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The orientation of fibroblasts and neutrophils on elastic substrata
Experimental Cell Research 146 (1983) 117-126
The Orientation
of Fibroblasts
and Neutrophils
Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/83/070117-10$02.00/0
on Elastic Substrata
WENDY S. HASTON, J. M. SHIELDS and P. C. WILKINSON Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow, GlI 6NT Scotland
SUMMARY The reaction of tibroblasts to the elastic properties of the substratum was studied using elastic collagen films. These films were stretched in one axis to give a substratum which was anisotropic in its elasticity and deformability. Analysis of the orientation of tibroblasts cultured on these substrata showed that they oriented along the axis of stretch which was also the axis of fibre alignment. This orientation was significantly reduced when the films were made less elastic by attachment to a glass slide and chemical fixation. Neither of these procedures appeared to alter the surface shape of these films, which suggests that the elastic properties of the substratum markedly influence the orientation of tibroblasts. The orientation of locomotion of neutrophil leukocytes on elastic collagen films was also analysed and no bias along the axis of stretch was observed. This was compared with neutrophil locomotion in 3-D stretched collagen gels, in which a strong bias along the axis of stretch and of tibre alignment was observed. The possible reasons for the response of these two cell types is discussed.
It has been proposed by Harris et al. [l, 2, 31 that the primary function of the excessive traction generated by locomoting fibroblasts is the rearrangement of extracellular matrices. This rearrangement results from the tension which cells place on deformable fibrous materials such as collagen gels. The same authors also suggested that libroblasts subsequently make use of such rearrangements to facilitate movement along paths of alignment, a phenomenon described as contact guidance by Weiss [4]. The mechanisms controlling the guidance of libroblasts, however, are not clear. In a system involving only one variable, namely curvature, Dunn & Heath [5] showed conclusively that chick heart tibroblasts were responding to the shape of the substratum. They demonstrated that certain threshold curvatures on a fine glass rod imposed discontinuities in the microlilament system. Cells would, therefore, tend to align along the axis of least curvature, since this was also the axis of minimal cytoskeletal distortion. Dunn & Ebendal [6] extended this hypothesis to explain the guidance response of fibroblasts on aligned 3-D collagen matrices. The aligned bands of collagen which result from the rearrangement of gels by fibroblasts from the application of external pulling forces are, however, under considerable tension. Thus the substratum is not isotropic in its deformability, being more deformable in the axis perpendicular to the axis of stretch. This deformability is to some extent reversible because of the elastic properties of collagen gels and therefore anisotropic elasticity and deformability should be considered as other, possibly major, determinants of fibroblast guidance on deformable, fibrous matrices. This possibility was predicted by Dunn [7]. In this paper comparing fibroblast orientation on substrata with identical surface shapes but varying in elasticity, we provide evidence that fibroblasts do show a guidance response on substrata which are anisotropically deformable. We
118 Haston. Shields and Wilkinson
Exp Cell Res 146 (1983)
compare this with the guidance response of neutrophil leukocytes which generate very low tractional forces when moving [l] and which may therefore exert insufficient tension to distort aligned collagen fibres and thus to respond to a field which is anisotropically deformable. We have made no distinction in this paper between deformability and elasticity, although the two properties are clearly distinct, i.e., an elastic substratum is deformable but a deformable substratum may not be very elastic. MATERIALS
AND METHODS
Cells. BHK and 3T3 cells were supplied by Dr J Edwards, Department of Cell Biology, Glasgow University, and maintained in serial passage in 50 ml Falcon flasks, in RPM1 1640 medium (Flow Laboratories) supplemented with 10% FCS and 100 IU penicillin/streptomycin. Fibroblasts in log phase were removed from flasks for use by incubating for 5 min with 0.1% bovine trypsin at 37°C and washing the resulting cell suspension in RPM1 1640 medium containing 20 % FCS. Neutrophil leukocytes were obtained by Dextran sedimentation of normal venous blood, followed by centrifugation through FicollfDiosil (Flow Laboratories, Irvine, Scotland). After two washes the neutrophils were finally suspended in Hanks-MOPS (morpholinopropane sulphonate) buffer at pH 7.2 with 10% autologous serum. Substrata. We experienced some difficulty in selecting a substratum which was elastic enough to stretch without forming surface wrinkles, with minimal surface shape, and both optically and physically suitable for cell culture. For example, the silicone rubber films used by Harris et al. [l] while shown to be deformable by fibroblasts, tended to shatter or wrinkle when placed under external tension. These films also form surface ‘stress wrinkles’ when tension is applied by cells growing out of tissue explants and therefore incidental changes in surface shape cannot be controlled [l]. We eventually prepared elastic substrata with minimal surface shape, by dehydrating thin collagen gels into films, in which the collagen tibres collapse together so that the 3-D gel structure is lost.
