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The effects of continuous and discontinuous groove edges on cell shape and alignment
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Experimental Cell Research 288 (2003) 177–188
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The effects of continuous and discontinuous groove edges on cell shape and alignment Ann-Sofie Andersson,* Petra Olsson, Ulf Lidberg, and Duncan Sutherland Department of Applied Physics, Chalmers University of Technology, SE-412 96, Go¨teborg, Sweden Received 10 December 2002, revised version received 19 February 2003
Abstract Nanofabricated model surfaces and digital image analysis of cell shape were used to address the importance of a continuous sharp edge in the alignment of cells to shallow surface grooves. The grooved model surfaces had either continuous or discontinuous edges of various depths (40 – 400 nm) but identical surface chemistry and groove/ridge dimensions (15 m wide). Epithelial cells were cultured on the model surfaces for 10 and 24 h. Fluorescence microscopy combined with image analysis were used to quantify cell area and alignment and to make cell shape classifications of individual cells. The degrees of alignment of cells and the percentages of elongated cell classes increased with groove depth on samples with continuous grooves. Two main differences, with regard to cell response, were observed between the continuous and discontinuous grooved surfaces. First, significantly fewer cells aligned to surface grooves with discontinuous edges than to grooves with continuous edges. Second, there were lower percentages of the elongated cell classes on discontinuous grooves than on continuous ones. We concluded that grooved surfaces with continuous edges are more potent in aligning and inducing elongated cells. The results from the present study suggest that a mechanism of alignment involving orientation along a continuous edge is likely. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Contact guidance; Nanostructures; Nanotopography; Surface topography; Surface grooves; Digital image analysis; Cell-surface interaction; Shape classification; Cell shape; Actin cytoskeleton
Introduction All cells in the human body are surrounded by topographic and chemical cues. These cues can be provided by other cells, the extracellular matrix (ECM), or synthetic materials. Examples of ECM-related cues are collagen and fibronectin fibers. These cues are also known for their ability to align and guide cells [1–3]. The ability to control and guide cells can be very useful in biomaterial and tissue engineering applications in order to create more advanced products [4,5]. However, the exact mechanism behind the cell guidance ability of, for example, a collagen fiber in vivo is unclear; in particular whether it is the topography and/or the chemistry that causes the guidance. Therefore, fabricated model surfaces with defined topographies (e.g.,
* Corresponding author. Fax: ⫹46-31-7723134. E-mail address: [email protected] (A.-S. Andersson).
grooves) or chemistries are used to study the cell-surface phenomenon called contact guidance. Substrates with parallel repeating step edges can impose directional constraints on cells (orientation and shape) and also affect the rate and direction of cell migration (i.e., contact guidance). In order to determine which controls topographically induced contact guidance, parameters such as groove/ridge width and groove depth have been systematically varied [1,6 –10]. The degree of contact guidance is also influenced by cellular parameters such as cell type and cell density [1,9,11–13]. Cell alignment is often reduced when the repeat spacing of ridges and grooves is increased, and when the repeat spacing is much larger than the breadth of the cell, alignment is diminished [9,14,15]. Deeper grooves have also been found to be more potent in inducing alignment than shallower ones [9,16,17]. Interestingly, macrophages and growth cones of neurons have been reported to change their orientation to surface grooves as shallow as 30 and 14 nm, respectively [17,18].
0014-4827/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4827(03)00159-9
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In order to elucidate the mechanism(s) behind contact guidance, more detailed questions regarding the changes in cellular morphology arise. Is the entire cell aligned or only the cytoskeleton? Which cytoskeleton components are aligned? How early in time, and in which order, are these features aligned? Experiments have been designed to answer such questions. Studies using fibroblasts and grooves have revealed that all or some of the following features become aligned: entire cells; actin microfilaments; microtubules; focal contacts, and/or ECM proteins [19 –23]. Oakley and colleagues reported that the first component to become oriented along grooves was the microtubules (20 min after seeding), followed by the other components investigated (actin filaments, focal contacts, and entire cells) [23]. Other researchers have observed that actin condensation, as well as vinculin staining, occurred along groove/ ridge boundaries 5 min after plating [24]. In this study the alignment of microtubules was not observed until after 30 min. Furthermore, it has been observed that entire cells can be aligned before a well-organised actin cytoskeleton has been developed [19]. The complexity of contact guidance is further illustrated by the fact that cells can align either with an intact actin microfilament system or with an intact microtubule system [21]. Finally, it has been reported that both endogenous and exogenous ECM proteins become oriented to the groove direction [20]. A couple of theories have emerged in an attempt to explain the phenomenon of contact guidance. The first theory proposes that the stiff rectangular structure of focal contacts is responsible for contact guidance [14,15,20,25]. The rectangular focal contacts become oriented along the groove direction and polarises the actin cytoskeleton originating from these contacts [26]. Eventually the entire cell becomes aligned, since the motility forces can only be exerted along the long axis of focal contacts. The second theory proposes that the dynamics of actin (or microtubule) polymerisation induce contact guidance [13,19,22,24,27,28]. The lamellipodia at the front edge of moving cells contain microspikes where the actin polymerisation takes place. Microspikes approaching a ridge perpendicularly are exposed to an unfavorable force so that actin polymerisation less likely occurs. Instead, polymerisation occurs at the ends of microspikes extending parallel to ridges. Another interpretation of this theory is that cells mechanically sense and adapt to their environment, as cells have been reported to do on flexible substrates [29 –31]. The third theory proposes that ECM proteins preferentially adsorb to the discontinuities present on grooved surfaces and are responsible for the subsequent alignment of focal contacts and contact guidance [32,33]. These theories are all based on the existence of a more or less “sharp” continuous groove/ridge edge. The hypothesis suggested in the present study is that a continuous edge is a prerequisite for contact guidance on surfaces with shallow grooves. We designed experiments to test this hypothesis, which, to our knowledge, has not been attempted before.
We fabricated model surfaces containing grooves with either continuous or discontinuous edges of various depths, but identical surface chemistry and groove/ ridge dimensions. The effect of the grooved samples on mammary epithelial cells was tested in vitro and cell area, alignment, and morphology were analysed.
Materials and methods Sample fabrication The starting substrate for all fabrication was polished silicon wafers that were partially cut into 6 ⫻ 6 mm pieces using a diamond saw (Loadpoint Microace 3⫹). During cutting, the surface of these substrates was protected by a polymethylacrylate (PMMA) sacrificial layer, which was subsequently removed by sequential ultrasonic agitation in acetone, isopropanol, and MilliQ water, each for 10 min. These substrates were subjected to an RF-induced oxygen plasma treatment (0.25 T, 50 W, 120 s) in a reactive ion etcher (PlasmaTherm Batchtop VII RIE/PE), and subsequently coated with 40 nm of thermally evaporated titanium (AVAC HVC-600). Titanium-coated wafers were used for all subsequent fabrication and is the control continuous sample (flat). Photolithography was used to fabricate the continuous, and the discontinuous, grooved topographies, as described previously [34]. Grooves of various depths were obtained by coating the patterned resists with 40, 110, 200, or 400 nm of titanium before lift-off. The pattern of the photomask resulted in 15-m-wide ridges and grooves. These samples were the continuous grooved ones (g40, g110, g200, and g400 in Fig. 1). Colloidal lithography was used to fabricate the discontinuous grooved samples (g110nano, g200nano, and g400nano) and the discontinuous control samples (nano) (Fig. 1) [34 –36]. The former samples were fabricated by applying colloidal lithography to grooved substrates (created by photolithography) and the latter samples by applying colloidal lithography to titanium-coated substrates. The substrates were exposed to polystyrene particles (107 ⫾ 4 nm diam.), which electrostatically self-assembled on the surface. The particle solution used for the preparation of the discontinuous control (nano) and the discontinuous grooved samples contained 0.4 and 0 mM NaCl, respectively. The substrates were subsequently coated with 110 nm titanium obtaining hemispherical pillars on the surface. Independent confirmation of the model surfaces used in the present study was obtained by a pilot study. The pilot experiments were performed with samples in TiO2 containing 15-m-wide regions of hemispherical pillars separated by 15-m-wide regions of a flat surface. These samples were prepared by a combination of photolithography and colloidal lithography. All wafers were oxidised to saturation by an oxygen plasma treatment (0.5 T, 200 W, 120 s). The precut fabri-
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Fig. 1. Schematic, illustrating the topography of the samples used in this study. The letter “g,” as in g40, stands for grooves and the number following indicates the groove depth in nanometers (for example, 40 nm). The word “nano,” as in g110nano, indicates that the surface contains hemispherical nanopillars. There are two control surfaces, one for the continuous grooves (g40, g1110, g200, and g400) entitled flat and another for the discontinuous grooves (g110nano, g200nano, and g400nano) entitled nano. On all grooved surfaces the ridges and grooves were 15-m wide.
