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Observation of fibroblast motility on a micro-grooved hydrophobic elastomer substrate with different geometric characteristics
Micron 38 (2007) 278–285 www.elsevier.com/locate/micron
Observation of fibroblast motility on a micro-grooved hydrophobic elastomer substrate with different geometric characteristics Wen-Ta Su a,c,*, Yung-Feng Liao b, I.-Ming Chu c b
a Department of Chemical Engineering, National Taipei University of Technology, Taipei 106, Taiwan Laboratory of Molecular Neurobiology, Institute of cellular and organismic biology, Academia Sinica, Taipei 115, Taiwan c Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan
Received 24 October 2005; received in revised form 19 April 2006; accepted 20 April 2006
Abstract We used a hydrophobic micro-textured poly-dimethylsiloxane (PDMS) in the presence of serum protein at 37 8C to study the motility of mouse stromal fibroblast on variant (15–100 mm) parallel ridge/groove with 30 mm depth. In this paper, we observed the temporal changes in cell morphology and locomotion by using time-lapse phase-contrast microscopy. When fibroblasts seeded onto the micro-grooved substrate, almost all of cells concentrated at the bottom of the grooves. Sequentially, the fibroblasts attached and spread on the surface, migrated toward the walls of the grooves, climbed up and down the ridges frequently, apparently, the 30 mm depth of groove did not hinder movement across the micro-grooves. Eventually, they stopped proliferating as a result of contact inhibition and formed a confluent monolayer on the ridges almost exclusively, with an orientation parallel to the direction of the ridge/groove. Cellular shape of fibroblast was enhanced with the micro-grooves, the form index of nucleus was 2.6-fold greater than that of cells on smooth surfaces. Further, we found that hydrophobic surfaces are more prone to direct cellular motility in comparison with hydrophilic surfaces. # 2006 Elsevier Ltd. All rights reserved. Keywords: Micro-groove; Silicone elastomer; Fibroblast; Migration; Hydrophobic
1. Introduction Directed cell movement is a process that is generally recognized as being crucial for the development of tissue engineering: new cells must be aligned and positioned correctly for wound healing and maintenance of tissue structure. Therefore, the understanding mechanism of cell movement is essential for basic morphogenetic processes. Most research on the directional control of cell motility has focused on the role of gradients of motility factors, such as chemically (Banyard and Zetter, 1998; Lauffenburger and Horwitz, 1996; Parent and Devreotes, 1999; Adams and Schwartz, 2000; Holly et al., 2000; del Pozo et al., 2000) or electrochemically (Kaji et al., 2004; Kimura et al., 2005) modified surfaces. However, it is well known that the topographical structure of an underlying substrate can control the locomotion and orientation of cultured cells and can affect cell morphology, cytoskeletal organization and motility
* Corresponding author. Tel.: +886 2 27712171x2554; fax: +886 2 27317117. E-mail address: [email protected] (W.-T. Su). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.04.008
(Curtis and Wilkinson, 1998; Walboomers et al., 1998), as well as changes in gene expression in vitro. Topographical substrates can be made through micro-fabrication technology (Brunette et al., 1983), and subsequently can be transferred into polymeric surface by molding, making them suitable for the study of cellsubstrate interaction with textured polymers. This versatile and flexible approach toward fabrication on the micro- or nanometer level provides excellent surface control for modifying and controlling cell behavior in vitro. Many mammalian cells are capable of undergoing a crawling movement, but, how cellular movement is controlled has long been of interest to developmental biologists and tissue engineers. When placed upon micro-grooves of particular dimensions, many cell types align themselves in the direction of the grooves, termed contact guidance (Weiss, 1954). We were interested to know what effects wider and deeper parallel micro-grooves with varying widths/repeat spaces (from 15 to 200 mm) and a 30-mm depth would have upon the migration of a fibroblast. We applied only physical topographical guidance, i.e., using the reactions of the cells to the topography of their substrate and the mechanical forces applied to them by their
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surrounding patterns, and used the motile properties of the cells themselves to move them into suitable positions on a hydrophobic substrate presenting parallel micro-grooves. We speculated that lower interaction of cell and hydrophobic biomaterial would have the capacity to increase cell motility as compared with migration over a hydrophilic surface and the extent of the increase would depend upon the topography of the biomaterials, because we believed that deeper grooves would provide a more macroscopic architecture having the capacity to increase the degree of cell migration, relative to the migration the occurs on planar surfaces. In additional, we believe that the distribution of oxygen gas in the culturing environment plays a significant role in this process, so we propose an oxygen transfer model to explain the phenomena and to determine a possible mechanism in this cellular motility process. 2. Materials and methods 2.1. Grooved substrates Textured silicon wafers were prepared using photolithography and etching techniques. All substrates were obtained by replica molding of PDMS (polydimethylsiloxane MDX 44210, Dow Corning) on the silicon wafer under vacuum for 2 h and then were put in an oven at 60 8C for 2 h. After polymerization, the silicone elastomer negative surface replicas were removed. Samples were cut into pieces (1 cm2) and washed in a series of cleaning solutions (Su et al., 2004). Finally, the samples were sterilized for 30 min in 70% ethanol, and then rinsed with sterile PBS prior to cell culturing. The wettability of substrates was measured with contact angle meter (FACE, CA-D). 2.2. Cell culture Mouse bone marrow stromal fibroblasts were purchased from Bioresource Collection and Research Center (BCRC60228) and maintained in medium RPMI 1640, supplemented with 10% FBS for routine culture, then suspension seeded onto both planar and micro-grooved substrates. The seeding density was ca. 1 105 cell/mL (2.5 mL/dish) in a 35-mm-wide dish. All images of culturing fibroblasts were obtained using a phase
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contrast microscope. Cell numbers in the grooves and ridges were counted and cellular orientation angles along the direction of ridge/groove were measured by Image-Pro Plus software (Media Cybernetics). Cell moving path with its migration speed was measured by time-lapse microscope (Zeiss Axiovert 200 M) for 12 different cells, which were scanned every 20 min up to 25 h and their moving path was tracked with metamorph software (Universal Imaging Corporation). 2.3. Immunofluorescence staining of the cell nucleus and cytoskeleton After fibroblasts had been cultured on a micro-grooved substrate for 1.5 days, all specimens were given two rinses with PBS to remove any unattached cells. Cells were fixed in situ for 10 min in 4% paraformaldehyde and then permeabilized with 1% Triton 100. Thereafter, vinculin was stained with mouse anti-vinculin (Sigma) followed by incubation with goat antimouse-FITC (Sigma); subsequently, filamentous actin was stained with phalloidin-TRITC (Sigma) and the nucleus with DAPI (Molecular Probes). The specimens were then examined under a fluorescence microscope (Zeiss Axioplan 2 image MOT; Axiovision software). From these images, Image-Pro Plus software (Media Cybernetics) was used to measure the longest and widest segments of nucleus so as to calculate the form index (FI). 3. Results 3.1. Surface characteristics of micro-textured substrates The micro-textured silicon wafer and the PDMS substrate surface pattern presented parallel ridge/groove patterns. Fig. 1A and B display top views of the silicon wafer and the PDMS substrate, respectively. The patterns and dimensions of the PDMS substrates were close to those of the silicon wafer molds – with respect to their depths, groove widths, ridge widths, and for the structure of the vertical wall – but, because the PDMS structures were negative surface replicas, the dimensions of their groove and ridge widths were opposite to those of the silicon wafers’ dimensions. Table 1 summarizes the dimensions of all substrates that we prepared. The results of the
Fig. 1. SEM micrographs of silicon wafer surfaces: (A) 30-mm groove, 60-mm ridge; micrographs of PDMS surfaces; (B) 60-mm groove, 30-mm ridge.
