Fluid shear induced endothelial cell detachment from modified polystyrene substrata

COLLOIDS AND SURFACES

B

Collolds

and Surfaces

B: Bmnterfaces

3 (1994) 147-l j8

Fluid shear induced endothelial cell detachment from modified polystyrene substrata T.G. van Kooten a, J.M. Schakenraad b, H.C. van der Mei a, A. Dekker ‘, C.J. Kirkpatrick d, M. Walter e, D. Korzec e, J. Engemann e, H.J. Busscher a** a Laboratory for

Materia Technica, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands b Centre for Biomedical Technology. University of Groningen, Oostersingel59. Gb 25, FO, 9713 EZ Groningen. The Netherlands ’ Institute of Pathology, Klinikum der R WTH Aachen, Pauwelstrasse 30, D-52074 Aachen. Germany d Institute of Pathology, Klinikum der Johannes Gutenberg Universitdt Main:, LangenbeckstraJe 1, D-55131 Mainz, Germany ’ Forschungszentrumfiir Mikrostrukturtechnik, Obere Lichtenplatzerstrafle 336. 42287 Wuppertal2. Germany

Received 5 October 1993; accepted 28 February 1994

Abstract In this study, human umbilical vein endothelial cells were seeded for 3 and 24 h on polystyrene (PS), tissue culture polystyrene (TCPS) and polystyrene modified by oxygen plasma treatment (PtPS) in order to investigate their detachment behaviour during exposure to fluid shear. All three materials have smooth surfaces at the submicron level. Equilibrium water contact angles were higher on PS (88’) than on TCPS (78’) and PtPS (79’). Furthermore, contact angle hysteresis, i.e. the difference between advancing and receding angles, was much larger on TCPS (39’) and PtPS (41”) than on PS (14”), indicating either a large surface heterogeneity or a possible reorientation of functional groups on TCPS and PtPS. X-ray photoelectron spectroscopy data revealed that both TCPS and PtPS had oxygen incorporated at their surfaces (18.6% and 9.9%, respectively), whereas only TCPS had a small amount of nitrogen (0.9%) at its surface. Cells seeded on these materials w&e exposed to various shear stresses in the range 88-352 dyn cm -’ in order to gain more insight into the influence of the specific physico-chemical surface properties of these polystyrene surfaces on cell retention, cell morphology and migration. The retention of cells having adhered during 24 h on TCPS and PtPS was not different from the retention of cells having adhered during 3 h, but retention on TCPS and PtPS was higher than on PS. At shear stresses of 88 and 176 dyn cm-‘, however, differences in the morphology of cells adhered to TCPS during 3 and 24 h were observed, indicating that between 3 and 24 h changes in cell-substratum interactions occurred. Migration on TCPS and PtPS was accompanied by the presence of fibrillike structures left behind on the surface. Summarizing, this study shows that cell retention is higher on modified polystyrenes than on polystyrene, the only major difference between the polystyrenes being the higher contact angle hysteresis and oxygen content of the modified polystyrene surfaces.

Keywords: Endothelial

* Corresponding

cell adhesion; Parallel plate flow chamber; Polystyrene; Surface modification;

author.

0927-7765,94,507.00 D 1994 Elsevier Science B.V. All rights reserved .SSDI 0927-7765(94)01133-P

