Polypyrrole - A Potential Candidate for Stimulated Nerve Regeneration
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aqueous solution (Milli-Q® ultra pure water) of 0.1 M pyrrole (Aldrich Chemical Co., Milwaukee, WI) containing 0.1 M sodium salt of poly(styrenesulfonate) (PSS) (Aldrich Chemical Co.). The sodium salt of poly(styrenesulfonate) served as both the dopant and electrolyte. A Pine Instruments AFRDE4 bipotentiostat (Pine Instruments, Grove City, PA) was used as the source of constant voltage. Films of two different thickness were synthesized: 0.10.15 gm (thin film) and 1.8-2.0 R.m (thick film). The film thickness was controlled by the passage of charge [10]. PP thick films were laminated with PLGA films (50:50 or 85:15) [11] to yield processible and suturable PP discs and tubes. In brief, the PP thick film was gently peeled off the ITO glass and then floated in distilled deionized water (Milli-Q0) and, subsequently transferred onto a clean glass slide. The PP film was then wetted with methylene chloride and layered with PLGA film of desired thickness. The laminated PP films were then cut into discs (5 mm diameter x 2 gtm in thickness) for cell growth studies and implantation in rat models. They were also fashioned into tubes (13 mm x 1.2 mm diameter) to be used as conduits for transected sciatic nerve regeneration studies in rat models. Cell Culture: Thin films of PP were examined for their ability to support nerve cell growth in vitro using the PC-12 cell line, which is a well-characterized, nerve-like line derived from a rat pheochromocytoma [12]. PC-12 cells respond reversibly to nerve growth factor (NGF) by induction of the neuronal phenotype (expression of neurites). PC- 12 cells were cultured in 85% high-glucose DMEM (GibcoBRL, Grand Island, NY), 10% heat-inactivated horse serum, and 5% fetal bovine serum (FBS). Cells were maintained in a humid, 7% CO 2 incubator and passaged via trituration at 1:2 dilution every other day. Cells were "primed" by the addition of 25 ng/ml NGF (Boehringer-Mannheim, Indianapolis, IN) 24 hr. prior to seeding cells for an experiment. During experiments, cells were maintained in culture medium supplemented with 25 ng/ml NGF. Cells were seeded into wells formed by the attachment of sterile Plexiglas wells (1 cm x 1.5 cm inner dimension) to PP-PSS films using autoclaved vacuum grease. In all experiments, I ml of a 2 x 104 cells/ml solution was used per well. Neurite Length Measurement: Thin PP films permitted the use of light microscopy and quantitative image analysis tools to study cell-material interactions in detail. Cells were viewed using an inverted phase contrast microscope and a 20x objective (Diaphot-TMD; Nikon Inc., Garden City, NY). Images from the microscope were acquired using a CCD video camera (HVC-20; Hitachi, Japan) and were subsequently digitized using NIH Image software (v 1.58; National Institutes of Health, Bethesda, MD) and a Scion image capture board (LG-3; Scion Corp., Frederick, MD). The lengths of the individual neurites for each cell were measured using the NIH image software. Length was defined as the straight-line distance from the tip of the neurite to the ju
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