Figure 1. Schematic representation of the experimental design.

To examine the effects of intercellular distance and cell distribution on the interactions between adipose-derived stem cells (ADSCs) and neonatal articular chondrocytes (NChons), three different in vitro co-culture models were used: (A) In the condition medium (CM-) model, each cell type was cultured alone with supplementation of conditioned medium (CM) from the other cell type. (B) In the bi-layered (bi-) co-culture, ADSCs and NChons were confined in separate hydrogel layers with no direct cell-cell contact, and soluble paracrine signals were allowed to diffuse into the adjacent layer. (C) In the mixed cell co-culture model, ADSCs and NChons were mixed together in 3D at 4 different cell ratios (NChon:ADSC: 75C:25A, 50C:50A, 25C:75A, and 10C:90A). The initial cell density was maintained constant at 15 million/ml in all co-culture models. (D) The concentration of paracrine factors decays rapidly with distance from the secreting cell. In all the co-culture models, human adult ADSCs and bovine NChons were encapsulated in 3D biomimetic hydrogels and cultured in vitro for 21 days in chondrogenic medium supplemented with TGF-β3 (10 ng/ml).