Differential Effect of ECM Molecules on Re-Expression of Cartilaginous Markers in Near Quiescent Human Chondrocytes LI-HSUAN CHIU, 1,2 SHIH-CHING CHEN, 3 KAI-CHEN WU, 2 CHARNG-BIN YANG, 4 CHIA-LANG FANG, 5 WEN-FU T. LAI, 6 ** AND YU-HUI TSAI 1,2,3 * 1 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Graduate Institute of Cell and Molecular Biology, College of Medicine, Taipei Medical University, Taipei, Taiwan 3 Department of Physical Medicine and Rehabilitation, Taipei Medical University Hospital, Taipei, Taiwan 4 Department of Orthopaedics, Taipei County Hospital, Taipei, Taiwan 5 Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 6 Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan The limited source of healthy primary chondrocytes restricts the clinical application of tissue engineering for cartilage repair. Therefore, method to maintain or restore the chondrocyte phenotype during in vitro expansion is essential. The objective of this study is to establish the beneficial effect of ECM molecules on restoring the re-expression of cartilaginous markers in primary human chondrocytes after extensive monolayer expansion. During the course of chondrocyte serial expansion, COL2A1, SOX9, and AGN mRNA expression levels, and GAG accumulation level were reduced significantly in serially passaged cells. Exogenous type II collagen dose-dependently elevated GAG level and induced the re-expression of cartilaginous marker mRNAs in P7 chondrocytes. Chondroitin sulfate did not show significant effect on P7 chondrocytes, while hyaluronic acid inhibited the expression of SOX9 and AGN mRNAs. Upon treatment with type II collagen, FAK, ERK1/2, and JNK were activated via phosphorylation in P7 chondrocytes within 15 min. Furthermore, GFOGER integrin blocking peptide, MEK inhibitor and JNK inhibitor, not p38 inhibitor, significantly reduced the type II collagen-induced GAG deposition level. Finally, in the presence of TGF-b1 and IGF-I, P7 chondrocytes cultured in 3D type II collagen matrix exhibited better cartilaginous features than those cells cultured in the type I collagen matrix. In conclusion, type II collagen alone can effectively restore cartilaginous features of expanded P7 human chondrocytes. It is probably mediated via the activation of FAK-ERK1/2 and FAK-JNK signaling pathways. The potential application of type II collagen in expanding a scarcity of healthy chondrocytes in vitro for further tissue engineering is implicated. J. Cell. Physiol. 226: 1981–1988, 2011. ß 2010 Wiley-Liss, Inc. Articular cartilage is an avascular tissue with limited capacity of self-repair. In the adult articular cartilage, chondrocytes are the only cell type dispersed among the dense cartilage-specific matrix, which is abundant of type II collagen and glycosaminoglycans. This histological feature makes that the articular cartilage bears only limited spontaneous healing ability (Hunter, 1995). One of the surgical attempts to restore normal cartilage surface of the joint is made by the subchondral drilling technique. Bleeding from the subchondral bone promotes wound healing and results in a hyaline-like but more cellular tissue at the defect site of the cartilage (Mainil-Varlet et al., 2003). During recent years, intensive efforts have been made to develop cell therapies for cartilage repair, such as the autologous transplantation of chondrocytes (Brittberg et al., 1994; Convery et al., 1996; Ghazavi et al., 1997). Large quantities of healthy cells from limited source are required in such cell-based therapies. Consequently, in vitro expansion of chondrocytes for cell implantation cannot be avoided. Abbreviations: COL2A1, type II collagen alpha 1 chain gene; COL1A1, type I collagen alpha 1 chain gene; AGN, aggrecan gene; SOX9, SRY-box 9 gene; ITGA2, integrin alpha 2 gene; ITGB1, integrin beta 1 gene; CS, chondroitin sulfate; HA, hyaluronic acid; COL II, type II collagen; COL I, type I collagen; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase. Li-Hsuan Chiu and Shih-Ching Chen contributed equally to this work. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. All authors declare that there is no conflict of interest with other people or organizations that could inappropriately influence their work. Contract grant sponsor: National Science Council, Taiwan, ROC; Contract grant numbers: NSC-92-2321-B-038-010, 98-3112-B- 038-003, and 97-2120-M038-001. Contract grant sponsor: Taipei City Government, Department of Health, Taipei, Taiwan; Contract grant number: 95001-62003. *Correspondence to: Yu-Hui Tsai, Graduate Institute of Medicine, College of Medicine, Taipei Medical University, 250 Wu-Xin Street, Taipei 110, Taiwan. E-mail: cmbyht18@tmu.edu.tw **Correspondence to: Wen-Fu T. Lai, Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, 250 Wu-Xin Street, Taipei 110, Taiwan. E-mail: laitw@tmu.edu.tw Received 13 January 2010; Accepted 28 October 2010 Published online in Wiley Online Library (wileyonlinelibrary.com), 16 December 2010. DOI: 10.1002/jcp.22530 ORIGINAL RESEARCH ARTICLE 1981 Journal of Journal of Cellular Physiology Cellular Physiology ß 2010 WILEY-LISS, INC.