacting polyurethanes, B. Butruk-Raszeja, M. Trzaskowski, T. Ciach, Journal of Biomaterials Applications, 2015, 29 (6) 801-812.

Cell membrane-mimicking coating for blood-contacting

polyurethanes.

Beata Butruk-Raszeja1, Maciej Trzaskowski1, Tomasz Ciach1*

1Laboratory of Biomedical Engineering, Faculty of Chemical and Process

Engineering, Warsaw University of Technology, Warynskiego 1, 00-645 Warsaw,

Poland

*CORRESPONDING AUTHOR:

Beata Butruk-Raszeja

Phone: +48 22 234 64 92

Fax: +48 22 825 14 40

e-mail address: [email protected]


Abstract

The aim of the presented work was to develop simple modification technique

for polyurethanes (PU) intended for use in blood-contacting implants (vascular grafts,

heart prosthesis, ventricular assist devices). Polyurethane surface was modified with

soybean-derived phosphatidylcholine (PC) via one-step dip coating technique. In

order to evaluate blood compatibility of the obtained materials, samples were

contacted with human blood under static and arterial flow-simulated conditions. The

PC-modified surfaces were thoroughly characterized and tested for fibrinogen

resistance, the ability to resist platelet adhesion and activation, hemolysis percentage

and plasma recalcification time. Results demonstrated significant, more than 3-fold

reduction in the amount of fibrinogen adsorbed to PC-modified materials as compared

to non-modified PU. Analysis of the samples’ surface after incubation with blood

showed high reduction in platelet adhesion. The results were confirmed by analysis of

blood samples collected after shear stress tests - the percentage of free (non-

aggregated) platelets remaining in blood samples contacted with PC-coated materials

exceeded 70%. The same parameter measured for non-modified PU was significantly

lower and equaled 28%.

KEYWORDS: polyurethane; surface modification; blood compatibility; platelet;

fibrinogen; phosphatidylcholine; biomimicry

1. Introduction

Many materials used for fabricating medical devices provoke adverse reactions

when exposed to tissues and body fluids. These reactions are particularly undesired in

terms of blood-contacting devices – the contact of material with blood causes protein

deposition followed by platelet adhesion, activation and aggregation. Few strategies

were developed to overcome this problem and improve biomaterial

hemocompatibility. Since the blood-biomaterial interactions are determined by

surface physicochemical properties, most of the material modification techniques are

focused on surface modification instead of bulk modification. One of the simplest yet

very effective methods is based on introduction of surface-passivating agents. It can

be performed either by increasing hydrophobicity or hydrophilicity of the surface.


The first strategy is based on the generation of hydrophobic, chemically inert

coatings, which imitate the “lotus effect” – the hydrophobic agent reduce wettability

of the surface and provide “self-cleaning” properties [1]. The second strategy aims to

increase surface hydrophilicity, which can be achieved by incorporation of a hydrogel

coating. In our previous study we developed a method to fabricate hydrogel-based

coating for polyurethanes [2]. We demonstrate that hydrogel coating significantly

improved polyurethane hemocompatibility [3].

Other strategies are based on introduction of bioactive compounds: heparin [4–

6], thrombomodulin [7,8] or nitric oxide [9–11]. There is also very promising group

of peptide coatings (mostly based on RGD sequence) that are design to promote

surface endothelialization [12–16].

Recently, there is a growing interest in phospholipids surfaces. The strategy is

based on the discovery that surfaces presenting phosphatidylcholine head groups

exhibit excellent hemocompatibility [17–19]. Research carried out in the 80's by a

group of prof. Chapman showed asymmetry in deposition of red cells’ and platelets’

membranes components [20]. The inner lipid layer contains lipids, e.g.

phosphatidylserine, which during contact with blood can cause blood coagulation,

whereas the outer layer exhibits excellent hemocompatibility. The outer lipid layer is

built mainly from phosphatidylcholine and sphingomyelin which possess the same

polar group - the phosphorylcholine group. It has been proved that the presence of

phosphorylcholine groups on the materials surface significantly reduces adsorption of

plasma-derived proteins, such as fibrinogen, fibronectin or von Willebrand factor,

which results in the high hemocompatibility of the PC-modified surfaces [21]. The

mechanism of protein repellency is based on the interactions between water and the

phosphorylcholine groups. The large amount of free water around the

phosphorylcholine group is thought to repel proteins and prevent conformational

changes in the adsorbed proteins [22].

