897
Influence of substratum wettability on the strength of adhesion of human fibroblasts T.G. van Kooten*, J.M. Schakenraadf, H. J. Busscher*
H.C. van der Mei* and
*Laboratory for Materia Technica, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; and +Centre for Biomedical Technology, University of Groningen, Oostersingel59, Gb 25, FO, 9713 EZ Groningen, The Netherlands
To determine the strength of adhesion and the detachment mechanisms of fibroblasts from substrata with different wettability, the behaviour of adhered ceils was studied in a parallel-plate flow chamber during exposure to shear. Adhered cells were observed in situ, i.e. in the flow chamber, by phase-contrast microscope and images were analysed semiautomatically. Detachment was found to be dependent both on shear stress and time, although a critical shear stress can be found below which no detachment occurs. On all substrata, cells round up before detachment and are approximately spherical immediately before detachment. The strength of adhesion calculated ranged from 0.6-3.5 X lo-” N per cell on FEP-Teflon@ (the least wettable material included) to 9.4 X lo-’ N per cell for glass (the most wettable). Ease of detachment seemed to decrease with increasing wettability. However, cells reacted more strongly with tissue culture polystyrene (TCPS) than expected on the basis of its wettability, probably due to surface chemistry. Keywords:
Cell-material
interactions,
fibroblasts,
cell adhesion,
wettability
Received 24 February 1992; accepted 26 March 1992
are frequently used in biomedical devices as, for example, vascular grafts, artificial skin, ocular lenses and bone prostheses. The interaction of these materials with surrounding tissues or body fluids is important. For example, a firm adhesion of bone to a hip prosthesis is required for optimal patency. Cellular interactions with biomaterials are an important area of research. Cell adhesion to and spreading on a biomaterial is, amongst other factors, dependent on the surface wettability of the biomaterial. Schakenraad et al.’ reported that cells spread poorly on hydrophobic substrata and more extensively on more hydrophilic substrata. Absolom et al.’ reported a similar dependence on substratum wettability for endothelial cell coverage of the substratum. Van Wachem et ale3 found that human endothelial cells adhered optimally (in terms of numbers) on moderately wettable polymers. In a well-defined hydroxyethylmethacrylate-methylmethacrylate copolymer series optimal spreading was also found for moderately wettable substrata4’ ‘. Most studies on cell adhesion and spreading are carried out under static conditions. In many biomedical applications, however, the strength of adhesion of cells to a biomaterial is more determinant for the ultimate Biomaterials
Correspondence
to Dr T.G. van
0 1992 Butterworth-Heinemann 0142-9612/92/130897-08
Ltd
Kooten.
patency than adhesion or spreading as such. A number of methods exist to study the strength of adhesion. In most studies a well-defined flow is used to quantify strength of adhesion, as, for example, the rotating disk6, the radialflow chamber7, s, capillaries93 lo or the parallel-plate flow chamber”-‘3. Other methods include micropipette suction’4* 15, and also controlled trypsinization’“. In most studies, the behaviour of cells exposed to flow cannot be followed continuously, due to limitations in the flow chamber design or the lack of image-processing equipment. In most cases, additional steps are necessary to observe the cells, often involving passages through a liquid-air interface, which may cause detachment or changes in cell morphology. We have developed a flow circuit in which we are able to monitor the behaviour of cells continuously in situ and process series of sequentially taken images semiautomatically’7. We have used this flow circuit to study the differences in behaviour during exposure to flow of cells adhered to hydrophobic fluomethylenepropylene-Teflon@ copolymer (FEP-Teflon) and hydrophilic glass” and found that cells adhered to FEP-Teflon detached much faster than cells adhered to glass. Furthermore it appeared that a passage through a liquid-air interface (for example with electron microscopy preparations] removed a substantial part of the cells adhered to FEP. Biomaterials
1992,
Vol. 13 No.
13
898
Influence
of wettability
This study aimed to determine the in situ behaviour of cells adhered to substrata with different wettabilities during exposure to flow.
MATERIALS AND METHODS Substrata Four materials with widely different wettabilities were used in this study: glass, tissue culture polystyrene (TCPS), Poly(methylmethacrylate) (PMMA) and FEPTeflon (FEP). Glass was cut out of 2.0 mm thick plates to 76 X 50 mm samples, matching the dimensions of the flow chamber. PMMA was obtained from M.A. Vink Kunststoffen B.V. (Didam, The Netherlands) as 2.0 mm thick plates and also machined to match the dimensions of the flow chamber. FEP was obtained from Fluorplast BV (The Netherlands] as a 0.1 mm film and attached to PMMA with double-sided sticky tape to obtain a plate with 2.0 mm thickness. TCPS was cut out of Greiner tissue culture-quality Petri dishes, being extremely careful not to touch the surface, therewith damaging the sensitive surface layer. All materials, except for TCPS, were sonicated in a strong detergent (5% RBS 35, Perstorp Analytical, The Netherlands, in demineralized water), thoroughly rinsed in demineralized water, and finally sonicated three times in demineralized water. Advancing type contact angles with water, measured on all materials with the sessile drop technique at room temperature, were smaller than 15” for glass, 70° for TCPS, 76” for PMMA, and 109’ for FEP.
Cell line Human skin fibroblasts (PK 84-l) were cultured in 25 or 75 cm2 T-flasks (Greiner) with HEPES-buffered RPMI1640 medium (Gibco) supplemented with 2 mM glutamine (Gibco], 100 units/ml penicillin/streptomycin and 10% fetal calf serum (FCS) at 37% in humidified air and 5% Cultures were subdivided by trypsinization Et w/w% trypsin 1:250 (Difco) in PBS (9.0 mM Na,HPO,, 1.3 mM KHzPOl, 140 mM NaCl)) when they reached confluency.
Flow system
and set-up of the experiments
The flow system is described in detail elsewhere17. It contains three basic modules: flow loop, heating system, and image analysis components. Medium is pumped through the flow loop by a peristaltic pump (WatsonMarlow 503U with 6 mm inner diameter tubing) into a damping vessel, This vessel dampens the pulsations of the flow into a steady flow. Then, the medium flows through a parallel-plate flow chamber mounted on a phase-contrast microscope stage. Medium is collected in a thermostated double-walled vessel. The flow chamber itself is heated directly with four power resistors (330 each), connected in parallel to a power supply (10 V) with feedback from a PtlOO thermocouple in the downstream compartment of the flow chamber. The chamber dimensions are 76 [length] X 38 (width] X 0.2 [height) mm. Cells were exposed to a single shear stress (singleshear experiments) or to an incrementally loaded shear stress (incremental-shear experiments). The single-shear Biomaterials
1992. Vol. 13 No. 13
on detachment
of fibroblasts:
T.G. van Kooten et al.
experiments were only carried out on glass, whereas the incremental-shear experiments were carried out on all substrata. The preparations for both types of experiments were the same: cells were suspended in 10% serum containing RPMI-1640 medium and seeded on the substratum under study. Tissue culture polystyrene (Greiner) was used as a control to observe the spreading activity of the individual cell cultures. The cells were incubated for 3 h at 37“C in 5% CO, to allow adhesion and spreading. Then cells were exposed to the desired flow regime. In the single-shear experiments, one shear stress was maintained during maximally 210 min. In the incremental-shear experiments the flow rate was increased every 15 min by approximately 3 ml/s to a maximum 33 ml/s.
Image analysis
and data handling
Cells were observed and processed with an image analysis system described elsewhere17. A CCD-camera (type MO, High Technology Holland BV) grabs a field of 0.27 mm2 through a light microscope [Olympus BH-2, Phase Contrast, X10 objective AlOPL, Olympus photoocular NFK X3.3 LD). The frame grabber (PCVISION Plus, Imaging Technology Incorporated), installed in an AT-IBM compatible personal computer, digitizes the information to images with 512 X 512 pixel resolution and 8 bit intensities and displays them on a video screen. In the single-shear experiments images were grabbed at least every 15 min. In the incremental-shear experiments images were grabbed just before increasing the flow rate and at the start of an experimental run. The images were saved on a hard disk. The series of sequentially saved images was then analysed with the image analysis software [TIM, Difa/T.E.A. Measuring Systems BV]. Maximally 50 cells were marked in the starting image. These cells were traced in the following images. Three parameters were determined for each cell in each image: area (A], perimeter, and a two-dimensional projected shape factor (S):
s=
perimeter 2(nA)“’
Because the shape is a function of the perimeter, only data for the area and shape of spreaded cells are presented. The incrementally loaded flow regime includes two parameters: time and shear stress. Provided that flow is laminar and established, the shear stress rw is a function of flow rate Q and chamber height h as follows:
.,=,x$ in whichp is the viscosity and w is the chamber width. Since the sample plates tend to bend under the influence of the pressure difference over the chamber, the chamber height h was measured regularly during the experiment. All shear stresses reported in this paper are corrected for this effect. Single-shear experiments were performed once for a single-shear stress. The incremental-shear experiments were done four times for each substratum with different
Influence
of wettability
on detachment
cell passages. Data of four different on a substratum were averaged.
of fibroblasts:
experimental
T.G. van Kooten et al.
