Langmuir 1998, 14, 5133-5138
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Effects of Organosilane Monolayer Films on the Geometrical Guidance of CNS Neurons Mieko Matsuzawa,*,† Shuzo Tokumitsu,† Wolfgang Knoll,†,‡ and Hiroyuki Sasabe† Frontier Research Program, RIKEN, Wako, Saitama, Japan, and Max-Planck-Institute fu¨ r Polymerforschung, D-55128 Mainz, Germany Received January 20, 1998. In Final Form: June 10, 1998 This work reports on the alignment of central nerve processes using surfaces of modified organosilane monolayer films. Two types of organosilane monolayer films, formed of decyldimethylsiloxane (DDMS) or trimethylsiloxane (TMS), were covalently formed on glass substrates. The films were pattern modified with a synthetic peptide (P1543) derived from mouse laminin, an extracellular matrix (ECM) protein, via a combination of ultraviolet lithography and chemical modification technique. The modification procedure generated a pattern of 10-µm-wide peptide stripes that were surrounded by either DDMS films or TMS films. A significant difference between the two patterned substrates (DDMS/P1543 and TMS/P1543) was in hydrophobic properties of the surrounding surfaces: whereas the DDMS surrounding surfaces were more hydrophobic compared with the peptide surfaces, the TMS surrounding surfaces showed an equivalent hydrophobic property to that of the peptide surfaces. Effects of the surrounding surfaces on the alignment of central nerve processes were investigated by growing neurons dissociated from embryonic rat hippocampi on the patterned DDMS/P1543 and TMS/P1543 substrates at a low density (40 cells/mm2) in a chemically defined culture medium. The time-lapse video microscopy revealed that although a similar bipolar morphology was developed by the hippocampal neurons grown on both patterned substrates, the growth behaviors of the nerve tips were highly affected by physicochemical characteristics of the surrounding surfaces. The growing tips advanced straightforward along the peptide stripes when the surrounding surfaces were formed of DDMS films, whereas those often explored the surrounding surfaces formed of TMS films. Our work directly shows that although the attachment of neurons on patterned substrates is affected by hydrophobic characteristics of surrounding surfaces, the hydrophobicity is not a necessary factor for the neurite guidance. We presume that localized chemical cues, such as laminin synthetic peptide, are rather crucial constituents that affect the directional outgrowth of central nervous system neurons.
Introduction During the development of the nervous system, neurons extend nerve processes to their target regions and form specific neuronal connections. One of the mechanisms underlying the formation of these specific neuronal wirings is that by the guidance of nerve tips, growth cones, by localized molecular cues.1 The growth cones can detect positive or negative guidance cues in their microenvironment and steer toward or away from the cues.1 Hence, the geometrical regulation of growing nerve tips by means of artificially deposited localized cues may affect the neuronal growth, leading to the modulation of neuronal network architecture in culture. Organosilane monolayer films are of great interest for chemically modifying surfaces of silicon-based substrates because of their high degree of organization and stability. A considerable advantage is that an ultraviolet lithographic technique can be applied to fabricate patterns on organosilane films. Then, the patterned films can be used as substrates for further chemical modification. For example, the technique has provided a reliable way to pattern surfaces of alkylsilane films with biomaterial substances, such as amines and synthetic peptides.2-5 A microstamping technique using organothiols has been recognized as an alternative way of fabricating chemical patterns on gold substrates.6 Previous studies have shown * Corresponding author. Current address The Johns Hopkins University, Applied Physics Laboratory, Johns Hopkins Road, Laurel, MD 20723-6099. † Frontier Research Program. ‡ Max-Planck-Institute fur Polymerforschung. (1) Dodd, J.; Jessell, T. M. Science 1992, 242, 692.
