Chondroitin sulfate sodium salt from shark cartilage

Pellets were digested at day 21 of chondrogenic differentiation using 1 mg/mL Proteinase K, 1 mM iodoacetamide, 10 μg/mL Pepstatin A in 50 mM Tris, and 1 mM EDTA buffer (pH 7.6; all Sigma-Aldrich) for 16 h at 56°C, followed by Proteinase K inactivation at 100°C for 10 min. Afterward, to determine the amount of DNA, cell lysates were treated with 0.415 IU heparin and 1.25 µg RNase for 30 min at 37°C, followed by the addition of 30 μL CYQUANT GR solution (Invitrogen). The samples were analyzed using a SpectraMax Gemini plate reader with an excitation of 480 nm and an emission of 520 nm. As a standard, DNA sodium salt from calf thymus (Sigma-Aldrich) was used. To determine the amount of GAG, cell lysates were incubated with 1, 9-dimethylmethylene blue (DMB), as previously described by Farndale et al. (1986 (link)), and analyzed with an excitation of 590 nm and 530 nm. The 530:590 nm ratio was used to determine the GAG concentration. As a standard, chondroitin sulfate sodium salt from shark cartilage (Sigma-Aldrich) was used.

Fish gelatin was extracted from the skin of Greenland Halibut (Reinhardtius hippoglossoides), a by-product of industrial fish processing for food, as described below. Standard commercial gelatin type A and B, chondroitin sulfate sodium salt from shark cartilage, hyaluronic acid sodium salt from Streptococcus equi, methacrylic anhydride and 2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone were purchased from Sigma Aldrich, St. Louis, MO, USA and used as received.

GAG content was quantified by the DMMB assay as described [23] (link). Briefly, samples (n = 10) were freeze-dried to a constant weight, and samples (10 mg) were digested in papain buffer (125 mg/ml papain, 5 mM cysteine–HCl, 5 mM disodium EDTA in PBS) at 60°C for 24 h. Then, 50 µl of each sample was mixed with 250 µl 1, 9-dimethyl-methylene blue (Sigma) in a 96-well microtiter plate and the absorbance was measured at 530 nm. The amount of GAG content was calculated by reference to a standard curve prepared using different concentrations of chondroitin sulfate sodium salt from shark cartilage (Sigma).