Alcian blue staining and quantification

At different time points, cultured chondrocytes grown on nanopillar arrays were fixed with 4% paraformaldehyde for 15 min at 4 °C. Thereafter, each well was incubated with 0.1% Alcian blue 8GX (Sigma Aldrich) dissolved in 0.1 N hydrochloric acid in phosphate buffered saline for 30 min. Subsequently, a flatbed scanner was used to take color images (at 1200 pixels per image) of the nanopillar arrays. White balance correction of nanopillar array image was performed before image deconvolution to extract the Alcian blue stain. Measurement of Alcian blue intensity was performed using the color deconvolution plugin for ImageJ (National Institutes of Health). All intensity measurements were normalized to those on Flat at day 7.