Otani Yushi, Komura Makoto, Komura Hiroko, Ishimaru Tetsuya, Konishi Kenichiro, Komuro Hiroaki, Hoshi Kazuto, Takato Tsuyoshi, Tabata Yasuhiko, Iwanaka Tadashi
1 Department of Pediatric Surgery, Graduate School of Medicine, University of Tokyo , Tokyo, Japan .
Tissue Eng Part A. 2015 Feb;21(3-4):627-36. doi: 10.1089/ten.TEA.2013.0655. Epub 2015 Jan 20.
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.
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.
(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.
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.
含缓慢释放碱性成纤维细胞生长因子（b-FGF）的明胶海绵可增强软骨形成。本研究探讨明胶海绵中b-FGF的最佳含量以制备工程化软骨。
制备含β-磷酸三钙（β-TCP）且浸渍有b-FGF（0、10、100、500、1000和2000μg/cm³）的明胶海绵。从C57B6J小鼠的耳廓软骨中分离软骨细胞并进行扩增。将扩增后的耳廓软骨细胞（10×10⁶个细胞/cm³）接种到用作支架的明胶海绵上。构建体组件通过同基因方式植入小鼠皮下空间。此后，在2、4或6周时取出构建体。
（1）形态学：在植入后4周和6周时，植入构建体的尺寸大于含500、1000和2000μg/cm³ b-FGF且浸渍有β-TCP的明胶海绵支架的尺寸。（2）构建体重量：在植入后2、4和6周时，构建体重量的增加大致与b-FGF浸渍支架体积的增加成比例，但2000μg/cm³ b-FGF浸渍构建体组除外。（3）组织学检查：在植入后4周时，在浸渍有超过100μg/cm³ b-FGF的明胶海绵中观察到构建体中心的细胞外基质。构建体中具有丰富细胞外基质的细胞区域对软骨特异性标志物Ⅱ型胶原呈阳性。（4）蛋白质测定：在植入浸渍有超过100μg/cm³ b-FGF的明胶海绵后4周和6周时，糖胺聚糖和Ⅱ型胶原表达显著增加。在植入后6周时，浸渍有100μg/cm³或更多b-FGF的构建体中Ⅱ型胶原与Ⅰ型胶原的比例高于小鼠耳廓软骨中的比例。
浸渍有超过100μg/cm³ b-FGF并结合β-TCP以及软骨细胞（10×10⁶个细胞/cm³）的明胶海绵在植入后4周可制备工程化软骨。