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Table 1 Dimensions of the substrates used in this study Substrate
Groove width (mm)
Ridge width (mm)
Repeat space (mm)
Groove depth (mm)
PDMS15 Si-wafer45 PDMS30 Si-wafer60 PDMS100 Si-wafer100
45 15 60 30 100 100
15 45 30 60 100 100
60 – 90 – 200 –
30 30 30 30 30 30
Table 2 Wettability of various substrate with water contact angle Substrate
Angle (8)
Peri dish Coverslips glass Si-wafer PDMS
86.4 4.39 43.25 2.21 54.25 6.34 106.25 4.35
wettability measurements are shown in Table 2. The data showed PDMS had a much high contact angle than other substrates, distinctly was a large hydrophobility. The sterilization method test was performed using both an autoclave and a 70% ethanol solution; the fibroblast appeared to have the same behavior in either case, with no obvious differences between the two sterilization methods. This ethanol solution did not appear to change the surface chemistry of the PDMS substrate and did not undergo any pattern degradation during sterilization, which
is consistent with the results reported by Mata et al. (2002). Since the procedure was more convenient, we performed all subsequent sterilizations using 70% ethanol solution. 3.2. Observation of cellular motility using time-lapse phase-contract microscopy When fibroblasts were seeded onto the cultured substrate, most cells settled to the bottom of the grooves as expected and only a small number of them landed on the ridges; Fig. 2A display this phenomenon for the PDMS30 substrates. In contrast, cells seeded on a planar surface were distributed evenly on the surface (image not shown). After incubation, the physical topography restricted and induced the direction of cell spreading. Fig. 2B displays the fibroblasts’ migration and location at the ridges and grooves of the PDMS100 after culturing for 6 h. Fibroblasts had a tendency to migrate to the walls of the grooves; subsequently,
Fig. 2. Cells settled down and concentrated on the grooves of PDMS30 initially (A). For 6 h culture, fibroblasts spread on the grooves and ridges of the PDMS100 (B). After 3.5 days culture, most of the cells stop proliferating and formed confluent monolayer on the ridges of PDMS30 (C) and PDMS100 (D). All images: 20 (arrow is the ridge).
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Fig. 3. The distribution of cellular orientation on micro-grooved PDMS. The oriented angle to the direction of ridge/groove was decreased to follow cultured period; therefore micro-grooves could induce fibroblast alignment along the direction of ridge/groove (data was plotted as mean standard deviation).
they aggregated to form clusters and whole clusters climbed up toward neighboring ridges. Most of these clusters then moved and changed their cellular morphology on the ridges constantly. Upon further steady incubation for 3.5 days, almost all of the fibroblast clusters remained concentrated on the upper surfaces of the ridges and with their cellular orientations distributed along the direction of the ridge/groove. At this time, fibroblasts had stopped proliferating due to contact inhibition and formed a confluent monolayer on the ridges. We observed these phenomena on both PDMS30 (Fig. 2C) and PDMS100 (Fig. 2D). From time-lapse recording, we observed that the fibroblasts were contractile and stretched their cellular bodies back and forth in the direction of their migration constantly during the culturing process, in a manner akin to how an amoeba might migrate. Furthermore, although it appeared that the fibroblasts preferred to aggregate with each other, some fibroblasts having very active motility would break away from one cluster to descend down the bottom of the grooves and thereafter climbed up neighboring ridge. Finally, these
Fig. 4. The percentage of cells that migrated upon the surfaces of ridges or stayed on the grooves relative to the culture time. (A) Cells located in the grooves. (B) Cells located on the ridges (data was plotted as mean standard deviation).
fibroblasts oriented parallel with the direction of the grooved surfaces of the elastomers, as Fig. 3 shows that is consistent with those of previous studies (Dunn and Brown, 1986; den Braber et al., 1996a, 1996b; Meyle et al., 1994; Curtis and
Fig. 5. Moving paths of fibroblast on surface of various substrates for 35 h culture. (A) PDMS15; (B) PDMS30; (C) PDMS100; (D) collagen-coated PDMS30; (E) collagen-coated PDMS100. Red point is beginning position; blue point is ending position. All images: 20.
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Fig. 6. The velocity of cells movement on various substrates. Cells migrated fast on the PDMS substrate than that on the collagen-coated PDMS and coverslips glass (data was plotted as mean standard deviation).
Clark, 1990; Clark et al., 1990; Dalton et al., 2001). The ridges of PDMS15 are, however, too narrow for the fibroblasts to spread fully flat upon them and, thus, the edges of the fibroblast membranes are forced to bend down and into the groove; indeed, the number of fibroblasts that migrated to the tops of the ridges from the bottom of each groove was less pronounced in this case; as shown in Fig. 4. In contrast, for PDMS100, which has wider ridges and grooves, but the cell clusters still aligned well along the ridges with very low angles of orientation. They did not freely spread as that observed on the planar surface.