Retention

1. rntr~oction

Endothelial cells constitute the natural inner lining of blood vessels, and as such possess antithrombogenic properties. These properties can be utilized in the application of vascular prostheses, which are used to replace occluded or otherwise malfunctioning blood vessels. Seeding of vascular prostheses with endothelial cells therefore is a widely studied field in vascular research, however still with a low efficiency. Materials which are frequently used for vascular prostheses include polyester Dacron’@ and (expanded) polytetrafluoroethylene. These materials demonstrate low adhesive properties. It has been demonstrated. however, that precoating with extracellular matrix proteins or fibronectin profoundly increases the adherence of endothelial cells to these materials [l--5]. Although fibronectin precoating increases initial cell adhesion, cell retention during exposure to shear stresses in the order of 10 dyn cm-‘, a physiologicahy relevant shear stress in veins, is stili disappointin~Iy low [6--S]. In general, the massive inefficiency of cell seeding is at the very root of the failure of seeded vascular prostheses made out of these hydrophobic materials [9,10]. It is known that substrata with low or intermediate hydrophobicity sustain optimal cell adhesion and spreading [11,12]. Therefore, it is surprising that these materials have not been used more frequently for vascular prostheses to be seeded with endothelia1 cells. Surface properties can be modified by, for exampIe, plasma treatment, which has been advocated to be a good way of improving the adhesion of endothelial cells to vascular graft materials [ 131. Enhancement of endothelial cell adhesion to plasma-treated grafts indeed is suggested by the formation of a neoartery within a polyetherurethaneurea-based vascular prosthesis with a plasma-treated inner surface after 5 weeks of implantation [ 143. The aim of this study is to determine whether the detachment behaviour, including the migration and morphological responses of human umbilical vein endothehaf cells on polystyrene. can be influenced by plasma treatment. To this end, experiments were performed on standard polystyrene

(PS), tissue culture polystyrene (TCPS) and polystyrene modified by oxygen plasma treatment ( PtPS) during exposure to different shear stresses.

2. Methods 2.1. SLibstrntn

Polystyrene and TCPS, y-irradiated during manufacture, were cut from Petri dishes (diameter, 14 cm; Greiner) to 76 x 50 x 1.05 mm3 (length x width x thickness) plates, taking care not to touch the surface. Ten polystyrene plates were modified by an oxygen plasma treatment (PtPS) in a 40 x 40 x 40 cm3 reactor chamber. First, a vacuum of 10-j mbar was achieved in the chamber by a combination of a Trivac DXBDS rotary pump (Leybold) and a Turbovac 450 (Leybold) turbo molecular pump. Oxygen gas (99.995% purity) was passed into the vacuum chamber at a partial vapour pressure of 8 x lo-” mbar. A plasma was generated by an IIFQ 1303-3 ion source, supplied by a Hiittinger TIS 0.5/13.56 MHz HF generator. The ion source is flanged at the vacuum chamber and is penetrated by a magnetic field, parallel to the cylinder axis. The sample is fixed onto a target holder in the centre of the vacuum chamber and located in the remote plasma at the rear side of the holder, which means that it does not “see” the ion source. The energy of the beam ions was about 60 eV, but the ions which reached the PS surface from the remote plasma had lower energies, of a few electron volts (below 10 eV). The exposure time to the oxygen plasma was 13-15 s, although sometimes up to 20 s were necessary to attain the desired equilibrium contact angles. The operating’ temperature for the target sample was kept at 2O’C. All substrata were exposed to UV light in a sterile laminar flow cabinet during 8 h for sterilization prior to further experimentation. 2.2. Slrrfirce chnmcteri:ntion Water contact angles on the different substrata were measured using the sessile drop technique. The equilibrium contact angle (f?,,) was measured when the water droplet rested on the solid surface

1. G. van Kooten rt al. :CoNords Surf&w

B. Blomtrrfaces 3 ! 1994) 147-158

149

after withdrawal of the micro-syringe needle. Advancing (0,) and receding (6,) contact angles were determined after the droplet was forced to advance over the surface, by moving the substratum horizontally while keeping the microsyringe needle in the droplet. Contact angle measurements were performed in triplicate. Surface roughness was measured with a surface tracing instrument, the Perthometer C5D (Perthen), equipped with a stylus of radius 2 urn. High pass filtered profiles were recorded and the stylus surface roughness R, was derived [ 151. The R, values presented are average values over 6 tracings per sample. X-ray photoelectron spectroscopy (XPS) was performed using an S-Probe apparatus (Surface Science Instruments) equipped with a monochromatic X-ray source at a photoemission angle of 35’. Relative atomic percentages of each traced element were estimated from peak areas of widescan spectra over a spot area of 250 x 1000 urn’ using sensitivity factors supplied by the manufacturer. Carbon and oxygen atoms in different functional groups were analyzed by decomposition of the C 1s and 0 1s peaks with reference to the C-C peak around 284.8 eV, imposing a full-width-athalf-maximum of 1.35 eV and 1.6 eV, respectively, and taking into account the binding energies of the various functional groups present. Three samples of each substratum were analyzed.