Currently, one of the most often applied monomers presenting

phosphorylcholine moiety is 2-methacryloyloxyethylphosphorylcholine (MPC). MPC,

designed by Nakabayashi and coworkers [23], has been used to modify polysulfone

[24], PET [25–27], PU [28–37]; PU/MPC blends was also used to modify Dacron

vascular prosthesis [38–40]. The PC-modified materials exhibit excellent blood


compatibility, however, most of the proposed modification technique is based on

multistep chemical grafting and application of synthetic methacrylate monomers [33–

35,41–43].

In this paper we report a simple and effective method of modifying

polyurethanes with phosphorylcholine compound using dip coating technique, which

can be a promising alternative for multistep chemical grafting. Instead of using

synthetic monomers we use natural-origin phosphatidylcholine – the commercially

available lecithin extracted from soya beans (PC). Lecithin is characterized by a high

content of phosphatidylcholine (22%), together with other phospholipids such as

phosphatidylethanolamine and phosphatidylinositol. Phospholipids found in lecithin

are natural components of the human body and play important physiological functions

– build the cytoplasmic membrane, ensure their proper fluidity and permeability, are

part of the natural surfactant present in alveoli, they are also involved in the digestive

process. Lecithin is non-toxic and highly biocompatible, commonly used as an

additive to drugs and cosmetics, and as an emulsifier in the food industry.

The resulting coating possesses self-organizing, cell membrane-mimicking

properties. The aim of the proposed studies was to determine the effect of the PC

molecules surface concentration on biological and physico-chemical properties of the

surface.

2. Materials and methods

2.1. Materials

Polyurethane (ChronoFlex C, 75D, AdvanSource Biomaterial, Wilmington,

U.S) was purchased in a form of pellets. Soybean-derived phosphatidylcholine was

purchased from Merck, Poland. Cyclohexanone and dimethylacetamide (DMAC),

both from Chempur, Poland, were used as received, without further purification. Cell

culture media and reagents, namely, Dulbecco’s modified Eagle’s medium (DMEM),

fetal bovine serum (FBS), glutamine, penicillin-streptomycin, Dulbecco’s phosphate-

buffered saline (D-PBS) and trypsin were purchased from Gibco, Poland. All other

chemicals were purchased from Sigma-Aldrich, Poland.


2.2. PU film preparation

PU films were prepared by solution casting and solvent evaporation technique.

Briefly, polyurethane pellets (ChronoFlex) were washed with alcohol/water solution,

dried and dissolved in DMAC at a concentration of 20% w/v. The solution was

poured onto clean glass and dried at 37°C until total solvent evaporation. The

obtained film was peeled-away, cut and used for further modification.

2.3. PU modification with phosphatidylcholine

PC-based coating was fabricated using one-step dip-coating method. In order to

prepare modifying solution, alcohol-washed and dried PU pellets were dissolved in

cyclohexanone at a concentration of 1% (w/v). Subsequently, PC at a given

concentration (0.5%, 1%, 1.5% or 2%) was added to the solution and stirred until total

dissolution. Samples of PU films (2 mm thick, 20 mm in diameter) were dipped in

modifying solution for a few seconds at RT (25°C) and dried for a given period of

time. Samples were then washed in 0.1% SDS solution (5 minutes) followed by

distilled water (24h) and lyophilized/dried at 37°C until total solvent evaporation.

2.4. Surface characterization

Wettability of the PC-modified PU films was determined using dynamic contact

angle measurements by placing 1 µl of distilled water onto analyzed surface.

Advancing and receding contact angles were analyzed with goniometer CAM 200

(KSV) with Attension Theta Software (Biolin Scientific). The average contact angle

was calculated according to the formula:

cos θ = (cos θA + cos θR)/2 [44]

where: θ – average contact angle, θA – advancing contact angle, θR – receding contact

angle.

Homogeneity of the PC coating the modifying surfaces was analyzed by

staining with rhodamine 6G as described elsewhere [45]. Briefly, samples were

immersed in rhodamine 6G aqueous solution (0.2mg/mL) for 30s, washed with

distilled water for 30s and dried at 37°C. The surfaces were observed with

fluorescence microscopy (Eclipse 80i, Nikon); obtained images were analyzed with

an Image-J program (National Institutes of Health, Washington, US).

https://www.researchgate.net/publication/224709865_Soft_contact_Measurement_and_interpretation_of_contact_angles?el=1_x_8&enrichId=rgreq-a8f35467-e86c-4d5e-a6ba-8a7fbf359d97&enrichSource=Y292ZXJQYWdlOzI2NTg2MjA0MztBUzoxODI5OTYwOTEwMjMzNjBAMTQyMDY0MTA3Njg3OQ==


Thickness of the PC coating was measured elipsometrically (UVISEL, Horiba-

Jobin-Yvon).