Therefore, even within deeper grooves, wider ridges still have an ability to induce fibroblast migration and orientation (compared Fig. 4A and B with Fig. 3). This finding distinct from previous studies with a few mm to 10 mm wide and deep (Brunette, 1986; Wojciak-Stothard et al., 1995; van Kooten et al., 1998), and can possibly be explained by the specific influence of surface free energy of the substrate on the amount of the adsorbed proteins, which altered the surface characteristics of the polymers so that cell migration is induced on the PDMS. Many cells were able to migrate significant distances on the micro-grooved PDMS through dynamic membrane extensions, as seen by direct visualization with time lapse microscope. Typical paths of movement on PDMS substrate as shown in Fig. 5. Fig. 5A–C clearly shows that cells migrated from grooves upon ridges, but cells were restricted in the interval of grooves on collagen-coated PDMS (shown in Fig. 5D and E). From these data we calculate cellular moving rate and found that cell motility on grooved and flat bare PDMS was faster than cells on collagen-coated PDMS and coverslips glass (Fig. 6). Rapid migration requires efficient mechanisms to release adhesions at the rear of the cell, probably because cell adhesion to collagen-coated PDMS and coverslips glass was too strong for efficient cell detachment, leaving cells unable to move quickly. However, serum protein adsorbed on PDMS is lower than that transferred via the micro-contact printing technique (Mrkish and Whiteside, 1995), like this intermediate cell-substrate interactions, contrarily promote cells as they wish movement.
Fig. 7. Immunofluorescence overlay images showing the actin (red); vinculin (green) and nucleus (blue) of the cells on the grooved PDMS substrates after incubation for 1.5 days. On the 15-mm ridges (A), and on the 30-mm ridge (B), the nucleus appeared oblong form; 100-mm ridges (C) and planar (D) were ellipse. The cells possess highly aligned actin parallel to the direction of the ridge/grooves, and the vinculin concentrated densely on the ridges (arrow is the ridge).
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Fig. 8. The FI of nucleus was affected to grooved substrate, and was an inverse proportion to width of ridges (data was plotted as mean standard deviation).
3.3. Immunofluorescence observation of the cell nucleus and cytoskeleton To further evaluate the behavior of fibroblasts on microgrooved substrates, we monitored the distributions of actin, vinculin, and the nuclei to describe micro-filaments with respect to their focal contacts and cell adherence. For each of the micro-grooved PDMS substrates that we used, the actin fibers appeared to be elongated with their long axes aligned parallel with the direction of the ridge/groove and demonstrated clearly that the vinculin of the focal contacts concentrated densely on the edges and top surfaces of the ridges, as indicated in Fig. 7A–C. This observation indicates that the alignment of the filaments of the cytoskeleton within the cell reflects the orientation of the cell as a whole. On smooth substrate (Fig. 7D), the fibroblasts spread fully with many bundles of actin filaments anchored to the plasma membrane at sites of extended focal contacts; there was obvious formation of focal contact at the cell periphery. These immunofluorescence observations indicate clearly that the nuclei possessed oblong forms on the ridges of PDMS15 (Fig. 7A) and PDMS30 (Fig. 7B), but they retained their elliptical configurations on the 100-mm-wide ridges of PDMS100 (Fig. 7C) and on the planar surface (Fig. 7D). Consequently, cell shape was changed by micro-grooves of substrate, cell elongated along the direction of ridge/groove, that promote nucleus form to become deformed. The form index of nucleus was shown in Fig. 8. The degree of deformation was proportional to the degree of discontinuity of surface. 4. Discussion Our aims of this study is that directed cells to their targets. We explore the influence of surface properties on cell migration. The surface characteristics including the surface chemistry and surface topography have a significant influence on adhesion, morphology, orientation, and motility. The wettability of the surface has been shown to be one of the important factors to cell behavior. Certain chemical groups present on the surface of the material can alter cell response (Ruardy et al., 1997), and topographically also induced wettability (Brochard-Wyart, 1995), it is to be expected that micro-grooved surfaces will exhibit heterogeneous surface energy even if the surface is chemically homogeneous. However, unlike a drop of water, cellular shape and motility are largely influenced by the cytoskeleton; the cell will tend to
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form focal adhesions in locations that balance cytoskeletal forces (Ingber and Tensegrity, 2003). On a high adhesive substrate, the overall cell-substrate bond is strong, causing a very slow rate of cell detachment with the result that cells become immobilized (as our result of Fig. 5D and E). Conversely, on a poor adhesive substrate, cell-substrate interaction is too weak and cells cannot generate enough traction for forward migration (Palecek et al., 1999). Therefore, it is postulated that maximal speed can be obtained by cells moving on surfaces at intermediate attachment strength (Dimilla et al., 1993). In general, hydrophobic surfaces are less amenable to cell adhesion than are their hydrophilic counterparts (Lindblad et al., 1997; Scotchford et al., 1998; Altankov and Groth, 1994). In our study, cells indicate poor cellular attachment initially, but still were exponential proliferating later on non-treated silicon rubber. This phenomenon was to ascribe to the surface of PDMS presents hydrophobic methyl groups, that could adsorb biomolecules in the serum, such as fibronectin, onto surface, and permit cell adhesion, then which induce intermediate cell–substrate bonding, therefore, it is conducive for the rapid movement and cellular motile traction was random during the entire culturing period, but the cells’ final goal was to reach the top surfaces of the ridges (shown in Fig. 5A–C). Our result contrasts those reported by Reyes et al. (2004) and Kaji et al. (2004) these authors used heterogeneous forces (chemical or electrochemical modification) on the surface of the substrate to render specific regions adhesive or non-adhesive and to promote cell migration parallel to specific locomotion. Other features of a material have been identified as important factor determinants of cell migration, such as the mechanical strength of a substrate (Kuntz and Saltzman, 1997). Mechanical forces between cells and their environment interaction are believed to provide the driving force for cell locomotion (Lauffenburger and Horwitz, 1996; Oliver et al., 1994). 3D structure appears many anchorage points that provide many opportunities for cells to have mechanical interactions. Dunn and Brown (1986) proposed that directed cell migration on a topographical substrate results from the substrate imposing mechanical restrictions on the formation of certain linear bundles of micro-filaments. The results of many studies have suggested that, during locomotion, intracellular forces generate mechanical tension on the underlying substrate. Dembo and Wang (1999) demonstrate that lamellipodium of the fibroblast is able to generate intense traction stress, and the traction is integral to the mechanism of fibroblast locomotion and that the mechanical energy for traction is derived from cytoskeletal contractile activity. For move forward, cells must adhere to the substrate and exert rearward traction forces, therefore, contraction of cytoskeleton causes tension to pull the frontal adhesions backward and push the tail adhesions forward. In fact, from time-lapse recordings, it is clear that the cellular cytoskeleton is not static – it is a dynamic structure – and that the cell processes are considerably flexible; the polymerization and depolymerization of cellular compounds is influenced by the geometrical topography of the substrate, which creates a mechanical stress that influences cell spreading
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Fig. 9. The oxygen transfer from the air above the medium to the surface of the substrate, when cells grown on the ridge (for example: 100-mm groove/ridge). The oxygen concentration contour on the cultured dish, the oxygen concentration (mM) around ridge was measured. The middle zone is the 30-mm-height ridge.