899
runs
RESULTS Results of the detachment study on glass in which one shear stress was applied during maximally 210 min are shown in Figures l-3. From Figure 1 it can be seen that cells are removed sooner at higher shear stresses. At lower shear stresses (4.4 and 8.8 N/m’) cells stay adhered to the surface and no significant detachment is obvious, whereas at higher shear stresses 50% detachment times were 135 and 38 min for 17.6 and 26.3 N/m2 respectively. From Figure 2 it is evident that, although the percentage of cells does not substantially decrease in time at the lower shear stresses of 4.4 and 8.8 N/m’, the mean spreading areas do decrease. At the higher shear stresses it can be seen that the decrease in area at 17.6 and 26.3 N/ m2 follows a similar time dependence, despite the fact that Figure 1 showed that the cells detached faster at 26.3 N/m2 compared with 17.6 N/m’. Figure 3 indicates
I
\
90
120
150
180
210
Time (min)
Figure 1 The percentage of cells present as a function of time in single-shear experiments on glass for four different shear stresses: A, 4.4; 0, 8.8; +, 17.6; B, 26.3 N/m2.
0
30
60
90
120
150
180
210
Time (min)
Figure2 The mean areas of the cells still present as a function of time in single-shear experiments on glass for four different shear stresses: A, 4.4; 0, 8.8; +, 17.6; W, 26.3 N/m2. Representative standard deviations over 50 individual cells are indicated by bars.
0.901
I 0
30
‘I
.
60
90
120
150
180
’ 210
Time [min)
Figure 3 The mean two-dimensional, projected shape factors of the cells still present as a function of time in single-shear experiments on glass for four different shear stresses: A, 4.4; 0, 8.8; +, 17.6; W, 26.3 N/m2. Representative standard deviations over 50 individual cells are indicated by bars.
that at the lower shear stresses, the decrease in spreading area is in general not accompanied by a return to a circular projected shape (S = 1.0) opposite to the observation done at the higher shear stresses showing a decrease of the shape factor. The mean shape factors of cells exposed to 17.6 and 26.3 N/m2 follow similar time dependences. Figure 4 shows a series of sequential images taken from a single-shear experiment at 17.6 N/m’. These show that, in general, cells round up before detachment and that there is great variability in cell appearances and in times of detachment. Furthermore, it is apparent from Figure 4 that both spread cells and circular cells move over appreciable distances in the flow direction. Visible cellular debris after detachment was only found occasionally in the single-shear as well as the incremental-shear experiments. In single-shear experiments more debris could be observed more frequently at the higher shear stresses as small vesicles. These vesicles, however, also detached as flow continued. When the vesicles were traced back to the starting image, it appeared that most vesicles were already present as vesicles at this stage. Results of the incremental-shear experiments on FEP, PMMA, TCPS and glass are shown in Figures 5-7. From Figure 5 it can be seen that the rate of cell detachment increases in the order TCPS, glass, PMMA, FEP. Also, detachment does not start instantaneously after the onset of flow, except for FEP. The time at which 50% of the cells has detached, t,,, was 3.5 f 1.5 min for FEP, 72 + 21 min for PMMA, 85 f 3 min for glass and >195 min for TCPS. Figure 6 shows that the areas of cells decrease under the influence of time and shear stress. The variability in spreading areas is large, the standard errors of the mean being about 20% of the average values. The spreading areas of cells at the start of an experiment reflect the situation after 3 h of spreading under static conditions. Testing for groups reveals that the mean initial spreading areas decrease in the order TCPS, glass and PMMA, FEP (P < 0.01). The decrease of the mean spreading areas is very gradual for cells adhered to TCPS and very fast for cells adhered to FEP. Biomaterials
1992, Vol. 13 No. 13
900
Influence
of wettability
on detachment
of fibroblasts:
T.G. van Kooten et al.
Figure4 Sequentially taken light micrographs of cells adhered to glass and exposed to a shear stress of 17.6 N/m*. Flow direction is from left to right, times (min) are indicated, bar = 50fim. Note the migration behaviour of the cell indicated in the starting image (arrow).
5
0
30
60
90
120
150
180
210
Time fmin)
200
-3--e 0
' 30
q-q_, 60
‘c
I
90
120
150
,
180
I
210
Time fmin)
Figure5 The percentage of cells present as a function of time and shear stress in incremental-shear experiments for four different substrata: A, glass; 4, tissue culture polystyrene (TCPS); 0, poly(methytmethac~late) (PMMA); n , fluoroethylene propylene (FEP). Representative standard errors of the mean over four experimental runs with different cell batches are indicated by bars.
Figure6 The mean areas of the cells still present as a function of time and shear stress in incremental-shear experiments for four different substrata: A, glass; tissue culture polystyrene (TCPS); 0, poly(methylmethac~late) (PMMA)~ n , fluoroethylene propylene (FEP). Representative standard errors of the mean over four experimental runs with different cell batches are indicated by bars.
The areas of cells adhered to PMMA decrease faster than those of cells adhered to glass. Smallest areas observed for circular cells were about 200 pm’, corresponding to a diameter of 16pm. Figure 7 shows the mean shape factors as a function of time and shear stress. Initial shape factors seem to decrease in the order TCPS, FEP, PMMA, glass, but the differences are not significant due to the large variability in shapes present on the surfaces. Shape factors for cells adhered to FEP drop to unity very
fast. Cells on PMMA first showed an increase in shape factor, followed by a decrease. Cells adhered to TCPS showed a similar pattern but with more gradual changes. Cells adhered to glass seemed to be rather stable with respect to their shape but the return to circular projected shapes usually occurred within several minutes as soon as a sufficiently high shear stress was applied, resulting in a rapid subsequent detachment. Figure 8 shows the influence of wettability (represented
Biomaterials
1992, Vol. 13 No. 13
+
,
Influence
of wettability
on detachment
T.G. van Kooten et al.
of fibroblasts:
901
T *
1.30
T / ,--F\
i
l
0 iFI
0
30
. .
l
*
.
‘q\
60
90
120
.
l
150
.
*
180
.
210
Time (min)
0
Figure 7 The mean two-dimensional, projected shape factors of the cells still present as a function of time and shear stress in incremental-shear experiments for four different substrata: A, glass; 4, tissue culture polystyrene (TCPS); 0, poly(methylmethacrylate) (PMMA); W, fluoroethylene propylene (FEP). Representative standard errors of the mean over four experimental runs with different cell batches are indicated by bars.
by the water contact angle) on several characteristic parameters of the incremental-shear experiments, including the times at which 90% (ts,,) and 50% (t,,) of the cells are still present (Figure Ba), the mean spreading areas (Figure Bb) and the projected shape factors (Figure Bc) at the start of the experiment as well as at t,,. It can be seen that, whereas in general the results correlate rather well with the wettability of the substrata, the data for TCPS do not fit into these relationships.
20
40 .
a
Water
60 contact
80 angle
100
T
120
’ 100
J..
0 120
’
’
’ 120
(degrees)
1100 -
2001 0
*
’ 20
b
.
8 40
Water
.
’ 60
contact
.
angle
’ 80 (degrees)
DISCUSSION The behaviour was investigated of human fibroblasts adhered to substrata with different wettability during exposure to a single shear and to an incrementally loaded shear stress. The parallel-plate flow chamber was used because in this device a well-known flow regime can be applied to the cells and analysis can be done in situ, i.e. without additional shear forces acting on the adhered cells during passage through liquid-air interfaces”.
