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CSPG is one of the major components of aggrecan in articular cartilage [37]?and?[38]. Production of extracellular CSPG is one of the features of chondrocytes. As shown in Fig.?4(A), punctate patterns of CSPG were observed for all cells. Co-localization of F-actin and the CSPG pattern revealed the deposition of CSPG in the extracellular space for cells on 40?��m, 80?��m patterns and the unpatterned surface. Although the presence of CSPG at the periphery of cells on 20?��m patterns was not obvious, it was found on the cell membrane via z-scan imaging (Fig. S2, Supporting Information). Synthesis and extracellular deposition of collagen II is a key phenotypic marker of differentiated chondrocytes [39]?and?[40]. At 5?h, collagen II was observed in all cells (Fig.?4(A)) but with different distribution patterns. For cells on patterns, collagen II immunofluorescence was concentrated in cytoplasm around the nucleus of the cell. In contrast, cells on unpatterned surfaces have scattered patches within and around the cell body. No obvious extracellular deposition of collagen II was shown at the cell periphery, suggesting that the synthesis of collagen II was mainly intracellular at this time point. For quantitative comparison of the matrix production by individual cells, the total immunofluorescence intensity of CSPG or collagen II staining per cell (FCSPG and Fcol II) was measured. FCSPG of cells on patterns are either comparable to (20?��m and 80?��m patterns) or higher than (40?��m patterns) that on unpatterned surface (Fig.?4(B)). In the case of Fcol-II, no significant difference was observed between cells on 40?��m, 80?��m and unpatterned surfaces, although the values were lower for cells on 20?��m patterns (1.5-fold, p? present on the majority of cells, which was accompanied by disappearance of both CSPG and collagen II (Fig. S3, Supplementary Information), indicating the loss of differentiated phenotype. The biomimetic 3D culture system confined cells on patterns for weeks thereby enabling the investigation of the long-term effects of cell geometries on chondrocyte dedifferentiation. At day 2, all cells showed CSPG immunofluorescence within their main body (Fig.?5(A) and Fig. S4, Supporting Information).