and alignment. Cell motility was very sensitive to the topography of substrate. Cells can use the edges of the ridge as footholds to gain mechanical adhesion, an additional source of traction that is absent on planar surfaces. Sheetz et al. (1998) described the migration of fibroblasts as occurring through a multistep process that requires dynamic interactions between the cell, substrate, and cytoskeleton. Thus, the cells continually creep and covet a state in which their internal and external forces are in equilibrium, and this process induces their aligned cellular shape. In this study, we observed that the clustering of cells in a parallel manner on top of narrow ridges is perhaps their optimal steady state environment. The most dramatic phenomenon we observed in this study was that fibroblasts rose to the tops of the ridges, and preferred to reside there rather than at the bottoms of the grooves. From our combined observations, we think that oxygen supply is one of the most important parameters for cell culture. When seeded the cells onto the surfaces of the substrates, oxygen required for cell growth from a constant temperature, constant humidity, 5% CO2/air atmosphere above the medium. We solved the oxygen supply equation using a molar flux of oxygen. Concentration profile is a linear function, oxygen is transferred gradually by diffusion from the upper parts of the medium to the surface of the elastomer; we might expect that the model for growth of the cells would be diffusion-controlled during the culturing period. We quantified oxygen concentration using FEMALAB23 MATLAB6.1 computer software; Fig. 9 displays the results of resolved expression. We found that zone of ridge has the lowest oxygen concentration, but it is only slightly different to that at the bottom of the groove and, thus, most of the cells do not descend to the bottom; rather, they are content to remain on the surfaces of ridges. 5. Conclusions In this study, we have found another method to induce and control migration and location of cells that does not involve
chemical or electrochemical modification of the surface of substrate to render specific regions adhesive or non-adhesive. There are three major conclusions from this study of the reactions between cells and grooved substrates: (1) fibroblasts tend to align along the direction of the grooves and attain longer cell bodies than they do on flat substrates; (2) fibroblasts spread readily and eventually climb up the walls of the grooves and onto the ridges; (3) the rate of cell motility on grooved and flat bare PDMS was faster than cells on collagen-coated PDMS and coverslips glass. Thus, culture substrates possessing various topographies can be designed to affect the behavior of bone marrow stromal fibroblasts; such effects may be particularly applicable when cells must be localized in specific areas or when cell migration must be directed. One practical application of the substrate-mediated control of cell behavior may be in the design of implanted devices that control cell migration and location; we expect that manipulating the healing response of regenerating tissues around such implants would improve future device performance. Acknowledgment This work was supported partly by the National Science Council (NSC) of Taiwan under contract no. NSC93-2113-M492-002. References Adams, J.C., Schwartz, M.A., 2000. Stimulationof fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42. J. Cell Biol. 150, 807– 822. Altankov, G., Groth, T., 1994. Reorganization of substratum-bound fibronectin on hydrophilic and hydrophobic materials is related to biocompatibility. J. Mater. Sci. Mater. Med. 5, 732–737. Banyard, J., Zetter, B.R., 1998. The role of cell motility in prostate cancer. Cancer Metast. Res. 17, 449–458. Brochard-Wyart, F., 1995. Droplets: capillarity and wetting. In: Daoud, C.E.W.M. (Ed.), Soft Matter Physics. Springer, Berlin, pp. 1–45. Brunette, D.M., 1986. Spreading and orientation of epithelial cells on grooved substrata. Exp. Cell Res. 167, 203–217. Brunette, D.M., Kenner, G.S., Gould, T.R.L., 1983. Grooved titanium surface orient growth and migration of cells from human gingival explants. J. Dent. Res. 62, 1045–1048. Clark, P., Connolly, P., Curtis, A.S.G., Dow, J.A.T., Wilkinson, C.D.W., 1990. Topographical control of cell behavior. II. Multiple grooved substrata. Development 108, 635–644. Curtis, A.S.G., Clark, P., 1990. The effects of topographic and mechanical properties of materials on cell behavior. Crit. Rev. Biocompat. 5, 343–362. Curtis, A.S.G., Wilkinson, C.D.W., 1998. Reaction of cells to topography. J. Biomater. Sci. Polymer. Edn. 9, 1313–1329. Dalton, B.A., Walboomers, X.F., Dziegielewski, M., Evans, M.D.M., Taylor, S., Jansen, J.S., Steele, J.G., 2001. Modulation of epithelial tissue and cell migration by micro-grooves. J. Biomed. Mater. Res. 56, 195–207. del Pozo, M.A., Price, L.S., Alderson, N.B., Ren, X.D., Schwartz, M.A., 2000. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19, 2008–2014. Dembo, M., Wang, Y.L., 1999. Stresses at the cell-to-substrate interface duringlocomotion of fibroblasts. Biophys. J. 76, 2307–2316. den Braber, E.T., de Ruijter, J.E., Smits, H.T.J., Ginsel, L.A., von Recum, A.F., Jansen, J.A., 1996a. Quantitative analysis of cell proliferation and orientation on substrata with uniform parallel surface micro-grooves. Biomaterials 17, 1093–1099.
