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The effects of hydrostatic pressure (HP) on the matrix synthesis by human articular chondrocytes have been reported elsewhere. In order to optimize the production of extracellular matrix, we aimed to clarify the effects of repetitive HP on metabolic function by human articular chondrocytes.The human articular chondrocytes were expanded and embedded within a collagen gel/sponge scaffold. We incubated these constructs with and without HP followed by atmospheric pressure (AP) and repeated the second HP followed by AP over 14 days. Genomic, biochemical, and histological evaluation were performed to compare the effects of each regimen on the constructs.The gene expressions of collagen type II and aggrecan core protein were significantly upregulated with repetitive HP regimens compared with a single HP or AP by 14 days (P < 0.01 or 0.05). Matrix metalloptoteinase-13 (MMP-13) in AP was upregulated significantly compared to other HP regimens at day 14 (P < 0.01). No significant difference was observed in tissue inhibitor of metalloproteinases-II. Immunohistology demonstrated that application of HP (both repetitive and single) promoted the accumulation of specific extracellular matrix and reduced a MMP-13. A single regimen of HP followed by AP significantly increased the amount of sulfated glycosaminoglycan than that of the AP, whereas repetitive HP remained similar level of that of the AP.Repetitive HP had a greater effect on anabolic activity by chondrocytes than a single HP regimen, which will be advantageous for producing a matrix-rich cell construct.