TGF beta affects collagen cross-linking independent of chondrocyte phenotype but strongly depending on physical environment

Transforming growth factor beta (TGF beta) is often used in cartilage tissue engineering to increase matrix formation by cells with various phenotypes. However, adverse effects of TGF beta, such as extensive cross-linking in cultured fibroblasts, have also been reported. Our goal was to study effects of TGF beta on collagen cross-linking and evaluating the role of cellular phenotype and physical environment. We therefore used four different cell populations in two very different physical environments: primary and expanded chondrocytes and fibroblasts embedded in alginate gel and attached to tissue culture plastic. Matrix production, collagen cross-linking, and alpha-smooth muscle actin (alpha SMA) were analyzed during 4 weeks with or without 2.5 ng/mL TGF beta 2. TGF beta 2 did not affect collagen deposition by primary cells. In expanded cells, TGF beta 2 increased collagen deposition. Chondrocytes and fibroblasts in monolayer produced more collagen cross-links with TGF beta 2. In alginate, primary and expanded cells displayed an unexpected decrease in collagen cross-linking with TGF beta 2. aSMA was not present in alginate cultures and barelay upregulated by TGF beta 2. Organized alpha SMA fibers were present in all monolayer cultures and became more pronounced with TGF beta 2. This study demonstrates that the physical environment determined by the substrate used co-determines the response of cells to TGF beta. The presence of mechanical stress, determined with alpha SMA-staining, is probably responsible for the increase in collagen cross-linking upon addition of TGF beta.