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Observation of fibroblast motility on a micro-grooved hydrophobic elastomer substrate with different geometric characteristics Wen-Ta Su a,c,*, Yung-Feng Liao b, I.-Ming Chu c b
a Department of Chemical Engineering, National Taipei University of Technology, Taipei 106, Taiwan Laboratory of Molecular Neurobiology, Institute of cellular and organismic biology, Academia Sinica, Taipei 115, Taiwan c Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan
Received 24 October 2005; received in revised form 19 April 2006; accepted 20 April 2006
Abstract We used a hydrophobic micro-textured poly-dimethylsiloxane (PDMS) in the presence of serum protein at 37 8C to study the motility of mouse stromal fibroblast on variant (15–100 mm) parallel ridge/groove with 30 mm depth. In this paper, we observed the temporal changes in cell morphology and locomotion by using time-lapse phase-contrast microscopy. When fibroblasts seeded onto the micro-grooved substrate, almost all of cells concentrated at the bottom of the grooves. Sequentially, the fibroblasts attached and spread on the surface, migrated toward the walls of the grooves, climbed up and down the ridges frequently, apparently, the 30 mm depth of groove did not hinder movement across the micro-grooves. Eventually, they stopped proliferating as a result of contact inhibition and formed a confluent monolayer on the ridges almost exclusively, with an orientation parallel to the direction of the ridge/groove. Cellular shape of fibroblast was enhanced with the micro-grooves, the form index of nucleus was 2.6-fold greater than that of cells on smooth surfaces. Further, we found that hydrophobic surfaces are more prone to direct cellular motility in comparison with hydrophilic surfaces. # 2006 Elsevier Ltd. All rights reserved. Keywords: Micro-groove; Silicone elastomer; Fibroblast; Migration; Hydrophobic
1. Introduction Directed cell movement is a process that is generally recognized as being crucial for the development of tissue engineering: new cells must be aligned and positioned correctly for wound healing and maintenance of tissue structure. Therefore, the understanding mechanism of cell movement is essential for basic morphogenetic processes. Most research on the directional control of cell motility has focused on the role of gradients of motility factors, such as chemically (Banyard and Zetter, 1998; Lauffenburger and Horwitz, 1996; Parent and Devreotes, 1999; Adams and Schwartz, 2000; Holly et al., 2000; del Pozo et al., 2000) or electrochemically (Kaji et al., 2004; Kimura et al., 2005) modified surfaces. However, it is well known that the topographical structure of an underlying substrate can control the locomotion and orientation of cultured cells and can affect cell morphology, cytoskeletal organization and motility
* Corresponding author. Tel.: +886 2 27712171x2554; fax: +886 2 27317117. E-mail address: [email protected] (W.-T. Su). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.04.008
(Curtis and Wilkinson, 1998; Walboomers et al., 1998), as well as changes in gene expression in vitro. Topographical substrates can be made through micro-fabrication technology (Brunette et al., 1983), and subsequently can be transferred into polymeric surface by molding, making them suitable for the study of cellsubstrate interaction with textured polymers. This versatile and flexible approach toward fabrication on the micro- or nanometer level provides excellent surface control for modifying and controlling cell behavior in vitro. Many mammalian cells are capable of undergoing a crawling movement, but, how cellular movement is controlled has long been of interest to developmental biologists and tissue engineers. When placed upon micro-grooves of particular dimensions, many cell types align themselves in the direction of the grooves, termed contact guidance (Weiss, 1954). We were interested to know what effects wider and deeper parallel micro-grooves with varying widths/repeat spaces (from 15 to 200 mm) and a 30-mm depth would have upon the migration of a fibroblast. We applied only physical topographical guidance, i.e., using the reactions of the cells to the topography of their substrate and the mechanical forces applied to them by their
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surrounding patterns, and used the motile properties of the cells themselves to move them into suitable positions on a hydrophobic substrate presenting parallel micro-grooves. We speculated that lower interaction of cell and hydrophobic biomaterial would have the capacity to increase cell motility as compared with migration over a hydrophilic surface and the extent of the increase would depend upon the topography of the biomaterials, because we believed that deeper grooves would provide a more macroscopic architecture having the capacity to increase the degree of cell migration, relative to the migration the occurs on planar surfaces. In additional, we believe that the distribution of oxygen gas in the culturing environment plays a significant role in this process, so we propose an oxygen transfer model to explain the phenomena and to determine a possible mechanism in this cellular motility process. 2. Materials and methods 2.1. Grooved substrates Textured silicon wafers were prepared using photolithography and etching techniques. All substrates were obtained by replica molding of PDMS (polydimethylsiloxane MDX 44210, Dow Corning) on the silicon wafer under vacuum for 2 h and then were put in an oven at 60 8C for 2 h. After polymerization, the silicone elastomer negative surface replicas were removed. Samples were cut into pieces (1 cm2) and washed in a series of cleaning solutions (Su et al., 2004). Finally, the samples were sterilized for 30 min in 70% ethanol, and then rinsed with sterile PBS prior to cell culturing. The wettability of substrates was measured with contact angle meter (FACE, CA-D). 2.2. Cell culture Mouse bone marrow stromal fibroblasts were purchased from Bioresource Collection and Research Center (BCRC60228) and maintained in medium RPMI 1640, supplemented with 10% FBS for routine culture, then suspension seeded onto both planar and micro-grooved substrates. The seeding density was ca. 1 105 cell/mL (2.5 mL/dish) in a 35-mm-wide dish. All images of culturing fibroblasts were obtained using a phase
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contrast microscope. Cell numbers in the grooves and ridges were counted and cellular orientation angles along the direction of ridge/groove were measured by Image-Pro Plus software (Media Cybernetics). Cell moving path with its migration speed was measured by time-lapse microscope (Zeiss Axiovert 200 M) for 12 different cells, which were scanned every 20 min up to 25 h and their moving path was tracked with metamorph software (Universal Imaging Corporation). 2.3. Immunofluorescence staining of the cell nucleus and cytoskeleton After fibroblasts had been cultured on a micro-grooved substrate for 1.5 days, all specimens were given two rinses with PBS to remove any unattached cells. Cells were fixed in situ for 10 min in 4% paraformaldehyde and then permeabilized with 1% Triton 100. Thereafter, vinculin was stained with mouse anti-vinculin (Sigma) followed by incubation with goat antimouse-FITC (Sigma); subsequently, filamentous actin was stained with phalloidin-TRITC (Sigma) and the nucleus with DAPI (Molecular Probes). The specimens were then examined under a fluorescence microscope (Zeiss Axioplan 2 image MOT; Axiovision software). From these images, Image-Pro Plus software (Media Cybernetics) was used to measure the longest and widest segments of nucleus so as to calculate the form index (FI). 3. Results 3.1. Surface characteristics of micro-textured substrates The micro-textured silicon wafer and the PDMS substrate surface pattern presented parallel ridge/groove patterns. Fig. 1A and B display top views of the silicon wafer and the PDMS substrate, respectively. The patterns and dimensions of the PDMS substrates were close to those of the silicon wafer molds – with respect to their depths, groove widths, ridge widths, and for the structure of the vertical wall – but, because the PDMS structures were negative surface replicas, the dimensions of their groove and ridge widths were opposite to those of the silicon wafers’ dimensions. Table 1 summarizes the dimensions of all substrates that we prepared. The results of the
Fig. 1. SEM micrographs of silicon wafer surfaces: (A) 30-mm groove, 60-mm ridge; micrographs of PDMS surfaces; (B) 60-mm groove, 30-mm ridge.