Preparation
of collagen films
Collagen, in aqueous solution, was prepared from rat tail tendons, as described previously [8]. This solution was brought to physiological pH and osmotic strength by diluting 8.8 ml collagen solution with 1 ml 10X RPM1 1640and adding 200 ul 1 M MOPS (pH 7.3) and then adjusting the pH to 7.0 with NaOH. 1.5 ml of this mixture was pipetted onto siliconized glass slides, spread evenly, and allowed to dry in a stream of sterile air. After drying, the slides were immersed in PBS which does not rehydrate the films, and the collagen film was peeled away from the slide with forceps. We found these films to be elastic, easy to manipulate, optically excellent and suitable for cell culture. The film was floated onto another slide and a piece of filter paper placed over each end of the film to stretch it to the required length (approx. 130% original). The film was fixed in position by placing a stainless steel slide, with a 16-mm diameter hole cut in the centre, over the film and sealing it in position with silicone grease. This left a chamber in which cells could be cultured on the upper surface of the collagen film. This type of fibrous matrix has complex mechanical properties and therefore isolating a single property such as elasticity presents a difficult problem. The gross elasticity of the gel, before dehydration, is a direct result of the random orientation of the collagen tibres which superficially resembles the organization of collagen tibres in skin [9], and like skin, the gel can stretch up to 200% before tearing, if it is freed from the substratum. In contrast, the individual collagen tibres can only stretch by about 10% before failing mechanically. Collagen films are good elastic substrata, but in order to test the hypothesis that anisotropic elasticity may determine guidance, then this elasticity must be removed or at least minimised. The films were treated as follows to modify their elasticity. (a) Some films were kept wet after stretching and were, therefore, prevented from adhering to the glass slide supporting them. These were the most elastic. (b) Some films were dried down onto the glass slide so that the films became adherent to the glass and thus lost their gross elasticity. (c) Since the films were composed of sheets of tibres, drying down the collagen film onto the supporting substratum (as described in b) might not necessarily immobihse tibres in the upper surface. We attempted to reduce the intrinsic elasticity of the individual tibres, and the deformability of the
Exp Cell Res 146(1983)
Orientation
offibroblasts
and neutrophils
on elastic substrata
Fig. 1. The orientation of fibroblasts on collagen and latex rubber films after 24 h in culture. (a-c) 3T3 cells on collagen films. In (a) the film was not attached to the glass substratum; in (b) the film was dried onto the glass; and in (c) the film was dried onto the glass and subsequently fixed with glutaraldehyde. (4 A BHK fibroblast oriented along the axis of stretch on a latex rubber film. Bars, 50 Irm. fibres relative to each other, by fixing the films with 2.5 % glutaraldehyde. Both the non-adherent (a) and adherent films (b) were fixed with glutaraldehyde, followed by overnight washing in PBS, and 2~ 10 min rinses in glycine buffer to block any remaining free aldehyde groups, before culturing cells on the films.
Preparation
of 3-D collagen gels for experiments
with neutrophil
leukocytes
Aligned hydrated collagen gels were prepared as described by Wilkinson et al. [IO]. Briefly, 2 ml of collagen solution containing lo6 neutrophils (adjusted to physiological pH and ionic strength) were pipetted onto a glass slide. The spread liquid was allowed to set and then loosened from the glass with an orange stick. A piece of filter paper was placed over each end of the loosened gel and then moved apart, stretching the gel to the required extent, about 30% greater than the original length.
Latex film We also found that thin layers of latex rubber in aqueous solution could be dried onto glass slides and then removed as a thin film which could be stretched to give a flat, unwrinkled substratum. Unfortunately, although some results were obtained, the optical properties were poor and cells only attached and spread in low numbers. Cell culture. 3T3 cells and BHK tibroblasts were cultured on the various substrata in RPM1 1640 medium plus 10% FCS for 24 h at 37°C. After 24 h the cultures were fixed in 2.5 % glutaraldehyde for measuring. Neutrophil leukocytes were pipetted onto the collagen films in the culture chamber, in RPM1 1640 medium plus 10% autologous plasma and filmed immediately on the heated stage of an inverted microscope, using a Nikon 16 mm tine camera and autotimer at one frame every 4 sec. Measurement. Fibroblast guidance was measured as described by Dunn & Heath [ll using nuclear orientation. The length of the nucleus along the axis of stretch (A) and at right angles (E) was taken for 50 cells on all of the substrata and orientation was expressed as the mean log,, A/B.
119
120 Haston, Shields and Wilkinson
Exp Cell Res 146(1983)
Fig. 2. SEM micrographs of BHK fibroblasts on collagen films after 24 h in culture. In (a) the fibroblast is oriented along the axis of stretch on a film which was dried onto the glass substratum; in (b) the film was dried onto the substratum and then fixed and (c) is a higher magnification of a fibroblast on an unfixed film, showing that the surface of the collagen film is essentially co-planar. Bars, (a, b) 10 pm; (c) 1 urn.
Since nuclear orientation of neutrophils cannot be measured, the orientation of neutrophil locomotion was measured as described by Wilkinson et al. [lo]. Briefly, the positions of neutrophils were tracked from the time-lapse film by marking the cell centre at 40 set intervals (10 frames) giving a cell track. The angle (between 0 and 90” and ignoring the sign) made to the axis of stretch by each 10frame section of the cell track was measured. Orientation of locomotion was assessed by summing the number of segments for each cell at angles between 0 and 44” to the axis of stretch (A), and the number of segments between 45 and 90” to that axis (B). A simple comparison can be made between different experiments by calculating C (A)/C (B) which should be 1.O where locomotion is random, and greater than 1.Oif the cells are orienting to the axis of stretch. Significance was measured by a x2 test. Scanning electron microscopy (SEM). Preparations were fixed at room temperature with 2.5% glutaraldehyde (Sigma, Poole, Dorset) in 0.1 M phosphate buffer, pH 7.0 for 30 min, followed by two
Exp Cell Res 146(1983)
Orientation
offibroblasts
and neutrophils
on elastic substrata
washes of 15 mitt in phosphate buffer with 0.2 M sucrose. The preparations were post-fixed with 1% 0~0~ in phosphate buffer for 30 min, washed twice in phosphate buffer for 5 min each and then rinsed in distilled water for 10 min before dehydrating through 30, 50, 70, 85 and 3 x 100% ethanol for 5 min each. The dehydrated preparations were critical point-dried in a Poloron CPD and then sputter-coated to give 10 nm coating from a 50/40% gold/palladium source using a stage cooled to - lO”C, using a longer coating cycle to reduce heat damage to the collagen films.
