title: Interactions between integrin ligand density and cytoskeletal integrity regulate BMSC chondrogenesis.
abstract: 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 alphavbeta3 integrin receptors. When cultured with the chondrogenic supplements, TGF-beta1 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-beta1 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.
authors.affiliation: George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Connelly
authors.forename: Andrés J
authors.initials: AJ
authors.lastname: García
authors.forename: Marc E
authors.initials: ME
authors.lastname: Levenston
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology; D002454:Cell Differentiation; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D020219:Chondrogenesis / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007091:Image Processing, Computer-Assisted; D016023:Integrins / Q000378:metabolism*; D008024:Ligands; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology
chemical_list: D016023:Integrins; D008024:Ligands; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid