Cartilage tissue-engineering strategies aim to produce a functional extracellular matrix similar to that of the native tissue. However, none of the myriad approaches taken have successfully generated a construct possessing the structure, composition, and mechanical properties of healthy articular cartilage. One possible approach to modulating the matrix composition and mechanical properties of engineered tissues is through the use of bioreactor-driven mechanical stimulation. In this Study, we hypothesized that exposing scaffold-free cartilaginous tissue constructs to 7 days of continuous shear stress at 0.001 or 0.1 Pa would increase collagen deposition and tensile mechanical properties compared to that of static controls. Histologically, type 11 collagen staining was evident in all construct groups, while a surface layer of type I collagen increased in thickness with increasing shear stress magnitude. The areal fraction of type I collagen was higher in the 0.1-Pa group (25.2 +/- 2.2%) than either the 0.001-Pa (13.6 +/- 3.8%) or the static (7.9 +/- 1.5%) group. Type II collagen content, as assessed by ELISA, was also higher in the 0.1-Pa group (7.5 +/- 2.1%) compared to the 0.001-Pa (3.0 +/- 2.25%) or static groups (3.7 +/- 3.2%). Temporal gene expression analysis showed a flow-induced increase in type I and type II collagen expression within 24 h of exposure. Interestingly, while the 0.1-Pa group showed higher collagen content, this group retained less sulfated glycosaminoglycans in the matrix over time in bioreactor Culture. Increases in both tensile Young's modulus and ultimate strength were observed with increasing shear stress, yielding constructs possessing a modulus of nearly 5 MPa and strength of 1.3 MPa. This study demonstrates that shear stress is a potent modulator of both the amount and type of synthesized extracellular matrix constituents in engineered cartilaginous tissue with corresponding effects on mechanical function. Biotechnol. Bioeng. 2009;104: 809-820. (C) 2009 Wiley Periodicals, Inc.