cated wafers were snapped into pieces and blown with N2 gas to remove any particulate contamination. All samples were treated with UV/ozone for 20 min and used for experiments with cells within 2 h. Sample characterisation The vertical dimensions of the grooves were measured with a profilometer (Tencor Alpha Step 500 Surface Profilometer). Average groove and ridge widths were calculated from scanning electron microscopy (SEM) micrographs. SEM images of the hemispherical pillars were analysed with Scion Image software (Scion Corporation) to obtain the average feature number density and diameter and to estimate the average feature spacing (centre to centre) [35,36]. Atomic force microscopy was used to measure the average height of the protrusions. X-ray photoelectron spectroscopy was used to analyse the surface chemistry after UV/ozone treatment. Cell culture All materials used for cell culture were purchased from GIBCO BRL unless stated otherwise. Mouse mammary epithelial cells (HC11, kindly provided by Dr. R Ball, Friedrich Miescher-Institut, Basel, Switzerland) were cultured in RPMI 1640 supplemented with 5 g/mL insulin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (EGF) (Sigma-Aldrich), 1% (w/v) penicillin–streptomycin (PEST), and 10% (v/v), fetal bovine serum (FBS) at 37°C in a 5% CO2/95% air atmosphere. Cell media were changed every 2 days. Confluent cells were split 1:6 every 4 days. The cells were split with trypsin (0.25%) to get a single cell suspension and counted. Cell experiments on model surfaces The nine experimental surfaces (flat, g40, g110, g200, g400, g400nano, g200nano, g110nano, and nano, Fig. 1) and the samples for the pilot study were placed in culture dishes (48-well tissue culture plates, Greiners). HC11 cells were seeded at a density of either 40,000 or 60,000 cells per
well in 0.8 mL. The cells were cultured for 10 and 24 h in RPMI 1640 supplemented with 5 g/mL insulin (SigmaAldrich), 10 ng/mL EGF (Sigma-Aldrich), 1% (w/v) PEST, and 10% (v/v) FBS at 37°C in a 5% CO2/95% air atmosphere. All experiments were in quadruplicate, except in the pilot study in which the experiments were in duplicate. At the end of the prescribed time periods, the cells were rinsed with 1⫻ phosphate-buffered saline (PBS; pH 7.0) and fixed in situ with 2% paraformaldehyde in PBS for 15 min. The cells were then rinsed with PBS and stored at 4°C for staining. The fixed cells were briefly rinsed once in PBS, treated with 10 mM ethanolamine in PBS and 0.1% Triton X-100 in PBS for 5 and 1 min, respectively, and incubated with 200 nM rhodamine-phalloidin (Molecular Probes) in PBS for 15 min. After rinsing, the nuclei of the cells were labeled with 300 nM DAPI (Sigma-Aldrich) in PBS for 8 min. Fluorescence microscopy and image analysis The stained cells were visualized under epifluorescence microscopy at 40⫻ using an Axioplan 2 Imaging (Zeiss) and Zeiss filter DAPI H365 for DAPI and Cy 3/TRITC for rhodamine. Digital images, acquired with a CCD camera (Axiocam b/w; Zeiss) attached to an imaging system (Axiovision 3.0; Zeiss), were stored on a computer. Four separate regions (each 345 ⫻ 273 m) of each sample were photographed. The images (1300 ⫻ 1030 pixels with 12 bits of greyscale) were analysed using a homewritten macro in the image analysis program KS 400 (Zeiss). The macro enabled interactive thresholding and measured morphological parameters of individual cells. The macro did not provide any other image processing, such as dilation and contrast enhancement. Descriptions of the measured parameters are given in Table 1. Three distinct cell shapes (specifically round, rectangular, and spool) were identified on the samples (Fig. 2A). Twenty cells were selected as representatives for each cell shape and constituted a training group [37]. The three training groups could be discriminated by the use of a combination of the parameters dispersion, elongation, and round-
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Table 1 Descriptions of the measured cell parameters Measured parameters
Description
Angle
Angle of the main axis of an ellipse with the same geometric moment of inertia as that of a cell. Describes cell orientation. Area of the convex circumscribed region that was used to calculate roundess (RN). Area of the filled region that describes the projected cell area. These two moments are invariant to rotation, position, and size of the object and were used to calculate dispersion (D) and elongation (E). These calculations were based on established theories [14,49]. D and E have been used successfully in other cell morphometric studies [11,14,50,51,61,62]. Perimeter of the convex circumscribed filled region. This value was used to calculate RN.
AreaC AreaF Hu’s first moment
Hu’s second moment
PerimC Shape descriptors Dispersion (D)
Elongation (E)
Roundness (RN)
D quantifies the difference between the shape of an object and an equimomental ellipse. D of an ellipse is zero and increases with the irregularity of the object (i.e., how stellate the object is). E entails how much the equimomental ellipse is lengthened or stretched out. E for a circle is zero, but for an ellipse, with the ratio of axes 1:2, E is equal to one. AreaC and PerimC were used to calculate roundness, RN ⫽ PerimC/[(4 ⫻ ⫻ AreaC)0.5] [37,49,51].
⫾ 0.4 m, respectively. The average groove depths for these samples are given in Table 2. The average number density and the average feature spacing (centre to centre) of the hemispherical protrusions on the discontinuous grooves were 13.2 ⫾ 0.4 per m2 and 230 nm, respectively. The average diameter of the protrusions was 160 ⫾ 7 nm. The average number density and the average feature spacing (centre to centre) of the hemispherical protrusions on the discontinuous control (nano) were 22.1 ⫾ 0.6 per m2 and 180 nm, respectively. The average diameter of the protrusions was 166 ⫾ 15 nm. The average feature height on all substrates with hemispherical protrusions was approximately 100 ⫾ 5 nm. It was reported earlier by our group that the distribution of the hemispherical structures on a surface has short range order (over one to three feature dimensions), but not long-range order [35,36]. The difference in height, on the samples for the pilot study, between the flat regions and the regions with hemispherical pillars was approximately 100 nm. All samples had the same surface chemistries, namely TiO2, with less than an atomic monolayer of hydrocarbon contaminants. Results from the pilot study Images of actin-stained cells were used to quantify alignment and preferential adsorption of individual cells after 10
ness (Table 1, Fig. 2B). Each training group was described by a three-dimensional volume, in parameter space, defined by the measured parameters’ mean values ⫾ 2 SD. The volumes for the spool and rectangular training groups partially overlapped, and this overlapping region, where cells fulfilled the criteria for both spool and rectangular shape classes, was identified as a separate cell shape class “rectangular and spool” (rect&spool). All individual cells were classified according to the classification criteria as “round”, rectangular (rect), “spool,” or rect&spool shaped. Statistical analysis The nonparametric Kruskal–Wallis test was used to test for differences between the groups of data, followed by the Dunn’s post test using GraphPad Instat (GraphPad Software). A p value less than 0.01 was considered significant.
Results Sample characterisation Representative SEM images of surfaces with continuous and discontinuous grooves are illustrated in Fig. 3. The average groove and ridge widths were 15.2 ⫾ 0.2 and 14.5
Fig. 2. Cell shapes and classification method. (A) Representative cell shapes observed on the experimental samples. The symbol above each cell corresponds to symbols used in the 3D graph in Fig. B. Fig. B. The circle, triangle, and square represents round, spool, and rect cells, respectively. (B) A 3D plot of the measured parameters, specifically, roundness, dispersion, and elongation for the cells in the training groups representing round-, spool-, or rect-shaped cells.
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Fig. 3. Scanning electron micrographs of TiO2 samples. Continuous grooves with (A) 40-, and (B) 200-nm-deep grooves (g40 and g200, respectively). (C and D) Discontinuous grooves with 400-nm-deep grooves (g400nano). Bar represents 10 m in A–C. Bar represents 100 nm in D.
and 24 h of culture on the samples tested in the pilot study. Cell orientation was measured as the angle between the main axis of an ellipse with the same moment of inertia as the cell and the direction of the surface grooves. For unaligned cells, with random orientation, a median of approximately 45° with a large dispersion is expected. The cells aligning with the surface grooves were characterized by a very low median angle of relatively small spread. The percentage of aligned cells (within 10° of the groove direction) was about 30% after 10 and 24 h (data not shown). Additionally, the cells adhered preferrentially to the flat areas (data not shown). The results from the pilot study had significant influence on the choice of model surface for discontinuous edges and the interpretation of resultant data (see discussion).
