Effects of contact guidance and gravity on L929 cell orientation

Transcription

1 Article Polymer Chemistry April 2011 Vol.56 No.10: doi: /s SPECIAL TOPICS: Effects of contact guidance and gravity on L929 cell orientation ZHOU Feng 1,2, YUAN Lin 2*, MEI Yan 1 & CHEN Hong 1,2 1 School of Materials Science and Engineering, Wuhan University of Technology, Wuhan , China; 2 College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou , China Received September 9, 2010; accepted September 29, 2010 Contact guidance and external force field are two important factors that influence cell orientation. Microgroove-like patterns on PDMS substrates were fabricated using soft-lithography, and the effects of gravitational field direction relative to the pattern, on L929 cell orientation were investigated. The majority of cells were aligned along the grooves when gravity was parallel to the groove direction, because of contact guidance. When gravity was perpendicular to the groove direction, cell alignment decreased noticeably. Although contact guidance induced by the pattern played a dominant role in determining cell orientation, gravity also had an important influence when the field direction was perpendicular to the groove direction. No synergistic influence on cell alignment was observed when the field direction was parallel to the groove direction. polydimethylsiloxane, contact guidance, gravity field, cell orientation Citation: Zhou F, Yuan L, Mei Y, et al. Effects of contact guidance and gravity on L929 cell orientation. Chinese Sci Bull, 2011, 56: , doi: / s Contact guidance is the phenomenon in which cells growing on submicrogroove patterns will adjust their orientation and alignment to those patterns [1,2]. With recent knowledge advances in micro-patterned surface effects on cell behavior, and increasing demand for tissue engineering, applications of contact guidance have expanded into oriented tissue growth, such as nerve and tendon regeneration. The physiological effects on cells caused by contact guidance can also control their interaction with materials [3 6]. Thus, contact guidance has received significant attention with regard to tissue engineering. For in vivo applications, cell orientation cannot be controlled solely by contact guidance. The human body is an extremely complex system, and during in vivo application, cells undergo a series of biochemical reactions. They are also subjected to both in vivo and in vitro environmental factors. An example is electromagnetism caused by electrical signal transduction between neurons [7] and the ubiquitous gravity field. The effects of simulated gravity (i.e. *Corresponding author ( yuanl@suda.edu.cn) hypergravity and microgravity) and microgrooved surface on cell orientation and physiological effects have been investigated [8 10]. So far, these studies have all been based on a gravitational field perpendicular to the microgrooved surface. When microgrooved surfaces are used to induce defined cell orientation to promote tissue regeneration (e.g. nerve conduits), such alignment may not be maintained, since the microgrooved pattern and gravitational field may exhibit various spatial relationships. Two typical relationships are when the field direction is parallel to the microgrooved surface and either parallel or perpendicular to the microgroove direction. Contact guidance direction induced by the microgroove pattern and field direction exhibits two different combinations in such cases: namely parallel superposition and perpendicular superposition. The effect of these two combinations on cell orientation, and which of them plays the dominant role in determining cell orientation are still unknown. Studying cell orientation under these two conditions is beneficial for optimizing biomaterial use on microgrooved patterns. In this study, we fabricated microgrooved patterns on The Author(s) This article is published with open access at Springerlink.com csb.scichina.com

