Articular cartilage with its inherently poor capacity for self-regeneration represents a primary target for tissue engineering strategies, with approaches focusing on the in vitro generation of neo-cartilage using chondrocyte-seeded 3D scaffolds subjected to mechanical conditioning. Although uniaxial compression regimens have significantly up-regulated proteoglycan synthesis, their effects on the synthesis of collagen have been modest. Articular cartilage is subjected to shear forces during joint motion. Accordingly, this study utilized an apparatus to apply biaxial loading to chondrocytes seeded within agarose constructs with endplates. The chondrocytes yielded a monotonic increase in proteoglycan synthesis both in free swelling culture up to day 8 and when the constructs were subjected to dynamic compression alone (15% amplitude at a frequency of 1Hz for 48h). However, when dynamic shear (10% amplitude at 1Hz) was superimposed on dynamic compression, total collagen synthesis was also up-regulated, within 3 days of culture, without compromising proteoglycan synthesis. Histological analysis revealed marked collagen deposition around individual chondrocytes. A significant proportion (50%) of collagen was released into the culture medium, suggesting that it had only been partially synthesized in its mature state. The overall biosynthetic activity was enhanced more when the biaxial stimulation was applied in a continuous mode as opposed to intermittent loading. Results of the present study strongly suggest that proteoglycan and collagen synthesis may be triggered by uncoupled mechanosensitive cellular responses. The proposed in vitro model and the prescribed conditioning protocols demonstrated that a short pre-culture period is preferable to long free swelling culture condition as it enables a significantly higher up-regulation of collagen. Biotechnol. Bioeng. 2017;114: 1614-1625. (c) 2017 Wiley Periodicals, Inc.