After 28 days of culture, constructs were digested with 1ml proteinase K 0.5 mg/ml at 56°C for 16 h. Total DNA content was measured spectrofluorometrically following reaction with Bisbenzimide Hoechst 33258 dye (Polysciences Inc., Warrington, PA, United States) with purified calf thymus DNA as standard (Lubio Science, Luzern, Switzerland) (Labarca and Paigen, 1980 (link)).
Sulfated glycosaminoglycans (GAG) retained within the scaffolds was determined by a direct spectrophotometric microassay according to the dimethylmethylene blue dye method (Sigma-Aldrich, Buchs, Switzerland) at pH 1.5, using bovine chondroitin 4-sulfate sodium salt from bovine trachea (Fluka, St. Louis, MO, United States) (Farndale et al., 1986 (link)). Total GAG content of the culture media was also measured to assess the release of matrix molecules from the constructs. All samples containing hyaluronan were blanked with media containing 0.2% hyluronan and DMMB at pH 1.5 was used to eliminate the noisy signal due to the residual interaction between DMMB and Hyaluronan.
Sulfated glycosaminoglycans (GAG) retained within the scaffolds was determined by a direct spectrophotometric microassay according to the dimethylmethylene blue dye method (Sigma-Aldrich, Buchs, Switzerland) at pH 1.5, using bovine chondroitin 4-sulfate sodium salt from bovine trachea (Fluka, St. Louis, MO, United States) (Farndale et al., 1986 (link)). Total GAG content of the culture media was also measured to assess the release of matrix molecules from the constructs. All samples containing hyaluronan were blanked with media containing 0.2% hyluronan and DMMB at pH 1.5 was used to eliminate the noisy signal due to the residual interaction between DMMB and Hyaluronan.