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Table 1 Dimensions of the substrates used in this study Substrate
Groove width (mm)
Ridge width (mm)
Repeat space (mm)
Groove depth (mm)
PDMS15 Si-wafer45 PDMS30 Si-wafer60 PDMS100 Si-wafer100
45 15 60 30 100 100
15 45 30 60 100 100
60 – 90 – 200 –
30 30 30 30 30 30
Table 2 Wettability of various substrate with water contact angle Substrate
Angle (8)
Peri dish Coverslips glass Si-wafer PDMS
86.4 4.39 43.25 2.21 54.25 6.34 106.25 4.35
wettability measurements are shown in Table 2. The data showed PDMS had a much high contact angle than other substrates, distinctly was a large hydrophobility. The sterilization method test was performed using both an autoclave and a 70% ethanol solution; the fibroblast appeared to have the same behavior in either case, with no obvious differences between the two sterilization methods. This ethanol solution did not appear to change the surface chemistry of the PDMS substrate and did not undergo any pattern degradation during sterilization, which
is consistent with the results reported by Mata et al. (2002). Since the procedure was more convenient, we performed all subsequent sterilizations using 70% ethanol solution. 3.2. Observation of cellular motility using time-lapse phase-contract microscopy When fibroblasts were seeded onto the cultured substrate, most cells settled to the bottom of the grooves as expected and only a small number of them landed on the ridges; Fig. 2A display this phenomenon for the PDMS30 substrates. In contrast, cells seeded on a planar surface were distributed evenly on the surface (image not shown). After incubation, the physical topography restricted and induced the direction of cell spreading. Fig. 2B displays the fibroblasts’ migration and location at the ridges and grooves of the PDMS100 after culturing for 6 h. Fibroblasts had a tendency to migrate to the walls of the grooves; subsequently,
Fig. 2. Cells settled down and concentrated on the grooves of PDMS30 initially (A). For 6 h culture, fibroblasts spread on the grooves and ridges of the PDMS100 (B). After 3.5 days culture, most of the cells stop proliferating and formed confluent monolayer on the ridges of PDMS30 (C) and PDMS100 (D). All images: 20 (arrow is the ridge).
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Fig. 3. The distribution of cellular orientation on micro-grooved PDMS. The oriented angle to the direction of ridge/groove was decreased to follow cultured period; therefore micro-grooves could induce fibroblast alignment along the direction of ridge/groove (data was plotted as mean standard deviation).
they aggregated to form clusters and whole clusters climbed up toward neighboring ridges. Most of these clusters then moved and changed their cellular morphology on the ridges constantly. Upon further steady incubation for 3.5 days, almost all of the fibroblast clusters remained concentrated on the upper surfaces of the ridges and with their cellular orientations distributed along the direction of the ridge/groove. At this time, fibroblasts had stopped proliferating due to contact inhibition and formed a confluent monolayer on the ridges. We observed these phenomena on both PDMS30 (Fig. 2C) and PDMS100 (Fig. 2D). From time-lapse recording, we observed that the fibroblasts were contractile and stretched their cellular bodies back and forth in the direction of their migration constantly during the culturing process, in a manner akin to how an amoeba might migrate. Furthermore, although it appeared that the fibroblasts preferred to aggregate with each other, some fibroblasts having very active motility would break away from one cluster to descend down the bottom of the grooves and thereafter climbed up neighboring ridge. Finally, these
Fig. 4. The percentage of cells that migrated upon the surfaces of ridges or stayed on the grooves relative to the culture time. (A) Cells located in the grooves. (B) Cells located on the ridges (data was plotted as mean standard deviation).
fibroblasts oriented parallel with the direction of the grooved surfaces of the elastomers, as Fig. 3 shows that is consistent with those of previous studies (Dunn and Brown, 1986; den Braber et al., 1996a, 1996b; Meyle et al., 1994; Curtis and
Fig. 5. Moving paths of fibroblast on surface of various substrates for 35 h culture. (A) PDMS15; (B) PDMS30; (C) PDMS100; (D) collagen-coated PDMS30; (E) collagen-coated PDMS100. Red point is beginning position; blue point is ending position. All images: 20.
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Fig. 6. The velocity of cells movement on various substrates. Cells migrated fast on the PDMS substrate than that on the collagen-coated PDMS and coverslips glass (data was plotted as mean standard deviation).
Clark, 1990; Clark et al., 1990; Dalton et al., 2001). The ridges of PDMS15 are, however, too narrow for the fibroblasts to spread fully flat upon them and, thus, the edges of the fibroblast membranes are forced to bend down and into the groove; indeed, the number of fibroblasts that migrated to the tops of the ridges from the bottom of each groove was less pronounced in this case; as shown in Fig. 4. In contrast, for PDMS100, which has wider ridges and grooves, but the cell clusters still aligned well along the ridges with very low angles of orientation. They did not freely spread as that observed on the planar surface.
