Protein synthesis of human articular chondrocytes cultured in vitro for autologous transplantation.

Chondrocytes in hyaline cartilage produce typical matrix proteins, the most abundant of them being collagen type II and aggrecan. Chondrocytes in monolayer cell culture dedifferentiate and gain fibroblastic phenotype. The cells gradually start to produce collagen type I while the production of collagen type II and aggrecan decreases. Transplantation of autologous chondrocytes cultured in vitro is used for treatment of aseptic articular cartilage lesions. For this purpose, cartilage biopsy is taken and isolated cells are subsequently proliferated in a monolayer cell culture. When implanted, the cells start to produce specific cartilaginous matrix that fills the defect. Prior to surgical procedure the cells can also be cryopreserved for longer periods of time after reaching appropriate numbers. We tested the influence of cultivation time and number of continuous culture passages as well as the influence of cryopreservation on the matrix protein synthesis of human articular chondrocytes. The ability of dedifferentiated chondrocytes to redifferentiate has been monitored by measuring matrix protein synthesis of the cells, re-seeded in agarose suspension culture. The results obtained show progressive dedifferentiation during monolayer cell culture procedures, facilitated by cryopreservation. Successful redifferentiation of cells re-seeded in suspension cultures was observed regardless of the previous level of chondrocyte dediferentiation.

Determination of optimal transport conditions for biopsies of human articular cartilage as well as for suspensions of cultured chondrocytes used in autologous transplantation.

Autologous transplantation of chondrocytes is currently being promoted as a novel approach for the treatment of deep cartilage lesions. Briefly, the method involves enzyme-mediated release of chondrocytes from cartilage biopsies, the expansion of cells by in vitro cultivation and their re-implantation into the defect. The success of this technique depends on many factors including transport conditions for both, cartilage biopsies from the operating hall to the laboratory and the return transport of final suspension of cultured chondrocytes. To determine the extent of cellular damage in biopsies, chondrocytes were enzymatically isolated following a few days of tissue preservation in different tissue culture media. The proportion of dead cells was assessed by Trypan blue staining and counting. The viability was not dependant of the type of the medium used and remained approximately 50% in all samples, even after 72 h. To develop optimal conditions for transport of final chondrocyte suspension, isolated cells were firstly grown in monolayer cultures. Cell suspensions in media with different additives were injected into special glass containers used for the transport and left at 4 degrees C or 25 degrees C for up to 3 days. During this period every 24 h the samples were taken and viability as well as apoptosis levels were assessed. Viability of cells in suspensions at 25 degrees C decreased significantly and became inadequate already after 48 h. In contrast to that, the proportion of viable cells at 4 degrees C remained above 80% even after 48 h. In the majority of the samples, culture medium containing serum and vitamin C provided the best conditions for long-term preservation of chondrocytes.