2.5. Coating degradation

The degradation test was performed according to ASTM F1635-04 ("Standard

Test Method for Testing In Vitro Degradation of Hydrolytically Degradable Polymer

Resins and Fabricated Forms for Surgical Implants"). Samples (disks with a diameter

of 18 mm) have been sterilized with ETO and placed in a solution of PBS (pH = 7.4)

containing sodium azide (0.1% w / v) and SDS (0.1% w / v). The solution:mass ratio

was maintained at 50:1. Containers were sealed and incubated at 37C. After a certain

time of degradation (7, 14, 21 and 28 days) samples were removed from the solution,

rinsed with distilled water, dried to constant weight and analyzed by contact angle

measurements and rhodamine 6G staining.

2.6. Hemocompatibility analysis

2.6.1. Fibrinogen adsorption

The plasma-derived fibrinogen adsorption to the test materials was analyzed

using platelet poor plasma (PPP). In order to prepare PPP 50 ml of blood was drawn

from a healthy, aspirin-free donor (female, 28 years) in the K2EDTA tubes using the

BD Vacutainer vacuum system. Blood was centrifuged at 300g for 30 minutes. The

obtained supernatant was transferred to clean tubes and centrifuged at 2000g for 20

minutes. After the centrifugation, the supernatant (PPP) was transferred to a new

sterile tube.

Materials’ samples (disks with a diameter of 18 mm) were placed in the 24-well

polystyrene plates and equilibrated with PBS at 37C overnight. Next, the

investigated materials were contacted with 100% PPP for 1 hour at 37C. Materials

with surface-adsorbed fibrinogen were analyzed with ELISA assay. The following

procedure was applied: after incubation with PPP material samples were rinsed (3 x 5

minutes) with washing buffer (PBS supplemented with 0.05% Tween 20, Sigma-

Aldrich, Poland), blocked with non-fat dry milk (5% solution in PBS, 1 hour, RT),

rinsed with washing buffer (3 x 5 minutes), incubated with primary antibodies (1

hour, RT), rinsed with washing buffer (3 x 5 minutes), incubated with secondary


antibodies (1 hour, RT), rinsed with washing buffer (6 x 5 minutes) and transferred to

fresh plates to eliminate the influence of the protein adsorbed to the well walls.

Materials were then incubated with the peroxidase substrate solution - SigmaFast

OPD (o-Phenylenediamine dihydrochloride, Sigma-Aldrich, Poland) in the dark at RT

for 30 minutes. After the reaction, part of the solution (200 μl) from each well was

transferred to 96-well plate; the optical density of the solution was read at the 450 nm.

During the test the following antibodies were used: primary polyclonal anti-

fibrinogen (Sigma-Aldrich, Poland, dilution 1:1000), primary monoclonal anti-

fibrinogen, clone 85D4 (Sigma-Aldrich, Poland, dilution 1:1000), secondary

antibodies conjugated to peroxidase (Sigma-Aldrich, Poland, dilution 1:20000)

Antibodies were diluted with the 2% solution of dry non-fat milk in washing buffer. A

set of samples (K_PU and K_PC 2) were subjected to all ELISA assay steps with the

elimination of the first step (materials incubation with PPP). These control materials

were used to evaluate the possibility of the non-specific binding of the antibodies to

the materials’ surfaces. As a blank a solution of o-phenylenediamine dihydrochloride

(a substrate for the antibody-conjugated peroxidase) was used.

2.6.2. Hemolysis assay

PC-modified samples were placed in Falcon tubes and incubated with 10 ml of

0.9% NaCl at 37ºC for 1 h. Next, saline was replaced with fresh NaCl solution (5 ml)

followed by addition of 0.1 ml of human blood (freshly collected in K2EDTA tubes)

and incubated at 37ºC for 2 h. Afterwards, all samples were centrifuged at 2500 rpm

for 5 min. The hemoglobin released from erythrocytes was characterized by a

spectrophotometer at the absorbency wavelength 545 nm.

The degree of hemolysis was calculated as follows:

%H = ((As-An)/(Ap-An))*100%

where H is the degree of hemolysis, As is the absorbance of the sample, Ap is the

absorbance of the positive control and An is the absorbance of negative control.

The sample for positive control was prepared by mixing 0.1ml of blood with 5 ml of

distilled water, while the sample for negative control was prepared by mixing 0.1ml

of blood with 5 ml of 0.9% NaCl.


