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The photochemistry of organosilanes was used to (1) create mixed monolayers having continuously adjustable surface free energies and (2) affect high resolution adhesion and spatial orientation of biological cells on silica substrates. Monolayers were formed from two materials, an aminoalkylsilane, NH2(CH2)2NH(CH2)3Si(OCH3)3 (EDA), and a perfluorinated alkylsilane, CF3(CF2)s(CH2)2Si(CH3)zC1 (1 3F), and were characterized by ellipsometry and water contact angle measurements. Deep UV (193 nm) radiation was used to induce photochemical changes in the cell-adhesive EDA monolayers. X-ray photoelectron spectroscopy indicated that the amine groups of EDA were removed by the exposure, leaving only Si-OH or alkyl fragments having 5 3 carbons. The exposed substrates were then reacted with 13F to form mixed monolayers or hydrophobic monolayers that inhibited cell adhesion in the irradiated regions. The degree of 13F reactivity with EDA in the unirradiated regions was observed to be solvent-dependent, suggesting that conformational states of the surface amine groups lead to a reduction in their accessibility. The selective photochemistry was exploited to produce high resolution molecular patterns, defined using patterned irradiation, that were mapped with scanning Auger electron spectroscopy. The patterns were used to spatially control the adhesion and direct the outgrowth of rat hippocampal neurons and porcine aortic endothelial cells in vitro. Patterns of controlled geometry may provide new approaches to the study of surface-directed growth, intercellular communication, and organogenesis or be used to control the alignment of individual cells with transducer elements in biosensors and implants. IntroductionRecent reports have indicated that monolayer systems can be targeted for a variety of applications, including cell adhesion and patterning.',2 Several techniques have been introduced in the past few years for the production of patterned self-assembled monolayers (SAMs) of organic functionalitie~.~~~ One approach utilizes conventional photoresist methodology to produce high resolution patterns for the adhesion and growth of cells.2 A fundamentally new technique involves direct modification of SAMs by patterned deep UV e x p o s~r e .~~~ Molecules in the exposed regions of a SAM absorb the radiation and undergo photocleavage to yield surface residues. These residues can then be modified with a second type of functionality. This technique offers several

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Chenaically moid glass substrates were used to denste dirental adheso, growth, and differention f endoel cells. Endothellal cells were eamine for Madei and growth on glass, glass treated with N-(2-aminethyl)-3-amnopropyl trimethoxysilane (EDA) Several approaches have also demonstrated the potential for controlling the spatial distribution of cells on the surface. Carter (8, 9) and later Harris (10), using acetate-coated glass, created palladium islands and channels for studying single fibroblast cell reactions and clone formation. Kleinfeld and coworkers (11) fabricated high-resolution patterns of selfassembled monolayers (SAMs) of amino-and perfluoroalkylsilanes, which resulted in dissociated neurons that preferentially adhered to and developed electrically active associations within the geometrically defined regions. The use of SAMs to create regions of defined chemical reactivity also allows selective attachment of fibroblasts and endothelial cells to regions modified by covalently bound peptides that contain the Arg-Gly-Asp (RGD) binding domain (12)(13)(14).In this study, a series of SAM-modified surfaces were examined for their ability to support adherence and facilitate growth and differentiation of endothelial cells. Patterned substrates, created by deep UV photolithography (15), were used to provide spatially defined regions that promoted adhesion, spreading ofendothelial cells, and ultimately spatially defined neovascular assemblies on a two-dimensional surface. MATERIALS AND METHODSReagents. Human fibronectin (hFN) was obtained from Collaborative Research. Heparin sulfate (HS) (47.5 kDa) was obtained from Sigma. N-(2-Aminoethyl)-3-aminopropyl trimethoxysilane (EDA) and (tridecafluoro-1,1,2,2-tetrahydrooctyl)-l-dimethylchlorosilane (13F) were used as procured from Hflls America (Bristol, PA).Substrate Preparation. Patterned deposition of EDA and 13F was accomplished by using the photolithography method of Dulcey et al. (15). Glass substrates were washed in methanol/HCl, 1:1 (vol/vol) for 1 hr and 18 M H2SO4 for 1 hr followed by multiple washes with deionized water (18 Mfl). After the final wash, the substrates were immersed in boiling deionized water for 20 min, removed, and immersed in 1% EDA/94% (vol/vol) methanol/i mM acetic acid/5% H20 for 15 min. The substrates were then rinsed at least three times in anhydrous methanol and baked at 1200C for 5 min.13F was deposited on either clean glass or patterned EDA surfaces by using a 1% (vol/vol) solution of the silane in anhydrous toluene for 1-3 hr, followed by at least four rinses in toluene, and baked 5 min at 120'C. EDA films were exposed to pulsed ArF excimer laser (193 nm) radiation (15 J/cm2, 30 Hz) through a lithographic mask. Deep UVirradiated regions of the substrate were resilanized with 13FAbbreviations: SAM, self-assembled monolayer; HUVE, human umbilical vein endothelial cell; EDA, N-(2-aminoethyl)-3-aminopropyl trimethoxysilane; 13F, (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-dimethylchlorosilane; bFGF, basic fibroblast growth factor; ...