Mechanical Forces Regulate Cartilage Tissue Formation by Chondrocytes via Integrin-mediated cell Spreading

In vitro grown cartilage is functionally inferior to native tissue, and improvements in its quality should be attempted so it can be used therapeutically. In these studies we investigated the effects of cell shape on tissue quality through alteration of substrate geometry and application of mechanical stimuli. Articular chondrocytes were isolated and cultured on the surface Ti-6Al-4V substrates with various geometries. When cultured on fully porous titanium alloy substrates, chondrocyte spreading was enhanced over those grown on substrates with solid bases. Chondrocytes which remained round did not synthesize significant amounts of matrix and were thus unable to form cartilaginous tissue. In contrast, chondrocytes which were directed to spread to a limited amount, resulting in a polygonal morphology, accumulated significantly more matrix molecules and in time formed cartilage-like tissue. Computational fluid dynamics analyses demonstrated that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on substrates with solid bases where media flow-through is restricted. Integrin-blocking experiments revealed that integrins are important regulators of cell shape, and appeared to influence the accumulation of collagen and proteoglycans by chondrocytes. Furthermore, compressive mechanical stimulation induced a rapid, transient increase in chondrocyte spreading by 10 minutes, followed by a retraction to pre-stimulated size within 6 hours. This has been shown to be associated with increased accumulation of newly synthesized proteoglycans. Blocking the α5β1 integrin, or its β1 subunit, inhibited cell spreading and resulted in a partial inhibition of compression-induced increases in matrix accumulation, thereby substantiating the role of β1 integrins in this process. These results suggest that both fluid induced shear forces and compressive forces regulate chondrocyte matrix accumulation by altering cell morphology, which is mediated by integrins. Identifying the molecular mechanisms that influence chondrocyte shape and thus tissue formation may ultimately lead to the development of a tissue that more closely resembles native articular cartilage.Ph