RESULTS Substrata
Collagen gels could be stretched up to 200% of their original length before tearing, as measured after stretching manually, but after dehydration stretchability was reduced to about 150%. This reduction in elasticity was presumably because a proportion of the fibres became adherent to each other and were therefore immobilised. Stretching the dehydrated films pulled many of the tibres into alignment along the axis of stretch (fig. 1a-c) but at the same time the surface of the film became essentially co-planar as a result of the fibres moving close together (see below, fig. 2a-c). By drying the stretched films onto the glass substratum, the gross elasticity of the film was obviously reduced but fibres in the upper surface, not adherent to the glass, presumably retained their intrinsic elasticity and some capability of movement relative to each other. It is this residual elasticity which we attempted to remove by fixation of the films with glutaraldehyde. Glutaraldehyde fixation of the non-adherent films made them brittle and we were unable to stretch them manually in excess of 120% of the original length without tearing them. However, we have observed by SEM that hydrated (3-D) collagen gels which have been glutaraldehyde-fixed, dehydrated and critical point-dried, can be deformed and even aligned when placed under external tension. It is probable, then, that glutaraldehyde fixation of the non-adherent films would have left them with some deformability, although the elasticity of individual collagen fibres was abolished. The least elastic substrata were produced by glutaraldehyde-fixing those films which had been dried onto the glass substratum. Table 1. Mean nuclear orientation
offibroblasts
relative to the axis of stretch on
dried collagen films Figures represent the mean of 50 randomly selected nuclei from l-2 day cultures of BHK cells and 3T3 cells Treatment of collagen film Cell type
Stretched wet
Stretched dried
Stretched wet-fixed
Stretched dried-fixed
BHK *SE
0.233 kO.015
0.212 kO.016
0.175 zko.019
0.104 kO.024
3T3 +SE
0.184 kO.029
0.081 kO.035
0.141 kO.024
-0.008 kO.041
The figures are log,, A/B (see text).
121
122 Haston, Shields and Wilkinson
Exp Cell Res 146 (1983)
The surface shape of these films was not visibly altered by either allowing them to adhere to a glass substratum or by glutaraldehyde fixation, as observed by SEM (see fig. 26). The response of 3T3 cells to the collagen substrata is shown in fig. 1a-c. The best guidance response was consistently observed on films which were not adherent to the glass, i.e. the most elastic substrata (fig. 1a, table 1). The fibroblasts still showed some response on the glass-adherent films (fig. 1 b, table 1) but the number of well-spread cells with ruffled margins was increased and orientation was significantly less than on the films which were not adherent to the glass. Glutaraldehyde fixation of the non-adherent films reduced the orientation of both fibroblast types significantly. Glutaraldehyde fixation of the adherent films (which produced the least deformable of the substrata) reduced the orientation of the BHK cells still further and no orientation of 3T3 cells could be demonstrated on these substrata (fig. 1 c, table 1). BHK fibroblasts showed guidance along the axis of stretch on thin latex rubber films (fig. 14, but it was not possible to measure nuclear orientation because of the poor optical properties of the latex rubber. Also, no practical method of immobilizing the rubber, to give an inelastic control, was found. We have previously reported that neutrophil leukocytes show an oriented response while moving inside a hydrated, 3-D collagen gel aligned by releasing the gel from the substratum and then stretching it [lo]. These gels had 3-D shape, but were also anisotropically elastic, because of the method of aligning the collagen fibres by placing the gel under tension. To test whether the neutrophils were responding to elasticity and deformability, we measured their orientation while moving on the surface of dehydrated collagen films. Fig. 3a shows the tracks of neutrophils moving inside a stretched, hydrated lattice (3-D), where the direction of locomotion was strongly biased along the axis of alignment of the collagen fibres. In contrast, no orientation of locomotion was observed on any of the 2-D films whether dehydrated, glutaraldehyde-fixed or unfixed. This is illustrated in fig. 3 b showing the tracks of neutrophils moving over the surface of the non-adherent collagen films. The angles made by individual segments of cell paths are shown in fig. 4a and b. Scanning electron micrographs of all of the collagen films showed that the collagen fibres were tightly packed and essentially co-planar. No difference in shape was detectable between any of the collagen films (fig. 2a, b). Although some fibroblasts were oriented along the axis of stretch on the adherent films, they were not markedly spindle-shaped. The main body of the cell was often elongated, but broad, ruffled and extremely flattened areas were seen at either end of the cell. These flattened areas were up to 20 urn wide, covering numerous collagen tibres (fig. 2a) providing further evidence that the substratum was essentially co-planar. In summary, fibroblasts showed a strong guidance response along the axis of stretch on elastic collagen films which were not attached to the substratum. This response was reduced but still significant when the collagen film was allowed to adhere to the substratum. Glutaraldehyde fixation of the adherent film reduced the response of BHK fibroblasts still further and abolished the response of 3T3
Exp Cell Res 146(1983)
Orientation
of fibroblasts
and neutrophils
on elastic substrata
a
b
\
/
Fig. 3. The tracks of neutrophil leukocytes moving through (a) the matrix of a hydrated, stretched collagen gel (3-D); (b) over the surface of a dehydrated stretched collagen film (2-D). In (a) the value for CA/CB is 1.7030 x2=11.7); (b) CA/CB=0.9060 (x2=0.027). Arrowed bar drawn along the axis of stretch, 100 pm.
