2.9. Biochemical Assays

To check the quantity of sulfated glycosaminoglycan (sGAG) secretion, spheroids transfected by miR-(140+21) and spheroids differentiated using the chondrogenic differentiation medium were examined using a Glycosaminoglycans Assay Kit (Chondrex, Inc., Redmond, WA). For each group, 10 spheroids were collected and rinsed with PBS weekly for four weeks. Proteinase K of 0.4 mL (Sigma Aldrich) at a final concentration of 0.5 mg/mL was first added to each Eppendorf tube with 10 spheroids and then incubated at 56 °C overnight for enzymatic lysis of cells and DNA release. After adding 1,9 dimethylmethlyene blue (DMB) solution for 3 min as per manufacturer’s instructions, the absorbance was measured at 525 nm using the microplate reader (PowerWaveX). sGAG content from each sample was normalized to the dsDNA content.

To check the quantity of ALP secretion, spheroids transfected by miR-148b and spheroids differentiated using osteogenic differentiation medium were used. Alkaline phosphatase (ALP) activity assay was conducted using an ALP assay kit (K412–500; BioVision, Inc., Mountain View, CA) according to the manufacturer’s instructions. 10 spheroids per sample were resuspended in an assay buffer and subsequently centrifuged at 13,000 Xg for 3 min at 4 °C to remove any insoluble material. The supernatant was mixed with p-nitrophenyl phosphate (pNPP) substrate and then incubated at 25 °C for 60 min. The optical density of the resultant pNPP was determined at 405 nm using the microplate reader (PowerWaveX).