Single-shear experiments
versus
$ m
5 1.10
incremental-shear
Two flow regimes were used. The commonly used method to investigate the behaviour of cells exposed to flow is to subject cells to a single shear stress during a certain period of time; this was used for a number of shear stresses for cells adhered to glass. From such experiments a critical shear stress can be derived, below which cells do not detach and above which the viability of the cells may be affectedlg. For shear stresses above this critical shear stress, detachment of adhering cells progresses with time. Detachment characteristics then include the shear stress applied and t,,, i.e. the time after which 50% of the initially adhered cells have detached. The other method used in this study involved an
-
c
0.901 0
C
’ 20
a
’
8
40 Water
’
0
60 contact
’
’
80 angle
100
(degrees)
Figure8 Detachment behaviour of human fibroblasts as a function of the water contact angle (degrees) on thesubstratum, as derived from the incremental-shear experiments. a, The timest,,andt,,(min)atwhich90%(0)and 50%(O)oftheinitial cell population is still present at the surface. Bars indicate the standard deviation over four experimental runs. b, The mean areas of spread cells at the start of the incremental-shear regime (0) and at t,, (0). Bars indicate standard errors of the mean of four experimental runs. c, The mean shape factors at the start of the incremental-shear regime (0) and at t,, (0). Bars indicate standard errors of the mean of four experimental runs. For tissue culture polystyrene (TCPS) t,, is taken instead of r,,, as rsOcould not be determined. Biomaterials
1992. Vol. 13 No. 13
902
Influence
of wettability
incrementally loaded shear stress. This method has also been used by Bowers et al. in micropipette suction experiments14. The incremental-shear method allowed the cells to adapt to a certain shear stress during 15 min after which the shear stress was increased. The rationale for this approach was that the number of experimental runs necessary to get a good understanding of the behaviour of the cells adhered to a substratum was less than the number of runs necessary when using single shear stresses. For this type of experiment, a 50% detachment level can also be given but its interpretation is more complex than for single-shear experiments. The in situ observation of the behaviour of the cells during exposure to flow together with the imageprocessing equipment is very important in order to get an understanding of the events preceding actual detachment, as demonstrated in Figure 4. This figure, for example, suggests that cells make new contacts during detachment and thereby resist the shear forces for longer.
Cellular responses to shear: influence substratum wettability
of
The response of cells during exposure to flow is different for substrata with different wettabilities. In general, cells adhere more firmly to substrata with high wettability than to substrata with low wettability (Figure Ba). This also applies to initial spreading areas, as shown before by Schakenraad et al.‘. The dependence of spreading area and adhesion strength on wettability does not seem to apply to TCPS. This can probably be explained by the presence of hydroxy groups on TCPSzol ‘I, known to enhance cell adhesion and spreading. Hydroxylation of poly(tetrafluoroethylene), for example, a polymer closely related to FEP, resulted in mean cell spreading areas similar to TCPS”. There is an optimal density for hydroxy groups, however, as shown by Horbett et al.23, who found that the presence of an excessive number of hydroxy groups resulted in a lower critical shear stress. The observation that cells adhered to TCPS seemed more resistant to flow than cells adhered to glass, might be explained by a more optimal hydroxy group density on TCPS relative to glass. The differences in cell behaviour on substrata with different wettability in the presence of flow persist despite the fact that the substrata will be covered rapidly by adsorbed proteins during the incubation, most likely even before the first cells make contact. This strongly suggests that proteins adsorb in various ways on the different substrata, possible differences being the distributionZ4, compositionz5, quantity” or conformation26-2g of the adsorbed proteins. Cell spreading, for example, is correlated with fibronectin5 and vitronectin adsorption30-32, both present in FCS33, 34.Fibronectin adsorption to TCPS has been found to be sixfold higher than to the more hydrophobic polystyrene3’, indicating that indeed fibronectin adsorption is influenced by wettability. It is also known that cells are able to adhere and spread by production of endogenous adhesion proteins replacing proteins adsorbed to the substrata33* 36-38, This is not possible, however, on hydrophobic substrata due to irreversible adsorption of albumin to these substrata3’. Biomaterials
1992, Vol. 13 No. 13
on detachment
Mechanisms
of fibroblasts:
of detachment
T.G.
van Kooten et al.
of cells
Cellular behaviour in response to shear stress is dependent on the substratum to which cells are adhered. In general, cells round up and detach. Filopodial networks can be present during the process of rounding up, as shown before on FEP and glass”. Scanning electron microscopical observations of cells adhered to glass and exposed to a single shear stress showed that those networks were most abundantly present at the lower shear stresses. Such networks were not observed on PMMA and TCPS after incrementally increased flow. Cells adhered to PMMA oriented themselves perpendicular to the direction of flow before rounding up, a feature not observed on the other substrata. Cells adhere through a collection of receptor-ligand bonds which may form or dissociate in response to shear. Cozens-Roberts et al. 3g have tried to mimic cellular adhesion by theoretically describing the kinetics of receptor-ligand bond formation and dissociation between a cell and a surface, using antigen-coated beads on antibody-coated surfacesa. Although their model proved useful to account for a number of detachment characteristics also found in this study, it did not include the dynamic nature of a cell - lateral diffusion of receptors over the cell surface into or out of the contact area4’, heterogenic surface properties, deformability, reassemblage of membranes, and a dynamically organized cytoskeleton coupled to the extracellular contacts4*. Besides dissociation of receptor-ligand bonds, it is also possible that a cell detaches through membrane rupture4’v 43, so-called ‘cohesive failure’23. The observations done in this study suggest that the flow regime applied influences the presence of visible cellular debris, and thus has important implications for whether membrane cohesive strength or adhesion strength is being assayed. Horbett et al.‘3 concluded that cohesive failure occurred, based on the appearance of 3T3 fibroblasts in a radial flow chamber detachment assay. However, Bowers et aLI4 did not find visible debris with micropipette suction, although cellular detachment took place within an average of 20 s. Cells respond in an active and time-dependent fashion to shear and therefore it is difficult to present values for the strength of adhesion of a cell. A rough estimate of the strength of adhesion can be given on the basis of the shear stress and spreading area at detachment. Since, at detachment, cells are mostly spherical with a diameter of around 16 pm, a shear force required for detachment in the range 0.6-3.5 X 10 -lo N can be calculated for FEP, 2.1-5.2 X lo-'N for PMMA, 3.3-9.4 X lo-'N for glass and >5.2 X lo-'N for TCPS. If we neglect the fact that these shear forces work parallel to the substratum surface, while adhesion forces operate perpendicularly, we may consider this force as a strength of adhesion. In a review, Hubbe listed values obtained from different studies ranging from lo-’ to lo-” N per ce1144. Other values reported are 3.6 X lo-’ N for NIL fibroblasts on glass45 and 1 X lo-’ N for red cells on polystyrene15. Our values thus correspond well with those reported in literature. The following conclusions can be drawn from the results of this study: Detachment
of fibroblasts
from substrata
with different
Influence
of wettability
on detachment
of fibroblasts:
T.G. van Kooten
becomes easier as the substratum wettability decreases. On TCPS, however, fibroblast adhesion was stronger than expected on the basis of its wettability, probably due to its specific surface chemistry. Fibroblasts do not detach when exposed to shear stresses below a critical threshold. Above this critical shear stress, cellular detachment progresses as a timedependent process. Under the flow regime used, spread cells round up before detachment and detach as spherical cells, without leaving visible debris. Adhesion strengths calculated range from 0.6X lo-" to 9 X lo-’ N per cell, dependent on the substratum, in agreement with reported literature values.
wettability
13
14
15
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17
REFERENCES 1
2
3
4
5
6
7
8
9
10
11
12
Schakenraad,
J.M., Busscher, H.J., Wildevuur, C.R.H. and Arends, J., The influence of substratum surface free energy on growth and spreading of human fibroblasts in the presence and absence of serum proteins, 1. Biomed. Mater. Res. 1986, 20, 773-784 Absolom, D.R., Hawthorn, L.A. and Chang, G., Endothelialization of polymer surfaces, 1. Biomed. Mater. Res. 1988, 22, 271-285 Van Wachem, P.B., Beugeling, T., Feijen, J,, Bantjes, A., Detmers, J.P. and Van Aken, W.G., Interaction of cultured human endothelial cells with polymeric surfaces of different wettabilities, Biomaterials 1985, 6, 403-408 Van Wachem, P.B., Hogt, A.H., Beugeling, T., Feijen, J., Bantjes, A., Detmers, J.P. and Van Aken, W.G., Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge, Biomaterials 1987, 8, 323-329 Horbett, T.A. and Schway, M.B., Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrylate-ethylmethacrylate copolymers, 1. Biomed. Mater. Res. 1988, 22, 763-793 Pratt, K.J., Williams, S.K. and Jarrell, B.E., Enhanced adherence of human adult endothelial cells to plasma discharge modified polyethylene terephtha1ate.l. Biomed. Mater. Res. 1989, 23, 1131-1147 Crouch, CF., Fowler, H.F. and Spier, R.E., The adhesion of animal cells to surfaces: the measurement of critical surface shear stress permitting attachment or causing detachment, 1. Chem. Tech. Biotech. 1985, 35B, 273-281 Cozens-Roberts, C., Quinn, J.A. and Lauffenburger, D.A., Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with the radialflow detachment assay, Biophys. J. 1990,58(4), 857-872 Andre, P., Capo, C., Benoliel, A.M., Bongrand, P., Rouge, F. and Aubert, C., Splitting cell adhesiveness into independent measurable parameters by comparing ten human melanoma cell lines, Cell Biophys. 1990, 17(2), 163-180 Bongrand, P. and Golstein, P., Reproducible dissociation of cellular aggregates with a wide range of calibrated shear forces: application to cytolytic lymphocyte-target cell conjugates, J. Immunol. Methods 1983,58,209-224 Koslow, A.R., Stromberg, R.R., Friedman, L.I., Lutz, R.J., Hilbert, S.L. and Schuster, P., A flow system for the study of shear forces upon cultured endothelial cells, J. Biomech. Eng. 1986, 108,338-341 Viggers, R.F., Wechezak, A.R. and Sauvage, L.R., An apparatus to study the response of cultured endothelium
18
19
20
21
22
23
24
25
26
27
28
29
30
903
et al.