123
124 Haston,
Shields and Wilkinson
Sector angle
Exp Cell Res 146 (1983)
between
0-W
Fig. 4. Alignment of 40-see segments of cell path related to the axis of alignment of collagen. The angle to that axis made by individual segments was measured (ignoring the direction of movement), i.e., they were scored between 0 and 90” ignoring the sign. Angles of segments of paths of cells moving (a) within a 3-D collagen gel (10 cells); (b) on the surface of a 2-D elastic collagen film (10 cells). Mean angle (a) 39.1”; (b) 43.5”.
fibroblasts. In contrast, neutrophil leukocytes showed orientation of locomotion moving inside 3-D-stretched collagen gels, but no orientation while moving on the surface of 2-D-stretched collagen films. Thus anisotropic deformability and elasticity, as well as surface shape, should be considered as possible determinants of guidance on aligned fibrous matrices. DISCUSSION The possible influences of various anisotropic properties of substrata on cell behaviour have been extensively reviewed by Dunn [7]. In the original experiments of Dunn & Heath [5] using glass rods of different diameter, surface curvature was the only variable property and therefore its effect on cell behaviour could be studied with some confidence. The elastic properties of a substratum are more complex, especially if the substratum is a fibrous matrix. An ideal substratum to measure the effects of anisotropic elasticity on cell behaviour would be one entirely without surface shape and unchanged chemically after stretching. Surface shape, however, can never be totally excluded and in the substrata used in these experiments collagen fibres could be distinguished with SEM. As the diameter of individual fibres is about 0.1 urn then we must assume that at least 0.05 urn of the collagen fibres can stand proud of the surface. This, however, is true for all the collagen films used in those experiments. In other words, shape is constant but deformability and elasticity are not. Chemical changes induced by stretching may alter the adhesiveness of the substratum to cells and thus influence cell behaviour. For example, the change in the conformation of natural rubber molecules after stretching alters the hydrophobicity of rubber. The colla-
Exp Cell Res 146 (1983)
Orientation
offibroblasts
and neutrophils
on elastic substrata
gen films used in this paper do not change chemically after stretching because the individual collagen fibres are not stretched [9]. Rather elasticity results from the deformability of the fibrous matrix. Stretching such a fibrous matrix pulls many of the fibres into alignment and thus the film becomes anisotropic in surface shape. However, although the tibres are pulled very close together and the surface is essentially co-planar, the alignment of the fibres may still give rise to a field of anisotropic adhesiveness. In such a case, the aligned tibres of collagen can be compared with the substrata used by Carter [ll] made up of adhesive strips with decreasing repeat spacing. It has been suggested by Dunn [7] from these experiments that if the repeat spacing of adhesive strips was small enough for a spread cell to span several strips then no orientation along the strips would occur. In the experiments reported here, the fibroblasts clearly spanned numerous collagen tibres suggesting that adhesive anisotropy is not detected by the cells. Glutaraldehyde fixation of the adherent collagen film removed its ability to elicit a guidance response with 3T3 cells. As there was no apparent difference in the shape of the glutaraldehyde-fixed adherent films and the glutaraldehyde-fixed non-adherent films, we would suggest that the loss of guidance by cells on the fixed, adherent fdms was because these substrata were isotropic in their deformability in contrast to the glutaraldehyde-fixed but non-adherent films which retained enough anisotropic deformability to elicit a response. In an anisotropic field of elasticity one would expect that orientation of cells would be preferred along the axis where the tractional forces exerted by the cell caused minimal distortion of the substratum. If an elastic substratum deforms as the result of traction exerted by a spreading fibroblast, then the substratum must be gathered underneath the cell, as demonstrated by Harris et al. [I], until the resistance to stretch equals the tractional force exerted by the tibroblast, and then the deformed substratum must be held in position by the fibroblast to prevent it recoiling as the cell moves forward. This would clearly present problems to a locomoting cell and it is not clear from the results of Stopak 8z Harris [3] whether the fibroblasts which pull a collagen gel into alignment between two explants are themselves able to move and take advantage of the guidance field which they have created. The response of fibroblasts to the anisotropic elasticity of a substratum must depend on the cell generating a tractional force which can distort the substratum. This in turn must depend on the adhesive interaction between the fibroblast and collagen and therefore one might predict that transformed fibroblasts would show significantly less response than normal tibroblasts on elastic substrata. Differences in the responses of BHK and 3T3 cells may also be a result of differences in their adhesive interactions with collagen. Neutrophil leukocytes show oriented locomotion while moving inside 3-Dhydrated collagen gels which have been aligned by stretching [lo]. The lack of orientation shown by neutrophils moving on the 2-D collagen films described here suggests that the forces exerted by these cells are not great enough to distort the substratum and therefore not great enough for them to detect, and respond to, these levels of anisotropic elasticity. Harris et al. [l] found that neutrophils were
125
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Exp Cell Res 146 (1983)
unable to distort silicone rubber sheets suggesting that the traction generated by moving neutrophils is indeed very small compared with tibroblasts. Neutrophils, which apparently lack microfilament bundles when moving, are also unlikely to respond to the 3-D shape of collagen gels by aligning along the axis of minimal cytoskeletal distortion, as has been suggested for fibroblasts [6]. It is interesting that the potential of fibroblasts to modify extracellular matrices by tractional structuring [3] may direct the locomotion of other cell types such as neutrophil leukocytes which probably respond to as yet undefined features of the 3-D matrix. This work was supported by the MRC. We thank Lawrence Tetly and Helen Hendry, Department of Zoology, Glasgow University, for their advice on scanning electron microscopy.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Harris, A K, Wild, P & Stopak, D, Science 208 (1980) 177. Harris, A K, Stopak, D & Wild, P, Nature 290 (1981) 249. Stopak, D & Harris, A K, Dev biol 90 (1982) 383. Weiss, P, J exp zoo1 68 (1934) 393. Dunn, G A & Heath, J P, Exp cell res 101 (1976) 1. Dunn, G A & Ebendal, T, Exp cell res 111 (1978) 475. Dunn, G A, Cell behaviour (ed R Bellairs, A S G Curtis & G A Dunn) pp. 247-280. Cambridge University Press, Cambridge (1982). Elsdale, T & Bard, J, J cell biol 54 (1972) 626. Trelstead, R L & Silver, F H, Cell biology of the extracellular matrix (ed Elisabeth Hay) pp. 179-215. Plenum Press, New York and London (1981). Wilkinson, P C, Shields, J M & Haston, W S, Exp cell res 140 (1982) 55. Carter, S B, Nature 213 (1967) 256.
