A collagen-coated surface enables quantitative evaluation of morphological behaviors of rabbit chondrocytes relating to cell differentiation in an early culture phase

A surface with collagen type I coating was used as a tool mimicking three dimensional environment for studying the behaviors of rabbit chondrocyte aggregates found in an early culture phase. The morphology of cell aggregates was evaluated quantitatively in terms of a morphology-relating parameter of specific branching factor, Zs, determined under varied culture conditions. Branching in aggregates increased along with proceeding of culture from 5 to 6 days when seeded at a lower density of X0 = 1.0 × 103 cells/cm2, giving Zs = 0.24 tips/cell at 6 days, the value of which was 1.7 times than that obtained from the culture with a higher seeding density of X0 = 1.0 × 104 cells/cm2 (Zs = 0.14 tips/cell). This value significantly reduced to Zs = 0.13 tips/cell in the culture with X0 = 1.0 × 103 cells/cm2 by using the conditioned medium prepared from the foregone culture with X0 = 1.0 × 104 cells/cm2, inducing the morphological architecture and collagen type II production of aggregated cells with the similarities to those in the standard culture seeded at 1.0 × 104 cells/cm2. The mRNA expressions of differentiation-marker genes (collagen types I and II) were confirmed to be regulated in accordance with the morphological analyses as well as the extracellular matrix formation of aggregates in the both cultures conducted with the standard and conditioned media. The present study suggested that soluble factor(s) secreted from the chondrocytes at the higher seeding density modulates the cell aggregation and the state of chondrogenic differentiation through encouraging cell–cell communications.