to shear stress, J. Biomech. Eng. 1986, 108, 332-337 Truskey, G.A. and Pirone, J.S., The effect of fluid shear stress upon cell adhesion to fibronectin-treated surfaces, J. Biomed. Mater. Res. 1990, 24, 1333-1353 Bowers, V.M., Fisher, L.R., Francis, G.W. and Williams, K., A micromechanical technique for monitoring cellsubstrate adhesiveness: measurements of the strength of red blood cell adhesion to glass and polymer test surfaces, J. Biomed. Mater. Res. 1989, 23, 1453-1473 Francis, G.W., Fisher, L.R., Gamble, R.A. and Gingell, D., Direct measurement of cell detachment force on single cells using a new electromechanical method, J. Cell Sci. 1987, 87, 519-523 Duval, J.L., Letort, M. and Sigot-Luizard, M.F., Comparative assessment of cell/substratum static adhesion using an in vitro organ culture method and computerized analysis system, Biomaterials 1988, 9, 155-161 Van Kooten, T.G., Schakenraad, J.M., Van der Mei, H.C. and Busscher, H.J., Development and use of a parallel plate flow chamber for studying cellular adhesion to solid surfaces, J. Biomed. Mater. Res. 1992, 26, 725-738
Van Kooten, T.G., Schakenraad, J.M., Van der Mei, H.C. and Busscher, H.J., Detachment of human fibroblasts from FEP-Teflon surfaces, Cells and Materials, 1991, I, 307-316 Kretzmer, G. and Schiigerl, K., Response of mammalian cells to shear stress, Appl. Microbial. Biotech. 1991, 34, 613-616 Bentley, K.L. and Klebe, R.J., Fibronectin binding properties of bacteriologic petri plates and tissue culture dishes, J. Biomed. Mater. Res. 1985, 19,757-769 Curtis, A.S.G., Forrester, J.V., McInnes, C. and Lawrie, F., Adhesion of cells to polystyrene surfaces, J. Cell Biol. 1983, 97, 1500-1506 Massia, S.P. and Hubbell, J.A., Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials, J. Biomed. Mater. Res. 1991, 25, 223-242 Horbett, T.A., Waldburger, J.J., Ratner, B.D. and Hoffman, A.S., Cell adhesion to a series of hydrophilic-hydrophobic copolymers studied with a spinning disc apparatus, J. Biomed. Mater. Res. 1968, 22, 383-404 Uyen, H.M.W., Schakenraad, J.M., Sjollema, J.. Noordmans, J., Jongebloed, W.L., Stokroos, I. and Busscher, H.J., Amount and surface structure of albumin adsorbed to solid substrata with different wettabilities in a parallel plate flow cell, J. Biomed. Mater. Res. 1990,24, 1599-1614 Horbett, T.A. and Weathersby, P.K., Adsorption of proteins from plasma to a series of hydrophilichydrophobic copolymers. I. Analysis with the in situ radioiodination technique, J. Biomed. Mater. Res. 1981, 15, 403-423 Soderquist, M.E. and Walton, A.G., Structural changes in proteins adsorbed on polymer surfaces, J. Coil. Interf. Sci. 1980, 75(2), 386-397 Rapoza, R.J. and Horbett, T.A., Postadsorptive transitions in fibrinogen: influence of polymer properties, J. Biomed. Mater. Res. 1990, 24, 1263-1287 Shiba, E., Lindon, J.N., Kushner, L., Matsueda, G.R., Hawiger, J., Kloczewiak, M., Kudryk, B. and Salzman, E.W., Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces, Am. J. Physiol. 1991, 260, C965-C974 Narasimhan, C. and Lai, C.-S., Conformational changes of plasma fibronectin detected upon adsorption to solid substrates: a spin-label study, Biochemistry, 1989, 28, 5041-5046 Underwood, P.A. and Bennett, F.A., A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin, J. Cell Sci. 1969, 93, 641-649 Biomaterials
1992. Vol. 13 No. 13
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32
33
34
35
36
37
38
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of wettability
Bale, M.D., Wohlfahrt, L.A., Mosher, D.F., Tomasini, B. and Sutton, R.C., Identification of vitronectin as a major plasma protein adsorbed on polymer surfaces of different copolymer composition, Blood 1989, 74(8), 26982706 Steele, J.G., Johnson, G., Norris, W.D. and Underwood, P.A., Adhesion and growth of cultured human endothelial cells on perfluorosulphonate: role of vitronectin and fibronectin in cell attachment, Biomaterials 1991, 12, 531-539 Ertel, S.I., Ratner, B.D. and Horbett, T.A., Radiofrequency plasma deposition of oxygen-containing films on polystyrene and poly(ethylene terephthalate) substrates improves endothelial cell growth, 1, Biomed. Mater. Res. 1990, 24, 1837-1659 Hayman, E.G., Pierschbacher, M.D., Suzuki, S. and Ruoslahti, E., Vitronectin - a major cell attachmentpromoting protein in fetal bovine serum, Exp. Ceil Res. 1985, 169, 245-258 Grinnell, F. and Feld, M.K., Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during celf adhesion in serumcontaining medium, J. Eiol. Chem. 1982, 257(g), 4888-4893 Haas, R. and Culp, L.A., Properties and fate of plasma fibronectin bound to the tissue culture substratum, J. Cell Physiol. 1982, 113, 289-297 Knox, P., Kinetics of cell spreading in the presence of different concentrations of serum or fibronectin-depleted serum, 1, Ceil Sci. 1984, 71,51-59 Dekker, A., Beugeling, T., Wind, I-I., Poot, A., Bantjes, A.,
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40
41
42
43
44 45
on detachment
of fibroblasts:
T.G. van Koofen et al.