2.6.3. Plasma recalcification time

Human blood, freshly collected in citrated tubes (3.8%, BD Vacutainer) was

centrifuged at 300g for 30 minutes. The supernatant was transferred to clean tubes

and centrifuged at 2000g for 20 minutes. The obtained PPP was applied onto the

polymer films equilibrated in 0.9% NaCl solution at 37°C for 2 h. Clotting in the

plasma was initiated by recalcification with 0.025 mol/L CaCl2 aqueous solution

added to the PPP. Clotting times were recorded at the first signs of any fibrin

formation.

2.6.4. Platelet adhesion

Before the experiments, all samples have been sterilized with ETO and

immersed into isotonic (pH 7.4) phosphate buffer solution (PBS) for 24 h. Fresh

blood, donated by healthy adult volunteer (female, age 27) was collected into

K2EDTA tubes (BD Vacutainer, Poland) without using stasis, the first sample tube

was rejected. Blood was centrifuged at 200 x g for 20 minutes to obtain platelet-rich

plasma (PRP). PBS-hydrogenated materials were placed in 24-well culture plate and

incubated with 1 ml of PRP for 1h at 37°C. After that time, materials were gently

rinsed with PBS and fixed with glutaraldehyde (2.5%/PBS, 2h, 4°C) followed by

fixation with OsO4 (1%/PBS, 1h, 4°C). Materials were then dehydrated through

graded concentrations of ethanol (50%, 60%, 70%, 80%, 90% and 100%, 5 minutes

each), dried, sputtered with carbon and analyzed with scanning electron microscope.

Obtained images (magnification 500x) were used to calculate the average percentage

of platelet-covered area; the number was calculated using ImageJ program from five

randomly chosen places on the same sample.

2.6.5. Blood-biomaterial interactions under shear stress

Blood-biomaterial interactions under flow conditions were evaluated using a

platelet analyzer (Impact-R, DiaMed) as described elsewhere [46]. Briefly, ETO-

sterilized materials in the form of discs with a diameter of 18 mm was placed in a

PTFE cones (part of the ImpactR analyzer) and covered with 130 µl of blood. Arterial

flow simulated shear stress (720 rpm) was applied for 5 minutes. After the test, blood

samples were collected and analyzed with flow cytometry. Each material was tested


in triplicate. Two additional blood samples were also studied: blood samples stored

under static conditions (negative control) and blood sample activated by adding ADP

at a concentration of 20 mM (positive control).

Blood samples incubated with materials were analyzed in order to calculate the

percentage of platelets remaining in blood after the shear-stress test in relation to free

platelets present in negative control:

RP [%] = (RPX/RPKN)*100%,

where: RP – the remaining platelets [%], RPX – number of platelets present in blood

sample after the shear stress test, RPKN – number of platelets present in the negative

control blood sample.

Additionally, we quantified the level of platelet activation as a percentage of

P-selectin positive platelets. The percentage ratio between free platelets and platelet

aggregates present in blood after shear stress test was also calculated.

Statistical analysis

Results of contact angle measurements and hemocompatibility studies were

expressed as means±SD. Statistical significance of differences was analyzed using

single factor analysis of variance (ANOVA) for p<0.05 with post-hoc Tukey’s test

(OriginPRO 8.0). Results of experiments with blood under shear stress were presented

using box charts; the following values are presented: data points (circle points), mean

value (square points), median (horizontal line inside boxes), standard deviation

(boxes).

3. Results and discussion

3.1. Coating homogeneity and thickness

The presence and the distribution of PC particles on the PU surfaces were

evaluated by staining the modified surfaces with rhodamine 6G. Rhodamine 6G, due

to its lipophilic character, became a suitable stain for lipids and PC-containing

materials, allowing to access coating coverage and homogeneity [45]. Fig.1

summarizes the microscopic images and 3D plots of fluorescence intensity of PC-

modified materials after staining with rhodamine. As expected, control materials that

do not contain PC (PU and PC 0) did not stain and fluorescence intensity was


negligible. On the contrary, all PC containing surfaces were stained with rhodamine

and yielded high intensity fluorescence. For all PC-coated samples the fluorescence

intensity increased together with the increase of PC content in the modifying layer.

Observation of all stained surfaces confirmed that PC was evenly deposited through

the whole material surface; areas without PC coating were not found.

All PC-coated surfaces exhibit a presence of bright points – probably

aggregates of PC molecules. Number of surface aggregates increases together with

the increase in PC concentration. The highest number of the bright points is presented

on PC 1.5 and PC 2 surfaces. In the case of these materials, in addition to the

formation of PC aggregates insoluble PC from modifying solution may settle on the

surface. It was noted that in the solutions with higher PC concentrations (1.5% and

2%) PC dissolves with difficulty, which could indicate that solutions close to

saturation were obtained. In this case, there may be an incomplete dissolution of PC

and its deposition on the surface of the material.