2 978 Zhou F, et al. Chinese Sci Bull April (2011) Vol.56 No.10 polydimethylsiloxane (PDMS), and investigated cell orientation under perpendicular and parallel superposition. 1 Materials and methods 1.1 Pattern preparation and device fabrication A 10:1 (w/w) solution of elastomer base and curing agent (Dow Corning, Midland, MI) was thoroughly mixed, poured onto the prepared silicon master, and degassed for 1 h. After solidifying at 70 C for 120 min, PDMS was carefully peeled off, leaving the surface pattern formed on the PDMS. The pattern was thoroughly rinsed with acetone, absolute ethanol and deionized H 2 O, before being dried under nitrogen. The device used in this study is illustrated in Figure 1, and the fabrication process was as follows: four pieces of the patterned PDMS were fixed on the inner surface of a disposable cuvette. Microgroove direction was parallel to the axis of the cuvette for two pieces, and perpendicular for the remaining two. The cuvette was then sterilized for 1 h with 4 ml of 75% ethanol, and rinsed thoroughly with sterilized DI H 2 O. A PDMS plug was subsequently used to seal the device, and a syringe needle used to penetrate the plug. The needles outer open end was then sealed with cotton wool, dampened with 75% ethanol. Two devices were fabricated, allowing an experimental and a control device. For the former, the device was rotated 90 along the cuvette axis, thus ensuring the fixed PDMS was parallel to the gravitational field direction. This allowed two different spatial orientations between the field and microgrooves. 1.2 The adsorption of fibronectin The native PDMS surface is hostile with respect to cell adhesion, so fibronectin (Sigma, Product No. F2006) was first adsorbed. Fibronectin (30 μl, 25 μg/ml) was added onto each PDMS sample and held at room temperature for 1 h. Loosely bound fibronectin was rinsed thoroughly with phosphate buffered saline (PBS) solution. 1.3 Cell culture cells/cm 2, and cultured in RPMI 1640 (Gibco) culture medium containing 10% fetal bovine serum. The devices were placed in an incubator at 37 C containing 5% CO 2 (Galaxy S, RS Biotech, UK). Following cell adhesion (~5 h), the experimental device was rotated by 90 and photographs taken with a microscope (XDS-1B, Chongqing Optical & Electrical Instrument Co, China) equipped with a CMOS camera (GDS310, Guang Di Optical & Electrical Instrument Co, China) every 24 h. 1.4 Data acquisition and analysis Fifteen photographs were taken for each PDMS sample, with five randomly selected for data analysis. ImageJ (NIH, USA) was used to measure the angle between cell axis and microgrooves, and also the corresponding cell numbers. 2 Results and discussion 2.1 PDMS surface characterization SEM (JSM-840, JEOL) was used to observe the microgroove pattern on PDMS, as shown in Figure 2. The surface was clear and regular, validating the high quality of the fabricated microgrooved PDMS. AFM (Nanoscope IV, Digital Instruments, Veeco, US) was then employed to characterize the microgrooved surface in greater detail, and to obtain accurate three dimensional measurements of the pattern (Figure 3). The ridge top and bottom were 3.9 and 6.7 μm wide, respectively. The groove width and depth were 11 and 0.92 μm, respectively. 2.2 Contact guidance testing (1) Contact guidance for microgrooved PDMS. To confirm if microgrooved PDMS could induce contact guidance, the orientations of cells on flat and microgroove PDMS were compared. Figure 4 (a) and (b) show cells adhered on flat and microgrooved patterns, respectively, 48 h after cell adhesion. Most cells were aligned along the microgrooves, while L929 cells were seeded into the device at a density of Figure 1 The device fabricated and used for this study. Figure 2 SEM image of microgrooved PDMS (scale bar: 10 μm).

