CULTIVATION OF HUMAN CARTILAGE IN A 3-DIM ENSIONAL PERFUSION CHAMBER - CHARACTERIZATION OF THE COLLAGEN-SYNTHESIS

In reconstructive head and neck surgery, there is a great need for car tilage transplants, Sufficient autologous graft is often not available , Heterologous cartilage is used frequently, although there is danger of transmitting viral infections and resorption rates are high. We hav e developed a three-dimensional model for the formation of cartilage i n vitro. The aim of this study was to characterize the collagen synthe sis under these culture conditions. Human chondrocytes were isolated b y digesting septal cartilage matrix in the presence of type II collage nase, hyaluronidase, and Dnase II in Ham's F12 medium. The resulting c ells were kept in monolayer culture for one week and then suspended in 2% ultra-low-melting agarose (1:1). The cell-agarose conglomerate was encapsulated with a 3% ultralow-melting agarose solution and placed i n a perfusion culture chamber. A permanent flow of fresh medium (Ham's F-12 supplemented with 50 mu g/ml ascorbic acid and 2% fetal calf ser um) was provided by a peristaltic pump which delivered 1 ml/h with on/ off intervals of 30 min. Samples were recovered after two weeks. Using electron microscopy abundant collagen fibril formation was shown. The collagen fibrils were identified histologically as cartilage specific type II collagen. No mRNA expression of collagen type X was observed using in situ hybridization. The cells appeared in a round cell shape with round nucleus and only slight variations in form and size. The pr esent results indicate that the chondrocytes maintain their differenti ated phenotype and continue to synthesize typical matrix products in t his three-dimensional perfusion culture chamber. The synthesis of cart ilage as graft material in reconstructive surgery might to be possible with this new culture system.