The flow circuit has been described in detail previously [17]. Briefly, it contains three basic modules: the flow loop, the heating system, and the image analysis components. The medium is pumped through the flow loop by a peristaltic pump (Watson-LMarlow 503U with 1.5, 4 or 8 mm i.d. tubing) into a damping vessel. This vessel dampens the pulsations of the flow into a steady flow. The medium then flows through a parallel plate flow chamber mounted on a phase-contrast microscope stage. Medium is collected in a thermostatted double-walled vessel. The flow chamber itself is heated directly with four power resistors (33 R each), connected in parallel with each other to a power supply (10 V) with feedback from a PtlOO thermocouple situated in the downstream compartment of the flow chamber. The chamber dimensions are 76 x 38 x 0.20 mm3 (length x width x height).

2.3. Cell chrre

2.5. Experimental

umbilical endothelial cells Human vein (HUVEC) were cultured as follows. Cells were freshly harvested from the umbilical cord vein essentially as described by Jaffe et al. [ 161. Vein segments were cannulated and rinsed with HEPES buffer (0.137 M NaCl, 4.0mM KCl, 10mM HEPES, 10 mM glucose; pH 7.55). Endothelial cells were isolated using 0.1% collagenase CLS I (Worthington)/O.Ol% EDTA in HEPES buffer supplemented with 0.25% bovine serum albumin (BSA) for 15 min at 37’C. The primary culture (PO) and subsequently passaged cultures were grown in Ham’s F12 nutrient medium with Lglutamine and 25 mM HEPES f 1:l). supplemented with 100 U ml-’ penicillin, 100 ug ml-’ strepto-

Cells were harvested by treatment with collagenase as described above and seeded on the substratum in medium with 20% HPS. Seeding densities were approximately 5 x 10’ cells ml-‘, resulting in After allowing the cells to 100-200 cells mm-‘. adhere and spread for 3 or 24 h, the substratum plate was placed in the flow chamber, which was then assembled in the flow circuit. Subsequently, cells were exposed to a shear stress created by a flow of serum-free culturing medium. Cells, which were spread at the start of the experiment, were observed and processed with an image analysis system as described previously [17]. The analysis of the detachment and migration behaviour was performed by marking 50 spread cells in

mycin (all from Gibco, Life Technologies) and 20% human pooled serum (HPS) at 37 ‘C in 5% CO2 and 95% humidity. At confluency, cells were passaged using 0.1% collagenase in a splitting ratio of 1:3. Cells were routinely grown on tissue culture quality plastic flasks (Greiner) precoated overnight with 0.2% gelatine in PBS. Cells at passage P2 were harvested for the experiments. 2.4. Flow circuit

set-up

150

T G van Kooten et al. Col1od.s Surfuces B. Biointrrfacrs 3

the starting image prior to exposure to flow. These cells were randomly selected, implying thus that detachment at any time is representative of the entire population. Changes in the following parameters were determined: cell numbers, spreading areas (A) and shape factors (S), with S being defined as S=-

P

(1)

2&z

where P is the perimeter of a cell. Furthermore, migration (D). defined as the displacement in the direction of flow, was followed by determining the position of the upstream border of the individual cells. The shear stress t, reported in this paper is derived from

6Q 5,=/1--y wh-

where p is the viscosity, Q is the flow rate, h is the chamber height and w is the chamber width. Occasionally, substratum plates were removed from the chamber and prepared for scanning electron microscopy as described previously [ 181.

