Aim: The healing capacity of human articular cartilage is very limited in the adult. Therefore tissue engineering techniques were developed to treat cartilage lesions. To it, autologous chondrocytes are harvested from the affected joint and expanded in vitro. During expansion chondrocytes may dedifferentiate, characterized by an increase in type I collagen and a decrease in type II collagen expression. Since high expression of type II collagen is of central importance for the properties of cartilage after transplantation, we investigated if the human platelet supernatants (hPS) containing PDGF and TGF-b or recombinant human bone morphogenetic protein-2 (BMP-2) may modulate the chondrogenic phenotype in monolayer cell cultures (2D) and in three-dimensional culture (3D) systems.

Methods: Chondrocytes from articular knee cartilage of 14 individuals (mean age 36.5 6.5 years) with no history of inflammatory joint disease were isolated and expanded under GMP conditions suitable for clinical purposes. The hPS was prepared from blood of 3 donors and pooled. Cells were seeded either in 2D cultures or embedded in alginate beads (3D) in presence or absence of hPS or recombinant human BMP-2 (generous gift of Dr. Hortschansky, Jena, FRG). After two weeks in culture, cells were harvested and analysis of the chondrogenic phenotype was performed using quantitative RT-PCR, immunocytochemistry and ELISA methods.

Results: Expansion of chondrocytes in primary culture (P0) did not yield populations of dedifferentiated or hypertrophic cells. Expanding cells in first subculture (P1) resulted in spontaneous reduction of type II collagen expression and increase in type I collagen mRNA amounts. Seeding P1 chondrocytes in 3D culture significantly reduced type I collagen, BMP-4 and IL-18 and maintained high type II collagen and BMP-2 encoding mRNA (p < 0.05). Reduction of IL-1b and elevation of IL-10 mRNA were noted but were statistically not significant. Addition of BMP-2 to 2D chondrocytes had no effect on type II collagen or IL-1b mRNA amounts (p < 0.05). In alginate cultures BMP-2 induced type II collagen and reduced IL-1b mRNA amounts. In contrast, addition of hPS containing PDGF and TGF-b, promoted mitotic activity in 2D and alginate cultures. The hPS reduced in 2D cultures type II and induced type I collagen expression. Even in alginate beads induction of type I collagen was detected.

Conclusions: We conclude that the chondrogenic phenotype is stabilized by BMP-2 more effectively in alginate beads but not in monolayer cultures. The hPS promotes proliferation of chondrocytes in vitro but induces elevated type I expression, an indicator of chondrocyte dedifferentiation.