Interactions between integrin ligand density and cytoskeletal integrity regulate BMSC chondrogenesis

Interactions with the extracellular matrix play important roles in regulating the phenotype and activity of differentiated articular chondrocytes; however, the influences of integrin‐mediated adhesion on the chondrogenesis of mesenchymal progenitors remain unclear. In the present study, agarose hydrogels were modified with synthetic peptides containing the arginine‐glycine‐aspartic acid (RGD) motif to investigate the effects of integrin‐mediated adhesion and cytoskeletal organization on the chondrogenesis of bone marrow stromal cells (BMSCs) within a three‐dimensional culture environment. Interactions with the RGD‐modified hydrogels promoted BMSC spreading in a density‐dependent manner and involved αvβ3 integrin receptors. When cultured with the chondrogenic supplements, TGF‐β1 and dexamethasone, adhesion to the RGD sequence inhibited the stimulation of sulfated‐glycosaminoglycan (sGAG) production in a RGD density‐dependent manner, and this inhibition could be blocked by disrupting the F‐actin cytoskeleton with cytochalasin D. In addition, interactions with the RGD‐modified gels promoted cell migration and aggrecanase‐mediated release of sGAG to the media. While adhesion to the RGD sequence inhibited BMSC chondrogenesis in the presence of TGF‐β1 and dexamethasone, osteocalcin and collagen I gene expression and alkaline phosphatase activity were enhanced by RGD interactions in the presence of serum‐supplemented medium. Overall, the results of this study demonstrate that integrin‐mediated adhesion within a three‐dimensional environment inhibits BMSC chondrogenesis through actin cytoskeleton interactions. Furthermore, the effects of RGD‐adhesion on mesenchymal differentiation are lineage‐specific and depend on the biochemical composition of the cellular microenvironment. J. Cell. Physiol. 217: 145–154, 2008. © 2008 Wiley‐Liss, Inc.