Area of cells Images of actin-stained cells were used to quantify the projected surface areas of individual cells at the end of the 10- and 24-h experiments. For each experimental condition, an average of 150 ⫾ 37 cells were analyzed. After 10 h, the adhering cells had similar median areas on all samples except the discontinuous samples (nano, g110nano, g200nano, and g400nano) for which the median areas were smaller by 75–100 m2 compared to the control flat surface (data not shown). After 24 h, the adhering cells on flat and g40 surfaces had similar median areas (Fig. 4). Compared to the results obtained on the flat sample, the median cell areas were significantly smaller on all other samples tested (Fig. 4).
Table 2 Measured depths of the grooved samples Continuous
Depth (nm)
Discontinuous
g40
g110
g200
g400
g400nano
g200nano
g110nano
37 ⫾ 3
120 ⫾ 4
206 ⫾ 6
346 ⫾ 4
370 ⫾ 6
198 ⫾ 5
102 ⫾ 7
Note. The letter, “g,” as in g40, stands for grooves and the number indicates the intended groove depth in nanometers (for example, 40 nm). The word “nano,” as in g110nano, indicates that the surface contained hemispherical nanopillars. On all the grooved surfaces the ridges and grooves were 15 m wide.
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Fig. 4. Cell area of individual epithelial cells after 24 h of culture on the samples tested in the present study. See legend to Fig. 1 for definitions. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles. The 5th and 95th percentiles are represented by filled circles. *p ⬍ 0.01 compared to flat.
Alignment of cells Images of actin-stained cells were used to quantify alignment of individual cells at the end of the 10- and 24-h experiments. Cell orientation was measured as the angle between the main axis of an ellipse with the same moment of inertia as the cell and the direction of the surface grooves. For unaligned cells, with random orientation, a median of approximately 45° with a large dispersion is expected. The cells aligning with the surface grooves were characterized by a very low median angle of relatively small spread. Cells with orientations within 10° of the surface grooves were considered aligned. For each experimental condition an average of 150 ⫾ 37 cells were analysed. The orientation of the adhering cells and the percentages of aligned cells, after 10 and 24 h, are presented in Fig. 5
and Table 3, respectively. On the continuous (flat) and discontinuous (nano) control samples, the cells were randomly oriented. Compared to the median angles for cells on the control samples, the median angles were significantly lower for cells on the g110, g200, g400, and g400nano samples after 10 h and on the g110, g200, and g400 samples after 24 h. The percentages of aligned cells increased, while the median angles decreased, with groove depth on the surfaces with continuous grooves. Furthermore, compared to the results obtained on discontinuous grooves of the same depth (e.g., g110 vs g110nano), the median angles were significantly lower for cells on the continuous grooves samples. On all samples that induced alignment, the adhering cells were less oriented along the grooves at 24 than at 10 h. Shape of cells Images of actin-stained cells were used to assess the shape of individual cells at the end of the 10- and 24-h experiments. The shape descriptors dispersion, elongation, and roundness were used to distinguish round; spool-, and rectangular-shaped cells (Table 1, Fig. 2). For each experimental condition an average of 150 ⫾ 37 cells were classified according to the criteria as round, rect, spool, or rect&spool shaped. The cell-shape classification results are presented in Fig. 6. Representative images of cells on the tested surfaces are presented in Fig. 7. The main differences, with regard to cell shape, between the two control surfaces were that more rectangular cells were present on the flat surface while more round cells could be found on the nano sample. There was approximately a 10-fold increase of the spool- and rect&spool-shaped cells on the g400 surface compared to the results obtained on flat surfaces, after 10 and 24 h. Compared to the flat samples the sum of these classes were also systematically increased on the g110 and g200 samples, at the time points tested in the present study. In
Fig. 5. Orientation of individual epithelial cells after (A) 10 and (B) 24 h of culture on the samples tested in the present study. Cells aligned with the surface grooves have small angles; unaligned cells have a median angle of 45°. See the legend to Fig. 4 for definitions. *p ⬍ 0.01 compared to flat. §p ⬍ 0.01 compared to g400. #p ⬍ 0.01 compared to g200. 1172 p ⬍ 0.01 compared to g110.
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Table 3 Percentage of cells aligned within 10° of the direction of the surface grooves Continuous
10 h 24 h
Discontinuous
Flat
g40
g110
g200
g400
g400nano
g200nano
g110nano
nano
11 9
12 12
51 33
75 53
68 56
47 18
23 9
16 13
11 8
Note. Values are percentages. Cell orientation was measured as the angle between the main axis of an ellipse with the same moment of inertia as the cell and the direction of the surface grooves. See footnote to Table 1 for definitions. There were two control surfaces, one for the continuous grooves (g40, g1110, g200, and g400) entitled flat and another for the discontinuous grooves (g110nano, g200nano, and g400nano) entitled nano. On all the grooved surfaces the ridges and grooves were 15-m wide.
contrast, compared to the flat samples, the increase of spooland rect&spool-shaped cells was only about 2- to 3-fold on the discontinuous g400nano samples, after 10 and 24 h. Furthermore, for the shallow g110nano and g200nano discontinuous grooves, the sums of these classes were not different from that of the flat surface, after 10 and 24 h. In addition there are no spool-shaped cells on the surfaces with discontinuous grooves. Finally, larger percentages of round cells were observed on discontinuous grooves than on surfaces with continuous grooves of the same depth; this difference was more pronounced on g110nano and g200nano samples after 24 h. Compared to the values after 10 h the sum of spool- and
rect&spool-shaped cells were reduced after 24 h of cell experiments (Fig. 6). Areas and alignment of spool- and rect&spool-shaped cells The major differences in the cell response to discontinuous and continuous grooved surfaces were the degree of cellular alignment and the percentages of spool- and rect&spool-shaped cells. A comparison of the median, and the dispersion of angles, for spool and rect&spool cells with round, rect, and unclassified cells showed that the former cell shapes were
Fig. 6. Cell-shape classification of individual epithelial cells after (A) 10 and (B) 24 h of culture on the samples tested in the present study. The scheme representing cell shapes is explained in the figure. See the legend to Fig. 1 for definitions.
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Fig. 7. Fluorescence images of actin-stained epithelial cells after 10 (A) and 24 h (B) of culture on flat, continuous grooves (depth 200 nm, g200) and discontinuous grooves (depth 200 nm, g200nano) surfaces. The symbols next to individual cells in the images correspond to their cell-shape classification. The arrow indicates the direction of the surface grooves. Original magnification, 400⫻. Bar represents 50 m.
more aligned than the latter ones on the g110, g200, and g400 samples. This result is exemplified in Fig. 8A and B for the sample g400. It should be noted however, that round, rect, and unclassified cells also were more aligned on the g400 than on the control surfaces (compare Fig. 5). This
observation revealed that cell shapes other than the spool and rect&spool also were aligned. A comparison of the median cell areas showed smaller values for spool and rect&spool cells than for round, rect, and unclassified cells. This result is exemplified in Fig. 8C
Fig. 8. Cell orientation (A and B) and area (B and C) of individual epithelial cells after 10 (A and C) and 24 h (B and D) of culture on surfaces with continuous grooves (depth 400 nm, g400). The scheme representing cell shapes is explained in the figure. Cells aligned with the surface grooves have small angles while unaligned cells have a median angle of 45°. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles. The 5th and 95th percentiles are represented by filled circles.