Therefore, even within deeper grooves, wider ridges still have an ability to induce fibroblast migration and orientation (compared Fig. 4A and B with Fig. 3). This finding distinct from previous studies with a few mm to 10 mm wide and deep (Brunette, 1986; Wojciak-Stothard et al., 1995; van Kooten et al., 1998), and can possibly be explained by the specific influence of surface free energy of the substrate on the amount of the adsorbed proteins, which altered the surface characteristics of the polymers so that cell migration is induced on the PDMS. Many cells were able to migrate significant distances on the micro-grooved PDMS through dynamic membrane extensions, as seen by direct visualization with time lapse microscope. Typical paths of movement on PDMS substrate as shown in Fig. 5. Fig. 5A–C clearly shows that cells migrated from grooves upon ridges, but cells were restricted in the interval of grooves on collagen-coated PDMS (shown in Fig. 5D and E). From these data we calculate cellular moving rate and found that cell motility on grooved and flat bare PDMS was faster than cells on collagen-coated PDMS and coverslips glass (Fig. 6). Rapid migration requires efficient mechanisms to release adhesions at the rear of the cell, probably because cell adhesion to collagen-coated PDMS and coverslips glass was too strong for efficient cell detachment, leaving cells unable to move quickly. However, serum protein adsorbed on PDMS is lower than that transferred via the micro-contact printing technique (Mrkish and Whiteside, 1995), like this intermediate cell-substrate interactions, contrarily promote cells as they wish movement.
Fig. 7. Immunofluorescence overlay images showing the actin (red); vinculin (green) and nucleus (blue) of the cells on the grooved PDMS substrates after incubation for 1.5 days. On the 15-mm ridges (A), and on the 30-mm ridge (B), the nucleus appeared oblong form; 100-mm ridges (C) and planar (D) were ellipse. The cells possess highly aligned actin parallel to the direction of the ridge/grooves, and the vinculin concentrated densely on the ridges (arrow is the ridge).
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Fig. 8. The FI of nucleus was affected to grooved substrate, and was an inverse proportion to width of ridges (data was plotted as mean standard deviation).
3.3. Immunofluorescence observation of the cell nucleus and cytoskeleton To further evaluate the behavior of fibroblasts on microgrooved substrates, we monitored the distributions of actin, vinculin, and the nuclei to describe micro-filaments with respect to their focal contacts and cell adherence. For each of the micro-grooved PDMS substrates that we used, the actin fibers appeared to be elongated with their long axes aligned parallel with the direction of the ridge/groove and demonstrated clearly that the vinculin of the focal contacts concentrated densely on the edges and top surfaces of the ridges, as indicated in Fig. 7A–C. This observation indicates that the alignment of the filaments of the cytoskeleton within the cell reflects the orientation of the cell as a whole. On smooth substrate (Fig. 7D), the fibroblasts spread fully with many bundles of actin filaments anchored to the plasma membrane at sites of extended focal contacts; there was obvious formation of focal contact at the cell periphery. These immunofluorescence observations indicate clearly that the nuclei possessed oblong forms on the ridges of PDMS15 (Fig. 7A) and PDMS30 (Fig. 7B), but they retained their elliptical configurations on the 100-mm-wide ridges of PDMS100 (Fig. 7C) and on the planar surface (Fig. 7D). Consequently, cell shape was changed by micro-grooves of substrate, cell elongated along the direction of ridge/groove, that promote nucleus form to become deformed. The form index of nucleus was shown in Fig. 8. The degree of deformation was proportional to the degree of discontinuity of surface. 4. Discussion Our aims of this study is that directed cells to their targets. We explore the influence of surface properties on cell migration. The surface characteristics including the surface chemistry and surface topography have a significant influence on adhesion, morphology, orientation, and motility. The wettability of the surface has been shown to be one of the important factors to cell behavior. Certain chemical groups present on the surface of the material can alter cell response (Ruardy et al., 1997), and topographically also induced wettability (Brochard-Wyart, 1995), it is to be expected that micro-grooved surfaces will exhibit heterogeneous surface energy even if the surface is chemically homogeneous. However, unlike a drop of water, cellular shape and motility are largely influenced by the cytoskeleton; the cell will tend to
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form focal adhesions in locations that balance cytoskeletal forces (Ingber and Tensegrity, 2003). On a high adhesive substrate, the overall cell-substrate bond is strong, causing a very slow rate of cell detachment with the result that cells become immobilized (as our result of Fig. 5D and E). Conversely, on a poor adhesive substrate, cell-substrate interaction is too weak and cells cannot generate enough traction for forward migration (Palecek et al., 1999). Therefore, it is postulated that maximal speed can be obtained by cells moving on surfaces at intermediate attachment strength (Dimilla et al., 1993). In general, hydrophobic surfaces are less amenable to cell adhesion than are their hydrophilic counterparts (Lindblad et al., 1997; Scotchford et al., 1998; Altankov and Groth, 1994). In our study, cells indicate poor cellular attachment initially, but still were exponential proliferating later on non-treated silicon rubber. This phenomenon was to ascribe to the surface of PDMS presents hydrophobic methyl groups, that could adsorb biomolecules in the serum, such as fibronectin, onto surface, and permit cell adhesion, then which induce intermediate cell–substrate bonding, therefore, it is conducive for the rapid movement and cellular motile traction was random during the entire culturing period, but the cells’ final goal was to reach the top surfaces of the ridges (shown in Fig. 5A–C). Our result contrasts those reported by Reyes et al. (2004) and Kaji et al. (2004) these authors used heterogeneous forces (chemical or electrochemical modification) on the surface of the substrate to render specific regions adhesive or non-adhesive and to promote cell migration parallel to specific locomotion. Other features of a material have been identified as important factor determinants of cell migration, such as the mechanical strength of a substrate (Kuntz and Saltzman, 1997). Mechanical forces between cells and their environment interaction are believed to provide the driving force for cell locomotion (Lauffenburger and Horwitz, 1996; Oliver et al., 1994). 3D structure appears many anchorage points that provide many opportunities for cells to have mechanical interactions. Dunn and Brown (1986) proposed that directed cell migration on a topographical substrate results from the substrate imposing mechanical restrictions on the formation of certain linear bundles of micro-filaments. The results of many studies have suggested that, during locomotion, intracellular forces generate mechanical tension on the underlying substrate. Dembo and Wang (1999) demonstrate that lamellipodium of the fibroblast is able to generate intense traction stress, and the traction is integral to the mechanism of fibroblast locomotion and that the mechanical energy for traction is derived from cytoskeletal contractile activity. For move forward, cells must adhere to the substrate and exert rearward traction forces, therefore, contraction of cytoskeleton causes tension to pull the frontal adhesions backward and push the tail adhesions forward. In fact, from time-lapse recordings, it is clear that the cellular cytoskeleton is not static – it is a dynamic structure – and that the cell processes are considerably flexible; the polymerization and depolymerization of cellular compounds is influenced by the geometrical topography of the substrate, which creates a mechanical stress that influences cell spreading
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Fig. 9. The oxygen transfer from the air above the medium to the surface of the substrate, when cells grown on the ridge (for example: 100-mm groove/ridge). The oxygen concentration contour on the cultured dish, the oxygen concentration (mM) around ridge was measured. The middle zone is the 30-mm-height ridge.
