Dnaqf

The sulfated glycosaminoglycan (sGAG) and DNA contents for papain digests of cell-hydrogel samples cultured for 7 days (n=5–6 per hydrogel formulation/cell type) were determined [41 (link), 66 (link)]. In brief, papain digests of each cell seeded HA-PEG hydrogel (10 units/mg papain: Sigma Aldrich #P4762, St. Louis, MO; 1X PBS, 5mM cysteine HCL, 5mM EDTA, pH=6.0; mixed for 2h at 37°C then filtered; digested for less than 24 hours at 60°C) were analyzed by a dimethylmethylene blue (DMMB) assay for sGAG content using commercially available chondroitin-4-sulfate as a standard (Sigma). The DNA content was measured in papain digests (10 µl) by mixing with solutions of Hoechst 33258 dye (200 µl, Sigma, #DNA-QF) followed by reading absorbance at 460 nm (355nm excitation, PerkinElmer, Waltham, MA) with calf thymus DNA as a standard (Sigma, #DNA-QF) [67 (link)].

For measurement of DNA and characterization of the cartilage extracellular matrix (n = 12 per group), cartilage and bone were separated, freeze dried, and digested in 100 mM sodium phosphate buffer/10 mM Na₂EDTA/10 mM L-cysteine/0.125 mg/mL papain overnight at 60 °C. DNA was analyzed by fluorescence assay using bisBenzimide (DNAQF, Sigma). Fluorescence was read using a Glomax Multi Jr Detection System (Promega, Madison, WI). DNA content was quantified by comparison to a standard curve generated using known amounts of calf thymus DNA. Cartilage GAG content was determined using the Blyscan Glycosaminoglycan Assay based on binding to dimethylmethylene blue dye (Biocolor, Carrickfergus, County Antrim, UK). Precise quantities were determined from a standard curve developed using the chondroitin sulphate supplied with the kit. Cartilage collagen content was determined using the chloramine-T hydroxyproline assay according to Reddy and Enwemeka [19 (link)]. Colorimetric results were obtained using a μQuant Microplate Spectrophotometer (Biotek, Winooski, VT). Hydroxyproline was used to develop a standard curve, and the amount of collagen was calculated assuming 12.5% of collagen is hydroxyproline. DNA, GAG, and collagen contents were normalized to tissue dry weight.