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Table 4 A summary of the major results obtained in the present study Continuous
Alignment to grooves Percentage of rect&spool or spool cells Percentage of round cells
Discontinuous
Flat
g40
g110
g200
g400
g400nano
g200nano
g110nano
nano
No Very small
No Very small
Medium Small
High Medium
High Large
Low Small
No Very small
No Very small
No Very small
Small
Small
Very small
Very small
Very small
Small
Medium
Large
Large
Note. The results presented in Figs. 5 and 6 and Table 3 are summarised, averaged for 10 and 24 h. A “high” alignment to grooves entails a low median angle in Fig. 5 and a large percentage in Table 3. A “no” alignment to grooves entails a median angle around 45° in Fig. 5 and a small percentage in Table 3. A “large” percentage of rect&spool or spool cells entails a large percentage of rect&spool- or spool-shaped cells in Fig. 6. A “large” percentage of round cells entails a large percentage of round cells in Fig. 6. See footnote to Table 1 for definitions.
and D for the sample g400. Furthermore, a comparison of the increase in cell area for all cells, from 10 to 24 h, showed a larger increase for flat samples than for surfaces with continuous grooves (data not shown). Discussion This work was designed to study how surface grooves, with continuous or discontinuous edges, of various depths, affect cell area, alignment, and shape. We were specifically interested to discern whether grooved surfaces with discontinuous edges could provide contact guidance for cells. A pilot study indicated that the choice of model surface is crucial when addressing such questions. The samples used in the pilot study had 15-m-wide ridges of hemispherical pillars separated by 15-m-wide regions of flat grooves. These samples could represent grooves with discontinuous edges and be compared to continuous grooves of the same dimensions. However, the results from the pilot study showed that, although about 30% of the cells were aligned, the cells adhered preferentially to the flat regions. Hence, the inhomogeneous topography (flat vs nanostructured) may affect the alignment of cells in addition to any influence of the discontinuous edge. There are also other reports of cell studies on surfaces with alternating, regular arrays of micron- or nanometer-sized pillars and flat regions, confirming that cells can adhere preferentially to specific regions [5,38 – 40]. Consequently, samples with different topography in the grooves and on the ridges cannot be used as a model system to study the effect of discontinuous edges on contact guidance. We have designed a model surface for discontinuous grooves where cells are exposed to the same topography in the grooves and on the ridges (Figs. 1 and 3). All grooved surfaces had identical surface chemistry and groove/ridge dimensions (15 m wide) (Figs. 1 and 3, Table 2). Mammary gland epithelial cells were seeded on the samples tested and cultured for 10 and 24 h. These cells are known to be sensitive to their microenvironment, specifically, changes in cell shape and integrin expression are involved in the differentiation of mammary epithelial cells [41– 44].
Variations in morphology of cells cultured on synthetic substrates have been described in numerous studies [7,8,10,45,46] and have also been suggested to control cell growth, differentiation, and apoptosis [47,48]. Cell area is a rather objective descriptor of morphology. Cell shape can quantitatively be described either by a single parameter, such as the aforementioned cell area, or as a combination of parameters, for example, of cell area and perimeter [37,49]. These combinations, called shape descriptors, for example, roundness (RN) normalises the cells to a circle with the same area as the cell, result in dimensionless parameters (Table 1). One problem with the RN approach is that cells adhering to grooved surfaces are often polarised. Another approach is to normalise cells to ellipses, with the same moment up to the second order [14,37,49]. The primary and secondary axis of this ellipse can then be used to calculate the dispersion (D) and elongation (E) for each cell, two parameters that are invariant to the position, size, and rotation of the cell [14,50,51]. In practical terms D and E represent how stellate-like and polarised the cell shape is, respectively (Table 1, Fig. 2). Fluorescent microscopy revealed that certain cell shapes were more frequent on some of the modified samples than on others; for example, elongated cells were more frequent on the surfaces with deep continuous grooves. Three characteristic cell shapes (round, rectangular, and spool) were identified and represented by training groups. The training groups were used to set up criteria for cell-shape classification. The cells were classified according to their dispersion, elongation, and roundness values as round, rect, spool, or rect&spool shaped (Fig. 2). The approach proved to be successful with a large percentage of cells being classified (Fig. 6). The major results of the present study are schematically summarized in Table 4. First, significantly more cells aligned to grooves with continuous edges than to those with discontinuous edges. Second, rect&spool- or spool-shaped cells were mainly present on the grooved samples: g110, g200, and g400. Third, round cells were mainly present on the discontinuous control and discontinuous groove samples. Taken together these results provided evidence that cell alignment and elongation increased with groove depth
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provided that the edge was continuous. Use of cell-shape classification (vs the single-parameter shape descriptors) made it possible to study not only variations across the cell population but also the properties of subpopulations of cultured cells. For example, a correlation between rect&spoolor spool-shaped cells and aligned cells was identified (Fig. 8A and B). This result suggests an alignment mechanism dependent on the continuous edges present on the surface. The result that round, rect, and unclassified cells also were partially aligned on surfaces with deeper continuous grooves suggests that more than one mechanism for cellular alignment may exist. On the other hand, cells were only aligned and slightly elongated on the samples with deep discontinuous grooves (g400nano) and almost no alignment was observed on shallower discontinuous grooves (g110nano and g200nano) (Table 3, Figs. 5 and 6). No spool-shaped cells were present on any of the surfaces with discontinuous grooves (Fig. 6). Furthermore, low percentages of spool- and/or rect&spoolshaped cells correlated with very little contact guidance (Figs. 5 and 6, Table 3). The degrees of cell alignment observed on samples with the deepest discontinuous grooves (g400nano) corresponded to those seen for the round, rect, and unclassified cells on the deepest continuous grooves (g400) (compare Figs. 5 and 8A and B). In the case for deeper discontinuous grooves where both a continuous edge and spool-shaped cells were absent, the mechanism for alignment might correspond to the mechanism of aligning the round, rect, and unclassified cells on surfaces with continuous grooves. The hypothesis suggested in the present study is that a continuous edge is a prerequisite for contact guidance on surfaces with shallow grooves. Our results provided evidence that, among grooves of the same depth, a continuous edge resulted in significantly more aligned cells and the presence of spool-shaped cells. Other researchers have speculated that the distance of separation between nanostructures could influence the formation of focal contacts and concomitantly cell adhesion and survival [52,53]. This could have important ramifications for the results obtained on the discontinuous surfaces in the present study, since these surfaces contain separated nanometer-sized pillars. In this study we did not measure cell adhesion and proliferation; however, we have previously cultured the mammary gland cells on TiO2 surfaces with hemispherical pillars (feature density, diameter, and spacing as for discontinuous grooves) and on control flat (unpublished results). The cells grew to confluence and had similar total protein contents up to 5 days in culture on the two surfaces, using the same culture conditions as in the present study. Recent studies showed similar uroepithelial cell adhesion and proliferation both on flat and on surfaces with hemispherical pillars; however, a star-shaped cell morphology and a lower cytokine production was observed on the latter surface [34]. Therefore, we conclude that the hemispherical pillars used in the present study to mimic
discontinuous edges do not, to the best of our knowledge, affect cell attachment and survival. Proposed mechanisms for cell contact guidance involve cytoskeletal components (actin microfilaments, microtubules, and focal contacts) and ECM proteins. Cell migration itself is a process involving dynamic and spatially regulated changes to the cytoskeleton and the cell adhesion process [54 –58]. In addition, it has recently been found that members of the Rho GTPase family are involved in the signaling for cell migration [54 –58]. How can one cytoskeleton component be responsible for contact guidance when cell migration itself is an extremely complex process? Current knowledge indicates that integrins (or integrin clusters) can activate Rac GTPase, which subsequently can regulate actin polymerisation [54 –56]. This process suggests that the binding of integrins on the cell membrane to ECM ligands could regulate actin polymerisation, and, consequently, the direction of lamellopodia movement. Such cross-talk also means that it is difficult to assign contact guidance to either actin polymerisation or integrin–ECM binding (followed by focal contact formation). Consequently, the phenomenon of contact guidance is probably based on a combination of preferential ECM protein deposition, restricted actin/microtubules polymerisation, and confined focal contact formation. This sequence of events does not rule out a mechanism in which mechanical forces may be involved. One influence of a continuous sharp edge on such events could be an altered ECM protein deposition. The influence of topographic nanostructures on the functional behaviour of proteins has been shown in two recent papers [59,60] The present study used a set of systematically varied nanofabricated model surfaces combined with digital image analysis of individual cells via shape descriptors and shape classification to address the issue of the importance of a continuous edge in the alignment of cells to shallow topographic structures (grooves). The results indicated that grooved surfaces with continuous or discontinuous edges have very different capabilites in inducing contact guidance. Grooved surfaces with continuous edges are potent in aligning and inducing elongated cells, while grooved surfaces with discontinuous edges do not align and elongate cells to a large extent. A mechansim of alignment involving orientation along a continuous edge is therefore likely. Finally, the combination of fabricated model surfaces and cell-shape classification is a powerful approach to study cell-surface interactions.
Acknowledgments This work was supported by the Swedish Foundation for Strategic Research through the Biocompatible Materials program and the Wallenberg Foundation. We also acknowledge valuable contributions from Prof. Rena Bizios and Dr. Karin Glasma¨star in preparing the manuscript.
