reactions of two hydrophilic surfaces with detergents, protein and whole human blood

COLLOIDS AND B SURFACES

ELSEVIER Colloids and Surfaces B: Biointerfaces 9 (1997) 67-79

Reactions of two hydrophilic surfaces with detergents, protein and whole human blood

Cecilia Eriksson 1, Eva Blomberg 2 Per Claesson 2, H~tkan Nygren 1 , ,

x Department of Anatomy and Cell Biology, University ofGdteborg, G6teborg, Sweden 2 Institute of Surface Chemistry and Royal Institute of Technology, Stockholm, Sweden

Received 27 September 1996; accepted 30 January 1997

Abstract

The surface chemistry of moscovite mica and hydrophilic glass was characterized by ESCA. The adsorption of detergents and lysozyme was measured by surface force methods, ellipsometry or ESCA. The results show that the protein has a higher affinity to the mica surface. A biological characterization of the surfaces was performed by exposure to whole blood, followed by detection of surface-adsorbed plasma proteins and cellular antigens by immunofluorescence. The brightness of the fluorescence was measured by computer-aided image analysis. The results show that hydrophilic glass was the most efficient activator of serine protease cascade enzymes. Platelets were activated at both surfaces, but were shown to leave the glass surface with time. The glass surface was more inflammatogenic than the mica surface as determined by the CDllb-integrin expression of PMN-cells. The results indicate that the nonself recognition of the surfaces is an array of subreactions, the kinetics of which determine the outcome of the blood-material contact. © 1997 Elsevier Science B.V.

Keywords: Mica; Glass; Plasma proteins; Platelets; PMN cells

1. Introduction

Almost all medical devices, implanted into the human body for short or long periods of time, will experience an initial exposure to blood. This exposure initiates a series of reactions at the b lood- material interface-interaction with water and salts, protein adsorption [1-4], activation of serine protease cascade systems [5-8] and adhesion and activation of cells [9-12]--collectively known as the nonself recognition of the material.

The causal relations between these different reactions are, of course, of utmost importance for our knowledge about biocompatibility of materials,

* Corresponding author.

0927-7765/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0927-7765 (97) 00010-6

and information on such relations can only be found in experiments using complex systems like whole blood, where all subreactions present them- selves simultaneously and in their natural order of appearance. The importance of surface energy for the outcome of plasma protein adsorption is widely recognized. Hydrophobic surfaces are known to enrich fibrinogen at the blood-mater ia l interface [ 13], a protein that serves as an adhesion mediator for platelets [9]. At hydrophilic surfaces, H M K and Factor XII may replace fibrinogen [13] and the contact activation system of coagulation is initiated, leading to direct formation of fibrin at the surface [14].

The outcome of protein adsorption is then thought to influence the adhesion and activation of cells arriving at the protein-coated surface.

68 C. Eriksson et al. / Colloids Surfaces B." Biointerfaces 9 (1997) 67-79

Although this assumption seems reasonable, there is little direct evidence of such a mechanism, apart from the causal relation between fibrinogen adsorption and platelet adhesion.

The results of the present and a previous [14] study indicate that a complex biofilm is formed at blood-material interfaces within a few seconds of exposure. The bulk coagulum is easily rinsed off, leaving no macroscopically visible traces of coagu- lated blood. The surface-adhering film is well defined, consisting of one to three cell-layers. Prolonged rinsing does not change the composition of the film [14].

A crucial question is: what plasma proteins or cellular antigens are exposed to the surrounding cells arriving to the surface at different times of exposure. The hypothesis behind the present study was that the composition of exposed antigens in this initial film would influence, not only the adhesion, but also the activity of inflammatory cells, arriving later. The ligands exposed to cellular receptors would also be available as binding sites for antibodies, making immunocytochemistry a valid test method. The aim of the present study is to elucidate the blood material interface at two hydrophilic surfaces with different chemistry. We show that the two hydrophilic surfaces, muscovite mica and glass, interact very differently with simple substances such as surfactants and proteins as well as with complex systems such as blood. This leads to differences in the initiation of the protease cascade systems, and consecutive cell adhesion and activation. We emphasize that it is not sufficient to classify systems in terms of hydrophilic and hydrophobic, but that a detailed surface chemical and biological characterization is needed.

