title: Differential effect of ECM molecules on re-expression of cartilaginous markers in near quiescent human chondrocytes.
abstract: 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-β1 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.
authors.affiliation: Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan.
authors.forename: Li-Hsuan
authors.initials: LH
authors.lastname: Chiu
authors.forename: Shih-Ching
authors.initials: SC
authors.lastname: Chen
authors.forename: Kai-Chen
authors.initials: KC
authors.lastname: Wu
authors.forename: Charng-Bin
authors.initials: CB
authors.lastname: Yang
authors.forename: Chia-Lang
authors.initials: CL
authors.lastname: Fang
authors.forename: Wen-Fu T
authors.initials: WF
authors.lastname: Lai
authors.forename: Yu-Hui
authors.initials: YH
authors.lastname: Tsai
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism; D024043:Collagen Type II / Q000096:biosynthesis / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D016023:Integrins / Q000037:antagonists & inhibitors; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D055758:SOX9 Transcription Factor / Q000096:biosynthesis; D023822:Tissue Engineering; D016212:Transforming Growth Factor beta / Q000378:metabolism
chemical_list: D015415:Biomarkers; C499177:COL2A1 protein, human; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D016023:Integrins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D016212:Transforming Growth Factor beta; D007334:Insulin-Like Growth Factor I; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D020930:MAP Kinase Kinase Kinases; D048670:MAP Kinase Kinase 4