Taken together, these data indicate that peptide-treated CS gels can better support neurite outgrowth than both untreated CS and HA gels. Peptides used in these studies were our site synthesized manually on Knorr resin (Synbiosci, SRK001) using standard FMOC chemistry in a specialized syringe filter system. Amino acids were coupled to the resin first using diisopropylcarbodiimide chemistry, followed by a second coupling step using o-(benzotriazol-1-yl)-N,N,N��,N��-tetramethyluronium hexafluorophosphate (Synbiosci, REAG2) and lutidine (Sigma-Aldrich, L3900). Cleavage from the resin was accomplished using a cocktail of trifluoroacetic acid (Acros Organics, 139725000) containing 2.5% water, 1.25% triisopropylsilane (TCI America, T1533), and 1.25% 1,2-ethanedithiol (Alfa Aesar, L12865) as scavengers.
The cleaved peptide was precipitated in a 10X volume excess of cold ethyl ether (Mallinckrodt Chemicals, 0848�C10), recovered by centrifugation, and then resolubilized in a solution of acetonitrile (Sigma-Aldrich, 34998) and water. Peptide samples were purified using reverse-phase chromatography utilizing a 22/250 Protein and Peptide C18 column (Grace-Davidson) on an ?KTA Explorer system (GE Healthcare). Purity of samples was confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) spectroscopy on a 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems). To allow covalent coupling of the peptide to the hydrogel matrix, the EKR peptide was synthesized with a c-terminal cysteine residue and a glycine spacer, to yield a final sequence of EKRIWFPYRRFGC.
The thiol group on the terminal cysteine can bind with PEG-DA via Michael-type addition.13 To facilitate this reaction, a solution of PEG-DA (Sunbio Systems, Inc., P2AC-3) and thiolated EKR was adjusted to a pH between 7.5 and 8, and incubated at 37��C for 30 min. After this reaction, thiolated CS or HA was added to the solution to form hydrogels, as described previously.5 For hydrogel rheological characterization, 2% hydrogels of CS or HA, crosslinked with PEG-DA, were prepared as described previously,5 with the exception that they were made containing 76.9 nM EKR peptide. Viscoelastic responses were determined using stress and frequency sweeps as described previously,4 utilizing an AR-G2 rheometer (TA Instruments) and a parallel plate geometry with a 20-mm diameter and a 585-��m gap.
30 min time sweeps at 0.5 Pa and 1 Hz were executed during gelation to ensure the sample had fully gelled before further testing was run. All tests were run in triplicate, and statistical analysis of rheological data was accomplished using ANOVA (�� = 0.05) in Origin Pro 8.0 (OriginLab). For cryo-SEM characterization, gel samples of 2% CS and Drug_discovery 2% HA containing 76.9 nM EKR peptide were made in specialized slit holders. Imaging methods were identical to those described previously,4 using an FEI NOVA nanoSEM field emission SEM (FEI Company).