Introduction: Human autologous chondrocyte transfer requires a small biopsy of articular cartilage (300–500 mg wet weight) obtained by arthroscopy from the patient’s knee joint. Chondrocytes are isolated and seeded at low density in monolayer culture to increase cell number. A common problem with this technique is that chondrocytes lose their phenotype by reverting to a fibroblast phenotype and synthesize a different matrix. Collagen type II and aggrecans are unique to hyaline cartilage-matrix. They form an extensive three-dimensional network of extracellular matrix in which other cell adhesion and growth factor molecules are integrated. It has been shown that a three dimension environment coupled with growth factors are important for the maintenance of the chondrocyte phenotype. Although cells cultured in alginate beads maintain their phenotype they do not proliferate well.

Aim of study: To develop and optimise a bovine chondrocyte culture system as a model for optimising human chondrocyte proliferation without dedifferentiation and their future transplantation. The optimum cell density determined for bovine chondrocyte cultures was used for human chondrocyte cultures. Cell proliferation and matrix synthesis of cultured human chondrocytes from normal as well as damaged knee joint articular cartilage obtained from debridement arthroscopy was investigated.

Methods: Bovine chondrocytes were seeded in collagen type I gels at various densities ranging from 104 to 106 cells/ml to obtain the minimal cell density required in a collagen gel culture system in which chondrocytes can proliferate and yet retain their unique phenotype. The media were supplemented with either bovine foetal calf serum (FCS) or a combination of three growth factors (3GFs), TGF-b1 + IGF-I + b-FGF. Cells and matrix were analysed on day 7, 14, and 21 of culture. Cell proliferation was determined by the trypan blue exclusion test. Cell morphology and matrix present were evaluated with both light and electron microscopy. A collagen type II specific antibody coupled with FITC conjugate was used to detect type II collagen neo-deposit in relation to the seeded type I collagen gels. The newly synthesised matrix was monitored after labelling cells with 35S-sulphate and 3H-proline. The collagen type was determined by SDS-PAGE Fluorography. Analysis of morphology and matrix synthesis was performed as

Results: Cell proliferation: Bovine chondrocytes cultured in collagen type I gels at low density proliferated up to 40 fold after 3 weeks while high density cultures proliferated only about 3 fold. There was no significant difference in cell numbers at day 21 in cultures supplemented with FCS or 3GFs. Therefore all human chondrocyte cultures were cultured at low density. Preliminary results from human chondrocyte cultures were obtained from 4 patients aged 59+19. After 4 weeks, human chondrocytes cultured at low density supplemented with FCS proliferated up to 10 fold in monolayer culture and up to 4 fold in collagen type I gels. Morphology: At all cell densities, the majority of bovine chondrocytes in the gels remained rounded while some cells near the surface of the gels were elongated. Human chondrocytes cultured at low density also demonstrated similar morphology. Matrix synthesis: For bovine chondrocyte culture, after 2 weeks in culture more than 70% of 35S-sulphate and 3H-proline incorporated matrix

Conclusions: This study has shown that bovine chondrocytes cultured at low density in collagen type I gels proliferated better than at high density and retained their phenotype. This low-density bovine chondrocyte culture model is applicable to human chondrocyte culture in vitro. Preliminary results shows that human chondrocytes obtained from patients aged 39–72 can proliferate both in gels and monolayer. Age of chondrocytes and growth factors may affect the growth of cells. This model system needs to be further investigated in normal human chondrocytes.