Received November 10, 1982 Revised version received January 25, 1983
The Orientation
of Fibroblasts
and Neutrophils
Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/83/070117-10$02.00/0
on Elastic Substrata
WENDY S. HASTON, J. M. SHIELDS and P. C. WILKINSON Department of Bacteriology and Immunology, University of Glasgow, Western Infirmary, Glasgow, GlI 6NT Scotland
SUMMARY The reaction of tibroblasts to the elastic properties of the substratum was studied using elastic collagen films. These films were stretched in one axis to give a substratum which was anisotropic in its elasticity and deformability. Analysis of the orientation of tibroblasts cultured on these substrata showed that they oriented along the axis of stretch which was also the axis of fibre alignment. This orientation was significantly reduced when the films were made less elastic by attachment to a glass slide and chemical fixation. Neither of these procedures appeared to alter the surface shape of these films, which suggests that the elastic properties of the substratum markedly influence the orientation of tibroblasts. The orientation of locomotion of neutrophil leukocytes on elastic collagen films was also analysed and no bias along the axis of stretch was observed. This was compared with neutrophil locomotion in 3-D stretched collagen gels, in which a strong bias along the axis of stretch and of tibre alignment was observed. The possible reasons for the response of these two cell types is discussed.
It has been proposed by Harris et al. [l, 2, 31 that the primary function of the excessive traction generated by locomoting fibroblasts is the rearrangement of extracellular matrices. This rearrangement results from the tension which cells place on deformable fibrous materials such as collagen gels. The same authors also suggested that libroblasts subsequently make use of such rearrangements to facilitate movement along paths of alignment, a phenomenon described as contact guidance by Weiss [4]. The mechanisms controlling the guidance of libroblasts, however, are not clear. In a system involving only one variable, namely curvature, Dunn & Heath [5] showed conclusively that chick heart tibroblasts were responding to the shape of the substratum. They demonstrated that certain threshold curvatures on a fine glass rod imposed discontinuities in the microlilament system. Cells would, therefore, tend to align along the axis of least curvature, since this was also the axis of minimal cytoskeletal distortion. Dunn & Ebendal [6] extended this hypothesis to explain the guidance response of fibroblasts on aligned 3-D collagen matrices. The aligned bands of collagen which result from the rearrangement of gels by fibroblasts from the application of external pulling forces are, however, under considerable tension. Thus the substratum is not isotropic in its deformability, being more deformable in the axis perpendicular to the axis of stretch. This deformability is to some extent reversible because of the elastic properties of collagen gels and therefore anisotropic elasticity and deformability should be considered as other, possibly major, determinants of fibroblast guidance on deformable, fibrous matrices. This possibility was predicted by Dunn [7]. In this paper comparing fibroblast orientation on substrata with identical surface shapes but varying in elasticity, we provide evidence that fibroblasts do show a guidance response on substrata which are anisotropically deformable. We
118 Haston. Shields and Wilkinson
Exp Cell Res 146 (1983)
compare this with the guidance response of neutrophil leukocytes which generate very low tractional forces when moving [l] and which may therefore exert insufficient tension to distort aligned collagen fibres and thus to respond to a field which is anisotropically deformable. We have made no distinction in this paper between deformability and elasticity, although the two properties are clearly distinct, i.e., an elastic substratum is deformable but a deformable substratum may not be very elastic. MATERIALS
AND METHODS
Cells. BHK and 3T3 cells were supplied by Dr J Edwards, Department of Cell Biology, Glasgow University, and maintained in serial passage in 50 ml Falcon flasks, in RPM1 1640 medium (Flow Laboratories) supplemented with 10% FCS and 100 IU penicillin/streptomycin. Fibroblasts in log phase were removed from flasks for use by incubating for 5 min with 0.1% bovine trypsin at 37°C and washing the resulting cell suspension in RPM1 1640 medium containing 20 % FCS. Neutrophil leukocytes were obtained by Dextran sedimentation of normal venous blood, followed by centrifugation through FicollfDiosil (Flow Laboratories, Irvine, Scotland). After two washes the neutrophils were finally suspended in Hanks-MOPS (morpholinopropane sulphonate) buffer at pH 7.2 with 10% autologous serum. Substrata. We experienced some difficulty in selecting a substratum which was elastic enough to stretch without forming surface wrinkles, with minimal surface shape, and both optically and physically suitable for cell culture. For example, the silicone rubber films used by Harris et al. [l] while shown to be deformable by fibroblasts, tended to shatter or wrinkle when placed under external tension. These films also form surface ‘stress wrinkles’ when tension is applied by cells growing out of tissue explants and therefore incidental changes in surface shape cannot be controlled [l]. We eventually prepared elastic substrata with minimal surface shape, by dehydrating thin collagen gels into films, in which the collagen tibres collapse together so that the 3-D gel structure is lost.