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The effects of continuous and discontinuous groove edges on cell shape and alignment Ann-Sofie Andersson,* Petra Olsson, Ulf Lidberg, and Duncan Sutherland Department of Applied Physics, Chalmers University of Technology, SE-412 96, Go¨teborg, Sweden Received 10 December 2002, revised version received 19 February 2003
Abstract Nanofabricated model surfaces and digital image analysis of cell shape were used to address the importance of a continuous sharp edge in the alignment of cells to shallow surface grooves. The grooved model surfaces had either continuous or discontinuous edges of various depths (40 – 400 nm) but identical surface chemistry and groove/ridge dimensions (15 m wide). Epithelial cells were cultured on the model surfaces for 10 and 24 h. Fluorescence microscopy combined with image analysis were used to quantify cell area and alignment and to make cell shape classifications of individual cells. The degrees of alignment of cells and the percentages of elongated cell classes increased with groove depth on samples with continuous grooves. Two main differences, with regard to cell response, were observed between the continuous and discontinuous grooved surfaces. First, significantly fewer cells aligned to surface grooves with discontinuous edges than to grooves with continuous edges. Second, there were lower percentages of the elongated cell classes on discontinuous grooves than on continuous ones. We concluded that grooved surfaces with continuous edges are more potent in aligning and inducing elongated cells. The results from the present study suggest that a mechanism of alignment involving orientation along a continuous edge is likely. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Contact guidance; Nanostructures; Nanotopography; Surface topography; Surface grooves; Digital image analysis; Cell-surface interaction; Shape classification; Cell shape; Actin cytoskeleton
Introduction All cells in the human body are surrounded by topographic and chemical cues. These cues can be provided by other cells, the extracellular matrix (ECM), or synthetic materials. Examples of ECM-related cues are collagen and fibronectin fibers. These cues are also known for their ability to align and guide cells [1–3]. The ability to control and guide cells can be very useful in biomaterial and tissue engineering applications in order to create more advanced products [4,5]. However, the exact mechanism behind the cell guidance ability of, for example, a collagen fiber in vivo is unclear; in particular whether it is the topography and/or the chemistry that causes the guidance. Therefore, fabricated model surfaces with defined topographies (e.g.,
* Corresponding author. Fax: ⫹46-31-7723134. E-mail address: [email protected] (A.-S. Andersson).
grooves) or chemistries are used to study the cell-surface phenomenon called contact guidance. Substrates with parallel repeating step edges can impose directional constraints on cells (orientation and shape) and also affect the rate and direction of cell migration (i.e., contact guidance). In order to determine which controls topographically induced contact guidance, parameters such as groove/ridge width and groove depth have been systematically varied [1,6 –10]. The degree of contact guidance is also influenced by cellular parameters such as cell type and cell density [1,9,11–13]. Cell alignment is often reduced when the repeat spacing of ridges and grooves is increased, and when the repeat spacing is much larger than the breadth of the cell, alignment is diminished [9,14,15]. Deeper grooves have also been found to be more potent in inducing alignment than shallower ones [9,16,17]. Interestingly, macrophages and growth cones of neurons have been reported to change their orientation to surface grooves as shallow as 30 and 14 nm, respectively [17,18].
0014-4827/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4827(03)00159-9
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In order to elucidate the mechanism(s) behind contact guidance, more detailed questions regarding the changes in cellular morphology arise. Is the entire cell aligned or only the cytoskeleton? Which cytoskeleton components are aligned? How early in time, and in which order, are these features aligned? Experiments have been designed to answer such questions. Studies using fibroblasts and grooves have revealed that all or some of the following features become aligned: entire cells; actin microfilaments; microtubules; focal contacts, and/or ECM proteins [19 –23]. Oakley and colleagues reported that the first component to become oriented along grooves was the microtubules (20 min after seeding), followed by the other components investigated (actin filaments, focal contacts, and entire cells) [23]. Other researchers have observed that actin condensation, as well as vinculin staining, occurred along groove/ ridge boundaries 5 min after plating [24]. In this study the alignment of microtubules was not observed until after 30 min. Furthermore, it has been observed that entire cells can be aligned before a well-organised actin cytoskeleton has been developed [19]. The complexity of contact guidance is further illustrated by the fact that cells can align either with an intact actin microfilament system or with an intact microtubule system [21]. Finally, it has been reported that both endogenous and exogenous ECM proteins become oriented to the groove direction [20]. A couple of theories have emerged in an attempt to explain the phenomenon of contact guidance. The first theory proposes that the stiff rectangular structure of focal contacts is responsible for contact guidance [14,15,20,25]. The rectangular focal contacts become oriented along the groove direction and polarises the actin cytoskeleton originating from these contacts [26]. Eventually the entire cell becomes aligned, since the motility forces can only be exerted along the long axis of focal contacts. The second theory proposes that the dynamics of actin (or microtubule) polymerisation induce contact guidance [13,19,22,24,27,28]. The lamellipodia at the front edge of moving cells contain microspikes where the actin polymerisation takes place. Microspikes approaching a ridge perpendicularly are exposed to an unfavorable force so that actin polymerisation less likely occurs. Instead, polymerisation occurs at the ends of microspikes extending parallel to ridges. Another interpretation of this theory is that cells mechanically sense and adapt to their environment, as cells have been reported to do on flexible substrates [29 –31]. The third theory proposes that ECM proteins preferentially adsorb to the discontinuities present on grooved surfaces and are responsible for the subsequent alignment of focal contacts and contact guidance [32,33]. These theories are all based on the existence of a more or less “sharp” continuous groove/ridge edge. The hypothesis suggested in the present study is that a continuous edge is a prerequisite for contact guidance on surfaces with shallow grooves. We designed experiments to test this hypothesis, which, to our knowledge, has not been attempted before.
We fabricated model surfaces containing grooves with either continuous or discontinuous edges of various depths, but identical surface chemistry and groove/ ridge dimensions. The effect of the grooved samples on mammary epithelial cells was tested in vitro and cell area, alignment, and morphology were analysed.
Materials and methods Sample fabrication The starting substrate for all fabrication was polished silicon wafers that were partially cut into 6 ⫻ 6 mm pieces using a diamond saw (Loadpoint Microace 3⫹). During cutting, the surface of these substrates was protected by a polymethylacrylate (PMMA) sacrificial layer, which was subsequently removed by sequential ultrasonic agitation in acetone, isopropanol, and MilliQ water, each for 10 min. These substrates were subjected to an RF-induced oxygen plasma treatment (0.25 T, 50 W, 120 s) in a reactive ion etcher (PlasmaTherm Batchtop VII RIE/PE), and subsequently coated with 40 nm of thermally evaporated titanium (AVAC HVC-600). Titanium-coated wafers were used for all subsequent fabrication and is the control continuous sample (flat). Photolithography was used to fabricate the continuous, and the discontinuous, grooved topographies, as described previously [34]. Grooves of various depths were obtained by coating the patterned resists with 40, 110, 200, or 400 nm of titanium before lift-off. The pattern of the photomask resulted in 15-m-wide ridges and grooves. These samples were the continuous grooved ones (g40, g110, g200, and g400 in Fig. 1). Colloidal lithography was used to fabricate the discontinuous grooved samples (g110nano, g200nano, and g400nano) and the discontinuous control samples (nano) (Fig. 1) [34 –36]. The former samples were fabricated by applying colloidal lithography to grooved substrates (created by photolithography) and the latter samples by applying colloidal lithography to titanium-coated substrates. The substrates were exposed to polystyrene particles (107 ⫾ 4 nm diam.), which electrostatically self-assembled on the surface. The particle solution used for the preparation of the discontinuous control (nano) and the discontinuous grooved samples contained 0.4 and 0 mM NaCl, respectively. The substrates were subsequently coated with 110 nm titanium obtaining hemispherical pillars on the surface. Independent confirmation of the model surfaces used in the present study was obtained by a pilot study. The pilot experiments were performed with samples in TiO2 containing 15-m-wide regions of hemispherical pillars separated by 15-m-wide regions of a flat surface. These samples were prepared by a combination of photolithography and colloidal lithography. All wafers were oxidised to saturation by an oxygen plasma treatment (0.5 T, 200 W, 120 s). The precut fabri-
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Fig. 1. Schematic, illustrating the topography of the samples used in this study. The letter “g,” as in g40, stands for grooves and the number following indicates the groove depth in nanometers (for example, 40 nm). The word “nano,” as in g110nano, indicates that the surface contains hemispherical nanopillars. There are two control surfaces, one for the continuous grooves (g40, g1110, g200, and g400) entitled flat and another for the discontinuous grooves (g110nano, g200nano, and g400nano) entitled nano. On all grooved surfaces the ridges and grooves were 15-m wide.
