Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials
Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials
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DOI：
10.1002/jbm.b.30648
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发表时间：
2007-04-01
影响因子：
3.4
通讯作者：
Hartmann, Daniel Jean
中科院分区：
文献类型：
--
作者：
Heinrich, Laurence;Freyria, Anne-Marie;Hartmann, Daniel Jean
这项工作的目的是在体外研究目前用于骨和软骨修复的两种生物材料的细胞定植，这一步对于了解工程组织的功能非常重要。目前使用组织学方法的方法并不总是适合组织工程分析。因此，我们建立了一个协议，以评估细胞分布，利用非侵入性共聚焦显微镜和荧光标记与远红发射波长，以优化支架的透明度和最小化光散射。硬（陶瓷替代物）和软（胶原海绵）生物材料分别接种，在支架的一侧，与人成纤维细胞和牛软骨细胞标记的碳菁染料（DiD和DiR）。DiR标记的成纤维细胞和软骨细胞在两种支架中的平均穿透深度约为270 μ m，大于DiD标记的细胞（136-218 μ m）。这些深度与细胞来源无关，但受支架性质的影响。胶原海绵是透明的，与陶瓷替代品相比，陶瓷替代品只能在开放的大孔中进行测量。除了设备的限制外，支架的限制还包括胶原海绵的扩散和陶瓷替代物的传输。因此，共聚焦显微镜技术可以用于解决多孔生物材料的细胞定植的问题，在一个非侵入性的方式。(c)2006 Wiley Periodicals，Inc.
The aim of this work was to study, in vitro, cell colonization of two biomaterials currently used for bone and cartilage repair, this step being important to understand the function of engineered tissues. Current methods that use histological approaches are not always suited to tissue-engineering analysis. We, therefore, set up a protocol to assess cell distribution, utilizing noninvasive confocal microscopy and fluorescent labels with a far red emission wavelength to optimize scaffold transparency and minimize light scattering. Hard (ceramic substitute) and soft (collagen sponge) biomaterials were seeded respectively, on one side of the scaffold, with human fibroblasts and bovine chondrocytes labelled with carbocyanine dyes (DiD and DiR). The mean penetration depth for DiR labelled fibroblasts and chondrocytes in the two scaffolds, around 270 mu m, was greater than for DiD (136-218 mu m) labelled cells. These depths were independent of cell origin but were influenced by the nature of the scaffolds. Collagen sponge is transparent in contrast to ceramic substitutes where measurements could only be made in opened macropores. Besides the limits of the equipment, the limits of the supports were diffusion for collagen sponges and transmission for ceramic substitutes. Confocal microscopy techniques could thus be used to address the question of cell colonization of porous biomaterials in a noninvasive manner. (c) 2006 Wiley Periodicals, Inc.