2. Materials and methods

2.1. Chemical characterization of surfaces

The chemical composition of muscovite mica and glass surfaces were determined using ESCA. The spectrometer employed, a Kratos (AXIS HS), utilized a monochromatic Mg X-ray source and a hemispherical analyser. The adsorbed amount of lysozyme on mica was also determined with this

instrument following the procedure developed by Herder et al. [15]. The adsorbed amount of lysozyme on SiO2 was determined by ellipsometry in situ using a Rudolph thin film ellipsometer, type 43603-200E [16]. The adsorption of cationic surfactants onto glass and onto muscovite mica surfaces were measured using two different surface force techniques [17]. The mica surfaces were studied employing the interferometric surface force technique developed by Israelachvili and Adams [18] whereas the glass surfaces were investigated using the so-called MASIF technique developed by Parker [18]. Detailed descriptions of these techniques can be found in [17-19].

2.2. Biological characterization

2.2.1. Surface preparation Glass slides were made hydrophilic by immer-

sion for 1 h in 70% ethanol containing 1% HCl(aq). After rinsing for 3 × 10 min in distilled water the slides were allowed to dry in air and to control the hydrophilic character of the glass a drop of water was spread on the surface. The mica was cut in squares of 9 × 9 mm. Each piece was split with a needle into thin layers which were stored in distilled water until use. The upper- and lowermost layers were not considered pure and therefore not used.

2.2.2. Exposure of capillary blood and immunofluorescence

Pieces of mica or glass were dried with an air current and put in a humified chamber at 37°C. Fresh capillary blood was taken from the fingertip of a healthy unmedicated donor, discarding the first two drops, and placed in drops of ca 20 111 on the surfaces. The blood-surface contact time varied from 5 s to 64 min. After the blood exposure mica pieces were washed with Dulbecco's phosphate buffered saline (D-PBS), containing physiological concentrations of Mg, Ca and K and placed on a cooling plate at 0°C. The hydrophilic glass slides were washed with D-PBS, fixed in absolute ethanol at - 2 0 ° C for 10 min and rehydrated before being put on the cooling plate. The specimen were incubated for 20 min with specific antibodies directed against fibrinogen, the complement factors C3c

C. Eriksson et al. / Colloids Surfaces B: Biointerfaces 9 (1997) 67-79 69

and Clq, prothrombin/thrombin, platelet membrane antigens (pan-antibody) and the von Willebrand factor (Dakopatts, Copenhagen, Denmark). To detect platelet vesicles the specimen were incubated with mouse monoclonal antibodies against thrombospondin (Brhringer, Mannheim, Germany). Mouse monoclonal antibodies against CD62 and CD1 lb (Serotec, Oxford, England) were used to detect cell activation. After 20 min incubation the specimen were rinsed with D-PBS and incubated with fluorescein-conjugated anti- rabbit or anti-mouse Ig-antibodies (Dakopatts, Copenhagen, Denmark) for another 20 min. The specimen were then rinsed again with D-PBS and mounted in Dabco (Fluka, Biochemica, Switzerland). Control experiments were performed by excluding the first antibody incubation. Acridine orange staining was used to detect leukocytes. Specimen were immersed in a solution of 0.01% acridine orange (Sigma, St Louis, MO) in 0.06 M phosphate buffer pH 6.0 for 3 min. These specimen were rinsed and mounted in phosphate buffer. Mica pieces were placed on glass slides prior to mounting. All specimen were covered with a glass cover slip and stored at -20°C until further analysis.

The specimen were photographed and examined in a fluorescence microscope (Zeiss, 3RS) where magnification, film speed and exposure time were kept constant. The photographs were scanned and digitized in a polaroid scanner and analysed in ADOBE Photoshop on a Power Macintosh 8100/80 desktop computer. To set zero levels background controls were used. Mean brightness or surface coverage of fluorescence in the pictures were used as quantitative measures of amount of bound antibodies.