that these patterned surfaces can serve as media to control the confinement and geometry of living biological cells in culture.2-6 Such techniques are of great benefit for biochemists and biophysicists in studying critical constituents between the biological cells and the substrates. Laminin is thought to be one of the extracellular matrix (ECM) molecules that are involved in influencing axon guidance and neuronal migration during embryonic morphogenesis of the nervous system.7 In vivo studies have revealed that laminin deposit is spatially and temporally associated with pioneer axon growth, suggesting that pathways formed of laminin may act as guidance cues for nerve growth pathfinding.8,9 In vitro studies have directly demonstrated roles of laminin in neuronal development, ranging from regulation of cell survival to neurite outgrowth, using both central and peripheral neurons.10 The extensive neuritic outgrowth by laminin is explained by the increased growth cone motility rather than growth cone-substratum adhe(2) Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. J. Neurosci. 1988, 8, 4098. (3) Dulcey, C. S.; Georger, J. H.; Krauthamer, V.; Stenger, D. A.; Fare, T. L.; Calvert, J. M. Science 1991, 252, 551. (4) Matsuzawa, M.; Potember, R. S.; Stenger, D. A.; Krauthamer, V. J. Neurosci. Meth. 1993, 50, 253. (5) Matsuzawa, M.; Umemura, K.; Beyer, D.; Sugioka, K.; Knoll, W. Thin Solid Films 1997, 305, 74. (6) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang, D. I. C.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696. (7) Sanes, J. Annu. Rev. Neurosci. 1989, 12, 491. (8) Letourneau, P. C.; Madsen, A.; Furcht, L. Dev. Biol. 1988, 125, 135. (9) Liesi, P.; Silver, J. Dev. Biol. 1988, 130, 774. (10) Manthorpe, M.; Engvall, E.; Ruoslahti, E.; Longo, F. M.; Davis, G. E.; Varon, S. J. Cell Biol. 1983, 97, 1882.
S0743-7463(98)00080-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/08/1998
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sion.11,12 Studies using proteolytic fragments of laminin have further suggested that the growth-promoting function of laminin can be substituted by its smaller domains.13 Synthetic peptides derived from these domains have been used to identify actual peptide sequences involved.14-16 Our study was motivated by a desire to characterize substrates that enable the geometrical guidance of neurons derived from mammalian central nervous tissues for forming simple neuronal connection in culture. We used organosilane monolayer films and fabricated patterns of growth-stripes (∼10 µm in width) formed of laminin synthetic peptide, P1543, that has a cell-adhesive and neurite-outgrowth promoting sequence.14 The growth pathways were formed so that they were surrounded with organosilane films having either higher or equivalent hydrophobicity. Effects of the surrounding surfaces on the nerve process alignment were directly examined by growing embryonic hippocampal neurons at a low density in a chemically defined culture medium. Optical microscopy together with a time-lapse video recording technique was used to directly observe responses of the hippocampal nerve tips to the patterned substrates. 2. Experimental Section 2-1. Materials. Glass coverslips (18 × 18 mm) were obtained from Matsunami, Japan. The coverslips were cleaned by immersing them in a 25% (v/v) sulfuric acid solution for overnight. The glass substrates were thoroughly rinsed with deionized water and dried prior to use. All silane chemicals (n-decyldimethylchlorosilane, trimethylchlorosilane, and aminopropyldimethylethoxysilane) were purchased from Chisso, Japan, and were used without further purification. N-(γ-Maleimidobutylyloxy)sulfosuccinimide ester (sulfo-GMBS) was purchased from Pierce (IL). A synthetic peptide, P1543, was a gift from Dr. P. Liesi. This peptide was modified from RDIAEIIKDI, which is derived from the γ-chain of mouse laminin, by adding glycine and cystein to the C-terminal.14 2-2. Surface Modification. 2-2-1. Organosilane Surfaces. A decyldimethylsiloxane (DDMS) film was covalently formed on an acid-cleaned glass coverslip via a siloxane linkage by immersing the substrate in toluene containing 10 mM decyldimethylchlorosilane. After certain time periods (ranging from 1 to 240 min), the substrate was removed from the solution and ultrasonicated in toluene for 1 min to remove unreacted materials from the surface. The sample was subsequently rinsed in ethanol and in water, then dried. A trimethylsiloxane (TMS) film was similarly formed on the glass coverslip by immersing the substrate in toluene containing 1 mM trimethylchlorosilane. 2-2-2. Peptide Surface. The acid-cleaned glass surface was reacted with aminopropyldimethylethoxysilane (APDMS) by immersing the substrate in a solution of 95% ethanol-5% deionized water containing 2% (v/v) aminopropyldimethylethoxysilane for 15 min. The substrate was thoroughly rinsed with ethanol and dried at 100 °C for 5 min to complete the condensation reaction. The amino-derivatized substrate was then transferred to a sterilized hood and chemically coupled with laminin synthetic peptide, P1543, using a heterobifunctional cross-linker, sulfoGMBS, as previously described.17 In brief, an APDMS-derivatized surface was reacted with 1 mM sulfo-GMBS in phosphate buffer, pH 8, for 1 h. The sulfo-GMBS-reacted surface was rinsed in sterilized water and sequentially reacted with cystein (carboxyl terminal) of P1543 (1 µM in phosphate buffer, pH 7) for 1 h. (11) Gunderson, R. W. J. Neurosci. Res. 1988, 21, 298. (12) Calof, A. L.; Lander, A. D. J. Cell Biol. 1991, 115, 779. (13) Paulsson, M. Crit. Rev. Biochem. Mol. Biol. 1992, 27, 93. (14) Liesi, P.; Na¨rva¨nen, A.; Soos, J.; Sariola, H.; Snounou, G. FEBS Lett. 1989, 244, 141. (15) Tashiro, K.; Sephel, G. C.; Weeks, B.; Sasaki, M.; Martin, G. R.; Kleinman, H. K.; Yamada, Y. J. Biol. Chem. 1989, 264, 16174. (16) Nomizu, M.; Kim, W. H.; Yamamura, K.; Utani, A.; Song, S.; Otaka, A.; Roller, P. P.; Kleinman, H. K.; Yamada, Y. J. Biol. Chem. 1995, 270, 20583. (17) Matsuzawa, M.; Liesi, P.; Knoll, W. J. Neurosci. Meth. 1996, 96, 189.
