Optimal Amount of Basic Fibroblast Growth Factor in Gelatin Sponges Incorporating β-Tricalcium Phosphate with Chondrocytes

Background: A gelatin sponge with slowly releasing basic fibroblast growth factor (b-FGF) enhances chondrogenesis. This study investigated the optimal amount of b-FGF in gelatin sponges to fabricate engineered cartilage. Materials and Methods: b-FGF (0, 10, 100, 500, 1000, and 2000 mu g/cm(3))-impregnated gelatin sponges incorporating beta-tricalcium phosphate (beta-TCP) were produced. Chondrocytes were isolated from the auricular cartilage of C57B6J mice and expanded. The expanded auricular chondrocytes (10x10(6) cells/cm(3)) were seeded onto the gelatin sponges, which served as scaffolds. The construct assembly was implanted in the subcutaneous space of mice through a syngeneic fashion. Thereafter, constructs were retrieved at 2, 4, or 6 weeks. Results: (1) Morphology: The size of implanted constructs was larger than the size of the scaffold with 500, 1000, and 2000 mu g/cm(3) b-FGF-impregnated gelatin sponges incorporating beta-TCP at 4 and 6 weeks after implantation. (2) The weight of the constructs increased roughly proportional to the increase in volume of the b-FGF-impregnated scaffold at 2, 4, and 6 weeks after implantation, except in the 2000 mu g/cm(3) b-FGF-impregnated constructs group. (3) Histological examination: Extracellular matrix in the center of the constructs was observed in gelatin sponges impregnated with more than 100 mu g/cm(3) b-FGF at 4 weeks after implantation. The areas of cells with an abundant extracellular matrix were positive for cartilage-specific marker type 2 collagen in the constructs. (4) Protein assay: Glycosaminoglycan and collagen type 2 expression were significantly increased at 4 and 6 weeks on implantation of gelatin sponges impregnated with more than 100 mu g/cm(3) b-FGF. At 6 weeks after implantation, the ratio of type 2 collagen to type 1 collagen in constructs impregnated with 100 mu g/cm(3) or more b-FGF was higher than that in mice auricular cartilage. Conclusion: Gelatin sponges impregnated with more than 100 mu g/cm(3) b-FGF incorporating beta-TCP with chondrocytes (10x10(6) cells/cm(3)) can fabricate engineered cartilage at 4 weeks after implantation.