Preparation
of collagen films
Collagen, in aqueous solution, was prepared from rat tail tendons, as described previously [8]. This solution was brought to physiological pH and osmotic strength by diluting 8.8 ml collagen solution with 1 ml 10X RPM1 1640and adding 200 ul 1 M MOPS (pH 7.3) and then adjusting the pH to 7.0 with NaOH. 1.5 ml of this mixture was pipetted onto siliconized glass slides, spread evenly, and allowed to dry in a stream of sterile air. After drying, the slides were immersed in PBS which does not rehydrate the films, and the collagen film was peeled away from the slide with forceps. We found these films to be elastic, easy to manipulate, optically excellent and suitable for cell culture. The film was floated onto another slide and a piece of filter paper placed over each end of the film to stretch it to the required length (approx. 130% original). The film was fixed in position by placing a stainless steel slide, with a 16-mm diameter hole cut in the centre, over the film and sealing it in position with silicone grease. This left a chamber in which cells could be cultured on the upper surface of the collagen film. This type of fibrous matrix has complex mechanical properties and therefore isolating a single property such as elasticity presents a difficult problem. The gross elasticity of the gel, before dehydration, is a direct result of the random orientation of the collagen tibres which superficially resembles the organization of collagen tibres in skin [9], and like skin, the gel can stretch up to 200% before tearing, if it is freed from the substratum. In contrast, the individual collagen tibres can only stretch by about 10% before failing mechanically. Collagen films are good elastic substrata, but in order to test the hypothesis that anisotropic elasticity may determine guidance, then this elasticity must be removed or at least minimised. The films were treated as follows to modify their elasticity. (a) Some films were kept wet after stretching and were, therefore, prevented from adhering to the glass slide supporting them. These were the most elastic. (b) Some films were dried down onto the glass slide so that the films became adherent to the glass and thus lost their gross elasticity. (c) Since the films were composed of sheets of tibres, drying down the collagen film onto the supporting substratum (as described in b) might not necessarily immobihse tibres in the upper surface. We attempted to reduce the intrinsic elasticity of the individual tibres, and the deformability of the
Exp Cell Res 146(1983)
Orientation
offibroblasts
and neutrophils
on elastic substrata
Fig. 1. The orientation of fibroblasts on collagen and latex rubber films after 24 h in culture. (a-c) 3T3 cells on collagen films. In (a) the film was not attached to the glass substratum; in (b) the film was dried onto the glass; and in (c) the film was dried onto the glass and subsequently fixed with glutaraldehyde. (4 A BHK fibroblast oriented along the axis of stretch on a latex rubber film. Bars, 50 Irm. fibres relative to each other, by fixing the films with 2.5 % glutaraldehyde. Both the non-adherent (a) and adherent films (b) were fixed with glutaraldehyde, followed by overnight washing in PBS, and 2~ 10 min rinses in glycine buffer to block any remaining free aldehyde groups, before culturing cells on the films.
Preparation
of 3-D collagen gels for experiments
with neutrophil
leukocytes
Aligned hydrated collagen gels were prepared as described by Wilkinson et al. [IO]. Briefly, 2 ml of collagen solution containing lo6 neutrophils (adjusted to physiological pH and ionic strength) were pipetted onto a glass slide. The spread liquid was allowed to set and then loosened from the glass with an orange stick. A piece of filter paper was placed over each end of the loosened gel and then moved apart, stretching the gel to the required extent, about 30% greater than the original length.
Latex film We also found that thin layers of latex rubber in aqueous solution could be dried onto glass slides and then removed as a thin film which could be stretched to give a flat, unwrinkled substratum. Unfortunately, although some results were obtained, the optical properties were poor and cells only attached and spread in low numbers. Cell culture. 3T3 cells and BHK tibroblasts were cultured on the various substrata in RPM1 1640 medium plus 10% FCS for 24 h at 37°C. After 24 h the cultures were fixed in 2.5 % glutaraldehyde for measuring. Neutrophil leukocytes were pipetted onto the collagen films in the culture chamber, in RPM1 1640 medium plus 10% autologous plasma and filmed immediately on the heated stage of an inverted microscope, using a Nikon 16 mm tine camera and autotimer at one frame every 4 sec. Measurement. Fibroblast guidance was measured as described by Dunn & Heath [ll using nuclear orientation. The length of the nucleus along the axis of stretch (A) and at right angles (E) was taken for 50 cells on all of the substrata and orientation was expressed as the mean log,, A/B.
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Fig. 2. SEM micrographs of BHK fibroblasts on collagen films after 24 h in culture. In (a) the fibroblast is oriented along the axis of stretch on a film which was dried onto the glass substratum; in (b) the film was dried onto the substratum and then fixed and (c) is a higher magnification of a fibroblast on an unfixed film, showing that the surface of the collagen film is essentially co-planar. Bars, (a, b) 10 pm; (c) 1 urn.
Since nuclear orientation of neutrophils cannot be measured, the orientation of neutrophil locomotion was measured as described by Wilkinson et al. [lo]. Briefly, the positions of neutrophils were tracked from the time-lapse film by marking the cell centre at 40 set intervals (10 frames) giving a cell track. The angle (between 0 and 90” and ignoring the sign) made to the axis of stretch by each 10frame section of the cell track was measured. Orientation of locomotion was assessed by summing the number of segments for each cell at angles between 0 and 44” to the axis of stretch (A), and the number of segments between 45 and 90” to that axis (B). A simple comparison can be made between different experiments by calculating C (A)/C (B) which should be 1.O where locomotion is random, and greater than 1.Oif the cells are orienting to the axis of stretch. Significance was measured by a x2 test. Scanning electron microscopy (SEM). Preparations were fixed at room temperature with 2.5% glutaraldehyde (Sigma, Poole, Dorset) in 0.1 M phosphate buffer, pH 7.0 for 30 min, followed by two
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Orientation
offibroblasts
and neutrophils
on elastic substrata
washes of 15 mitt in phosphate buffer with 0.2 M sucrose. The preparations were post-fixed with 1% 0~0~ in phosphate buffer for 30 min, washed twice in phosphate buffer for 5 min each and then rinsed in distilled water for 10 min before dehydrating through 30, 50, 70, 85 and 3 x 100% ethanol for 5 min each. The dehydrated preparations were critical point-dried in a Poloron CPD and then sputter-coated to give 10 nm coating from a 50/40% gold/palladium source using a stage cooled to - lO”C, using a longer coating cycle to reduce heat damage to the collagen films.
