Differential Effect of ECM Molecules on Re-Expression of Cartilaginous Markers in Near Quiescent Human Chondrocytes.

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, andAGNmRNAexpression levels,
and GAG accumulation level were reduced significantly in serially passaged cells. Exogenous type II collagen dose-dependently elevated
GAGlevel and induced the re-expression of cartilaginous markermRNAsin 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.