Type a chondroitin sulfate

A DMMB assay was performed to measure GAG content within the constructs. We followed our previously established protocol [28 (link)], which was developed by combining and modifying several protocols [54 (link),55 (link),56 (link),57 (link)]. Briefly, after transferring 50 µL aliquots of each digested solution to a 96-well plate, 200 µL of DMMB (Sigma, 341088) solution was added to each well and mixed by pipetting. Plates were incubated for 1 h at room temperature, and absorbance was measured by a microplate reader at 525 nm (Thermo Fisher). Based on the optical density (OD) values of the samples, concentrations of GAGs were quantified using a linear standard curve generated from various known concentrations of type A chondroitin sulfate (from the bovine trachea, Sigma-Aldrich). GAG content measured from digested solutions was normalized with hydrogel weights measured before the digestion step.

Several protocols were combined and modified to generate a DMMB assay for our study [60 (link),61 (link),62 (link),63 (link)]. A mixture was prepared by dissolving 21 mg of DMMB (Sigma, 341088) in 5 mL of absolute ethanol, followed by the addition of 2 g of sodium formate (Sigma). Then, the solution was mixed with 800 mL of double distilled water. Formic acid (95%, Sigma) was added dropwise to the solution until pH was 1.5, and then, distilled water was added to make 1L of final solution.
The process involved transferring 50 µL of centrifuged and digested samples into a 96-well plate. To this, 250 µL of DMMB was added to each well, and the contents were mixed by pipetting up and down. After incubation at room temperature for 1 h, a spectrophotometric plate reader (Thermo Fisher) was used to measure optical density (OD) of each well at 525 nm. A linear standard curve was also generated using various known concentrations of type A chondroitin sulfate (from bovine trachea, Sigma-Aldrich), and the GAG content of the hydrogel samples was extrapolated from the standard curve.