Molecular Microscopy, Chalmers University of Technology, Göteborg, Sweden.
Tissue Eng Part C Methods. 2012 Mar;18(3):227-34. doi: 10.1089/ten.TEC.2011.0211. Epub 2011 Dec 9.
Microscopy techniques based on laser-induced nonlinear optical processes allow for chemically specific imaging of unmodified samples at high spatial resolution in three dimensions and provide powerful tools for characterization of tissue-engineering constructs. This is highlighted by the simultaneous imaging of scaffold material, cells, and produced extracellular matrix collagen in samples consisting of osteoprogenitor MC3T3-E1 cells seeded on microporous bacterial cellulose (BC), a potential scaffold material for synthesis of osseous tissue. BC and collagen have been visualized by second harmonic generation (SHG) microscopy, and verification of collagen identification on cellulose scaffolds has been carried out on sectioned samples by comparison with the conventional histological staining technique. Both methods showed similar collagen distributions and a clear increase in the amount of collagen when comparing measurements from two time points during growth. For investigations of intact cellulose scaffolds seeded with cells, SHG was combined with simultaneous coherent anti-Stokes Raman scattering (CARS) microscopy for visualization of cell arrangement in three dimensions and to be correlated with the SHG data. Results showed that the osteoprogenitor cells were able to produce collagen already during the first days of growth. Further on, developed collagen fiber networks could be imaged inside compact regions of cells located in the cellulose micropores. Collagen production, the initial step of tissue mineralization, demonstrates the potential of BC as a scaffold material for bone tissue engineering. Furthermore, the noninvasive in situ monitoring of collagen inside compact tissue clearly manifests the benefits of nonlinear microscopy techniques, such as SHG and CARS, for use in tissue engineering.
基于激光诱导非线性光学过程的显微镜技术允许在三维空间以高空间分辨率对未经修饰的样品进行化学特异性成像，并为组织工程构建体的表征提供了强大的工具。这在同时对由接种在微孔细菌纤维素（BC）上的成骨前体细胞 MC3T3-E1 细胞组成的样品中的支架材料、细胞和产生的细胞外基质胶原蛋白进行成像中得到了强调，BC 是合成骨组织的潜在支架材料。BC 和胶原蛋白已通过二次谐波产生（SHG）显微镜进行了可视化，并且通过与传统组织学染色技术比较对纤维素支架上的胶原蛋白鉴定进行了验证。两种方法均显示出相似的胶原蛋白分布，并且在生长过程中的两个时间点进行比较时，胶原蛋白的数量明显增加。对于用细胞接种的完整纤维素支架的研究，SHG 与同时的相干反斯托克斯拉曼散射（CARS）显微镜相结合，用于三维细胞排列的可视化，并与 SHG 数据相关联。结果表明，成骨前体细胞在生长的最初几天就能产生胶原蛋白。此外，还可以在位于纤维素微孔内的细胞的致密区域中成像已开发的胶原蛋白纤维网络。胶原蛋白的产生是组织矿化的初始步骤，证明了 BC 作为骨组织工程支架材料的潜力。此外，在紧凑组织内对胶原蛋白的非侵入性原位监测清楚地表明了 SHG 和 CARS 等非线性显微镜技术在组织工程中的优势。