Feijen, J. and Van Aken, W.G., Deposition of cellular fibronectin and desorption of human serum albumin during adhesion and spreading of human endothelial cells on polymers, J. Mater. Sci.: Mater. Med. 1991, 2, 227-233 Cozens-Roberts, C., Lauffenburger, D.A. and Quinn, J.A., Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis, Biophys. J. 1990,58(4), 841-856 Chan. P.-Y., Lawrence, M.B., Dustin, M.L., Ferguson, L.M., Golan, D.E. and Springer, T.A., Influence of receptor lateral mobility on adhesion st~ngthening between membranes containing LFA-3 and CD2 J. Ceii Biol. 1991, 115(l), 245-255 Burridge, K., Fath, K., Kelly, T., Nuckolls, G. and Turner, C., Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton, Ann. Rev. Ceil Viol. 1988, 4, 487-525 Weiss, L., Studies on cellular adhesion in tissue culture IV. The alteration of substrata by cell surfaces, Exp. Cell Res.1961,25,504-517 Weiss, L. and Coombs, R.R.A.. The demonstration of rupture of cell surfaces by an immunological technique, Exp. CellRes. 1963,30,331-338 Hubbe, M.A., Adhesion and detachment of biological cells in vitro, Prog. Surface Sci. 1981, 11, 65-138 Lotz, M.M., Burdsal, CA., Erickson, H.P. and McClay, D.R., Cell adhesion to fibronectin and tenascin: Quantitative measurements of initial binding and subsequent strengthening response, 1. Cell Biol. 1989, 109, 1795-1805
Influence of substratum wettability on the strength of adhesion of human fibroblasts T.G. van Kooten*, J.M. Schakenraadf, H. J. Busscher*
H.C. van der Mei* and
*Laboratory for Materia Technica, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; and +Centre for Biomedical Technology, University of Groningen, Oostersingel59, Gb 25, FO, 9713 EZ Groningen, The Netherlands
To determine the strength of adhesion and the detachment mechanisms of fibroblasts from substrata with different wettability, the behaviour of adhered ceils was studied in a parallel-plate flow chamber during exposure to shear. Adhered cells were observed in situ, i.e. in the flow chamber, by phase-contrast microscope and images were analysed semiautomatically. Detachment was found to be dependent both on shear stress and time, although a critical shear stress can be found below which no detachment occurs. On all substrata, cells round up before detachment and are approximately spherical immediately before detachment. The strength of adhesion calculated ranged from 0.6-3.5 X lo-” N per cell on FEP-Teflon@ (the least wettable material included) to 9.4 X lo-’ N per cell for glass (the most wettable). Ease of detachment seemed to decrease with increasing wettability. However, cells reacted more strongly with tissue culture polystyrene (TCPS) than expected on the basis of its wettability, probably due to surface chemistry. Keywords:
Cell-material
interactions,
fibroblasts,
cell adhesion,
wettability
Received 24 February 1992; accepted 26 March 1992
are frequently used in biomedical devices as, for example, vascular grafts, artificial skin, ocular lenses and bone prostheses. The interaction of these materials with surrounding tissues or body fluids is important. For example, a firm adhesion of bone to a hip prosthesis is required for optimal patency. Cellular interactions with biomaterials are an important area of research. Cell adhesion to and spreading on a biomaterial is, amongst other factors, dependent on the surface wettability of the biomaterial. Schakenraad et al.’ reported that cells spread poorly on hydrophobic substrata and more extensively on more hydrophilic substrata. Absolom et al.’ reported a similar dependence on substratum wettability for endothelial cell coverage of the substratum. Van Wachem et ale3 found that human endothelial cells adhered optimally (in terms of numbers) on moderately wettable polymers. In a well-defined hydroxyethylmethacrylate-methylmethacrylate copolymer series optimal spreading was also found for moderately wettable substrata4’ ‘. Most studies on cell adhesion and spreading are carried out under static conditions. In many biomedical applications, however, the strength of adhesion of cells to a biomaterial is more determinant for the ultimate Biomaterials
Correspondence
to Dr T.G. van
0 1992 Butterworth-Heinemann 0142-9612/92/130897-08
Ltd
Kooten.
patency than adhesion or spreading as such. A number of methods exist to study the strength of adhesion. In most studies a well-defined flow is used to quantify strength of adhesion, as, for example, the rotating disk6, the radialflow chamber7, s, capillaries93 lo or the parallel-plate flow chamber”-‘3. Other methods include micropipette suction’4* 15, and also controlled trypsinization’“. In most studies, the behaviour of cells exposed to flow cannot be followed continuously, due to limitations in the flow chamber design or the lack of image-processing equipment. In most cases, additional steps are necessary to observe the cells, often involving passages through a liquid-air interface, which may cause detachment or changes in cell morphology. We have developed a flow circuit in which we are able to monitor the behaviour of cells continuously in situ and process series of sequentially taken images semiautomatically’7. We have used this flow circuit to study the differences in behaviour during exposure to flow of cells adhered to hydrophobic fluomethylenepropylene-Teflon@ copolymer (FEP-Teflon) and hydrophilic glass” and found that cells adhered to FEP-Teflon detached much faster than cells adhered to glass. Furthermore it appeared that a passage through a liquid-air interface (for example with electron microscopy preparations] removed a substantial part of the cells adhered to FEP. Biomaterials
1992,
Vol. 13 No.
13
898
Influence
of wettability
This study aimed to determine the in situ behaviour of cells adhered to substrata with different wettabilities during exposure to flow.
MATERIALS AND METHODS Substrata Four materials with widely different wettabilities were used in this study: glass, tissue culture polystyrene (TCPS), Poly(methylmethacrylate) (PMMA) and FEPTeflon (FEP). Glass was cut out of 2.0 mm thick plates to 76 X 50 mm samples, matching the dimensions of the flow chamber. PMMA was obtained from M.A. Vink Kunststoffen B.V. (Didam, The Netherlands) as 2.0 mm thick plates and also machined to match the dimensions of the flow chamber. FEP was obtained from Fluorplast BV (The Netherlands] as a 0.1 mm film and attached to PMMA with double-sided sticky tape to obtain a plate with 2.0 mm thickness. TCPS was cut out of Greiner tissue culture-quality Petri dishes, being extremely careful not to touch the surface, therewith damaging the sensitive surface layer. All materials, except for TCPS, were sonicated in a strong detergent (5% RBS 35, Perstorp Analytical, The Netherlands, in demineralized water), thoroughly rinsed in demineralized water, and finally sonicated three times in demineralized water. Advancing type contact angles with water, measured on all materials with the sessile drop technique at room temperature, were smaller than 15” for glass, 70° for TCPS, 76” for PMMA, and 109’ for FEP.
Cell line Human skin fibroblasts (PK 84-l) were cultured in 25 or 75 cm2 T-flasks (Greiner) with HEPES-buffered RPMI1640 medium (Gibco) supplemented with 2 mM glutamine (Gibco], 100 units/ml penicillin/streptomycin and 10% fetal calf serum (FCS) at 37% in humidified air and 5% Cultures were subdivided by trypsinization Et w/w% trypsin 1:250 (Difco) in PBS (9.0 mM Na,HPO,, 1.3 mM KHzPOl, 140 mM NaCl)) when they reached confluency.
Flow system
and set-up of the experiments
The flow system is described in detail elsewhere17. It contains three basic modules: flow loop, heating system, and image analysis components. Medium is pumped through the flow loop by a peristaltic pump (WatsonMarlow 503U with 6 mm inner diameter tubing) into a damping vessel, This vessel dampens the pulsations of the flow into a steady flow. Then, the medium flows through a parallel-plate flow chamber mounted on a phase-contrast microscope stage. Medium is collected in a thermostated double-walled vessel. The flow chamber itself is heated directly with four power resistors (330 each), connected in parallel to a power supply (10 V) with feedback from a PtlOO thermocouple in the downstream compartment of the flow chamber. The chamber dimensions are 76 [length] X 38 (width] X 0.2 [height) mm. Cells were exposed to a single shear stress (singleshear experiments) or to an incrementally loaded shear stress (incremental-shear experiments). The single-shear Biomaterials
1992. Vol. 13 No. 13
on detachment
of fibroblasts:
T.G. van Kooten et al.
experiments were only carried out on glass, whereas the incremental-shear experiments were carried out on all substrata. The preparations for both types of experiments were the same: cells were suspended in 10% serum containing RPMI-1640 medium and seeded on the substratum under study. Tissue culture polystyrene (Greiner) was used as a control to observe the spreading activity of the individual cell cultures. The cells were incubated for 3 h at 37“C in 5% CO, to allow adhesion and spreading. Then cells were exposed to the desired flow regime. In the single-shear experiments, one shear stress was maintained during maximally 210 min. In the incremental-shear experiments the flow rate was increased every 15 min by approximately 3 ml/s to a maximum 33 ml/s.
Image analysis
and data handling
Cells were observed and processed with an image analysis system described elsewhere17. A CCD-camera (type MO, High Technology Holland BV) grabs a field of 0.27 mm2 through a light microscope [Olympus BH-2, Phase Contrast, X10 objective AlOPL, Olympus photoocular NFK X3.3 LD). The frame grabber (PCVISION Plus, Imaging Technology Incorporated), installed in an AT-IBM compatible personal computer, digitizes the information to images with 512 X 512 pixel resolution and 8 bit intensities and displays them on a video screen. In the single-shear experiments images were grabbed at least every 15 min. In the incremental-shear experiments images were grabbed just before increasing the flow rate and at the start of an experimental run. The images were saved on a hard disk. The series of sequentially saved images was then analysed with the image analysis software [TIM, Difa/T.E.A. Measuring Systems BV]. Maximally 50 cells were marked in the starting image. These cells were traced in the following images. Three parameters were determined for each cell in each image: area (A], perimeter, and a two-dimensional projected shape factor (S):
s=
perimeter 2(nA)“’
Because the shape is a function of the perimeter, only data for the area and shape of spreaded cells are presented. The incrementally loaded flow regime includes two parameters: time and shear stress. Provided that flow is laminar and established, the shear stress rw is a function of flow rate Q and chamber height h as follows:
.,=,x$ in whichp is the viscosity and w is the chamber width. Since the sample plates tend to bend under the influence of the pressure difference over the chamber, the chamber height h was measured regularly during the experiment. All shear stresses reported in this paper are corrected for this effect. Single-shear experiments were performed once for a single-shear stress. The incremental-shear experiments were done four times for each substratum with different
Influence
of wettability
on detachment
cell passages. Data of four different on a substratum were averaged.
of fibroblasts:
experimental
T.G. van Kooten et al.