cated wafers were snapped into pieces and blown with N2 gas to remove any particulate contamination. All samples were treated with UV/ozone for 20 min and used for experiments with cells within 2 h. Sample characterisation The vertical dimensions of the grooves were measured with a profilometer (Tencor Alpha Step 500 Surface Profilometer). Average groove and ridge widths were calculated from scanning electron microscopy (SEM) micrographs. SEM images of the hemispherical pillars were analysed with Scion Image software (Scion Corporation) to obtain the average feature number density and diameter and to estimate the average feature spacing (centre to centre) [35,36]. Atomic force microscopy was used to measure the average height of the protrusions. X-ray photoelectron spectroscopy was used to analyse the surface chemistry after UV/ozone treatment. Cell culture All materials used for cell culture were purchased from GIBCO BRL unless stated otherwise. Mouse mammary epithelial cells (HC11, kindly provided by Dr. R Ball, Friedrich Miescher-Institut, Basel, Switzerland) were cultured in RPMI 1640 supplemented with 5 g/mL insulin (Sigma-Aldrich), 10 ng/mL epidermal growth factor (EGF) (Sigma-Aldrich), 1% (w/v) penicillin–streptomycin (PEST), and 10% (v/v), fetal bovine serum (FBS) at 37°C in a 5% CO2/95% air atmosphere. Cell media were changed every 2 days. Confluent cells were split 1:6 every 4 days. The cells were split with trypsin (0.25%) to get a single cell suspension and counted. Cell experiments on model surfaces The nine experimental surfaces (flat, g40, g110, g200, g400, g400nano, g200nano, g110nano, and nano, Fig. 1) and the samples for the pilot study were placed in culture dishes (48-well tissue culture plates, Greiners). HC11 cells were seeded at a density of either 40,000 or 60,000 cells per
well in 0.8 mL. The cells were cultured for 10 and 24 h in RPMI 1640 supplemented with 5 g/mL insulin (SigmaAldrich), 10 ng/mL EGF (Sigma-Aldrich), 1% (w/v) PEST, and 10% (v/v) FBS at 37°C in a 5% CO2/95% air atmosphere. All experiments were in quadruplicate, except in the pilot study in which the experiments were in duplicate. At the end of the prescribed time periods, the cells were rinsed with 1⫻ phosphate-buffered saline (PBS; pH 7.0) and fixed in situ with 2% paraformaldehyde in PBS for 15 min. The cells were then rinsed with PBS and stored at 4°C for staining. The fixed cells were briefly rinsed once in PBS, treated with 10 mM ethanolamine in PBS and 0.1% Triton X-100 in PBS for 5 and 1 min, respectively, and incubated with 200 nM rhodamine-phalloidin (Molecular Probes) in PBS for 15 min. After rinsing, the nuclei of the cells were labeled with 300 nM DAPI (Sigma-Aldrich) in PBS for 8 min. Fluorescence microscopy and image analysis The stained cells were visualized under epifluorescence microscopy at 40⫻ using an Axioplan 2 Imaging (Zeiss) and Zeiss filter DAPI H365 for DAPI and Cy 3/TRITC for rhodamine. Digital images, acquired with a CCD camera (Axiocam b/w; Zeiss) attached to an imaging system (Axiovision 3.0; Zeiss), were stored on a computer. Four separate regions (each 345 ⫻ 273 m) of each sample were photographed. The images (1300 ⫻ 1030 pixels with 12 bits of greyscale) were analysed using a homewritten macro in the image analysis program KS 400 (Zeiss). The macro enabled interactive thresholding and measured morphological parameters of individual cells. The macro did not provide any other image processing, such as dilation and contrast enhancement. Descriptions of the measured parameters are given in Table 1. Three distinct cell shapes (specifically round, rectangular, and spool) were identified on the samples (Fig. 2A). Twenty cells were selected as representatives for each cell shape and constituted a training group [37]. The three training groups could be discriminated by the use of a combination of the parameters dispersion, elongation, and round-
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Table 1 Descriptions of the measured cell parameters Measured parameters
Description
Angle
Angle of the main axis of an ellipse with the same geometric moment of inertia as that of a cell. Describes cell orientation. Area of the convex circumscribed region that was used to calculate roundess (RN). Area of the filled region that describes the projected cell area. These two moments are invariant to rotation, position, and size of the object and were used to calculate dispersion (D) and elongation (E). These calculations were based on established theories [14,49]. D and E have been used successfully in other cell morphometric studies [11,14,50,51,61,62]. Perimeter of the convex circumscribed filled region. This value was used to calculate RN.
AreaC AreaF Hu’s first moment
Hu’s second moment
PerimC Shape descriptors Dispersion (D)
Elongation (E)
Roundness (RN)
D quantifies the difference between the shape of an object and an equimomental ellipse. D of an ellipse is zero and increases with the irregularity of the object (i.e., how stellate the object is). E entails how much the equimomental ellipse is lengthened or stretched out. E for a circle is zero, but for an ellipse, with the ratio of axes 1:2, E is equal to one. AreaC and PerimC were used to calculate roundness, RN ⫽ PerimC/[(4 ⫻ ⫻ AreaC)0.5] [37,49,51].
⫾ 0.4 m, respectively. The average groove depths for these samples are given in Table 2. The average number density and the average feature spacing (centre to centre) of the hemispherical protrusions on the discontinuous grooves were 13.2 ⫾ 0.4 per m2 and 230 nm, respectively. The average diameter of the protrusions was 160 ⫾ 7 nm. The average number density and the average feature spacing (centre to centre) of the hemispherical protrusions on the discontinuous control (nano) were 22.1 ⫾ 0.6 per m2 and 180 nm, respectively. The average diameter of the protrusions was 166 ⫾ 15 nm. The average feature height on all substrates with hemispherical protrusions was approximately 100 ⫾ 5 nm. It was reported earlier by our group that the distribution of the hemispherical structures on a surface has short range order (over one to three feature dimensions), but not long-range order [35,36]. The difference in height, on the samples for the pilot study, between the flat regions and the regions with hemispherical pillars was approximately 100 nm. All samples had the same surface chemistries, namely TiO2, with less than an atomic monolayer of hydrocarbon contaminants. Results from the pilot study Images of actin-stained cells were used to quantify alignment and preferential adsorption of individual cells after 10
ness (Table 1, Fig. 2B). Each training group was described by a three-dimensional volume, in parameter space, defined by the measured parameters’ mean values ⫾ 2 SD. The volumes for the spool and rectangular training groups partially overlapped, and this overlapping region, where cells fulfilled the criteria for both spool and rectangular shape classes, was identified as a separate cell shape class “rectangular and spool” (rect&spool). All individual cells were classified according to the classification criteria as “round”, rectangular (rect), “spool,” or rect&spool shaped. Statistical analysis The nonparametric Kruskal–Wallis test was used to test for differences between the groups of data, followed by the Dunn’s post test using GraphPad Instat (GraphPad Software). A p value less than 0.01 was considered significant.
Results Sample characterisation Representative SEM images of surfaces with continuous and discontinuous grooves are illustrated in Fig. 3. The average groove and ridge widths were 15.2 ⫾ 0.2 and 14.5
Fig. 2. Cell shapes and classification method. (A) Representative cell shapes observed on the experimental samples. The symbol above each cell corresponds to symbols used in the 3D graph in Fig. B. Fig. B. The circle, triangle, and square represents round, spool, and rect cells, respectively. (B) A 3D plot of the measured parameters, specifically, roundness, dispersion, and elongation for the cells in the training groups representing round-, spool-, or rect-shaped cells.
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Fig. 3. Scanning electron micrographs of TiO2 samples. Continuous grooves with (A) 40-, and (B) 200-nm-deep grooves (g40 and g200, respectively). (C and D) Discontinuous grooves with 400-nm-deep grooves (g400nano). Bar represents 10 m in A–C. Bar represents 100 nm in D.
and 24 h of culture on the samples tested in the pilot study. Cell orientation was measured as the angle between the main axis of an ellipse with the same moment of inertia as the cell and the direction of the surface grooves. For unaligned cells, with random orientation, a median of approximately 45° with a large dispersion is expected. The cells aligning with the surface grooves were characterized by a very low median angle of relatively small spread. The percentage of aligned cells (within 10° of the groove direction) was about 30% after 10 and 24 h (data not shown). Additionally, the cells adhered preferrentially to the flat areas (data not shown). The results from the pilot study had significant influence on the choice of model surface for discontinuous edges and the interpretation of resultant data (see discussion).