RESULTS Substrata
Collagen gels could be stretched up to 200% of their original length before tearing, as measured after stretching manually, but after dehydration stretchability was reduced to about 150%. This reduction in elasticity was presumably because a proportion of the fibres became adherent to each other and were therefore immobilised. Stretching the dehydrated films pulled many of the tibres into alignment along the axis of stretch (fig. 1a-c) but at the same time the surface of the film became essentially co-planar as a result of the fibres moving close together (see below, fig. 2a-c). By drying the stretched films onto the glass substratum, the gross elasticity of the film was obviously reduced but fibres in the upper surface, not adherent to the glass, presumably retained their intrinsic elasticity and some capability of movement relative to each other. It is this residual elasticity which we attempted to remove by fixation of the films with glutaraldehyde. Glutaraldehyde fixation of the non-adherent films made them brittle and we were unable to stretch them manually in excess of 120% of the original length without tearing them. However, we have observed by SEM that hydrated (3-D) collagen gels which have been glutaraldehyde-fixed, dehydrated and critical point-dried, can be deformed and even aligned when placed under external tension. It is probable, then, that glutaraldehyde fixation of the non-adherent films would have left them with some deformability, although the elasticity of individual collagen fibres was abolished. The least elastic substrata were produced by glutaraldehyde-fixing those films which had been dried onto the glass substratum. Table 1. Mean nuclear orientation
offibroblasts
relative to the axis of stretch on
dried collagen films Figures represent the mean of 50 randomly selected nuclei from l-2 day cultures of BHK cells and 3T3 cells Treatment of collagen film Cell type
Stretched wet
Stretched dried
Stretched wet-fixed
Stretched dried-fixed
BHK *SE
0.233 kO.015
0.212 kO.016
0.175 zko.019
0.104 kO.024
3T3 +SE
0.184 kO.029
0.081 kO.035
0.141 kO.024
-0.008 kO.041
The figures are log,, A/B (see text).
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The surface shape of these films was not visibly altered by either allowing them to adhere to a glass substratum or by glutaraldehyde fixation, as observed by SEM (see fig. 26). The response of 3T3 cells to the collagen substrata is shown in fig. 1a-c. The best guidance response was consistently observed on films which were not adherent to the glass, i.e. the most elastic substrata (fig. 1a, table 1). The fibroblasts still showed some response on the glass-adherent films (fig. 1 b, table 1) but the number of well-spread cells with ruffled margins was increased and orientation was significantly less than on the films which were not adherent to the glass. Glutaraldehyde fixation of the non-adherent films reduced the orientation of both fibroblast types significantly. Glutaraldehyde fixation of the adherent films (which produced the least deformable of the substrata) reduced the orientation of the BHK cells still further and no orientation of 3T3 cells could be demonstrated on these substrata (fig. 1 c, table 1). BHK fibroblasts showed guidance along the axis of stretch on thin latex rubber films (fig. 14, but it was not possible to measure nuclear orientation because of the poor optical properties of the latex rubber. Also, no practical method of immobilizing the rubber, to give an inelastic control, was found. We have previously reported that neutrophil leukocytes show an oriented response while moving inside a hydrated, 3-D collagen gel aligned by releasing the gel from the substratum and then stretching it [lo]. These gels had 3-D shape, but were also anisotropically elastic, because of the method of aligning the collagen fibres by placing the gel under tension. To test whether the neutrophils were responding to elasticity and deformability, we measured their orientation while moving on the surface of dehydrated collagen films. Fig. 3a shows the tracks of neutrophils moving inside a stretched, hydrated lattice (3-D), where the direction of locomotion was strongly biased along the axis of alignment of the collagen fibres. In contrast, no orientation of locomotion was observed on any of the 2-D films whether dehydrated, glutaraldehyde-fixed or unfixed. This is illustrated in fig. 3 b showing the tracks of neutrophils moving over the surface of the non-adherent collagen films. The angles made by individual segments of cell paths are shown in fig. 4a and b. Scanning electron micrographs of all of the collagen films showed that the collagen fibres were tightly packed and essentially co-planar. No difference in shape was detectable between any of the collagen films (fig. 2a, b). Although some fibroblasts were oriented along the axis of stretch on the adherent films, they were not markedly spindle-shaped. The main body of the cell was often elongated, but broad, ruffled and extremely flattened areas were seen at either end of the cell. These flattened areas were up to 20 urn wide, covering numerous collagen tibres (fig. 2a) providing further evidence that the substratum was essentially co-planar. In summary, fibroblasts showed a strong guidance response along the axis of stretch on elastic collagen films which were not attached to the substratum. This response was reduced but still significant when the collagen film was allowed to adhere to the substratum. Glutaraldehyde fixation of the adherent film reduced the response of BHK fibroblasts still further and abolished the response of 3T3
Exp Cell Res 146(1983)
Orientation
of fibroblasts
and neutrophils
on elastic substrata
a
b
\
/
Fig. 3. The tracks of neutrophil leukocytes moving through (a) the matrix of a hydrated, stretched collagen gel (3-D); (b) over the surface of a dehydrated stretched collagen film (2-D). In (a) the value for CA/CB is 1.7030 x2=11.7); (b) CA/CB=0.9060 (x2=0.027). Arrowed bar drawn along the axis of stretch, 100 pm.