Tab. 1 summarizes the results of the coating thickness measurements (d) for

materials with different PC content. The results showed that the thickness of the

coating is approximately 10 μm and is independent of the concentration of PC. This

result is consistent with expectations, since in the used modification technique, the

main factor influencing the coating thickness is the concentration of PU in the

modifying solution. For all materials the same concentration of PU was applied (1%),

thus the thickness of the coating is maintained at a constant level.

3.2. Surface wettability

Wettability analysis of the modified surfaces is an important study confirming

the proper course of the modification process. The increase in surface hydrophilicity

indicates not only the successful coating of PU with phospholipids, but also the

correct orientation of phospholipids molecules (hydrophilic chains oriented toward

the polymer / water interface).

The surface wettability measurements revealed that introduction of PC

molecules significantly increased surface hydrophilicity. The values of average

contact angle were not influenced by the concentration of PC molecules and were


approximately 20° (detailed values shown in Tab. 1). The values were significantly

lower (p<0.0001) compared to both control materials.

Tab. 1 shows the values of decreasing and increasing contact angle obtained

during the dynamic contact angle measurements. It is considered that the contact

angle hysteresis, defined as the difference between the value of increasing and

decreasing contact angle, is a good indicator of the reorganization or migration of the

functional groups present on the surface [47]. It has been shown that materials

covered with phospholipids may present large contact angle hysteresis values, which

indicates a reorganization of the molecules present on dry surfaces during contact

with water [24,47]. In case of presented PC-coated materials, the contact angle

hysteresis is relatively low and does not differ from the values obtained for control

materials (PU and PC 0). Consequently, we concluded that the structure of

phospholipid layer is stable - during process of drying and wetting there is no

significant change in the surface wettability. In conclusion, the low values of contact

angles of PC-modified samples proved the presence and proper conformation of PC

molecules present onto material surfaces. Additionally, low contact angle hysteresis

indicated that the conformation of PC molecules onto the surface is stable and does

not change during drying/hydrating processes.

3.3. Coating degradation

In the case of dip coating surface modification technique, when there is no

chemical bond between the substrate and the coating, important parameter to be

assessed is durability of the coating. For this purpose, PC-modified materials were

incubated with solution of detergent for a specified amount of time (7,14 or 21 days).

Surface wettability decreased slightly along with increasing time of degradation

(Tab.1). After 7-day degradation the contact angle of analyzed surfaces increased,

reaching a value of greater than 30°. The change in the wettability of the subsequent

time point (14-day degradation) was lower, the value of the contact angle were

aproximately 40° and comparable to those measured at the last time point (21-day

degradation). Results have shown that the increase of contact angle was highest in the

early stage of degradation process, what can be an effect of washing-out the loosely

bound PC particles from the material’ surfaces. In a later stage of degradation, the


surface wettability has not changed significantly, reflecting the stability of the

obtained coatings.

Analysis of rhodamine-stained surface confirmed good durability of PC

coatings. The results of microscopic observation of stained surfaces are presented in

Fig. 1 - left column shows the pictures of materials before degradation, the right

column - materials incubated with the detergent solution for a period of 21 days. A

slight decrease in fluorescence intensity can be observed, which is consistent with the

results obtained from the surface wettability analysis. Nevertheless, materials

subjected to 21-day degradation process presented strong and uniform staining with

rhodamine, which confirmed the presence of PC molecules on their surface.

3.4. Hemocompatibility analysis under static conditions

3.4.1. Fibrinogen adsorption

Surface-adsorbed fibrinogen has been shown to be essential for platelet

adhesion to biomaterials [48]. Platelets bind to fibrinogen thorough interactions of

GPIIbIIIa receptor [49,50] with active platelet-binding epitopes expressed in protein

molecule. It has been found that surface properties clearly impact the conformational

changes in fibrinogen adsorbed on surfaces [51]. Thus, the platelet attachment to

biomaterial surface may be modulated by adsorption-induced conformational changes

of fibrinogen.

In the performed ELISA assay two types of anti-fibrinogen antibodies were

applied: polyclonal antibodies (pAb), enabling to assess the overall amount of the

adsorbed protein, and monoclonal antibodies (mAb) recognizing the D domain of the

fibrinogen molecule. The fibrinogen D domain is involved in the binding of platelets,

therefore, plays an important role in platelet adhesion and aggregation. The binding of

mAb indicates the correct domain conformation resulting in platelet-binding activity.