899
runs
RESULTS Results of the detachment study on glass in which one shear stress was applied during maximally 210 min are shown in Figures l-3. From Figure 1 it can be seen that cells are removed sooner at higher shear stresses. At lower shear stresses (4.4 and 8.8 N/m’) cells stay adhered to the surface and no significant detachment is obvious, whereas at higher shear stresses 50% detachment times were 135 and 38 min for 17.6 and 26.3 N/m2 respectively. From Figure 2 it is evident that, although the percentage of cells does not substantially decrease in time at the lower shear stresses of 4.4 and 8.8 N/m’, the mean spreading areas do decrease. At the higher shear stresses it can be seen that the decrease in area at 17.6 and 26.3 N/ m2 follows a similar time dependence, despite the fact that Figure 1 showed that the cells detached faster at 26.3 N/m2 compared with 17.6 N/m’. Figure 3 indicates
I
\
90
120
150
180
210
Time (min)
Figure 1 The percentage of cells present as a function of time in single-shear experiments on glass for four different shear stresses: A, 4.4; 0, 8.8; +, 17.6; B, 26.3 N/m2.
0
30
60
90
120
150
180
210
Time (min)
Figure2 The mean areas of the cells still present as a function of time in single-shear experiments on glass for four different shear stresses: A, 4.4; 0, 8.8; +, 17.6; W, 26.3 N/m2. Representative standard deviations over 50 individual cells are indicated by bars.
0.901
I 0
30
‘I
.
60
90
120
150
180
’ 210
Time [min)
Figure 3 The mean two-dimensional, projected shape factors of the cells still present as a function of time in single-shear experiments on glass for four different shear stresses: A, 4.4; 0, 8.8; +, 17.6; W, 26.3 N/m2. Representative standard deviations over 50 individual cells are indicated by bars.
that at the lower shear stresses, the decrease in spreading area is in general not accompanied by a return to a circular projected shape (S = 1.0) opposite to the observation done at the higher shear stresses showing a decrease of the shape factor. The mean shape factors of cells exposed to 17.6 and 26.3 N/m2 follow similar time dependences. Figure 4 shows a series of sequential images taken from a single-shear experiment at 17.6 N/m’. These show that, in general, cells round up before detachment and that there is great variability in cell appearances and in times of detachment. Furthermore, it is apparent from Figure 4 that both spread cells and circular cells move over appreciable distances in the flow direction. Visible cellular debris after detachment was only found occasionally in the single-shear as well as the incremental-shear experiments. In single-shear experiments more debris could be observed more frequently at the higher shear stresses as small vesicles. These vesicles, however, also detached as flow continued. When the vesicles were traced back to the starting image, it appeared that most vesicles were already present as vesicles at this stage. Results of the incremental-shear experiments on FEP, PMMA, TCPS and glass are shown in Figures 5-7. From Figure 5 it can be seen that the rate of cell detachment increases in the order TCPS, glass, PMMA, FEP. Also, detachment does not start instantaneously after the onset of flow, except for FEP. The time at which 50% of the cells has detached, t,,, was 3.5 f 1.5 min for FEP, 72 + 21 min for PMMA, 85 f 3 min for glass and >195 min for TCPS. Figure 6 shows that the areas of cells decrease under the influence of time and shear stress. The variability in spreading areas is large, the standard errors of the mean being about 20% of the average values. The spreading areas of cells at the start of an experiment reflect the situation after 3 h of spreading under static conditions. Testing for groups reveals that the mean initial spreading areas decrease in the order TCPS, glass and PMMA, FEP (P < 0.01). The decrease of the mean spreading areas is very gradual for cells adhered to TCPS and very fast for cells adhered to FEP. Biomaterials
1992, Vol. 13 No. 13
900
Influence
of wettability
on detachment
of fibroblasts:
T.G. van Kooten et al.
Figure4 Sequentially taken light micrographs of cells adhered to glass and exposed to a shear stress of 17.6 N/m*. Flow direction is from left to right, times (min) are indicated, bar = 50fim. Note the migration behaviour of the cell indicated in the starting image (arrow).
5
0
30
60
90
120
150
180
210
Time fmin)
200
-3--e 0
' 30
q-q_, 60
‘c
I
90
120
150
,
180
I
210
Time fmin)
Figure5 The percentage of cells present as a function of time and shear stress in incremental-shear experiments for four different substrata: A, glass; 4, tissue culture polystyrene (TCPS); 0, poly(methytmethac~late) (PMMA); n , fluoroethylene propylene (FEP). Representative standard errors of the mean over four experimental runs with different cell batches are indicated by bars.
Figure6 The mean areas of the cells still present as a function of time and shear stress in incremental-shear experiments for four different substrata: A, glass; tissue culture polystyrene (TCPS); 0, poly(methylmethac~late) (PMMA)~ n , fluoroethylene propylene (FEP). Representative standard errors of the mean over four experimental runs with different cell batches are indicated by bars.
The areas of cells adhered to PMMA decrease faster than those of cells adhered to glass. Smallest areas observed for circular cells were about 200 pm’, corresponding to a diameter of 16pm. Figure 7 shows the mean shape factors as a function of time and shear stress. Initial shape factors seem to decrease in the order TCPS, FEP, PMMA, glass, but the differences are not significant due to the large variability in shapes present on the surfaces. Shape factors for cells adhered to FEP drop to unity very
fast. Cells on PMMA first showed an increase in shape factor, followed by a decrease. Cells adhered to TCPS showed a similar pattern but with more gradual changes. Cells adhered to glass seemed to be rather stable with respect to their shape but the return to circular projected shapes usually occurred within several minutes as soon as a sufficiently high shear stress was applied, resulting in a rapid subsequent detachment. Figure 8 shows the influence of wettability (represented
Biomaterials
1992, Vol. 13 No. 13
+
,
Influence
of wettability
on detachment
T.G. van Kooten et al.
of fibroblasts:
901
T *
1.30
T / ,--F\
i
l
0 iFI
0
30
. .
l
*
.
‘q\
60
90
120
.
l
150
.
*
180
.
210
Time (min)
0
Figure 7 The mean two-dimensional, projected shape factors of the cells still present as a function of time and shear stress in incremental-shear experiments for four different substrata: A, glass; 4, tissue culture polystyrene (TCPS); 0, poly(methylmethacrylate) (PMMA); W, fluoroethylene propylene (FEP). Representative standard errors of the mean over four experimental runs with different cell batches are indicated by bars.
by the water contact angle) on several characteristic parameters of the incremental-shear experiments, including the times at which 90% (ts,,) and 50% (t,,) of the cells are still present (Figure Ba), the mean spreading areas (Figure Bb) and the projected shape factors (Figure Bc) at the start of the experiment as well as at t,,. It can be seen that, whereas in general the results correlate rather well with the wettability of the substrata, the data for TCPS do not fit into these relationships.
20
40 .
a
Water
60 contact
80 angle
100
T
120
’ 100
J..
0 120
’
’
’ 120
(degrees)
1100 -
2001 0
*
’ 20
b
.
8 40
Water
.
’ 60
contact
.
angle
’ 80 (degrees)
DISCUSSION The behaviour was investigated of human fibroblasts adhered to substrata with different wettability during exposure to a single shear and to an incrementally loaded shear stress. The parallel-plate flow chamber was used because in this device a well-known flow regime can be applied to the cells and analysis can be done in situ, i.e. without additional shear forces acting on the adhered cells during passage through liquid-air interfaces”.