3. Results In Table 1, surface characteristics including contact angles, surface compositions by XPS and roughnesses are shown for PS, TCPS and PtPS. The data indicate that TCPS and PtPS are similar Table 1 Surface characteristics of PS, TCPS and PtPS, roughness R, and elemental surface composition Substratum

PS TCPS PtPS

0,” (deg)

90* 1 82 & 2 76 k 2

Q, Meg)

76+ 1 43 * I 35 + 6

includmg

f-4qa (deg)

88 i: 2 78 i. 3 79 * 2

a Standard deviattons are gtven over 3 samples. b Standard deviations are given over 6 tracings per sample. ’ Neglecting hydrogen.

advancing,

/ 1991) 147-158

with respect to their wettability. PS is slightly more hydrophobic than TCPS and PtPS and presents considerably less contact angle hysteresis. The surface roughnesses RA of the various polystyrenes vary over a factor of two, but are all well in the submicron range. Both PtPS and TCPS had oxygen incorporated at their surfaces, whereas TCPS contains some nitrogen. For PS, oxygen was not found on the surface. In Table 2, the contributions of the different kinds of functional groups to the C 1s and 0 1s peaks are presented (see also Fig. 1). TCPS and PtPS contain similar percentages of the different functional groups associated with carbon and oxygen, although absolute atomic percentages of oxygen for PtPS were found to be twice as low. In Fig. 2, the percentage of cells adhered to TCPS, PtPS and PS is shown as a function of time for cells adhered during 3 and 24 h and subsequently exposed to various shear stresses. As cells adhered to TCPS only showed minor detachment at shear stresses of 88 and 176 dyn cm-‘, it was decided to test PS and PtPS only at the higher shear stresses. No significant differences in cell detachment after 150 min exist between the seeding times of 3 and 24 h for TCPS and PtPS, whereas on PS cells more readily detached after 3 h adhesion than after 24 h adhesion (~~0.05; StudentNewman-Keuls (SNK) test). In addition, cells adhered to PS showed significantly more detachment than cells adhered to TCPS and PtPS (p c 0.05; SNK test). In Fig. 3, the mean residual areas of cells adhered

recedin g and equilibrium

RAb (urn)

0.010 f 0.001 0.02 1 + 0.008 0.019 + 0.007

Elemental

surface

water

contact

angles,

the surface

composition”

C (at.%‘)

0 (at.%‘)

N (at.%‘)

100 80.5 k 0.3 90.1 t 0.4

0 18.6 + 0.2 9.9 f 0.1

0 0.9+0.1 0

T.G Table 3 Decompositions Sample

PS TCPS PtPS

WI Kooten et al.:Colloicis

of C 1s and 0 1s peaks into the contributton

Surfuces

B Blomrerfkces

of dtfferent functronal

C 1s peak

3 I19941 147-153

groups

( percentage

151

of total) for PS. TCPS and PtPS

0 Is peak

284.8 eV”

286.3 eV

287.7 eV

533.5 eV

533.5 eV

100 93.7 * 0.3 93.5 i 0.6

0 5.8 + 0.2. 5.7 + 0.5

0 0.5 * 0.3 0.8 + 0.3

0 59.3 f 2.2 63.5 + 6.’

0 JO.7 & 1.1 37 2 _t 6.2

Standard deviations are given over 3 samples. ’ The binding energy of the C 1s peak component

representing

to TCPS, PtPS and PS are shown as a function of time for cells adhered during 3 and 24 h and subsequently exposed to various shear stresses. Mean areas at the onset of flow were similar for TCPS (4505 um2) and PtPS (4589 urn’), but significantly lower for PS (3449 urn’, p cO.05; f-test). All curves display a relatively fast decrease during the first 30 min of exposure to shear stress, followed by a more transient decrease later on. In Fig. 4, the mean shape factors of cells adhered to the different substrata are shown as a function of time for cells adhered during 3 and 24 h and subsequently exposed to various shear stresses. Mean shape factors at the onset of flow were similar for all three substrata. However. shape factors increase faster for cells exposed to higher shear stresses than for cells exposed to lower shear stresses. Furthermore, in the first 30 min after the onset of flow, increases in shape factors occur faster for cells having adhered during 24 h than for cells having adhered only 3 h for TCPS and PtPS and all shear stresses. Elongation of cells observed in these experiments could not be correlated with any preferential orientation with respect to the direction of flow at any stage of the experiment. In Fig. 5, the mean migration distances of cells adhered to the different substrata are shown to be a linear function of time (linear correlation coefficients greater than 0.985). From Fig. 5, it can be seen that migration rates increase as shear stress increases up to a maximum value and are higher for cells having adhered during 24 h than for cells having adhered during 3 h. In Fig. 6, light micrographs are shown of the representative behaviour of HUVEC adhered to TCPS, PtPS and PS during exposure to shear

