Optimal amount of basic fibroblast growth factor in gelatin sponges incorporating β-tricalcium phosphate with chondrocytes

Abstract

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 μg/cm(3))-impregnated gelatin sponges incorporating β-tricalcium phosphate (β-TCP) were produced. Chondrocytes were isolated from the auricular cartilage of C57B6J mice and expanded. The expanded auricular chondrocytes (10×10(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 μg/cm(3) b-FGF-impregnated gelatin sponges incorporating β-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 μg/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 μg/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 μg/cm(3) b-FGF. At 6 weeks after implantation, the ratio of type 2 collagen to type 1 collagen in constructs impregnated with 100 μg/cm(3) or more b-FGF was higher than that in mice auricular cartilage.

Conclusion: Gelatin sponges impregnated with more than 100 μg/cm(3) b-FGF incorporating β-TCP with chondrocytes (10×10(6) cells/cm(3)) can fabricate engineered cartilage at 4 weeks after implantation.

FIG. 1.

Photographs of the constructs at 2, 4, or 6 weeks after implantation of a scaffold with 0, 10, 100, 500, 1000, or 2000 μg/cm³ b-FGF -impregnated gelatin sponge incorporating β-TCP. At 2 weeks after implantation, constructs were covered by white fibrous tissue and the size of the implanted constructs was approximately the same as the size of the scaffolds. At 4 weeks and 6 weeks after implantation, the size of the implanted constructs was larger than the size of the scaffold with 500, 1000, and 2000 μg/cm³ b-FGF-impregnated gelatin sponges incorporating β-TCP. b-FGF, basic fibroblast growth factor; β-TCP, β-tri-calcium-phosphate. Color images available online at www.liebertpub.com/tea

FIG. 2.

Weight of the constructs at 2, 4, or 6 weeks after implantation of a scaffold with 0, 10, 100, 500, 1000, or 2000 μg/cm³ b-FGF -impregnated gelatin sponge incorporating β-TCP. At 2 weeks after implantation (a), the weight of the constructs increased roughly proportional to the volume of the b-FGF-impregnated scaffold. These weights were maintained at 4 weeks (b) and 6 weeks (c) after implantation except in the 2000 μg/cm³ b-FGF-impregnated constructs group. Color images available online at www.liebertpub.com/tea

FIG. 3.

Microscopic images of the constructs. At 2 weeks after implantation, the presence of metachromasia and chondrogenic tissue on the surface of the engineered constructs was confirmed in the 100 μg/cm³ or more b-FGF-impregnated constructs groups. There was a mucinous solution in the middle portion of the engineered constructs in the 2000 μg/cm³ impregnated constructs group. Extracellular matrix in the center of the constructs was observed using gelatin sponges impregnated with 100 μg/cm³ or more b-FGF at 4 weeks after implantation. Color images available online at www.liebertpub.com/tea

FIG. 4.

Immunohistochemical staining findings at 6 weeks (a) Type 1 collagen staining, (b) Type 2 collagen staining, and (c) Type 10 collagen staining. At 6 weeks after implantation, the circumference of the engineered cartilage matrix was positive for type 1 collagen. The amount of impregnated b-FGF did not affect the area that was positive for type 1 collagen. The areas of cells with abundant extracellular matrix were positive for cartilage-specific marker type 2 collagen in the constructs. A few areas of collagen type 10 expression were observed in each group impregnated with 1000 μg/cm³ or more b-FGF at 6 weeks.

FIG. 5.

Glycosaminoglycan (GAG) expression in the engineered constructs at 2, 4, and 6 weeks after implantation (a) 2 weeks, (b) 4 weeks, and (c) 6 weeks. GAG expression was significantly higher in the groups impregnated with 100 μg/cm³ or more b-FGF than in the non-b-FGF-impregnated group at 4 and 6 weeks after implantation. Furthermore, the level of glycosaminoglycan expression in each group impregnated with 100 μg/cm³ or more b-FGF at 4 and 6 weeks after implantation was higher than that in auricular cartilage of 4-week-old mice, which served as the control. Color images available online at www.liebertpub.com/tea

FIG. 6.

Collagen type 2 expression in the engineered constructs at 2, 4, and 6 weeks after implantation (a) 2 weeks, (b) 4 weeks, and (c) 6 weeks. The level of collagen type 2 expression was also significantly higher in each group impregnated with 500 μg/cm³ or more b-FGF than in the non-b-FGF-impregnated group at 4 weeks after implantation. At 6 weeks after implantation, it was significantly higher in each group impregnated with 100 μg/cm³ or more b-FGF than in the non-b-FGF-impregnated group. The level of collagen type 2 expression in the auricular cartilage of normal mice, which served as a control, is lower than that in each group impregnated with 100 μg/cm³ or more b-FGF at 4 weeks and 6 weeks after implantation. Color images available online at www.liebertpub.com/tea

FIG. 7.

The ratio of type 2 collagen to type 1 collagen at 6 weeks after implantation. The ratio of type 2 collagen to type 1 collagen in native auricular cartilage, which served as the control, was around 0.4. The ratio in 100 μg/cm³ or more b-FGF-impregnated constructs was higher than that in native auricular cartilage. The ratio of type 2 collagen to type 1 collagen was positively correlated with the amount of impregnated b-FGF.