2.2.3. Statistical evaluation The differences between means were compared

by using Students' t-test. Experiments were repeated six times (n = 6).

3. Results and discussion

3.1. Muscovite mica and glass surfaces

Naturally occurring muscovite mica is a layered aluminosilicate mineral with the idealized formula

KA1/(AISi3)Olo(OH)/; its structure is schemati- cally illustrated in Fig. 1. Mica consists of 10 ~, thick sheets. Each sheet is essentially two silicate layers joined together by aluminium atoms. Isomorphic substitution of aluminium for silicon in the silicate layers results in a negative lattice charge that is neutralized by potassium ions (and to a much lesser extent sodium ions) present between the aluminosilicate sheets. The electrostatic interaction between potassium and oxygen is weaker than the covalent bonds between the aluminosilicate layers and muscovite mica can, therefore, easily be cleaved along the basal plane creating a molecularly smooth surface.

Upon cleavage along the basal plane the potassium ions are evenly distributed between the two surfaces, and in air the mica surfaces are neutralized by these potassium ions. In water the potassium ions will dissociate from the mica surface. The surface, that consists of Si, O and AI joined together by Si-O bonds and A1-O bonds, acquires a negative charge, and if all of the potassium ions dissociate from the mica basal plane this charge corresponds to one negative charge per 48 ~2 [20], or 2.1 x 1014 charges per c m 2, corresponding to a surface charge density of - 0 . 3 3 C m -2. It is important to notice that the mica surface charge arises from isomorphous substitution and that no OH-groups are present on the mica basal plane. (The OH-groups that are present reside inside the crystal and do not react with water.) In aqueous solution the surface charge density of mica is normally orders of magnitude lower than the lattice charge of -0.33 C m -2. The reason for this is that protons and other cations present in the solution adsorb, and thus decrease the net charge [211.

In contrast to the mica surface, the glass surface is not equally well defined. It contains siloxane bonds as well as silanol (Si-OH) groups and sialic acid (Si-O-) groups that may extend from the surface as short polymeric chains (see Fig. 2). It is the presence of sialic acid groups that gives the glass surface its negative charge. The ratio of different surface groups vary between different glasses and thus glass prepared and cleaned in different ways may have different surface properties. ESCA spectra for muscovite mica and the

70 C. Eriksson et al. / Colloids Surfaces B." Biointerfaces 9 (1997) 67-79

C l e a v e ~ O O 0 O O O O

:hnet

C l e a v e l i S ~

O Po ta s s ium

0 Oxygen

• Silicon • A l u m i n i u m

Fig. 1. A schematic illustration o f the crystalline structure of muscovite mica. Note that hydrogen a toms are not shown. Hydroxyl groups are not located at the mica basal plane, instead they are situated below the surface oxygen inside mica lattice.

i / I / :iiiii: y ~ ~ ~,~i

Fig. 2. A schematic illustration of the glass surface structure.

• Hydrogen

• Silicon

O Oxygen

glass used in the present study of adsorption from blood is illustrated in Fig. 3. The main peaks in the spectrum from mica show A1, Si, O and K, as expected from the crystal structure. The glass spectrum shows the presence of silicon, oxygen and sodium in the approximate molar ratio 1:2.4:0.95. Some carbon contaminants adsorbed from the surrounding air is also seen on both the muscovite mica and the glass surfaces.

By directly measuring the forces acting between mica surfaces and between glass surfaces in electro- lyte solutions using so-called surface force techniques [16-18] it is possible to determine the surface potential and surface charge density under various conditions. Much data from such studies are available in the literature [18,19,22] and some examples from KC1 and KBr solutions at pH 5.5-6.0 are shown in Fig. 4. Clearly, muscovite mica is more negatively charged than glass and this affects the adsorption of surfactants and proteins as shown below.