and alignment. Cell motility was very sensitive to the topography of substrate. Cells can use the edges of the ridge as footholds to gain mechanical adhesion, an additional source of traction that is absent on planar surfaces. Sheetz et al. (1998) described the migration of fibroblasts as occurring through a multistep process that requires dynamic interactions between the cell, substrate, and cytoskeleton. Thus, the cells continually creep and covet a state in which their internal and external forces are in equilibrium, and this process induces their aligned cellular shape. In this study, we observed that the clustering of cells in a parallel manner on top of narrow ridges is perhaps their optimal steady state environment. The most dramatic phenomenon we observed in this study was that fibroblasts rose to the tops of the ridges, and preferred to reside there rather than at the bottoms of the grooves. From our combined observations, we think that oxygen supply is one of the most important parameters for cell culture. When seeded the cells onto the surfaces of the substrates, oxygen required for cell growth from a constant temperature, constant humidity, 5% CO2/air atmosphere above the medium. We solved the oxygen supply equation using a molar flux of oxygen. Concentration profile is a linear function, oxygen is transferred gradually by diffusion from the upper parts of the medium to the surface of the elastomer; we might expect that the model for growth of the cells would be diffusion-controlled during the culturing period. We quantified oxygen concentration using FEMALAB23 MATLAB6.1 computer software; Fig. 9 displays the results of resolved expression. We found that zone of ridge has the lowest oxygen concentration, but it is only slightly different to that at the bottom of the groove and, thus, most of the cells do not descend to the bottom; rather, they are content to remain on the surfaces of ridges. 5. Conclusions In this study, we have found another method to induce and control migration and location of cells that does not involve
chemical or electrochemical modification of the surface of substrate to render specific regions adhesive or non-adhesive. There are three major conclusions from this study of the reactions between cells and grooved substrates: (1) fibroblasts tend to align along the direction of the grooves and attain longer cell bodies than they do on flat substrates; (2) fibroblasts spread readily and eventually climb up the walls of the grooves and onto the ridges; (3) the rate of cell motility on grooved and flat bare PDMS was faster than cells on collagen-coated PDMS and coverslips glass. Thus, culture substrates possessing various topographies can be designed to affect the behavior of bone marrow stromal fibroblasts; such effects may be particularly applicable when cells must be localized in specific areas or when cell migration must be directed. One practical application of the substrate-mediated control of cell behavior may be in the design of implanted devices that control cell migration and location; we expect that manipulating the healing response of regenerating tissues around such implants would improve future device performance. Acknowledgment This work was supported partly by the National Science Council (NSC) of Taiwan under contract no. NSC93-2113-M492-002. References Adams, J.C., Schwartz, M.A., 2000. Stimulationof fascin spikes by thrombospondin-1 is mediated by the GTPases Rac and Cdc42. J. Cell Biol. 150, 807– 822. Altankov, G., Groth, T., 1994. Reorganization of substratum-bound fibronectin on hydrophilic and hydrophobic materials is related to biocompatibility. J. Mater. Sci. Mater. Med. 5, 732–737. Banyard, J., Zetter, B.R., 1998. The role of cell motility in prostate cancer. Cancer Metast. Res. 17, 449–458. Brochard-Wyart, F., 1995. Droplets: capillarity and wetting. In: Daoud, C.E.W.M. (Ed.), Soft Matter Physics. Springer, Berlin, pp. 1–45. Brunette, D.M., 1986. Spreading and orientation of epithelial cells on grooved substrata. Exp. Cell Res. 167, 203–217. Brunette, D.M., Kenner, G.S., Gould, T.R.L., 1983. Grooved titanium surface orient growth and migration of cells from human gingival explants. J. Dent. Res. 62, 1045–1048. Clark, P., Connolly, P., Curtis, A.S.G., Dow, J.A.T., Wilkinson, C.D.W., 1990. Topographical control of cell behavior. II. Multiple grooved substrata. Development 108, 635–644. Curtis, A.S.G., Clark, P., 1990. The effects of topographic and mechanical properties of materials on cell behavior. Crit. Rev. Biocompat. 5, 343–362. Curtis, A.S.G., Wilkinson, C.D.W., 1998. Reaction of cells to topography. J. Biomater. Sci. Polymer. Edn. 9, 1313–1329. Dalton, B.A., Walboomers, X.F., Dziegielewski, M., Evans, M.D.M., Taylor, S., Jansen, J.S., Steele, J.G., 2001. Modulation of epithelial tissue and cell migration by micro-grooves. J. Biomed. Mater. Res. 56, 195–207. del Pozo, M.A., Price, L.S., Alderson, N.B., Ren, X.D., Schwartz, M.A., 2000. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19, 2008–2014. Dembo, M., Wang, Y.L., 1999. Stresses at the cell-to-substrate interface duringlocomotion of fibroblasts. Biophys. J. 76, 2307–2316. den Braber, E.T., de Ruijter, J.E., Smits, H.T.J., Ginsel, L.A., von Recum, A.F., Jansen, J.A., 1996a. Quantitative analysis of cell proliferation and orientation on substrata with uniform parallel surface micro-grooves. Biomaterials 17, 1093–1099.
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