Redifferentiation of dedifferentiated human chondrocytes in high-density cultures

Abstract.

High-density cultures are widely used as an in vitro model for studies of embryonic cartilage formation. In the present study we investigated the suitability of high-density cultures for the redifferentiation of dedifferentiated chondrocytes. When primary human chondrocytes were cultured in alginate beads, some cells emigrated into Petri dishes. These cells were cultured for one to eight passages (each passage lasting about 3 days) in monolayer culture. At each passage, monolayer cells were removed and allowed to grow in high-density cultures at the medium-air interface and subsequently investigated with morphological, immunolocalization and biochemical methods for the production of cartilage-specific markers, i.e. collagen type II and cartilage-specific proteoglycans. When such dedifferentiated chondrocytes from monolayer passages P1–P4 were introduced in high-density culture, they regained a chondrocyte phenotype and formed cartilage nodules surrounded by fibroblast-like cells. Cells were interconnected by typical gap junctions and after a few days in culturing produced cartilage-specific extracellular matrix, notably collagen type II and cartilage-specific proteoglycans. In contrast, cells taken from monolayer passages P5–P8 did not produce these chondrocyte-specific extracellular materials when grown in high-density culture. We conclude that the growth of dedifferentiated chondrocytes in high-density culture promotes their redifferentiation and reveals their chondrogenic potential. Such high-density cultures might serve as a model system to initiate and promote the redifferentiation of chondrocytes and to provide sufficient quantities of differentiated chondrocytes for autologous chondrocyte transplantation.