In cartilage, tissue engineering bioreactors can create a controlled environment to study chondrocyte behavior under mechanical stimulation or produce chondrogenic grafts of clinically relevant size. Here we present a novel bioreactor that combines mechanical stimulation with a two compartment system through which nutrients can be supplied solely by diffusion from opposite sides of a tissue-engineered construct. This design is based on the hypothesis that creating gradients of nutrients, growth factors, and growth factor antagonists can aid in the generation of zonal tissue-engineered cartilage. Computational modeling predicted that the design facilitates the creation of a biologically relevant glucose gradient. This was confirmed by quantitative glucose measurements in cartilage explants. In this system, it is not only possible to create gradients of nutrients, but also of anabolic or catabolic factors. Therefore, the bioreactor design allows control over nutrient supply and mechanical stimulation useful for in vitro generation of cartilage constructs that can be used for the resurfacing of articulated joints or as a model for studying osteoarthritis disease progression.