The obtained results of ELISA assay demonstrated significantly, more than 3-

fold, reduction in the amount of fibrinogen adsorbed at the PC-modified materials

compared to unmodified materials - PU and PC 0 (analysis using pAb) (Fig.2).

Similar results were obtained for the analysis of fibrinogen conformation (analysis

using mAb) - on the surface of the unmodified material (PU) the amount of active D

domain was 2-fold higher as compared with the PC-modified material. No effect of


the surface concentration of PC on the quantity and conformation of adsorbed

fibrinogen was observed.

The decrease in fibrinogen adsorption is highly correlated with the increase in

surface wettability and those results are in accordance with the similar ones already

presented in literature [52].

3.4.2. Hemolysis percentage and plasma recalcification time

Investigation of materials’ hemolytic effect was performed using direct method; the

results are given in Tab.2. In case of materials designed for contact with blood, the

hemolysis percentage cannot exceed 1%. All analyzed materials were characterized

by insignificant percentage of hemolysis (<1%).

The plasma recalcification times (PRTs) of the surface-modified PUs are

shown in Tab.2. The PRT of a PU surface was slightly increased by the PC coating.

There was no significant PRT difference observed between materials coated with

different concentration of PC molecules.

3.4.3. Platelet adhesion

SEM images of analyzed surfaces after incubation with PRP are shown in

Fig.3. Microscopic observation revealed thrombogenic potential of both control

materials: PU and PC 0. The percentage of surface area coated with platelets was

relatively high and equaled 9%±2% for PU and 11%±2% for PC 0 (Tab.2). Platelets

were evenly deposited through the whole analyzed area; many of them formed

aggregates, what indicated their previous activation. The platelet aggregates were

quite extensive and reached a diameter of about 30 µm. Most of the adhered platelets

presented morphology typical for the activated cells - flattened with multiple

pseudopodia.

In case of PC-modified materials the percentage of platelet-coated area was

significantly lower compared to the control materials. Among all PC-coated materials

the number of adhered platelets was the biggest for the samples with the lowest

concentration of PC (PC 0.5) – the percentage of platelet-coated area equaled

5%±4%. However, it should be stressed that in case of PC 0.5 the platelet deposition

was uneven; both, areas with high concentration of adhered platelets and areas with


negligible number of platelets were observed. In case of materials with higher PC

concentration (PC 1, PC 1.5, PC 2), the percentage of platelet-coated area was below

1%. The number of aggregates formed onto all PC-modified surfaces was very low;

the existing aggregates were relatively small with a diameter up to 10 µm. The

majority of adhered platelets presented morphology typical for non-activated cells –

spherical, with no visible pseudopodia.

3.5. Hemocompatibility analysis under dynamic conditions

3.5.1. Platelet consumption under shear stress

The number of platelets remaining in blood sample after shear stress test is an

important parameter during evaluation of material hemocompatibility. Thrombogenic

materials cause high platelet consumption in blood sample, which is due to platelet

activation followed by platelet aggregation and adhesion to material surface. The

numbers of platelets remaining in the analyzed blood samples after shear-stress test in

relation to the number of platelets present in negative control (RP) are shown in Fig

4A. In the case of the positive control P (blood sample activated by the addition of

ATP) the RP equaled 22%±5%. A similar value obtained for the unmodified material

- PU (28%± 24%). In the case of PC-modified materials obtained values exceeded

70%. The high values of RP indicated that the PC-modified surfaces did not provoke

platelets to adhere and aggregate. The study revealed no significant differences

between materials coated with different concentration of PC molecules. Statistical

analysis allowed to select three materials (PC 0.5, PC 1 and PC 1.5) that reached the

RP value which was significantly different (p<0.05) compared to the result obtained

for PU.

3.5.2. Platelet activation under shear stress

Activated platelets release protein accumulated in cytoplasmic vesicles. This

leads to appearance of a number of proteins onto the platelet surface, among which P-

selectin is a specific marker of platelet activation. Therefore, the percentage of

platelets expressing P-selectin is a good indicator of platelet activation. The

percentage of P-selectin-positive platelets present in blood samples incubated with the

test materials are shown in Fig 4B. In the case of non-modified PU the analyzed


parameter reached the value of 29% ± 13%. For most PC-modified materials the

percentage was slightly lower and amounted to 12% ± 10% (PC 0.5), 10% ± 4% (PC

1) and 9% ± 3% (PC 1.5). In the case of PC 2 the percentage of P-selectin-positive

platelet was similar to the values obtained for PU and equaled 21% ± 16%.