Area of cells Images of actin-stained cells were used to quantify the projected surface areas of individual cells at the end of the 10- and 24-h experiments. For each experimental condition, an average of 150 ⫾ 37 cells were analyzed. After 10 h, the adhering cells had similar median areas on all samples except the discontinuous samples (nano, g110nano, g200nano, and g400nano) for which the median areas were smaller by 75–100 m2 compared to the control flat surface (data not shown). After 24 h, the adhering cells on flat and g40 surfaces had similar median areas (Fig. 4). Compared to the results obtained on the flat sample, the median cell areas were significantly smaller on all other samples tested (Fig. 4).
Table 2 Measured depths of the grooved samples Continuous
Depth (nm)
Discontinuous
g40
g110
g200
g400
g400nano
g200nano
g110nano
37 ⫾ 3
120 ⫾ 4
206 ⫾ 6
346 ⫾ 4
370 ⫾ 6
198 ⫾ 5
102 ⫾ 7
Note. The letter, “g,” as in g40, stands for grooves and the number indicates the intended groove depth in nanometers (for example, 40 nm). The word “nano,” as in g110nano, indicates that the surface contained hemispherical nanopillars. On all the grooved surfaces the ridges and grooves were 15 m wide.
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Fig. 4. Cell area of individual epithelial cells after 24 h of culture on the samples tested in the present study. See legend to Fig. 1 for definitions. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles. The 5th and 95th percentiles are represented by filled circles. *p ⬍ 0.01 compared to flat.
Alignment of cells Images of actin-stained cells were used to quantify alignment of individual cells at the end of the 10- and 24-h experiments. Cell orientation was measured as the angle between the main axis of an ellipse with the same moment of inertia as the cell and the direction of the surface grooves. For unaligned cells, with random orientation, a median of approximately 45° with a large dispersion is expected. The cells aligning with the surface grooves were characterized by a very low median angle of relatively small spread. Cells with orientations within 10° of the surface grooves were considered aligned. For each experimental condition an average of 150 ⫾ 37 cells were analysed. The orientation of the adhering cells and the percentages of aligned cells, after 10 and 24 h, are presented in Fig. 5
and Table 3, respectively. On the continuous (flat) and discontinuous (nano) control samples, the cells were randomly oriented. Compared to the median angles for cells on the control samples, the median angles were significantly lower for cells on the g110, g200, g400, and g400nano samples after 10 h and on the g110, g200, and g400 samples after 24 h. The percentages of aligned cells increased, while the median angles decreased, with groove depth on the surfaces with continuous grooves. Furthermore, compared to the results obtained on discontinuous grooves of the same depth (e.g., g110 vs g110nano), the median angles were significantly lower for cells on the continuous grooves samples. On all samples that induced alignment, the adhering cells were less oriented along the grooves at 24 than at 10 h. Shape of cells Images of actin-stained cells were used to assess the shape of individual cells at the end of the 10- and 24-h experiments. The shape descriptors dispersion, elongation, and roundness were used to distinguish round; spool-, and rectangular-shaped cells (Table 1, Fig. 2). For each experimental condition an average of 150 ⫾ 37 cells were classified according to the criteria as round, rect, spool, or rect&spool shaped. The cell-shape classification results are presented in Fig. 6. Representative images of cells on the tested surfaces are presented in Fig. 7. The main differences, with regard to cell shape, between the two control surfaces were that more rectangular cells were present on the flat surface while more round cells could be found on the nano sample. There was approximately a 10-fold increase of the spool- and rect&spool-shaped cells on the g400 surface compared to the results obtained on flat surfaces, after 10 and 24 h. Compared to the flat samples the sum of these classes were also systematically increased on the g110 and g200 samples, at the time points tested in the present study. In
Fig. 5. Orientation of individual epithelial cells after (A) 10 and (B) 24 h of culture on the samples tested in the present study. Cells aligned with the surface grooves have small angles; unaligned cells have a median angle of 45°. See the legend to Fig. 4 for definitions. *p ⬍ 0.01 compared to flat. §p ⬍ 0.01 compared to g400. #p ⬍ 0.01 compared to g200. 1172 p ⬍ 0.01 compared to g110.
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Table 3 Percentage of cells aligned within 10° of the direction of the surface grooves Continuous
10 h 24 h
Discontinuous
Flat
g40
g110
g200
g400
g400nano
g200nano
g110nano
nano
11 9
12 12
51 33
75 53
68 56
47 18
23 9
16 13
11 8
Note. Values are percentages. Cell orientation was measured as the angle between the main axis of an ellipse with the same moment of inertia as the cell and the direction of the surface grooves. See footnote to Table 1 for definitions. There were two control surfaces, one for the continuous grooves (g40, g1110, g200, and g400) entitled flat and another for the discontinuous grooves (g110nano, g200nano, and g400nano) entitled nano. On all the grooved surfaces the ridges and grooves were 15-m wide.
contrast, compared to the flat samples, the increase of spooland rect&spool-shaped cells was only about 2- to 3-fold on the discontinuous g400nano samples, after 10 and 24 h. Furthermore, for the shallow g110nano and g200nano discontinuous grooves, the sums of these classes were not different from that of the flat surface, after 10 and 24 h. In addition there are no spool-shaped cells on the surfaces with discontinuous grooves. Finally, larger percentages of round cells were observed on discontinuous grooves than on surfaces with continuous grooves of the same depth; this difference was more pronounced on g110nano and g200nano samples after 24 h. Compared to the values after 10 h the sum of spool- and
rect&spool-shaped cells were reduced after 24 h of cell experiments (Fig. 6). Areas and alignment of spool- and rect&spool-shaped cells The major differences in the cell response to discontinuous and continuous grooved surfaces were the degree of cellular alignment and the percentages of spool- and rect&spool-shaped cells. A comparison of the median, and the dispersion of angles, for spool and rect&spool cells with round, rect, and unclassified cells showed that the former cell shapes were
Fig. 6. Cell-shape classification of individual epithelial cells after (A) 10 and (B) 24 h of culture on the samples tested in the present study. The scheme representing cell shapes is explained in the figure. See the legend to Fig. 1 for definitions.
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Fig. 7. Fluorescence images of actin-stained epithelial cells after 10 (A) and 24 h (B) of culture on flat, continuous grooves (depth 200 nm, g200) and discontinuous grooves (depth 200 nm, g200nano) surfaces. The symbols next to individual cells in the images correspond to their cell-shape classification. The arrow indicates the direction of the surface grooves. Original magnification, 400⫻. Bar represents 50 m.
more aligned than the latter ones on the g110, g200, and g400 samples. This result is exemplified in Fig. 8A and B for the sample g400. It should be noted however, that round, rect, and unclassified cells also were more aligned on the g400 than on the control surfaces (compare Fig. 5). This
observation revealed that cell shapes other than the spool and rect&spool also were aligned. A comparison of the median cell areas showed smaller values for spool and rect&spool cells than for round, rect, and unclassified cells. This result is exemplified in Fig. 8C
Fig. 8. Cell orientation (A and B) and area (B and C) of individual epithelial cells after 10 (A and C) and 24 h (B and D) of culture on surfaces with continuous grooves (depth 400 nm, g400). The scheme representing cell shapes is explained in the figure. Cells aligned with the surface grooves have small angles while unaligned cells have a median angle of 45°. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers above and below the box indicate the 90th and 10th percentiles. The 5th and 95th percentiles are represented by filled circles.
