Fig. 2.

Alginate beads culture and production of 3D-cultured constructs. a) Alginate bead culture. ATDC5 cells were cultured in alginate beads with growth medium containing insulin-transferrin-selenium and BMP-2 for 6 days. Toluidine blue staining showed increased cartilage matrix synthesis, especially as proteoglycan in the alginate beads. b) After dissolving with sodium citrate and Na₂EDTA, cells were seeded within collagen gels and incorporated into type-I collagen scaffold (Koken, Osaka, Japan) using a 27-gauge needle syringe. ATDC5 cells were distributed within the scaffold. c) Collagen type-II mRNA expression in the scaffold was significantly increased by the addition of recombinant BMP-2. d) In toluidine blue staining of 3D-cultured constructs, extracellular matrix was stained violet. e) In immunostaining, positive cells for the cartilage matrix components chondroitin 4-sulfate and keratan sulfate were present