C-C

bonds was set to 25-l 8 eV.

stresses of 132 dyn cmW2, 264 dyn cm-? and 264 dyn cm-‘, respectively. At a shear stress of 132 dyn cm-‘. cells remained spread on TCPS despite considerable changes in morphology (Fig. 6a). Sometimes, cells are obstructed by others during their migration (Fig. 6a), but in these cases the faster moving cells pass along the slower moving ones. Apart from migration and maintenance of a spread morphology, occasionally respreading of cells at the point of detachment is observed on TCPS (Fig. 6a) and PtPS (Fig. 6b), even at a shear stress of 264 dyn cm-‘. Cells adhered to PS do not show this response, but adapt a more nonspread morphology after exposure to shear (Fig. 6~). In Fig. 7. scanning electron micrographs are shown of HUVEC adhered to TCPS after exposure to shear stresses of 176 dyn cm-’ (Fig. 7a) and 352 dyn cm-’ (Fig. 7b) and to PtPS (Fig. 7c) after exposure to a shear stress of 352 dyn cm-‘. PS was not included as the cell numbers after exposure to shear were too small. Fibrillar structures aligned in the direction of flow were present upstream of migrating cells at the higher shear stresses on both TCPS and PtPS.

4. Discussion In this study, human umbilical vein endothelial cells were exposed to shear stress in a parallel plate flow chamber. The cell behaviour with respect to cell numbers, shape and migration was determined for cells adhered to polystyrenes with different surface properties as created by plasma treatment and for adhesion times of 3 and 24 h.

TCPS -,

2

295

291

287

283

279

273

/

,

345

541

537

533

329

525

529

525

Binding Energy (eV)

Binding Energy (eV)

PtPs

tL__.--295

291

287

283

279

275

545

I

1 I

541

537

533

Binding Energy (ev)

Binding Energy (eV)

PS

295

291

287

283

279

275

Binding Energy (eV) Fig. 1 C Is and 0 groups is shown.

Is peaks obtatned from XPS for PS, TCPS and PtPS. The result of decomposttlon

mto the several functtonal

T.G vantYoorenetal.:CoNordsSurfaces B. Biomrerfuces3 / 1994)f#LlS

u-l =:

'

cn

60 L 0

30

60 Time

90

120

150

c?

153

0; 0

30

60 Time

(minutes)

90

120

150

120

150

120

150

(m:nutes)

h

2z

ul

0 z

60 0

30

60 Time

90

120

05 0

150

30

60 Time

(minutes)

90 krwures)

c

z

2 Time

(mnutes)

Fig, 2. Percentage of ceIIs present as a function of time for HUVEC having adhered to (A) TCPS, (B) PtPS or (Cl PS during 3 h (filled symbols) or 24 h (empty symbols) and exposed to shear stresses of (&a) 88 dyn cm-‘, (Ir,O) I76 dyn cm-‘, (M,Cl) 264 dyn cm-? or (+,O) 351 dyn cmeL. Average values of 3 experimental runs on different HUVEC isolates are presented. Standard deviations are about 7% on average. Note the dtfferent scales of the 4’ axes.

