Chondrocytes were applied to a custom double-diffusion biochamber with porous (10 µm pore diameter) polyester membrane (PET 1009030; Sterlitech, Kent, WA), which had been precoated with 5 µg/ml fibronectin (BD Bioscience, San Jose, CA), as previously described (11 (link)), at a density of 3.13 × 106 cells/cm2. The cells were cultured under the same O2 condition as in expansion with bioreactor medium (serum-free DMEM with 4.5 g/L glucose (Invitrogen, Grand Island, NY), containing 1% ITS-premix (BD Bioscience, San Jose, CA), 100 nM dexamethasone (Sigma-Aldrich), 37.5 µg/mL L-ascorbate-2-phosphate (Wako chemicals, Osaka, Japan), and 1% sodium pyruvate, 1% non-essential amino acid, 1% glutamax, and 1% Penicillin/Streptomycin (all from Invitrogen, Grand Island, NY)). The medium was changed every other day. After 3 weeks, cartilage sheets were removed from the biochamber and allowed to free float until 6.5 weeks. Punches (5 mm in diameter) were taken for the mechanical testing from each sheet. Combined GAG/DNA analysis was made in triplicate (3 mm diameter punches), as was collagen/collagen cross-link analysis. Histology was performed on a single neutral buffered formalin fixed 3 mm punch from each sheet. The sheets were made in duplicate from 4 donors.