A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm(2) at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type 11 collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.