The modified polystyrenes TCPS and PtPS differ from native polystyrene in two aspects: (i) the modified materials have approximately a two- to three-fold larger water contact angle hysteresis predominantly due to smaller receding angles; (ii) the oxygen content of the modified surfaces is

oL----0 30

’

’

60

90

Time

(minutes)

Fig. 3. Mean residuai areas of cells present as a function of time for HUVEC having adhered to (A] TCPS, (B) PtPS or (C) PS during 3 h (fiiled symbols) or 24 h (empty symbols) and exposed to shear stresses of (A,n) 8s dyncm-2, (0.9) 176 dyn cmm2, (11.3) 264 dyncm-’ or (+,O) 352 dyncm-s. Average values of 3 experimental runs on different HUVEC tsolates are presented. Standard errors of the mean are about 15% on average.

considerable. The equilibrium water contact angles for TCPS are higher than values reported by Schakenraad et al. [ 191, who reported a value of 60’ which could be due to differences between batches. No data have been found for water contact angle hysteresis. The larger contact angle hysteresis observed on TCPS and PtPS as compared to PS

1.00

i

0

30

60 Time

B

150

90

:23

150

60

Time

90

?20

150

t20

150

720

?50

(mrwtes)

i

6

o.

30

k?:nutes)

H ,

0

!m

200

;; a

100

i--.-.__

0

30

60 Time

90

120

150

b

r

0 0

30

it? nutes)

60 Time

90

immutes)

too -0

30

60 Time

90

:20

150

irr-utes)

Fig. 4. Mean shape factors of cells present as a functmn of time for HUVEC havmg adhered to (?I) TCPS, (B) PtPS or (C) PS during 3 h (filled symbols) or 31 h (empty symbols) and exposed to shear stresses of (A,Ll) SY dyn cm-‘, 10.3) 176 or (+,3) 351 dyncm-“. dyn cm-“, (~,Cl) 764 dyncm-’ Average values of 3 experimental runs on dlfferent HUVEC isolates are presented. Standard errors of the mean are about 0.03 on average.

is unlikely to be due to roughness effects, as all R, values of the materials are within the submicron range. Thus this hysteresis either points to a surface chemical heterogeneity due to the modification treatment or to a reo~entat~on of functional groups on TCPS and PtPS in an aqueous phase [20]. The surface chemical composition data of native

_

z.

0

30

60 Time

90 (minutes)

Fig. 5. Mean migration distances of cells as a function of time for HUVEC havmg adhered to (A) TCPS, (Bi PtPS or(C) PS during 3 h (filled symbols) or 24 h (empty symbols) and exposed to shear stresses of (A,C) SSdyn cm-‘, (O.‘S,) 176 dyn cm-‘. (#,c?) 26-I dyn cm-* or (+.G ) 332 dqn cm-‘. Aberage values of 3 experimental runs on different HUVEC isolates are presented. Standard errors of the mean are about 1% on average.

PS do not fully agree with literature data [21 J, which report a small percentage of oxygen (1.5 at%). It is suggested. however, that exposure to mere daylight might give rise to oxygen incorporation in the surface due to UV radiation [X!]. The surface chemical composition of TCPS partly

agrees with literature data [23] ( 11.6 at% 0. 56.6 at9 C and 1.7% T. as in this study). but it should be noted that in an earlier study the same research group did not detect any nitrogen on TCPS from the same manufacturer [11] I IJ.Sslc 0: S5.1 at% C’). The C Is peak decompositions for TCPS and PtPS sllggest simi!ar functional groups on both materials dcspitz the presence of nitrogen on TCPS. Based upon rhe decomposition of the C 1s peak ( the przsencs of a significant C I ‘i component at 236.3 eV) and rhe 0 1s peak ! the component set at 532.5 eV is larger than the onr: at 533.8 rV). most of the oxygen in TCPS and PtPS secmj to be incorporated in hydrost-1 groups. i-1 Iarg2 variety of oxidation products on electron-beam-trz~~ted

polystyrene has recently been described by Onyiriukn [3]. including alcohol. ether. epoxide. carbonqi. acid and ester surfaces species. in an attempt to improve the tissue compatibility of potystyene with a single-step procedure. In general. hydrophilic materials give rise to larger cell spreading areas than hydrophobic materials [ 171. but thz differences in equilibrium contact angles on the present choice of substrata are too small to account for the obserl-ed differences in cell-material interactions. Thus. the lower celi retention on PS as compx’ed to retention on the slightly more h~dropIlilic modified polystyrenes probably is rt direct consequence of the larger contxt angle hysteresis and higher osygen content