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Table 4 A summary of the major results obtained in the present study Continuous
Alignment to grooves Percentage of rect&spool or spool cells Percentage of round cells
Discontinuous
Flat
g40
g110
g200
g400
g400nano
g200nano
g110nano
nano
No Very small
No Very small
Medium Small
High Medium
High Large
Low Small
No Very small
No Very small
No Very small
Small
Small
Very small
Very small
Very small
Small
Medium
Large
Large
Note. The results presented in Figs. 5 and 6 and Table 3 are summarised, averaged for 10 and 24 h. A “high” alignment to grooves entails a low median angle in Fig. 5 and a large percentage in Table 3. A “no” alignment to grooves entails a median angle around 45° in Fig. 5 and a small percentage in Table 3. A “large” percentage of rect&spool or spool cells entails a large percentage of rect&spool- or spool-shaped cells in Fig. 6. A “large” percentage of round cells entails a large percentage of round cells in Fig. 6. See footnote to Table 1 for definitions.
and D for the sample g400. Furthermore, a comparison of the increase in cell area for all cells, from 10 to 24 h, showed a larger increase for flat samples than for surfaces with continuous grooves (data not shown). Discussion This work was designed to study how surface grooves, with continuous or discontinuous edges, of various depths, affect cell area, alignment, and shape. We were specifically interested to discern whether grooved surfaces with discontinuous edges could provide contact guidance for cells. A pilot study indicated that the choice of model surface is crucial when addressing such questions. The samples used in the pilot study had 15-m-wide ridges of hemispherical pillars separated by 15-m-wide regions of flat grooves. These samples could represent grooves with discontinuous edges and be compared to continuous grooves of the same dimensions. However, the results from the pilot study showed that, although about 30% of the cells were aligned, the cells adhered preferentially to the flat regions. Hence, the inhomogeneous topography (flat vs nanostructured) may affect the alignment of cells in addition to any influence of the discontinuous edge. There are also other reports of cell studies on surfaces with alternating, regular arrays of micron- or nanometer-sized pillars and flat regions, confirming that cells can adhere preferentially to specific regions [5,38 – 40]. Consequently, samples with different topography in the grooves and on the ridges cannot be used as a model system to study the effect of discontinuous edges on contact guidance. We have designed a model surface for discontinuous grooves where cells are exposed to the same topography in the grooves and on the ridges (Figs. 1 and 3). All grooved surfaces had identical surface chemistry and groove/ridge dimensions (15 m wide) (Figs. 1 and 3, Table 2). Mammary gland epithelial cells were seeded on the samples tested and cultured for 10 and 24 h. These cells are known to be sensitive to their microenvironment, specifically, changes in cell shape and integrin expression are involved in the differentiation of mammary epithelial cells [41– 44].
Variations in morphology of cells cultured on synthetic substrates have been described in numerous studies [7,8,10,45,46] and have also been suggested to control cell growth, differentiation, and apoptosis [47,48]. Cell area is a rather objective descriptor of morphology. Cell shape can quantitatively be described either by a single parameter, such as the aforementioned cell area, or as a combination of parameters, for example, of cell area and perimeter [37,49]. These combinations, called shape descriptors, for example, roundness (RN) normalises the cells to a circle with the same area as the cell, result in dimensionless parameters (Table 1). One problem with the RN approach is that cells adhering to grooved surfaces are often polarised. Another approach is to normalise cells to ellipses, with the same moment up to the second order [14,37,49]. The primary and secondary axis of this ellipse can then be used to calculate the dispersion (D) and elongation (E) for each cell, two parameters that are invariant to the position, size, and rotation of the cell [14,50,51]. In practical terms D and E represent how stellate-like and polarised the cell shape is, respectively (Table 1, Fig. 2). Fluorescent microscopy revealed that certain cell shapes were more frequent on some of the modified samples than on others; for example, elongated cells were more frequent on the surfaces with deep continuous grooves. Three characteristic cell shapes (round, rectangular, and spool) were identified and represented by training groups. The training groups were used to set up criteria for cell-shape classification. The cells were classified according to their dispersion, elongation, and roundness values as round, rect, spool, or rect&spool shaped (Fig. 2). The approach proved to be successful with a large percentage of cells being classified (Fig. 6). The major results of the present study are schematically summarized in Table 4. First, significantly more cells aligned to grooves with continuous edges than to those with discontinuous edges. Second, rect&spool- or spool-shaped cells were mainly present on the grooved samples: g110, g200, and g400. Third, round cells were mainly present on the discontinuous control and discontinuous groove samples. Taken together these results provided evidence that cell alignment and elongation increased with groove depth
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provided that the edge was continuous. Use of cell-shape classification (vs the single-parameter shape descriptors) made it possible to study not only variations across the cell population but also the properties of subpopulations of cultured cells. For example, a correlation between rect&spoolor spool-shaped cells and aligned cells was identified (Fig. 8A and B). This result suggests an alignment mechanism dependent on the continuous edges present on the surface. The result that round, rect, and unclassified cells also were partially aligned on surfaces with deeper continuous grooves suggests that more than one mechanism for cellular alignment may exist. On the other hand, cells were only aligned and slightly elongated on the samples with deep discontinuous grooves (g400nano) and almost no alignment was observed on shallower discontinuous grooves (g110nano and g200nano) (Table 3, Figs. 5 and 6). No spool-shaped cells were present on any of the surfaces with discontinuous grooves (Fig. 6). Furthermore, low percentages of spool- and/or rect&spoolshaped cells correlated with very little contact guidance (Figs. 5 and 6, Table 3). The degrees of cell alignment observed on samples with the deepest discontinuous grooves (g400nano) corresponded to those seen for the round, rect, and unclassified cells on the deepest continuous grooves (g400) (compare Figs. 5 and 8A and B). In the case for deeper discontinuous grooves where both a continuous edge and spool-shaped cells were absent, the mechanism for alignment might correspond to the mechanism of aligning the round, rect, and unclassified cells on surfaces with continuous grooves. The hypothesis suggested in the present study is that a continuous edge is a prerequisite for contact guidance on surfaces with shallow grooves. Our results provided evidence that, among grooves of the same depth, a continuous edge resulted in significantly more aligned cells and the presence of spool-shaped cells. Other researchers have speculated that the distance of separation between nanostructures could influence the formation of focal contacts and concomitantly cell adhesion and survival [52,53]. This could have important ramifications for the results obtained on the discontinuous surfaces in the present study, since these surfaces contain separated nanometer-sized pillars. In this study we did not measure cell adhesion and proliferation; however, we have previously cultured the mammary gland cells on TiO2 surfaces with hemispherical pillars (feature density, diameter, and spacing as for discontinuous grooves) and on control flat (unpublished results). The cells grew to confluence and had similar total protein contents up to 5 days in culture on the two surfaces, using the same culture conditions as in the present study. Recent studies showed similar uroepithelial cell adhesion and proliferation both on flat and on surfaces with hemispherical pillars; however, a star-shaped cell morphology and a lower cytokine production was observed on the latter surface [34]. Therefore, we conclude that the hemispherical pillars used in the present study to mimic
discontinuous edges do not, to the best of our knowledge, affect cell attachment and survival. Proposed mechanisms for cell contact guidance involve cytoskeletal components (actin microfilaments, microtubules, and focal contacts) and ECM proteins. Cell migration itself is a process involving dynamic and spatially regulated changes to the cytoskeleton and the cell adhesion process [54 –58]. In addition, it has recently been found that members of the Rho GTPase family are involved in the signaling for cell migration [54 –58]. How can one cytoskeleton component be responsible for contact guidance when cell migration itself is an extremely complex process? Current knowledge indicates that integrins (or integrin clusters) can activate Rac GTPase, which subsequently can regulate actin polymerisation [54 –56]. This process suggests that the binding of integrins on the cell membrane to ECM ligands could regulate actin polymerisation, and, consequently, the direction of lamellopodia movement. Such cross-talk also means that it is difficult to assign contact guidance to either actin polymerisation or integrin–ECM binding (followed by focal contact formation). Consequently, the phenomenon of contact guidance is probably based on a combination of preferential ECM protein deposition, restricted actin/microtubules polymerisation, and confined focal contact formation. This sequence of events does not rule out a mechanism in which mechanical forces may be involved. One influence of a continuous sharp edge on such events could be an altered ECM protein deposition. The influence of topographic nanostructures on the functional behaviour of proteins has been shown in two recent papers [59,60] The present study used a set of systematically varied nanofabricated model surfaces combined with digital image analysis of individual cells via shape descriptors and shape classification to address the issue of the importance of a continuous edge in the alignment of cells to shallow topographic structures (grooves). The results indicated that grooved surfaces with continuous or discontinuous edges have very different capabilites in inducing contact guidance. Grooved surfaces with continuous edges are potent in aligning and inducing elongated cells, while grooved surfaces with discontinuous edges do not align and elongate cells to a large extent. A mechansim of alignment involving orientation along a continuous edge is therefore likely. Finally, the combination of fabricated model surfaces and cell-shape classification is a powerful approach to study cell-surface interactions.
Acknowledgments This work was supported by the Swedish Foundation for Strategic Research through the Biocompatible Materials program and the Wallenberg Foundation. We also acknowledge valuable contributions from Prof. Rena Bizios and Dr. Karin Glasma¨star in preparing the manuscript.
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