3 Zhou F, et al. Chinese Sci Bull April (2011) Vol.56 No Figure 3 AFM imaging and geometric analysis of the microgrooved PDMS. justing the pattern position, we could compare and analyze cell orientation under different spatial arrangements of gravity and pattern direction. Figure 5 illustrates that cells adhered on surfaces when gravity was perpendicular to the microgrooved surfaces (denoted gravity surface, used as the control device), and aligned parallel to the surface but perpendicular (gravity microgrooves) and parallel (gravity// microgrooves) to microgroove direction. Cell density on all surfaces increased with time. For gravity surface, most cells aligned along the microgrooves resulting in contact guidance. For gravity microgrooves, the number of cells deviating from the microgroove direction increased, because they experienced the combined effects that were perpendicular to each other (i.e. attenuated contact guidance). For gravity//microgrooves, cells still exhibited noticeable contact guidance, possibly because gravity and contact guidance were directionally aligned and caused a synergistic effect. To accurately investigate the effect of gravity on cell orientation, statistical analysis on cell distribution for each alignment was undertaken. The angle between cell axis and microgroove direction was measured, and the corresponding cell numbers calculated. The results are shown in Figures 6 8. These results indicated that under the three different experimental conditions, while the angle between cell axis and microgrooves was similarly distributed (0 90 ), most cells still aligned along the microgrooves (0 10 ). For gravity microgrooves, the proportion of cells aligned along the microgrooves decreased significantly compared with both the control and gravity//microgrooves, indicating that gravity attenuated contact guidance. However, no significant synergistic effect was observed for gravity//microgrooves. Recent reports have shown that contact guidance is closely related to three-dimensional pattern size [11,12]. Therefore, the pattern size should be optimized to weaken the unfavorable effect of gravitational field by inducing more pronounced contact guidance. 3 Conclusion Figure 4 Light microscope photos of cells on flat (a) and microgrooved (b) PDMS. cell orientation on the flat surface was randomly distributed. Thus, the microgrooved pattern could induce pronounced contact guidance. (2) The effect of gravity on contact guidance. By ad- Contact guidance induced by the pattern played a dominant role in determining cell orientation. However, contact guidance was significantly decreased when the direction of gravity was perpendicular to the microgrooves. When the direction was aligned, no remarkable synergistic effect was observed. The usually omitted effect of gravity should be considered when employing patterned tissue regeneration materials based on contact guidance. Negative effects of gravity may be able to be attenuated by optimizing pattern size to induce more enhanced contact guidance.

4 980 Zhou F, et al. Chinese Sci Bull April (2011) Vol.56 No.10 Figure 5 Light microscope photos of cells on surfaces under conditions where: gravity surface((a), (d), (g)), gravity microgrooves((b), (e), (h)) and gravity // microgrooves ((c), (f), (i)). Figure 6 The distribution of angles between cells axis and microgrooves after 24 h. Figure 7 The distribution of angles between cells axis and microgrooves after 48 h.

5 Zhou F, et al. Chinese Sci Bull April (2011) Vol.56 No Figure 8 The distribution of angles between cells axis and microgrooves after 72 h. This work was supported by the National Natural Science Foundation of China ( ) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education ( ). 1 Zhou F, Yuan L, Huang H, et al. Phenomenon of contact guidance on the surface with nano-micro-groove-like pattern and cell physiological effects. Chinese Sci Bull, 2009, 54: Weiss P. Experiments on cell and axon orientation in vitro: The role of colloidal exudates in tissue organization. J Exp Zool, 1945, 100: Quigley A F, Razal J M, Thompson B C, et al. A conducting-polymer platform with biodegradable fibers for stimulation and guidance of axonal growth. Adv Mater, 2009, 21: Nisbet D R, Rodda A E, Horne M K, et al. Neurite infiltration and cellular response to electrospun polycaprolactone scaffolds implanted into the brain. Biomaterials, 2009, 30: Nisbet D R, Forsythe J S, Shen W, et al. Review paper: A review of the cellular response on electrospun nanofibers for tissue engineering. J Biomater Appl, 2009, 24: Sukmana I, Vermette P. Polymer fibers as contact guidance to orient microvascularization in a 3D environment. J Biomed Mater Res A, 2010, 92A: Levin M. Bioelectromagnetics in morphogenesis. Bioelectromagnetics, 2003, 24: Loesberg W A, Walboomers X F, van Loon J, et al. The effect of combined hypergravity and microgrooved surface topography on the behaviour of fibroblasts. Cell Motil Cytoskelet, 2006, 63: Loesberg W A, Walboomers X F, Bronkhorst E M, et al. The effect of combined simulated microgravity and microgrooved surface topography on fibroblasts. Cell Motil Cytoskelet, 2007, 64: Loesberg W A, Walboomers X F, van Loon J, et al. Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography. Cell Motil Cytoskelet, 2008, 65: Loesberg W A, te Riet J, van Delft F, et al. The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion. Biomaterials, 2007, 28: Teixeira A I, Abrams G A, Bertics P J, et al. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J Cell Sci, 2003, 116: Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.