Fig

7. Sconning

electron

100 I.UTI for the ovrrcirws rsposrd dyn

cm-’

Ileft)

of HUVEC

during

150 min. Arrows to a shear btress

cells

adhered

and 35 hrm for the higher

to a shear stress of 176 d-n cm -’

~lnd exposed migratinp

microgrxphs

indicate

to Is).

during

15Omin.

fibrillx

structures

cm-’ of 353 d~+n

during

f bl

TCPS

mngnilication

t bt

HLT:EC

orizntttted

145 min. Irrtws

and (c) PtPS.

micrographs adhered

durrng

in the Jireztion indicate

tibriilx

Flow

rrighri.

I‘: from

left to right.

(31 HYVEC

.idhered

3 h ~tnd eYpoWd ol’ tio\r

t0 a Jhex

ICI HCTEC

struc‘tur,x a- kl’t lxhind

Bars represent during

sdhsred on III:

? h and

SCRSS

during

jurfrxce

Of

351

2-L h by the

of the

modified poIystyrene surfaces, as these predifferences with native PS. The large contact angle hysteresis on TCPS and PtPS may be the result of a microdomain structure of oxygen-rich and oxygen-poor areas. Cell retention may then be influenced by the oxygen-rich domains. Most of the oxygen of TCPS and PtPS seems to be incorporated in hydroxyl groups, which have been shown to promote cell adhesion in the presence of serum proteins to a variety of hydroxytated polymers E25.261 and an optima1 density for cell adhesion has been suggested to exist [27,28]. The differences in cetl retention on the different polystyrene surfaces are mediated indirectly by differences in adsorption of adhesive proteins as, for example, fibronectin and vitronrctin. These differences include the ability of material surfaces to exchange non-adhesive proteins as, for example, albumin with fibronectin or vitronectin [29]. Despite the small difference in wettabiiity, this exchange of proteins is much more favoured on TCPS than on PS [21,30-331, resulting in the relative inability of cells to deposit their own fibronectin and v~trone~t~n on PS [x6,33]. The influence of adhesion time prior to exposure to shear (i.e. 3 or 24 h) is most obvious in the cellular behaviour on TCPS, especially at the lower shear stresses of 88 and 176 dyn cm-” (larger plateau values of spreading areas, less pronounced increase in shape factors and slower migration of cells), suggesting that cell-substratum interactions change over this time period. This could be due to the development of different cell-substratum contacts or changes in the proteinaceous conditioning film on the substrata by mechanical removal of the adsorbed proteins by adherent ceffs [34], followed by the deposition of endageneous proteins C331” The migration observed in this study probably involves physical tearing as indicated by the presence of fib&like structures left behind on the surface (Figs. 7b and 7~). We assume that the fibrils are attached to oxygen-rich microdomains on the TCPS and PtPS surfaces, in line with a heterogenous surface as indicated by the large contact angle hysteresis. The fib&like material may be ~brone~t~n depositions, as the structures show seat the major

striking similarities with endog~neously produced fibronectin fib& [35,36-J. HUVEC have been shown to produce these fibrils already within 3 h after contacting a solid surface [35]. Apart from physical tearing, enzymatic turnover of integrin aggregates may also be involved in migration, a process which only may take 10 min for rapidly advancing cells [37].

The authors a~kno~v~edge the assistance of F. Dijk (Department of Histology and Cell Biology, University of Groningen) in preparing the scanning micrographs and of 3. de Vries for carrying out the XPS measurements. References J.M. Seeger and N. Kiingman. 3. Surg. Res., 38 ( 1985) 64I. K.A. Kester, M.B. Herring. M.P. Arnold, J.L. Gtover, H.-M. Park, M.N. Hetmus and Ph.J. Bendick, J. Vast. Surg,, 3 ( 1956) 58. J. Kaehier, P. Zifta. R. Fasoi, Mi. Deutsch and bf. Kadletz, J. Vase. Surg., Q (1989) 535.

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