Center for Biomaterials, University of Connecticut Health Center, Farmington, Connecticut 06030-1615, USA.
Tissue Eng Part C Methods. 2010 Dec;16(6):1357-66. doi: 10.1089/ten.TEC.2009.0701. Epub 2010 May 4.
Transgenic mice with a Col1a1-promoter-driven transgene pOBCol2.3GFP were previously developed to visually identify mature osteoblasts through fluorescent expression. Our goal was to determine if this technology could be used to nondestructively evaluate the in vitro differentiation of osteoprogenitor cells on biomaterials such as biomimetic carbonated hydroxyapatite (cHA). Primary osteoprogenitor cells were harvested from calvaria of neonatal Col2.3GFP transgenic mice and cultured on cHA and a tissue culture polystyrene (TCPS) control. The distribution of intensities and area percentage of green fluorescent protein (GFP)-positive cells were quantified using fluorimetry and image analysis of fluorescent microscopy. At 14 days, an increased area and higher mean intensity of GFP-positive cells was observed on cHA as compared to TCPS, indicating more rapid differentiation on cHA. Notably, there were large continuous regions of GFP-positive osteoblasts on cHA, in contrast to the sparse, nodules of osteoblasts on TCPS, implying that cHA provides an osteogenic cue to cells. Xylenol orange staining was capable of distinguishing osteoblast-initiated mineral from the cHA substrate. With this method the unique pattern of osteoblast differentiation on cHA was clearly observed for the first time. Importantly, the generalized method can be used for rapid, high-throughput, nondestructive screening of biomaterials intended to enhance osteogenic differentiation.
先前开发了一种带有 Col1a1 启动子驱动的转基因 pOBCol2.3GFP 的转基因小鼠，通过荧光表达来直观地识别成熟的成骨细胞。我们的目标是确定这项技术是否可用于非破坏性地评估成骨细胞前体细胞在生物材料（如仿生碳酸化羟基磷灰石（cHA））上的体外分化。从新生 Col2.3GFP 转基因小鼠的颅骨中采集原代成骨细胞前体细胞，并在 cHA 和组织培养聚苯乙烯（TCPS）对照物上进行培养。通过荧光计和荧光显微镜的图像分析，对绿色荧光蛋白（GFP）阳性细胞的强度和面积百分比的分布进行定量。在第 14 天，与 TCPS 相比，cHA 上 GFP 阳性细胞的面积增加且平均强度更高，表明 cHA 上的分化更快。值得注意的是，与 TCPS 上稀疏的成骨细胞结节相比，cHA 上有连续的 GFP 阳性成骨细胞大片区，这表明 cHA 为细胞提供了成骨信号。二甲氧唑橙染色能够区分成骨细胞起始的矿化与 cHA 基质。通过这种方法，首次清晰地观察到 cHA 上独特的成骨细胞分化模式。重要的是，这种通用方法可用于快速、高通量、非破坏性地筛选旨在增强成骨细胞分化的生物材料。
