High concentration rat tail type 1 collagen

To generate the PEG/Collagen IPNs, 3D collagen gels were first generated as previously described[41 (link),42 (link)]. Briefly, High Concentration Rat Tail Type I Collagen (Corning Life Sciences, Corning, NY) was combined in a 1:1 volumetric ratio with collagen neutralizing buffer (100 mM HEPES in 2x PBS, pH 7.3) and diluted to the appropriate working concentration in 1x PBS; in this study the collagen gel concentration was kept constant at 2 mg/mL. The collagen solution was then gelled by placing the solution in an incubator at 37°C for 1 hour. Once the collagen had fully polymerized, a solution containing the PEG monomers (10 kD PEGnorbornene (PEG4NB) (Sigma-Aldrich, St. Louis, MO) and 1 kD PEGdithiol (Creative PEGWorks, Chapel Hill, NC)), in a constant 1:2 molar ratio of PEG4NB:PEGdithiol, and 0.1 mM of the photo-initiator Eosin Y was added and allowed to diffuse into the collagen gels. Upon reaching diffusive equilibrium, excess PEG solution was removed and the surfaces of the collagen gels were washed one time with 1x PBS. The PEG network was then formed by irradiating the gels with visible laser light (514 nm, 500 mW cm⁻²) for 120 s.

Rat tail high concentration collagen type I was obtained from Corning (Bedford, MA). Sodium hydroxide, fluorescein isothiocyanate-dextran of 150K MW (FITC-dextran), sodium acetate, cysteine hydrochloride, sodium phosphate monobasic, sodium phosphate dibasic, papain suspension, cytochalasin D, and (–)-blebbistatin were procured from Sigma Aldrich (St. Louis, MO). Y-27632 and Na₂EDTA were purchased from EMD Millipore (Kankakee, IL). Dulbecco’s Phosphate Buffer Saline (DPBS), RPMI Medium 1640, Penicillin-Streptomycin (Pen Strep), 0.25% Trypsin-EDTA, fetal bovine serum (FBS), and collagenase Type I were acquired from Gibco by Life Technologies (Grand Island, NY). D-(–)-Ribose, Hoechst 33342, and Alexa Fluor™ 488 phalloidin were obtained from Thermofisher Scientific (Waltham, MA). AlamarBlue™ cell viability reagent was purchased from Invitrogen by Thermo Fisher Scientific (Eugene, OR). Acetone, methanol, and glacial acetic acid were acquired from Fisher Chemical (New Jersey). Endothelial cell basal medium-2 (EBM-2) and EGM-2 SingleQuots growth factors were obtained from Lonza (Walkersville, MD).