3.2. Adsorption of CTAB

Cetyltrimethylammonium bromide (CTAB) is a cationic surfactant that adsorbs strongly to both mica and glass. When CTAB adsorbs to the surfaces the negative interfacial potential is first reduced and at higher CTAB concentrations the interfacial potential becomes positive (Fig. 5). As can be seen from Fig. 5, the potentials from surface force measurements obtained on mica and glass are very different in all CTAB solutions. This is due to their different surface chemistry, particu- larly the larger negative charge on bare mica compared to on bare glass. The initial surfactant adsorption is electrostatically driven and the electrostatically bound surfactants are oriented with the nonpolar part towards the solution. When the charge neutralization point has been reached (at 0.0033 mM for mica and at 0.0459 mM for glass) electrostatic forces oppose further adsorption. The driving force for adsorption is now a hydrophobic

C Eriksson et al. / Colloids Surfaces B." Biointerfaces 9 (1997) 67-79 71

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Binding energy (eV) Fig. 3. Overview photoelectron spectra for muscovite mica (A), and for glass (B). The carbon signal emanates from adsorbed airborn contaminants whereas the other peaks originate from the substrate surfaces.

72 C. Eriksson et al. / Colloids Surfaces B: Biointerfaces 9 (1997) 67-79

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a n d g lass ( [ 3 ) p l o t t e d a g a i n s t t he sa l t c o n c e n t r a t i o n .

interaction between the surfactant tails and the adsorbed layer of surfactants already present at the surface. At the charge neutralization concentration (cnc) more surfactants are adsorbed to the more negatively charged mica than to glass. This results in a higher contact angle on mica surfaces, ca 90 °, compared to 65 ° for glass at the cnc [23]. A consequence of the higher hydrophobicity of mica at the cnc is that the driving force for further adsorption is higher on mica than on glass, explain- ing why the interfacial potential becomes more

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positive on mica than on glass at high CTAB concentrations.

3.3. Adsorption of lysozyme

Chicken egg white lysozyme is a small compact protein with dimensions of ca 4.5 × 3.0 × 3.0 nm. With an isoelectric point of about 11 it is positively charged at pH 5.6. Lysozyme carries a net charge

C. Eriksson et al. / Colloids Surfaces B." Biointerfaces 9 (1997) 67-79 73

of + 9 per molecule at this pH, with most of the positive charges located around the active site cleft which in turn is situated on a side parallel to the molecule's long axis [24]. On the side opposite the active cleft is a relatively large hydrophobic patch that may be involved in the dimerization of lysozyme, known to take place in concentrated solutions [24]. The adsorption isotherms on mica and silica are very different (Fig. 6) due to the difference in surface chemistry. The adsorption is much larger and more of a high affinity type on mica than on silica. This is likely due to the higher surface charge density on bare mica as compared to on bare silica surfaces. On mica the adsorption is high, ca 3 mg m-2 already at the lowest lysozyme concentration investigated, 0.001 mg m1-1, corresponding to nearly a hexagonal close packed end-on monolayer (3.10 mg m-2). We note that on mica the adsorbed amount increases with increasing lysozyme concentration and reaches a value of close to 4 mg m -2 at a lysozyme concentration of 0.2 mgm1-1. This adsorbed amount is more than can be accommodated in a monolayer and it thus appears that a partial bilayer does form on the surface, in agreement with data obtained from surface force measurements. The adsorption

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Fig. 6. The amount of lysozyme adsorbed on mica (O) as a function of concentration measured by means of ESCA. O, Amount of chicken egg white lysozyme adsorbed on silica measured by means of ellipsometry. In both cases the solution also contained 1 mM NaC1 and the pH was 5.6.

isotherm of lysozyme on glass displays a gradual increase of the adsorbed amount with increased protein concentration. About 20% of the lysozyme is desorbed from the silica surface upon rinsing, whereas no desorption is observed from the mica surfaces [16], indicating that the binding of lysozyme to silica is weaker than to mica.

