Bovine chondrocytes were cultured in monolayers and alginate beads with or without ascorbic acid (Asc) for 16 days. Cell proliferation was examined every 4 days by staining with Hoechst 33258 dye. The gene expression of aggrecan, and collagen type I and II was analyzed at 16 days by reverse transcription and polymerase chain reaction.Cell morphology and the production of extracellular matrix (ECM) were evaluated by cytochemical, immunocytochemical and electron microscopical methods.Cells were continuously cultured in alginate beads with Asc for 2 months, and the cell morphology and ECM were examined. The proliferation of chondrocytes was significantly stimulated with Asc in both monolayers and alginate beads at 16 days. Expression of the collagen type I gene in both cultures was increased, and that of the collagen type II gene in alginate beads was decreased, by Asc. There were no significant cytochemical and immunocytochemical differences between the cultures in alginate beads with or without Asc at 16 days.In alginate beads cultured with Asc for 2 months, proliferating cells were observed mainly at the periphery of the beads, and glycosaminoglycan and collagen type II were found around the cells. These results suggest that Asc stimulated the proliferation of chondrocytes and maintained the chondrogenic properties of the cells in an alginate beads culture.

This study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells (fetal fibroblasts, adult skin and muscle cells, and cumulus cells) after culture under a variety of conditions ; 1 ) growth to 60- 70% confluency (cycling) , 2) serum starvation, 3) culture to confluency. Cell -cycle phases were determined by flow cytometry with propidium iodide staining enabling the calculation of percentages of cells in GO /G1, S and G2 /M. The majority was in GO /GI regardless of cell type and treatment. Serumstarved or confluent cultures contained higher percentages of cells in GO /G1 (89.5-95.4% ; P <0.05) . Percentages of cells in GO /G1 increased as cell size decreased regardless of the cell type and treatment. In the serum-starved and confluent cultures, about 98% of small cells were in GO /G1. Serum-starved cultures contained higher percentages of small cells (38.5-66.9%) than cycling and confluent cultures regardless of cell type (P < 0.05) . After trypsinization of fetal fibroblasts and adult skin cells that were serum-starved and cultured to confluency, the percentages of cells in GO /G1 increased (P < 0.05) on incubation for 1.5 (95.7-99.5%) or 3 hr (95.9-98.6%). These results verify that serum starvation and culture to confluency are efficient means of synchronizing bovine somatic cells in GO /G1, and indicate that a more efficient synchronization of the cells in GO /G1 can be established by incubation for a limited time period after trypsinization of serum-starved or confluent cells.