1 9 dimethylmethylene blue dmb binding

Transwell discs were stained as described (Barter et al., 2015 (link)). Chondrogenic pellets and transwell discs were digested with papain (10 U/ml) at 60°C (Murdoch et al., 2007 (link)). The sulphated glycosaminoglycan (GAG) content was measured by 1,9-dimethylmethylene blue (DMB) binding (Sigma) using chondroitin sulphate (Sigma) as standard (Farndale et al., 1982 (link)), and the DNA content was measured with PicoGreen (Invitrogen) intercalating dye following the manufacturer's instructions. Cells undergoing osteoblast differentiation were fixed in 70% cold ethanol (5 min, −20°C). After drying the wells to reveal calcium-rich mineralisation deposits, the cells were incubated at room temperature with a solution of Alizarin Red (Sigma) (40 mM, pH 4.2) for 20-30 min. For quantification the staining was extracted with 10% (w/v) cetylpyridinium (Sigma) solubilised in 10 mM sodium phosphate buffer (pH 7) and the absorbance measured at 620 nM. Cells undergoing adipogenesis were fixed with formalin for 1 h, washed with distilled water and 60% isopropanol then dried. To reveal the presence of lipid droplets, the cells were stained with a 21% (w/v) solution of Oil Red O for 10 min. For quantification the staining was extracted with 100% isopropanol and the absorbance measured at 500 nM. Stained cells were washed with distilled water prior to image acquisition.

After culture, scaffolds were rinsed in PBS (Invitrogen), and frozen at −80 °C. For analysis, samples were digested in proteinase K (1 mg/ml in 50 mM Tris with 1 mM EDTA, 1 mM iodoacetamide and 10 mg/ml pepstatin A—all from Sigma-Aldrich) for 16 h at 56 °C. The sulphated GAG content was measured by 1,9 dimethylmethylene blue (DMB) binding (Sigma-Aldrich) using chondroitin sulfate (Sigma-Aldrich) as standard. The DNA content was determined using the CyQuant cell proliferation assay kit (Invitrogen) with supplied bacteriophage λ DNA as standard. Chondro-induction is defined as previously described.^{41 (link)} Based on experimentally measured GAG contents of pure MC seeded scaffolds (GAG_{100% MCs}) and pure ASC seeded scaffolds (GAG_{100% ASC}), the expected total GAG (GAG_expected) in the coculture cell-seeded scaffolds was calculated as a linear function of the proportion (%) of MC using the following equation: $${\mathrm{GAG}}_{\mathrm{expected}}={\mathrm{GAG}}_{100\mathrm{\%}\mathrm{MC}}+\left({\mathrm{GAG}}_{100\mathrm{\%}\mathrm{MC}}-{\mathrm{GAG}}_{100\mathrm{\%}\mathrm{ASC}}\right)\times \mathrm{\%}\mathrm{MC}$$
The interaction index was then calculated as the ratio of the GAG measured in the cocultured cell-seeded scaffolds (GAG_measured) to the GAG_expected. When the interaction index is higher than 1, then chondro-induction is considered to have occurred.^{20 (link),41 (link)}