3.5.3. Platelet aggregates vs. free platelets

The analysis also determined the percentage content of free platelets and

platelet aggregates present in blood samples after shear-stress test. Additionally small

aggregates (consisting of 2-3 platelets) and big aggregates (more than 3 platelets)

were distinguished.

The percentage of platelet aggregates obtained for the unmodified material was

high and exceeded the value for the positive control (PU: 41% ± 5%, P: 34% ± 6%)

(Fig.4C). All PC-coated materials were characterized by a reduced number of

aggregates, the values were as follows: 17% ± 9% (PC 0.5), 12% ± 7% (PC 1), 7% ±

3% (PC 1.5), 19% ± 6% (PC 2). Statistical analysis revealed that the values reached

for PC 1 PC 1.5 were significantly lower compared to the value for unmodified

material PU (p <0.05).

The number of big platelet aggregates is a particularly important parameter

during the assessment of surface hemocompatibility. The formation of big aggregates

shows strong platelet activation and initiates the process of blood clotting. The

percentage of big aggregates obtained for the control samples equaled 1%±1%

(negative control, N) and 14%±3% (positive control, P) (Fig.4D). For the unmodified

material (PU) the value was large and amounted to 31%±2%. For all PC-coated

materials a percentage of big aggregates significantly (p <0.05 vs. PU) decreased and

reached the following values: 6%±1% (PC 0.5), 2%±1% (PC 1), 1%±1% (PC 1.5),

11%±7% (PC 2). The study showed no significant differences (p> 0.05) in the

percentage content of free platelet and platelet aggregates between the modified

materials with different PC content.

Fig.4E summarizes the percentage of free platelets, big and small platelet

aggregates in analyzed blood samples. As can be seen, in the case of all materials PC-

modified a high percentage of free platelets was obtained, together with a low

percentage of big platelet aggregates. Each PC-coated material was characterized by a


higher number of free platelets as compared to both the unmodified material (PU) and

positive control (P). In addition, the percentage of free platelets and platelet

aggregates for PC-modified materials reached values close to the values obtained for

the negative control (N, the blood sample stored in a static condition without any

contact with coagulation factors). This result indicates very good non-thrombogenic

properties of the modified materials.

3.5.4. Hemocompatibility ranking

To summarize the results of hemocompatibility analysis under shear stress

conditions we constructed a plot where the percentage of platelet aggregates is shown

as a function of RP (the percentage of platelets remaining in blood after shear stress

test in relation to the platelets present in negative control) (Fig.4F). In this graph

materials with the best non-thrombogenic properties (low platelet consumption and

low number of platelet aggregates formed during the shear stress test) are placed in

lower right corner. At the same time, the most thrombogenic materials are located in

the upper left corner (high platelet consumption and high number of platelet

aggregates formed during the shear stress test). As shown in the chart, the unmodified

material (PU) exhibits a fairly high thrombogenic potential, exceeding the values that

characterize positive control. The whole group of PC-modified materials occupies a

separate place on the chart, in the area representing materials with high

hemocompatibility. The values of the analyzed parameters obtained by this group are

close to the values for the negative control, which confirms high biocompatibility of

these materials.

The preliminary results are promising, however, it should be stressed that there

are some limitations concerning shear stress test. Small volume of the tested blood

sample (130μl) causes its high sensitivity to surface defects. In case of any cracks or

other surface imperfections a large deviation from mean value of analyzed parameter

occurs. Thus, the analyzed surface must be thoroughly checked to limit this effect.

Moreover, due to the high morphological variation in blood morphology, the adequate

number of replicates must be tested. In the presented study three replicates of each

material were analyzed. Therefore, experiments with more replicates are planned in

the course of further research.


4. Conclusions

A simple and effective technique of polyurethane surface modification is

proposed. Method is based on one-step dip-coating technique. As a modifying agent

a soybean-derived lecithin were used. The modification results in formation of self-

organizing phosphatidylcholine layer onto the substrate surface. Studies have shown

that the modification significantly increases the hydrophilicity of the surface. The

phosphatidylcholine molecules are evenly distributed through the coating. Incubation

of the modified surfaces with blood under both static and dynamic conditions allowed

evaluating the thrombogenic potential of the surfaces. Results showed a significant

reduction in fibrinogen adsorption and platelet adhesion to the surface of modified

materials compared to the unmodified polyurethane. Analysis of blood incubated with

materials confirmed the improved hemocompatibility of the modified surfaces.

Acknowledgment

Authors sincerely thank Prof. Marek Sanak, Dr. Hanna Plutecka and Dr.