3.4. Initial reactions with blood

The amount of surface-adsorbed fibrinogen, prothrombin/thrombin, C lq and C3c was measured as mean brightness of the picture after blood exposure for 5 s (Fig. 7A). Fibrinogen was the most abundant protein found on both surfaces. When staining with anti-fibrinogen the mean brightness was ca 40 on both surfaces and there were no significant difference (P > 0.05) in brightness between the two surfaces. On the hydrophilic glass surface staining with antibodies against prothrombin/thrombin, C lq and C3c showed a mean brightness of ca 20 and there were no signifcant differences (P>0.05) in brightness between the three antibodies. When staining with antibodies against prothrombin/thrombin, Clq and C3c on mica the mean brightness was ca 8 and there were no significant differences in brightness between the three antibodies (P>0.05) .

The results show that the main difference between the surfaces was in the higher surface concentration of serine proteases at the hydrophilic glass surface. This finding is in agreement with results of others, showing that glass is an efficient activator of serine proteases [ 13].

The amount of platelets and von Willebrand factor was measured as fluorescence coverage of the surface after blood exposure for 5 s (Fig. 7B). When staining with anti-platelet membrane antigens mica had a coverage of 3 .2_ 0.4% compared to 2.4_+0.3% on the glass surface. Antibodies against von Willebrand factor showed a very low coverage on the mica surface while the hydrophilic glass had a 16 times higher coverage, 0.6_+0.2%. The von Willebrand factor was found localized to the platelet surfaces. The main difference between the two surfaces was thus in the higher levels of von Willebrand factor binding to platelets adhering to hydrophilic glass. This finding is in agreement

74 C Eriksson et al. / Colloids Surfaces B." Biointerfaces 9 (1997) 67-79

t5 gi -H

Ix) ¢-

m

50

40

30

20

10

T " ~ hydrofil ic glass

(a)

4

3 .5

r ; 2 . 5 U~ +1

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1.5 0 t j

1

0 .5

Fibr inogen P r o t h r o m b i n C l q C3c

mica

[] hydrofil ic glass

P l a t e l e t s von Wi l lebrand

(b)

Fig. 7. (A)Amount of surface-adsorbed fibrinogen, prothrombin/thrombin, Clq and C3c on mica and hydrophilic glass after blood exposure for 5 s. Brightness in pictures was measured by immunofluorescence and computer-aided image analysis (Adobe Photoshop), where 256 represented maximum brightness. Background levels were substracted from the presented data. Mean and SD are given (n = 6). (B) Coverage above background of platelet membrane antigens and yon Willebrand factor on mica and hydrophilic glass measured by immunofluorescence and computer-aided image analysis (Adobe Photoshop) after 5 s of blood exposure. Mean and SD are given (n=6).

with a s ta tement made by Soberano et al. [25], showing that only th rombin-ac t iva ted von Wi l l ebrand factor binds to platelets. In a previous

study, using longer exposure time, the t h rombin activity was no t detected, bu t the b ind ing of von Wi l lebrand factor to platelets was reported [26].

C. Eriksson et al. / Colloids Surfaces B. Biointerfaces 9 (1997) 67-79 75

This phenomenon illustrates the need for kinetic measurements, since some important reactions may occur transiently.

3.5. Platelet activation and adhesion

After blood exposure for 2, 4 and 8 min the amounts of CD62, thrombospondin and platelet membrane antigens was measured as percentage fluorescence coverage of the surface (Fig. 8). When staining with anti-CD62 on mica the coverage was low, 0.05 _+ 0.02%, the first 4 min but increased to 0.6_+0.1% after 8 min. The coverage of CD62 on the hydrophilic glass surface increased the first 4 rain to 0.4_+0.04% but decreased to 0.2_+0.03% after 8 min, a coverage lower than seen after 2 min.

The coverage of thrombospondin increased on both surfaces with time. Thrombospondin was always present in higher amounts on the hydrophilic glass surface than on mica. When using anti- thrombospondin on mica the coverage increased slowly the first 4 min, but between 4 and 8 min it increased seven times to 0.9_+ 0.2%. On the hydrophilic glass surface the increase in coverage was more continuous over the time measured.

