Effect of Transforming Growth Factor-β1 on Morphological Characteristics Relating to Migration and Differentiation of Rabbit Chondrocytes Cultured in Collagen Gels

The influence of transforming growth factor-β1 (TGFβ1) on the behavior of rabbit chondrocytes embedded in collagen gels was examined in terms of cell migration and consequent architecture of cell aggregation. In a low-seeding density culture (X0 = 2.0×105 cells/cm3) TGFβ1 (0–10.0 ng/ml) was added and observed during a 14-d culture period. Stereoscopic observation was performed on 5 d employing the morphology-related parameter of sphericity (Sc) for individual cells in the gels. The frequency of migrating cells with Sc less than 0.95 increased in a dose-dependent manner in response to TGFβ1. Moreover, the frequency of migrating cells in the culture with 10.0 ng/ml TGFβ1 was 0.32, two times higher than that in the reference culture without TGFβ1, while the frequency of dividing cells in the same culture was less than half of that in the reference culture. The histological observation of cultured gels on 14 d revealed that the starburst and loose aggregates with the spindle-shaped cells emerged in the TGFβ1-free culture, accompanying the poor production of collagen type II by the cells. On the other hand, the spherical-shaped cells were observed in the starburst aggregates with rich excretion of collagen type II in the culture with 5.0 ng/ml TGFβ1. Moreover, the mRNA levels of differentiation-marker genes (collagen types I and II) were regulated in accordance with the morphological analyses concerning the cell migration and aggregation in the cultures with and without TGFβ1. From these results, it was concluded that TGFβ1 had a culture time-dependent effect on the morphological characteristics relating to the migration and differentiation of the chondrocytes in the collagen gel-embedded cultures seeded at low density, that is, the growth factor promotes cell migration with deteriorated proliferation in the early culture phase, and accelerates the transformation of spindle-shaped cells to spherical-shaped ones in the prolonged culture.