Tissue samples for biochemical analysis were obtained from an adjacent slice to the histological section; anterior AF, NP, and posterior AF were harvested, that is, from MRI ROIs 1, 3, and 5, respectively. Samples were freeze-dried (speedvac) and subsequently digested in 1.5 mL papain-digestion buffer containing 0.1 M sodium acetate, 0.01 M L-cysteine, and 0.01 M EDTA, after which pH was titrated to 6.6 using 1 M NaOH and finally 0.33% (w/v) papain was added to the solution (all Merck Millipore). Samples were digested overnight in a continuously shaking water bath at 65 °C. papain digestion solutions were diluted and glycosaminoglycan (GAG) content was analyzed using a DMMB assay (Biocolor Ltd.) according to the manufacturer's protocol. The collagen content, expressed as the total amount of hydroxyproline (HYP), was quantified using a DMBA hydroxyproline assay, as described by Paul et al.35 (link) Measured amounts of GAG and HYP content were expressed in micrograms per milligram tissue dry weight.
Free full text: Click here