Single-shear experiments
versus
$ m
5 1.10
incremental-shear
Two flow regimes were used. The commonly used method to investigate the behaviour of cells exposed to flow is to subject cells to a single shear stress during a certain period of time; this was used for a number of shear stresses for cells adhered to glass. From such experiments a critical shear stress can be derived, below which cells do not detach and above which the viability of the cells may be affectedlg. For shear stresses above this critical shear stress, detachment of adhering cells progresses with time. Detachment characteristics then include the shear stress applied and t,,, i.e. the time after which 50% of the initially adhered cells have detached. The other method used in this study involved an
-
c
0.901 0
C
’ 20
a
’
8
40 Water
’
0
60 contact
’
’
80 angle
100
(degrees)
Figure8 Detachment behaviour of human fibroblasts as a function of the water contact angle (degrees) on thesubstratum, as derived from the incremental-shear experiments. a, The timest,,andt,,(min)atwhich90%(0)and 50%(O)oftheinitial cell population is still present at the surface. Bars indicate the standard deviation over four experimental runs. b, The mean areas of spread cells at the start of the incremental-shear regime (0) and at t,, (0). Bars indicate standard errors of the mean of four experimental runs. c, The mean shape factors at the start of the incremental-shear regime (0) and at t,, (0). Bars indicate standard errors of the mean of four experimental runs. For tissue culture polystyrene (TCPS) t,, is taken instead of r,,, as rsOcould not be determined. Biomaterials
1992. Vol. 13 No. 13
902
Influence
of wettability
incrementally loaded shear stress. This method has also been used by Bowers et al. in micropipette suction experiments14. The incremental-shear method allowed the cells to adapt to a certain shear stress during 15 min after which the shear stress was increased. The rationale for this approach was that the number of experimental runs necessary to get a good understanding of the behaviour of the cells adhered to a substratum was less than the number of runs necessary when using single shear stresses. For this type of experiment, a 50% detachment level can also be given but its interpretation is more complex than for single-shear experiments. The in situ observation of the behaviour of the cells during exposure to flow together with the imageprocessing equipment is very important in order to get an understanding of the events preceding actual detachment, as demonstrated in Figure 4. This figure, for example, suggests that cells make new contacts during detachment and thereby resist the shear forces for longer.
Cellular responses to shear: influence substratum wettability
of
The response of cells during exposure to flow is different for substrata with different wettabilities. In general, cells adhere more firmly to substrata with high wettability than to substrata with low wettability (Figure Ba). This also applies to initial spreading areas, as shown before by Schakenraad et al.‘. The dependence of spreading area and adhesion strength on wettability does not seem to apply to TCPS. This can probably be explained by the presence of hydroxy groups on TCPSzol ‘I, known to enhance cell adhesion and spreading. Hydroxylation of poly(tetrafluoroethylene), for example, a polymer closely related to FEP, resulted in mean cell spreading areas similar to TCPS”. There is an optimal density for hydroxy groups, however, as shown by Horbett et al.23, who found that the presence of an excessive number of hydroxy groups resulted in a lower critical shear stress. The observation that cells adhered to TCPS seemed more resistant to flow than cells adhered to glass, might be explained by a more optimal hydroxy group density on TCPS relative to glass. The differences in cell behaviour on substrata with different wettability in the presence of flow persist despite the fact that the substrata will be covered rapidly by adsorbed proteins during the incubation, most likely even before the first cells make contact. This strongly suggests that proteins adsorb in various ways on the different substrata, possible differences being the distributionZ4, compositionz5, quantity” or conformation26-2g of the adsorbed proteins. Cell spreading, for example, is correlated with fibronectin5 and vitronectin adsorption30-32, both present in FCS33, 34.Fibronectin adsorption to TCPS has been found to be sixfold higher than to the more hydrophobic polystyrene3’, indicating that indeed fibronectin adsorption is influenced by wettability. It is also known that cells are able to adhere and spread by production of endogenous adhesion proteins replacing proteins adsorbed to the substrata33* 36-38, This is not possible, however, on hydrophobic substrata due to irreversible adsorption of albumin to these substrata3’. Biomaterials
1992, Vol. 13 No. 13
on detachment
Mechanisms
of fibroblasts:
of detachment
T.G.
van Kooten et al.
of cells
Cellular behaviour in response to shear stress is dependent on the substratum to which cells are adhered. In general, cells round up and detach. Filopodial networks can be present during the process of rounding up, as shown before on FEP and glass”. Scanning electron microscopical observations of cells adhered to glass and exposed to a single shear stress showed that those networks were most abundantly present at the lower shear stresses. Such networks were not observed on PMMA and TCPS after incrementally increased flow. Cells adhered to PMMA oriented themselves perpendicular to the direction of flow before rounding up, a feature not observed on the other substrata. Cells adhere through a collection of receptor-ligand bonds which may form or dissociate in response to shear. Cozens-Roberts et al. 3g have tried to mimic cellular adhesion by theoretically describing the kinetics of receptor-ligand bond formation and dissociation between a cell and a surface, using antigen-coated beads on antibody-coated surfacesa. Although their model proved useful to account for a number of detachment characteristics also found in this study, it did not include the dynamic nature of a cell - lateral diffusion of receptors over the cell surface into or out of the contact area4’, heterogenic surface properties, deformability, reassemblage of membranes, and a dynamically organized cytoskeleton coupled to the extracellular contacts4*. Besides dissociation of receptor-ligand bonds, it is also possible that a cell detaches through membrane rupture4’v 43, so-called ‘cohesive failure’23. The observations done in this study suggest that the flow regime applied influences the presence of visible cellular debris, and thus has important implications for whether membrane cohesive strength or adhesion strength is being assayed. Horbett et al.‘3 concluded that cohesive failure occurred, based on the appearance of 3T3 fibroblasts in a radial flow chamber detachment assay. However, Bowers et aLI4 did not find visible debris with micropipette suction, although cellular detachment took place within an average of 20 s. Cells respond in an active and time-dependent fashion to shear and therefore it is difficult to present values for the strength of adhesion of a cell. A rough estimate of the strength of adhesion can be given on the basis of the shear stress and spreading area at detachment. Since, at detachment, cells are mostly spherical with a diameter of around 16 pm, a shear force required for detachment in the range 0.6-3.5 X 10 -lo N can be calculated for FEP, 2.1-5.2 X lo-'N for PMMA, 3.3-9.4 X lo-'N for glass and >5.2 X lo-'N for TCPS. If we neglect the fact that these shear forces work parallel to the substratum surface, while adhesion forces operate perpendicularly, we may consider this force as a strength of adhesion. In a review, Hubbe listed values obtained from different studies ranging from lo-’ to lo-” N per ce1144. Other values reported are 3.6 X lo-’ N for NIL fibroblasts on glass45 and 1 X lo-’ N for red cells on polystyrene15. Our values thus correspond well with those reported in literature. The following conclusions can be drawn from the results of this study: Detachment
of fibroblasts
from substrata
with different
Influence
of wettability
on detachment
of fibroblasts:
T.G. van Kooten
becomes easier as the substratum wettability decreases. On TCPS, however, fibroblast adhesion was stronger than expected on the basis of its wettability, probably due to its specific surface chemistry. Fibroblasts do not detach when exposed to shear stresses below a critical threshold. Above this critical shear stress, cellular detachment progresses as a timedependent process. Under the flow regime used, spread cells round up before detachment and detach as spherical cells, without leaving visible debris. Adhesion strengths calculated range from 0.6X lo-" to 9 X lo-’ N per cell, dependent on the substratum, in agreement with reported literature values.
wettability
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15
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REFERENCES 1
2
3
4
5
6
7
8
9
10
11
12
Schakenraad,
J.M., Busscher, H.J., Wildevuur, C.R.H. and Arends, J., The influence of substratum surface free energy on growth and spreading of human fibroblasts in the presence and absence of serum proteins, 1. Biomed. Mater. Res. 1986, 20, 773-784 Absolom, D.R., Hawthorn, L.A. and Chang, G., Endothelialization of polymer surfaces, 1. Biomed. Mater. Res. 1988, 22, 271-285 Van Wachem, P.B., Beugeling, T., Feijen, J,, Bantjes, A., Detmers, J.P. and Van Aken, W.G., Interaction of cultured human endothelial cells with polymeric surfaces of different wettabilities, Biomaterials 1985, 6, 403-408 Van Wachem, P.B., Hogt, A.H., Beugeling, T., Feijen, J., Bantjes, A., Detmers, J.P. and Van Aken, W.G., Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge, Biomaterials 1987, 8, 323-329 Horbett, T.A. and Schway, M.B., Correlations between mouse 3T3 cell spreading and serum fibronectin adsorption on glass and hydroxyethylmethacrylate-ethylmethacrylate copolymers, 1. Biomed. Mater. Res. 1988, 22, 763-793 Pratt, K.J., Williams, S.K. and Jarrell, B.E., Enhanced adherence of human adult endothelial cells to plasma discharge modified polyethylene terephtha1ate.l. Biomed. Mater. Res. 1989, 23, 1131-1147 Crouch, CF., Fowler, H.F. and Spier, R.E., The adhesion of animal cells to surfaces: the measurement of critical surface shear stress permitting attachment or causing detachment, 1. Chem. Tech. Biotech. 1985, 35B, 273-281 Cozens-Roberts, C., Quinn, J.A. and Lauffenburger, D.A., Receptor-mediated cell attachment and detachment kinetics. II. Experimental model studies with the radialflow detachment assay, Biophys. J. 1990,58(4), 857-872 Andre, P., Capo, C., Benoliel, A.M., Bongrand, P., Rouge, F. and Aubert, C., Splitting cell adhesiveness into independent measurable parameters by comparing ten human melanoma cell lines, Cell Biophys. 1990, 17(2), 163-180 Bongrand, P. and Golstein, P., Reproducible dissociation of cellular aggregates with a wide range of calibrated shear forces: application to cytolytic lymphocyte-target cell conjugates, J. Immunol. Methods 1983,58,209-224 Koslow, A.R., Stromberg, R.R., Friedman, L.I., Lutz, R.J., Hilbert, S.L. and Schuster, P., A flow system for the study of shear forces upon cultured endothelial cells, J. Biomech. Eng. 1986, 108,338-341 Viggers, R.F., Wechezak, A.R. and Sauvage, L.R., An apparatus to study the response of cultured endothelium
18
19
20
21
22
23
24
25
26
27
28
29
30
903
et al.
