Papain

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.

Chondrogenic differentiation was performed in pellet cultures. First, 2.5 × 10⁵ hES-MP cells were seeded in hMSC Chondrogenic Differentiation Media (BulletKit^®, Lonza) in 1.5 mL microtubes (Sarstedt, Nümbrech, Germany). The tubes were then centrifuged at 150× g for 5 min to generate a cell pellet. The tube lids were then punctured with a 2 mm sterile needle (Misawa, Tokyo, Japan) to facilitate gas exchange. The medium was fully changed every 2 to 3 days. Chondrogenic pellets were harvested after 0, 7, 14, 28, and 35 days for both gene expression analysis and the evaluation of glycosaminoglycan (GAG) concentration. For GAG analysis, the pellets were washed with PBS three times and then digested for 3 h at 65 °C in a papain extraction reagent containing 0.1 M sodium acetate (Sigma-Aldrich), 0.01 M Na₂EDTA (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), 0.005 M cystein HCl (Sigma), and 8 µL of crystallized papain (Sigma-Aldrich). After papain digestion, the GAG concentration was evaluated with a Blyscan assay (Biocolor, Carrickfergus, UK) following the manufacturer’s instructions. The pellets were stained with hematoxylin and eosin staining and Masson’s trichrome staining after 28 days of differentiation.