Effects of Charged Substrates on Attachment of Primary Bone Marrow Stromal Cells
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ABSTRACT The effects of charged substrates on the attachment of rat primary bone marrow stromal cells have been investigated using a novel cell culture chamber. Cells were cultured on the surfaces of a conductive and optically transparent indium tin oxide (ITO) coating. When a voltage was applied to the ITO electrodes, positive and negative charges were induced on the ITO electrodes. After 24 hours exposure to 0.8V, more cells attached to the anode than either the cathode or control both in presence or absence of serum in the culture medium. Protein desorption profiles also indicated that the enhanced attachment of cells to the anode was not controlled by the adsorption of serum proteins. The surface texture of ITO was analyzed using atomic force microscopy, the thickness by transmission electron microscopy, and crystallinity using glancing angle x-ray diffraction. INTRODUCTION Cell attachment is a complex cascade of early events at the cell-substrate surface and is critical to the establishment of later cellular responses. Various surface characteristics of the substrate have been reported to significantly influence the nature of cell attachment"23' ' 45 ' 6 . Among those investigations, the effects of various proteins, typically fibronectin 7' and vitronectin 9 , on cell attachment have been extensively investigated and reported to enhance cell
attachment. In addition, hydroxyl and other charged surfaces have also been found to enhance cell attachment 2 . We have previously reported that in a serum supplemented culture medium, more primary rat bone marrow stromal cells attach to the positively charged ITO surface (the anode) than the negatively charged surface (the cathode) and control after being exposed to an applied voltage of 0.8V for 24 hours using a novel cell culture chamber'°. Furthermore, after 3 days exposure to 0.8V, cells on the anode remain rounded in shape whereas those on the cathode and control exhibit a spreading morphology. One day after the removal of the voltage, cells on the anode start to send out cell processes and exhibit spreading morphology. In this paper, the surface roughness, crystallinity, and thickness of the ITO coating have been analyzed using atomic force microscopy (AFM), glancing angle x-ray diffraction (XRD), and scanning transmission electron microscopy (STEM). Since some proteins play an important role in the enhancement of cell adhesion, proteins adsorbed from the serum onto the anode, the cathode, and the control surfaces were analyzed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). The attachment of rat primary bone marrow stromal cells to the charged surfaces and controls in a serum free culture medium has also been investigated and compared with attachment of cells to the charged surfaces in the presence of serum. 141 Mat. Res. Soc. Symp. Proc. Vol. 414 01996 Materials Research Society
EXPERIMENT Culture Chamber Design Culture chambers designed for studies of the growth of cells on charged surfaces have been described previously'0 . Briefly,
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