to shear stress, J. Biomech. Eng. 1986, 108, 332-337 Truskey, G.A. and Pirone, J.S., The effect of fluid shear stress upon cell adhesion to fibronectin-treated surfaces, J. Biomed. Mater. Res. 1990, 24, 1333-1353 Bowers, V.M., Fisher, L.R., Francis, G.W. and Williams, K., A micromechanical technique for monitoring cellsubstrate adhesiveness: measurements of the strength of red blood cell adhesion to glass and polymer test surfaces, J. Biomed. Mater. Res. 1989, 23, 1453-1473 Francis, G.W., Fisher, L.R., Gamble, R.A. and Gingell, D., Direct measurement of cell detachment force on single cells using a new electromechanical method, J. Cell Sci. 1987, 87, 519-523 Duval, J.L., Letort, M. and Sigot-Luizard, M.F., Comparative assessment of cell/substratum static adhesion using an in vitro organ culture method and computerized analysis system, Biomaterials 1988, 9, 155-161 Van Kooten, T.G., Schakenraad, J.M., Van der Mei, H.C. and Busscher, H.J., Development and use of a parallel plate flow chamber for studying cellular adhesion to solid surfaces, J. Biomed. Mater. Res. 1992, 26, 725-738
Van Kooten, T.G., Schakenraad, J.M., Van der Mei, H.C. and Busscher, H.J., Detachment of human fibroblasts from FEP-Teflon surfaces, Cells and Materials, 1991, I, 307-316 Kretzmer, G. and Schiigerl, K., Response of mammalian cells to shear stress, Appl. Microbial. Biotech. 1991, 34, 613-616 Bentley, K.L. and Klebe, R.J., Fibronectin binding properties of bacteriologic petri plates and tissue culture dishes, J. Biomed. Mater. Res. 1985, 19,757-769 Curtis, A.S.G., Forrester, J.V., McInnes, C. and Lawrie, F., Adhesion of cells to polystyrene surfaces, J. Cell Biol. 1983, 97, 1500-1506 Massia, S.P. and Hubbell, J.A., Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials, J. Biomed. Mater. Res. 1991, 25, 223-242 Horbett, T.A., Waldburger, J.J., Ratner, B.D. and Hoffman, A.S., Cell adhesion to a series of hydrophilic-hydrophobic copolymers studied with a spinning disc apparatus, J. Biomed. Mater. Res. 1968, 22, 383-404 Uyen, H.M.W., Schakenraad, J.M., Sjollema, J.. Noordmans, J., Jongebloed, W.L., Stokroos, I. and Busscher, H.J., Amount and surface structure of albumin adsorbed to solid substrata with different wettabilities in a parallel plate flow cell, J. Biomed. Mater. Res. 1990,24, 1599-1614 Horbett, T.A. and Weathersby, P.K., Adsorption of proteins from plasma to a series of hydrophilichydrophobic copolymers. I. Analysis with the in situ radioiodination technique, J. Biomed. Mater. Res. 1981, 15, 403-423 Soderquist, M.E. and Walton, A.G., Structural changes in proteins adsorbed on polymer surfaces, J. Coil. Interf. Sci. 1980, 75(2), 386-397 Rapoza, R.J. and Horbett, T.A., Postadsorptive transitions in fibrinogen: influence of polymer properties, J. Biomed. Mater. Res. 1990, 24, 1263-1287 Shiba, E., Lindon, J.N., Kushner, L., Matsueda, G.R., Hawiger, J., Kloczewiak, M., Kudryk, B. and Salzman, E.W., Antibody-detectable changes in fibrinogen adsorption affecting platelet activation on polymer surfaces, Am. J. Physiol. 1991, 260, C965-C974 Narasimhan, C. and Lai, C.-S., Conformational changes of plasma fibronectin detected upon adsorption to solid substrates: a spin-label study, Biochemistry, 1989, 28, 5041-5046 Underwood, P.A. and Bennett, F.A., A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin, J. Cell Sci. 1969, 93, 641-649 Biomaterials
1992. Vol. 13 No. 13
904 31
32
33
34
35
36
37
38
Influence
of wettability
Bale, M.D., Wohlfahrt, L.A., Mosher, D.F., Tomasini, B. and Sutton, R.C., Identification of vitronectin as a major plasma protein adsorbed on polymer surfaces of different copolymer composition, Blood 1989, 74(8), 26982706 Steele, J.G., Johnson, G., Norris, W.D. and Underwood, P.A., Adhesion and growth of cultured human endothelial cells on perfluorosulphonate: role of vitronectin and fibronectin in cell attachment, Biomaterials 1991, 12, 531-539 Ertel, S.I., Ratner, B.D. and Horbett, T.A., Radiofrequency plasma deposition of oxygen-containing films on polystyrene and poly(ethylene terephthalate) substrates improves endothelial cell growth, 1, Biomed. Mater. Res. 1990, 24, 1837-1659 Hayman, E.G., Pierschbacher, M.D., Suzuki, S. and Ruoslahti, E., Vitronectin - a major cell attachmentpromoting protein in fetal bovine serum, Exp. Ceil Res. 1985, 169, 245-258 Grinnell, F. and Feld, M.K., Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during celf adhesion in serumcontaining medium, J. Eiol. Chem. 1982, 257(g), 4888-4893 Haas, R. and Culp, L.A., Properties and fate of plasma fibronectin bound to the tissue culture substratum, J. Cell Physiol. 1982, 113, 289-297 Knox, P., Kinetics of cell spreading in the presence of different concentrations of serum or fibronectin-depleted serum, 1, Ceil Sci. 1984, 71,51-59 Dekker, A., Beugeling, T., Wind, I-I., Poot, A., Bantjes, A.,
Biomaterials
1992, Vol. 13 No. 13
39
40
41
42
43
44 45
on detachment
of fibroblasts:
T.G. van Koofen et al.
Feijen, J. and Van Aken, W.G., Deposition of cellular fibronectin and desorption of human serum albumin during adhesion and spreading of human endothelial cells on polymers, J. Mater. Sci.: Mater. Med. 1991, 2, 227-233 Cozens-Roberts, C., Lauffenburger, D.A. and Quinn, J.A., Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis, Biophys. J. 1990,58(4), 841-856 Chan. P.-Y., Lawrence, M.B., Dustin, M.L., Ferguson, L.M., Golan, D.E. and Springer, T.A., Influence of receptor lateral mobility on adhesion st~ngthening between membranes containing LFA-3 and CD2 J. Ceii Biol. 1991, 115(l), 245-255 Burridge, K., Fath, K., Kelly, T., Nuckolls, G. and Turner, C., Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton, Ann. Rev. Ceil Viol. 1988, 4, 487-525 Weiss, L., Studies on cellular adhesion in tissue culture IV. The alteration of substrata by cell surfaces, Exp. Cell Res.1961,25,504-517 Weiss, L. and Coombs, R.R.A.. The demonstration of rupture of cell surfaces by an immunological technique, Exp. CellRes. 1963,30,331-338 Hubbe, M.A., Adhesion and detachment of biological cells in vitro, Prog. Surface Sci. 1981, 11, 65-138 Lotz, M.M., Burdsal, CA., Erickson, H.P. and McClay, D.R., Cell adhesion to fibronectin and tenascin: Quantitative measurements of initial binding and subsequent strengthening response, 1. Cell Biol. 1989, 109, 1795-1805