Matsuzawa et al. Unreacted materials were removed from the surface by thoroughly rinsing the substrate with sterilized water. In some experiments, glass substrates modified with DDMS and TMS films were used to go through the same modification steps already described. 2-3. Patterning. A glass substrate covered with either DDMS film or TMS film was directly covered with a lithographic mask and exposed to a deep ultraviolet (UV) laser beam as descried elsewhere.3,4 The exposure formed a pattern of irradiated stripes (10 µm in width) on the organosilane film surface. The patterned surface regions were consequently reacted with APDMS as already described. The procedure resulted in the formation of NH2-stripes that were separated by surrounding organosilane surfaces. 2-4. Goniometry and XPS Analysis. Water contact angle (WCA) and nitrogen signal (N1s) intensity were detected by goniometry and X-ray photoelectron spectroscopy. The WCA was measured using a sessile drop technique with an Elmer contact angle goniometer. All the WCA reported here are static angles. The N1s signals were analyzed using a VG ESCA lab MKII equipped with a Mg KR source (1253.6 eV). 2-5. Dissection and Culture. Hippocampal tissues were obtained from 18-day-old rat fetuses and dissociated into single cells by trypsin treatment and mechanical trituration as described elsewhere.18 The dissociated cells were placed onto the modified glass substrate at a low density (40 cells/mm2) in a chemically defined serum-free medium19 to directly examine effects of growth substrates on the neurite outgrowth. The cells were allowed to attach onto the substrate in a SANYO incubator in an atmosphere of 95% air/5% CO2 at 36.5 °C for 45 min. Unattached materials were then removed from the surface by gently rinsing the substrate and exchanging the culture medium. After 2 days of incubation, we evaluated the growth of hippocampal neurons using an Olympus optical microscope equipped with phasecontrast optics. 2-6. Time-Lapse Video Microscopy. The outgrowth behavior of the patterned nerve tips was examined by time-lapse video microscopy after ∼1.5 days in culture. A culture dish containing patterned neurons was placed on the microscope stage in a CO2 chamber at 36.5 °C. Time-lapse video images were then recorded every 1-10 min with a Sony-Olympus time-lapse video recording system. The recorded images were transferred to a Power PC computer. Changes in the neurite alignment along the recording time were examined by analyzing advancing angles of neuritic tips with an NIH image software. The advancing angles were determined with respect to the stripe direction. Growing tips ∼10 µm in length from the tip ends were used for the angle determination, and the movement of filopodia was neglected.
3. Results and Discussion 3-1. Substrate Modification and Cell Growth. Time periods for the DDMS and TMS reactions ranged from 1 to 240 min. Time-dependent changes in the surface hydrophobicity were quantitatively expressed by means of static WCA (Figure 1). Although the DDMS surfaces showed a rapid change in the surface hydrophobicity, gradual increases in WCA occurred on the TMS substrates. The angles reached to saturated values of 91° within 60 min on the DDMS surface and of 60° after 180 min on the TMS surface (Figure 1). We used a DDMS surface of WCA 91°, at which angle the surface was presumably covered with a highly packed DDMS monolayer, and a TMS surface of WCA 40°, at which angle the surface was partially covered with TMS, as substrates for the later modification. Through the peptide modification, a clean glass surface showed changes in WCA from