Wojciech Węgrzyn, Department of Internal Medicine, Jagiellonian University,

Krakow, for performing hemocompatibility studies.

Authors contribution

BBR: the conception and design of the study, acquisition of data, analysis and

interpretation of data, drafting the article, MT: acquition of data, TC: interpretation of

data, revising of the article.

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Figures and captions:

Fig. 1: Fluorescence images and fluorescence intensity plots of surfaces with different PC

content (PC 0.5, PC 1, PC 1.5 and PC 2) stained with rhodamine 6G, left – materials before

degradation (t = 0), right - materials subjected to 21-day degradation (t = 21) .


Tab. 1: Values of coating thickness and contact angle of unmodified PU (PU), control material

(PC 0) and PC-modified materials (PC 0.5, PC 1, PC 1.5, PC 2); d- coating thickness, θA –

advancing contact angle, θR – receding contact angle, θ – average contact angle, θ7/θ14/θ21 – final

contact angle after 7-/14-/21-day degradation,* p<0.0001 vs. PU; MV±SD, n=15

d [µm] θA [°] θR [°] θ [°] θ7 [°] θ14 [°] θ21 [°]

PU n/a 75.0±6.7 70.4±5.4 72.7±5.9 78.3±3.2 80.33.4 79.1±3.2

PC 0 10.1 0.1 68.1±5.6 62.9±4.5 65.5±4.9 n/a n/a n/a

PC

0.5 10.2 0.1 20.9±6.0* 19.1±5.3* 20.1±5.3* 33.9±4.2* 42.5±1.9* 45.3±0.3*

PC 1 10.1 0.1 22.0±5.5* 17.9±2.6* 20.1±3.9* 35.3±5.3* 40.0±0.6* 44.7±2.3*

PC

1.5 10.2 0.1 21.3±7.6* 16.5±7.3* 19.1±7.3* 34.9±3.8* 43.5±1.4* 45.4±2.7*

PC 2 10.2 0.2 23.6±3.8* 20.3±4.0* 22.0±3.8* 33.7±1.5* 39.9±2.3* 41.2±0.8*

Tab.:2. Platelet-coated area, hemolysis percentage (H) and plasma recalcification time (PRT) for

PC-modified materials, MV±SD, n=4.

Platelet-coated area

[%] H [%]

PRT [s]

PU 92 0.22 0.06 63025

PC 0 112 0.08 0.05 64830

PC 0.5 54 0.08 0.06 79850

PC 1 <1 0.12 0.06 78025

PC 1.5 <1 0.03 0.04 81010

PC 2 <1 0.03 0.04 73860


Fig. 2: Adsorption of plasma fibrinogen to the PC-modified materials: pAb - test using

polyclonal anti-fibrinogen antibody, mAb - test using monoclonal antibodies recognizing the D

domain of the fibrinogen molecule; MV ± SD, n = 4, * p <0.5 vs. PC 0, ** p <0.0001 vs. PC 0.


Fig. 3: Platelet adhesion to unmodified polyurethane (PU), control material (PC 0) and PC-

modified materials (PC 0.5, PC 1, PC 1.5, PC 2) after incubation with PRP under static

conditions at 37ºC for 1h (magnification 1000x and 5000x).

PC 0

PC 0.5 PC 1

PC 1.5 PC 2

K

Mag = 1000x20 μm

Mag = 5000x100 μm

Mag = 5000x100 μm

Mag = 5000x100 μm

Mag = 5000x100 μm

Mag = 5000x100 μm

Mag = 5000x100 μm

Mag = 1000x20 μm

Mag = 1000x20 μm

Mag = 1000x20 μm

Mag = 1000x20 μm

Mag = 1000x20 μm


Fig. 4: Results of hemocompatibility analysis under dynamic conditions: (A) the percentage of platelets remaining in blood samples after shear-stress test in relation to the platelets present in negative control, (B) the percentage of P-selectin-positive platelet present in blood samples after shear-stress test, (C) the percentage of platelet aggregates present in blood samples after test, (D) the percentage of big platelet aggregates present in blood samples after test, (E) the percentage ratio between free platelet, big platelet aggregates and small platelet aggregates present in blood samples after test, (F) hemocompatibility ranking of tested materials - the percentage of platelet aggregates is plotted as a function of the percentage of platelets remaining in blood after shear stress test (RP); N – negative control, P – positive control, PU – non-modified material, PC 0.5, PC 1, PC 1.5, PC 2 – PC-coated materials.

Cell membrane-mimicking coating for blood-contacting polyurethanes

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