When staining with platelet membrane antigens on mica the coverage was 2 .7+0.4% after 2 min and increased to 4.8 + 1.1% after 4 min. There was no further significant increase seen in coverage after that (P > 0.05) On hydrophilic glass the coverage of platelet membrane antigens was 1.6___ 0.3% after 2 min. The coverage did not change significantly between 2 and 4 m i n (P>0 .05) but had increased to 2.6 +0.3% after 8 min.

3.6. Normalization of CD62 values

The coverage of CD62 was normalized to coverage of platelet membrane antigens after 2, 4 and 8 min of blood exposure (Fig. 9) to see how platelet expression of CD62 changed with time. When only looking at coverage of CD62 the differences with time on one surface and differences between two surfaces does not take into account the fact that more coverage of CD62 on one surface can be a direct result of more platelets present secreting CD62 than on the other surface. Normalized values of CD62 showed that at 2 and 4 m i n secreted CD62 per platelet lay constant at 0.2 units but after 8 min the secretion per cell was reduced

r~

-H

O O)

== O

2 0

16

12

[ ] platelets mica

O platelets hydrofilic glass

CD62 mica

[] CD62 hydrofilic glass

• thrombospondin mica

[ ] thrombospondin hydrofilic glass

2 4 8 Time (rain)

Fig. 8. Coverage above background of platelet membrane antigens, CD62 and thrombospondin on mica and hydrophilic glass after blood exposure for 2, 4 and 8 min. Coverage was measured by immunofluorescence and computer-aided image analysis (Adobe Photoshop). Mean and SD are given (n=6) .

76 C Eriksson et al. / Colloids Surfaces B: Biointerfaces 9 (1997) 67-79

0.3

0.25

0 .2 O~ .m

0 o 0.15 "0

m E 0.1 0 z

0 . 0 5

E~ CD62/p la te le t hydrofilic glass

[ ] CD62/p la te le t mica

2 4 8 T i m e (rain)

Fig. 9. Normalized coverage of CD62 per platelet on mica and hydrophilic glass as a function of time of blood-material contact. Immunofluorescence and computer-aided image analysis (Adobe Photoshop) were used to measure coverage. Mean and SD are given (n=6 ) .

by 50% (Table 1). On mica normalized values of CD62 showed a low level of secretion per cell at 2 and 4 min of blood exposure, 0.02 and 0.01 units, respectively, but between 4 and 8 min the normalized value increased 10 times to 0.1 units (Table 1 ). Thus, the kinetics of platelet adhesion and activation differed between the surfaces. The results indicate that activated platelets leave the glass surface, and has a longer staytime at the mica surface. This result is in agreement with those of previous studies [26].

3.7. Adhesion and activation of neutrophils

Anti-CD 11 b was used to detect leukocyte activation after blood exposure for 8, 16, 32 and 64 min

Table 1 Normalized coverage of hydrophilic glass and mica with CD62/platelet membrane antigens after 2, 4 and 8 min of blood exposure (n = 6)

Time (min) Hydriphilic glass Mica

2 0.2 0.02 4 0.2 0.01 8 0.1 0.10

and was measured as percentage fluorescence coverage of the surfaces (Fig. 10). On the mica surface CD1 lb was present in small amounts after 8 min. During the first 32 min the coverage increased and reached 1.8_+0.4% after 32 min. At 64 min, how- ever, the coverage had decreased to 0.6 _+ 0.1%. On the hydrophilic glass surface C D l l b showed low coverage after 8 min. The coverage did not change significantly during the first 16 min, but thereafter the coverage increased with time to 7.4_+0.4% after 64 min. Comparing the two surfaces there was no difference in coverage of C D l l b after 8 min. At 16 min hydrophilic glass had more than twice the coverage of mica and this difference in coverage increased to > 12 times after 64 min. The surfaces were stained with acridine orange to detect leukocytes after 8, 16, 32 and 64min of blood exposure. The amount of stained cell nuclei was measured as percentage fluorescence coverage of the surface (Fig. 10). After 8 min of blood exposure 1.9_+0.6% of the mica surface was covered with acridine orange stained leukocytes and the coverage did not change significantly (P>0.05) during the following 56 min. On the hydrophilic surface acridine orange stained cells increased with

