2.7 Sulfated Glycosaminoglycan (sGAG) and Hydroxyproline Quantification

Cartilage ECM deposition was quantified by measuring sGAG and total collagen production. Constructs were homogenized and then digested for 18 h in 700 μl/construct of a papain solution (125 μg/ml papain, 50 mM sodium phosphate buffer, 2 mM N-acetyl cysteine (Sigma), pH 6.5). An aliquot of the digest was assayed for sGAG content using the Blyscan kit (Accurate Chemical & Scientific Corp, Westbury, NY) according to the manufacturer’s instruction. Another aliquot of the digest was assayed for DNA content using the QuantiT PicoGreen dsDNA Assay Kit (Invitrogen).

A third aliquot was used to quantify collagen deposition by measuring hydroxyproline levels using a modified hydroxyproline assay with bovine collagen type I as a standard. Briefly, 200 μl of each sample and standard were hydrolyzed with 200 μl of 4 N sodium hydroxide (Fisher) at 121 °C for 20 min. 200 μl of 4N HCl (Fisher) was added and the solution was titrated to a neutral pH. 1.2 mL of chloramine-T solution (Sigma) (14.1 g/L chloramine-T, 50 g/L citric acid, g/L sodium acetate trihydrate, 34 g/L NaOH, 0.21 M acetic acid) was incubated at room temperature for 20 min. Then, 1.2 mL of 15 g/L p-dimethylaminobenzaldehyde in 2:1 isopropanol:perchloric acid was added and the solution was placed in a 60 °C water bath for 20 min. Finally, 200 μl of each sample in triplicate was added into a 96 well plate and absorbance was read at 550 nm.