title: Characterization of layered chondrocyte sheets created in a co-culture system with synoviocytes in a hypoxic environment.
abstract: Endeavouring to repair and regenerate articular cartilage using cell sheets, we have previously established a co-culture system of chondrocytes and synoviocytes, and have reported the successful and rapid production of chondrocyte sheets. In the present study, to examine the effects of oxygen concentration on the chondrocyte sheets, we co-cultured human articular chondrocytes and human synoviocytes in 2%, 5% and 21% oxygen, and measured chondrocyte metabolic activity and proliferation activities under each condition for 14 days in culture. Layered chondrocyte sheets were also created under each condition and the proteoglycan (PG) level was compared with the gene expression of type I collagen (COL1), COL2, COL27, tissue metallopeptidase inhibitor 1 (TIMP1), fibronectin-1 (FN1), SRY-related HMG Box 9 (SOX9), aggrecan-1 (ACAN), integrin-α10 (ITGα10), matrix metalloproteinase 3 (MMP3), MMP13 and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5). Compared with 5% and 21% oxygen, the 2% condition caused significantly greater cell metabolic activity and proliferation (p < 0.05). The 2% condition produced a 10% greater PG level compared with 21% oxygen (p < 0.05). All conditions increased the expression of chondrocyte-specific genes, such as COL2, and were associated with low expression levels of catabolic factors, such as MMP3 and MMP13. These observations indicated that the specificity of the chondrocyte sheets was maintained under all conditions. The culture times did not differ between the 5% and 21% conditions. Compared with 21% oxygen, layered chondrocyte sheets rich in extracellular matrix were created 2.85 days earlier in 2% oxygen, which is similar to the level found in deep cartilage. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Institute of Advanced Biomedical Engineering and Science Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Kokubo
authors.affiliation: Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Kanagawa, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Sato
authors.affiliation: Institute of Advanced Biomedical Engineering and Science Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.affiliation: Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Kanagawa, Japan.
authors.forename: Genya
authors.initials: G
authors.lastname: Mitani
authors.affiliation: Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Kanagawa, Japan.
authors.forename: Yoshiyasu
authors.initials: Y
authors.lastname: Uchiyama
authors.affiliation: Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Kanagawa, Japan.
authors.forename: Joji
authors.initials: J
authors.lastname: Mochida
authors.affiliation: Institute of Advanced Biomedical Engineering and Science Tokyo Women's Medical University, Tokyo, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
mesh_terms: D000293:Adolescent; D000328:Adult; D015687:Cell Hypoxia; D049109:Cell Proliferation; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D018920:Coculture Techniques / Q000379:methods*; D024042:Collagen Type I / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008875:Middle Aged; D013194:Staining and Labeling; D000070918:Synoviocytes / Q000166:cytology*; D055815:Young Adult
chemical_list: D024042:Collagen Type I; D024043:Collagen Type II
keywords: human chondrocyte; hypoxia; layered chondrocyte sheets; regeneration; temperature-responsive culture dishes