Insulin-like growth factors (IGFs) plays various roles, including differentiation and mitogenesis, and IGFs are reported to regulate the bone growth and maintenance. This study was performed to analyze the enhancing effects of IGF-2 on osteogenic differentiation and the mineralization of stem cells cultured on deproteinized bovine bone mineral. Stem cell loaded bone graft material was cultured in the presence of the IGF-2 at final concentrations of 10 and 100 ng/mL and the morphology of the cells was observed on Days 1, 3, and 7. The commercially available, two-color assay based on plasma membrane integrity and esterase activity was also used for qualitative analyses on Days 1, 3, and 7. The level of alkaline phosphatase activity and anthraquinone dye assay were used to evaluate osteogenic differentiation on Days 7 and 14. Real-time polymerase chain reaction was applied in order to identify the mRNA expression of BGLAP, Runx2, and β-catenin. The stem cells were well-attached with fibroblast morphology and most of the stem cells produced a high intensity of green fluorescence, indicating that there were live cells on Day 1. The relative cellular viability assay values for IGF-2 groups at 0, 10, and 100 ng/mL on Day 1 were 0.419 ± 0.015, 0.427 ± 0.013, and 0.500 ± 0.030, respectively (p < 0.05). The absorbance values at 405 nm for alkaline phosphatase activity on Day 7 for IGF-2 at 0, 10, and 100 ng/mL were 2.112 ± 0.152, 1.897 ± 0.144, and 2.067 ± 0.128, respectively (p > 0.05). The mineralization assay results at Day 7 showed significantly higher values for IGF-2 groups at 10 and 100 ng/mL concentration when compared to the control (p < 0.05). The application of IGF-2 groups of 10 and 100 ng/mL produced a significant increase of BGLAP. Conclusively, this study indicates that the use of IGF-2 on stem cell loaded bone graft increased cellular viability, Alizarin red staining, and BGLAP expression of stem cells. This report suggests the combined approach of stem cells and IGF-2 with scaffold may have synergistic effects on osteogenesis.

中文翻译：

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胰岛素样生长因子2在脱蛋白牛骨矿物上培养的干细胞的成活谱系和成骨谱系和矿化中对细胞活力和分化的作用

胰岛素样生长因子（IGF）发挥各种作用，包括分化和有丝分裂，据报道，IGF可以调节骨骼的生长和维持。进行这项研究以分析IGF-2对成骨细胞分化和在脱蛋白牛骨矿物质上培养的干细胞矿化的增强作用。在IGF-2存在下以10和100 ng / mL的最终浓度培养负载干细胞的骨移植材料，并在第1、3和7天观察细胞的形态。市售的双色在第1、3和7天也使用了基于质膜完整性和酯酶活性的检测方法进行定性分析。在第7和14天，使用了碱性磷酸酶活性水平和蒽醌染料检测方法来评估成骨分化。应用实时聚合酶链反应以鉴定BGLAP，Runx2和β-catenin的mRNA表达。干细胞与成纤维细胞形态紧密结合，大多数干细胞产生高强度的绿色荧光，表明在第1天有活细胞。IGF-2组在0、10，第1天的100 ng / mL和100 ng / mL分别为0.419±0.015、0.427±0.013和0.500±0.030（p＜0.05）。在第7天，IGF-2在0、10和100 ng / mL的碱性磷酸酶活性在405 nm处的吸光度值分别为2.112±0.152、1.897±0.144和2.067±0.128（p> 0.05）。与对照组相比，在第7天的矿化试验结果显示，在10和100 ng / mL浓度下，IGF-2组的值明显更高（p <0.05）。使用10和100 ng / mL的IGF-2组可显着提高BGLAP。结论是，这项研究表明在负载干细胞的骨移植物中使用IGF-2可以提高细胞活力，茜素红染色和干细胞BGLAP的表达。该报告表明干细胞和IGF-2与支架的联合方法可能对成骨作用具有协同作用。