Engineering of cartilage tissue using bioresorbable polymer fleeces and perfusion culture

Replacement of injured or diseased skeletal tissues by either autograft or allograft cartilage has increased steadily during recent decades. The ideal method is to use autologous cartilage; however, this is extremely limited due to the scarcity of donor sites. We present a new approach to the in vitro formation of cartilage grafts for autologous grafting in reconstructive surgery. Bioresorbable polymer fleeces of polylactic acid were used as temporary cell carrier matrices to establish three-dimensional cultures of human chondrocytes. The polymer surface was coated with poly-L-lysine before cell integration. These cell-polymer tissue constructs were encapsulated with low melting point agarose and then placed in perfusion culture chambers to provide a constant supply of nutrients into the cultures. The culture medium consisted of Ham's F12 supplemented with 2% fetal calf serum and 50 micrograms/ml ascorbic acid. The cell-polymer tissues were harvested and frozen for toloudine and alcian blue staining as well as electron microscopic examination after different periods of time in culture. A monoclonal antibody specific for collagen type II was used to characterize the cell phenotype. With this culture procedure chondrocytes maintained a differentiated phenotype with synthesis of collagen and proteoglycan. Collagen fibrils with clear cross-striation were evident in electron microscopic images. The results show that our organotypic cell culture method allows the in vitro production of bioartificial cartilage for transplantation.