C. Eriksson et aL / Colloids Surfaces B." Biointerfaces 9 (1997) 67-79 77

u~ 44

.m

> 0 0

12 ~q E) CD11b mica -4

1 0 (3 C D 1 1 b hydrofilic glass

acridine orange mica

[ ] acridine orange hydrofilic glass

8 16 32 64 T ime (min)

Fig. 10. Coverage of CD 11 b and acridine orange stained leukocytes between 8 and 64 min of blood exposure on mica and hydrophilic glass, measured by immunofluorescence and computer-aided image analysis (Adobe Photoshop). Mean and SD are given (n = 6).

time and after 64 min reached a coverage of 10.5+ 1.3%. The difference in coverage between the surfaces increased with time and after 64 min four times more coverage of acridine orange stained cells was found on the hydrophilic glass surface than on mica.

The results show that P M N cells leave the mica surface, but accumulate on the glass surface.

3.8. Normalized values of CDllb

had four times more C D l l b per cell than hydrophilic glass. After 32 min, on the other hand, the hydrophilic glass surface had 1.4 times more CD1 lb per cell than mica and this difference had increased to 3.5 times after 64min. The results show that hydrophilic glass is more inflammatogenic than mica, measured as the expression of the CDl lb- in tegr in (complement receptor), and that activated cells are downregulated at the mica surface (Table 2).

Coverage of CD 11 b was normalized to coverage of acridine orange stained leukocytes after blood exposure for 8, 16, 32 and 64min to see how CD1 l b expression in leukocytes varied with time and between the two surfaces (Fig. 11 ). On hydrophilic glass the normalized values of CD1 lb lay constant at 0.1 during the first 16min of blood exposure, increased to 1.0 after 32 and then decreased to 0.7 after 64 min. On mica normalization of CD1 l b values showed that the coverage increased during the first 32 min to 0.7 units but decreased between 32 and 64 min of blood exposure to 0.2 units. Compar ing the surfaces equal amounts of C D l l b per cell was seen on both surfaces after 8 min but at 16 min the mica surface

4. Conclusions

Two negatively charged and hydrophilic surfaces, muscovite mica and glass, were found to bind detergents and protein with different affinity, reflecting the importance of surface properties other than hydrophilicity. The two surfaces also induce different responses when in contact with whole blood. The most striking difference in this respect is that P M N cells show a higher degree of integrin CD 11 b expression on the hydrophilic glass than on mica surfaces. The underlying process is an array of subreactions, the kinetics of which,

78 C. Eriksson et al. / Colloids Surfaces B: Biointerfaces 9 (1997) 67 79

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0

CD11b/a.o hydrofilic g lass

CDllb/a.o mica

_

8 16 32 64 T i m e ( r a i n )

Fig. 11. Coverage of CDl lb normalized to coverage of acridine orange stained leukocytes after 8, 16, 32 and 64 min of blood exposure. Immunofluorescence and computer-aided image analysis (Adobe Photoshop) were used as measurements of coverage. Mean and SD are given (n = 6).

Table 2 Normalized coverage of hydrophilic glass and mica with CD1 lb/acridine orange after blood exposure for 8, 16, 32 and 64 min (n = 6)

Time (min) Hydrophilic glass Mica

8 0.1 0.I 16 0.1 0.4 32 1.0 0.7 64 0.7 0.2

rather than qualitative differences, will govern the final result.

Acknowledgment

This study was supported by grants from the Swedish Medical Research Council (06235), The Swedish Natural Science Research Council and The Research Council of Engineering Sciences. We would like to thank Mikael Sundin for operating the ESCA spectrometer.

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reactions of two hydrophilic surfaces with detergents, protein and whole human blood

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