ENGINEERING HUMAN CARTILAGE TISSUE USING A PERFUSION CHAMBER

In the field of otolaryngology cartilage grafting is commonly performe d to reconstruct skeletal defects. Knowledge of chondrocyte growth and differentiation call now be used to engineer cartilage tissue for gra fting. The first condition is that chondrocytes maintain their differe ntiated phenotype besides being able to produce a new cartilage matrix . The target of this study was to evelop a three-dimensional culture s ystem for in-vitro formation of vital cartilage transplants. Chondrocy tes were isolated by digesting the cartilage matrix with collagenase a nd hyaluronidase. After embedding in ''low-melting'' agarose, the chon drocytes were placed into a perfusion culture chamber to provide a con stant supply of nutrients to the cultures. The peristaltic pump was op erated with on/off intervals of 30 min. Ham's F12 supplemented with 2% FCS and 50 mu g/ml ascorbic acid was employed as culture medium. Mono clonal antibodies specific to collagens type I and type II were used t o characterise cells and matrix synthesis. Synthesis of proteoglycans and collagens was achieved using toluidine blue and azan staining. Und er the described culture conditions, the chondrocytes maintained a dif ferentiated phenotype (expression of collagen type II) with synthesis of collagens and proteoglycans. An accumulation of matrix products was achieved pericellularly. After 2-8 weeks the obtained tissue exhibite d an excellent histological appearance showing the typical features of cartilage tissue. The results show that the perfusion chamber allows a quick in-vitro fabrication of a piece of pure cartilage tissue for t ransplantation.