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Sector angle
Exp Cell Res 146 (1983)
between
0-W
Fig. 4. Alignment of 40-see segments of cell path related to the axis of alignment of collagen. The angle to that axis made by individual segments was measured (ignoring the direction of movement), i.e., they were scored between 0 and 90” ignoring the sign. Angles of segments of paths of cells moving (a) within a 3-D collagen gel (10 cells); (b) on the surface of a 2-D elastic collagen film (10 cells). Mean angle (a) 39.1”; (b) 43.5”.
fibroblasts. In contrast, neutrophil leukocytes showed orientation of locomotion moving inside 3-D-stretched collagen gels, but no orientation while moving on the surface of 2-D-stretched collagen films. Thus anisotropic deformability and elasticity, as well as surface shape, should be considered as possible determinants of guidance on aligned fibrous matrices. DISCUSSION The possible influences of various anisotropic properties of substrata on cell behaviour have been extensively reviewed by Dunn [7]. In the original experiments of Dunn & Heath [5] using glass rods of different diameter, surface curvature was the only variable property and therefore its effect on cell behaviour could be studied with some confidence. The elastic properties of a substratum are more complex, especially if the substratum is a fibrous matrix. An ideal substratum to measure the effects of anisotropic elasticity on cell behaviour would be one entirely without surface shape and unchanged chemically after stretching. Surface shape, however, can never be totally excluded and in the substrata used in these experiments collagen fibres could be distinguished with SEM. As the diameter of individual fibres is about 0.1 urn then we must assume that at least 0.05 urn of the collagen fibres can stand proud of the surface. This, however, is true for all the collagen films used in those experiments. In other words, shape is constant but deformability and elasticity are not. Chemical changes induced by stretching may alter the adhesiveness of the substratum to cells and thus influence cell behaviour. For example, the change in the conformation of natural rubber molecules after stretching alters the hydrophobicity of rubber. The colla-
Exp Cell Res 146 (1983)
Orientation
offibroblasts
and neutrophils
on elastic substrata
gen films used in this paper do not change chemically after stretching because the individual collagen fibres are not stretched [9]. Rather elasticity results from the deformability of the fibrous matrix. Stretching such a fibrous matrix pulls many of the fibres into alignment and thus the film becomes anisotropic in surface shape. However, although the tibres are pulled very close together and the surface is essentially co-planar, the alignment of the fibres may still give rise to a field of anisotropic adhesiveness. In such a case, the aligned tibres of collagen can be compared with the substrata used by Carter [ll] made up of adhesive strips with decreasing repeat spacing. It has been suggested by Dunn [7] from these experiments that if the repeat spacing of adhesive strips was small enough for a spread cell to span several strips then no orientation along the strips would occur. In the experiments reported here, the fibroblasts clearly spanned numerous collagen tibres suggesting that adhesive anisotropy is not detected by the cells. Glutaraldehyde fixation of the adherent collagen film removed its ability to elicit a guidance response with 3T3 cells. As there was no apparent difference in the shape of the glutaraldehyde-fixed adherent films and the glutaraldehyde-fixed non-adherent films, we would suggest that the loss of guidance by cells on the fixed, adherent fdms was because these substrata were isotropic in their deformability in contrast to the glutaraldehyde-fixed but non-adherent films which retained enough anisotropic deformability to elicit a response. In an anisotropic field of elasticity one would expect that orientation of cells would be preferred along the axis where the tractional forces exerted by the cell caused minimal distortion of the substratum. If an elastic substratum deforms as the result of traction exerted by a spreading fibroblast, then the substratum must be gathered underneath the cell, as demonstrated by Harris et al. [I], until the resistance to stretch equals the tractional force exerted by the tibroblast, and then the deformed substratum must be held in position by the fibroblast to prevent it recoiling as the cell moves forward. This would clearly present problems to a locomoting cell and it is not clear from the results of Stopak 8z Harris [3] whether the fibroblasts which pull a collagen gel into alignment between two explants are themselves able to move and take advantage of the guidance field which they have created. The response of fibroblasts to the anisotropic elasticity of a substratum must depend on the cell generating a tractional force which can distort the substratum. This in turn must depend on the adhesive interaction between the fibroblast and collagen and therefore one might predict that transformed fibroblasts would show significantly less response than normal tibroblasts on elastic substrata. Differences in the responses of BHK and 3T3 cells may also be a result of differences in their adhesive interactions with collagen. Neutrophil leukocytes show oriented locomotion while moving inside 3-Dhydrated collagen gels which have been aligned by stretching [lo]. The lack of orientation shown by neutrophils moving on the 2-D collagen films described here suggests that the forces exerted by these cells are not great enough to distort the substratum and therefore not great enough for them to detect, and respond to, these levels of anisotropic elasticity. Harris et al. [l] found that neutrophils were
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unable to distort silicone rubber sheets suggesting that the traction generated by moving neutrophils is indeed very small compared with tibroblasts. Neutrophils, which apparently lack microfilament bundles when moving, are also unlikely to respond to the 3-D shape of collagen gels by aligning along the axis of minimal cytoskeletal distortion, as has been suggested for fibroblasts [6]. It is interesting that the potential of fibroblasts to modify extracellular matrices by tractional structuring [3] may direct the locomotion of other cell types such as neutrophil leukocytes which probably respond to as yet undefined features of the 3-D matrix. This work was supported by the MRC. We thank Lawrence Tetly and Helen Hendry, Department of Zoology, Glasgow University, for their advice on scanning electron microscopy.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Harris, A K, Wild, P & Stopak, D, Science 208 (1980) 177. Harris, A K, Stopak, D & Wild, P, Nature 290 (1981) 249. Stopak, D & Harris, A K, Dev biol 90 (1982) 383. Weiss, P, J exp zoo1 68 (1934) 393. Dunn, G A & Heath, J P, Exp cell res 101 (1976) 1. Dunn, G A & Ebendal, T, Exp cell res 111 (1978) 475. Dunn, G A, Cell behaviour (ed R Bellairs, A S G Curtis & G A Dunn) pp. 247-280. Cambridge University Press, Cambridge (1982). Elsdale, T & Bard, J, J cell biol 54 (1972) 626. Trelstead, R L & Silver, F H, Cell biology of the extracellular matrix (ed Elisabeth Hay) pp. 179-215. Plenum Press, New York and London (1981). Wilkinson, P C, Shields, J M & Haston, W S, Exp cell res 140 (1982) 55. Carter, S B, Nature 213 (1967) 256.
Received November 10, 1982 Revised version received January 25, 1983