Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I
Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulin-like growth factor-I
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DOI：
10.1002/jor.1100170403
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发表时间：
1999-07-01
影响因子：
2.8
通讯作者：
Nixon, AJ
中科院分区：
文献类型：
--
作者：
Fortier, LA;Lust, G;Nixon, AJ
在外植体和单层研究中，已知向软骨培养物中加入胰岛素样生长因子-I可刺激蛋白聚糖和II型胶原的合成。本研究的目的是确定长期补充胰岛素样生长因子-I在纤维蛋白盘中培养的软骨细胞中的作用，作为对软骨细胞/胰岛素样生长因子-I/纤维蛋白移植物在关节软骨修复过程中的体内应用的初步调查。软骨细胞-纤维蛋白培养物维持14天，加入不同浓度的胰岛素样生长因子-I，浓度为0、10、50或100 ng/ml培养基。培养物补充50或100 ng/ml的生长因子增加了聚集蛋白聚糖和IIB型前胶原mRNA的水平，并翻译成聚集蛋白聚糖和IIB型胶原蛋白，证实了染料结合试验的总蛋白多糖，II型胶原蛋白免疫组化，并测定胶原蛋白含量的高效液相色谱法。通过常规染色的圆形细胞形态、原位杂交的II型前胶原mRNA表达、免疫细胞化学的细胞周II型胶原产生的证据、CL-2B柱层析的大分子大小聚集蛋白聚糖单体的合成以及北方印迹杂交的I型或IIA型胶原缺乏明显的信息表达，证实了在培养的14天期间软骨细胞表型的维持。胰岛素样生长因子-I对软骨细胞基质成分表达的剂量反应效应在每毫升培养基中50和100 ng生长因子时最明显。这些数据证实：（a）在纤维蛋白的三维培养物中长时间培养软骨细胞保持软骨细胞表型，和（B）补充增加浓度的胰岛素样生长因子-I增强软骨细胞基质合成，并可提供在移植过程中增强软骨细胞表型稳定性和功能的方法。
The addition of insulin-like growth factor-I to cartilage cultures is known to stimulate the synthesis of proteoglycan and type-II collagen in explant and monolayer studies. The purpose of this study was to determine the effects of long-term supplementation with insulin-like growth factor-I in chondrocytes cultured in fibrin discs as a preliminary investigation to in vivo application of chondrocyte/insulin-like growth factor-I/fibrin grafts to articular-cartilage repair procedures. Chondrocyte-fibrin cultures were maintained for 14 days, with insulin-like growth factor-I added at varying concentrations of 0, 10, 50, or 100 ng/ml medium. Cultures supplemented with 50 or 100 ng of growth factor/ml had increased levels of aggrecan and type-IIB procollagen mRNA, and translation to aggrecan and type-:IIB collagen was confirmed by dye-binding assay of total proteoglycan, type-II collagen immunohistochemistry, and determination of collagen content by highperformance liquid chromatography. Maintenance of the chondrocyte phenotype during the 14 days of culture was confirmed by round cell morphology on routine staining,expression of type-II procollagen mRNA on in situ hybridization, evidence of production of pericellular type-II collagen on immunocytochemistry, synthesis of large-molecular-size aggrecan monomer on CL-2B column chromatography, and lack of appreciable message expression for type I or IIA collagen on Northern blot hybridization. Dose-response effects of insulin-like growth factor-I on the expression of chondrocyte matrix constituents were most pronounced at 50 and 100 ng of growth factor per milliliter of medium. These data confirm that (a) culture of chondrocytes for extended periods in three-dimensional cultures of fibrin maintains the chondrocyte phenotype and (b) supplementation with increasing concentrations of insulin-like growth factor-I enhances chondrocyte matrix synthesis and may provide a means to enhance chondrocyte phenotypic stability and function during transplantation grafting procedures.