1 9 dimethyl methylene blue zinc chloride double salt dye

NP and AF tissue were isolated from the IVDs using an 8 mm biopsy punch (#05.275.38; Kai Medical, Seki, Japan, distributed by Polymed Medical Center, Glattbrugg, Switzerland) and a #10 scalpel blade (#CE20.1; Carl Roth Inc.; Karlsruhe, Germany). Then, approximately one third of each tissue sample was dried overnight at 60 °C. On the following day, the tissue dry weight was determined, and all samples were subsequently digested overnight in a 5 mM L-cysteine hydrochloride (#20119; Sigma-Aldrich) enriched papain solution (3.9 U/mL; #P3125; Sigma-Aldrich) at 60 °C. To quantify the sulphated GAG, 1,9-dimethyl-methylene blue zinc chloride double salt dye (#341088; Sigma-Aldrich) was applied to the digested tissue and the culture medium. The absorbance was then read at 600 nm using an ELISA reader (Spectramax M5, Molecular Devices, distributed by Bucher Biotec, Basel, Switzerland). Chondroitin sulfate sodium salt from bovine cartilage (#6737, Sigma-Aldrich) served as a standard to calculate the GAG concentration. In the end, the amount of GAG in the tissue was put in relation either to the dry weight of the isolated tissue or to the amount of DNA in the tissue. The GAG content that was released into the culture medium was normalised to the IVD volume after its isolation from the bovine tail.

The amount of sulphated glycosaminoglycan (GAG) was determined in the digested AF tissue as well as in the culture medium, which was collected at day 13, three days after the last medium change. Therefore, 1,9-dimethyl-methylene blue zinc chloride double salt dye (#341088; Sigma-Aldrich) was added to each sample and to the standard, which was based on chondroitin sulfate sodium salt from bovine cartilage (#6737, Sigma-Aldrich) [33 (link)]. The absorbance was measured within five minutes at a wavelength of 600 nm. Once the amount of GAG was determined, the content in the culture medium was normalized to the IVD’s initial volume after its dissection, and the tissue’s GAG was normalized to its dry weight and DNA content.