Department of Physics, National Taiwan University, Taipei, Taiwan.
Tissue Eng Part C Methods. 2010 Oct;16(5):913-20. doi: 10.1089/ten.TEC.2009.0596.
We used the combined imaging modality of multiphoton autofluorescence and second-harmonic generation microscopy to investigate the chondrogenic process of human mesenchymal stem cells cultured in chitosan scaffold. Isolated human mesenchymal stem cells seeded onto chitosan scaffold were induced to undergo chondrogenesis by addition of the transforming growth factor-β3. After continuous culturing, the engineered tissues at the same scaffold location were imaged at different time points for up to 49 days. Using the acquired images of the chondrogenic process, we quantify tissue morphogenesis by monitoring the changes in multiphoton autofluorescence and second-harmonic generation signals from the engineered tissues. We found that the extracellular matrix generation can be modeled by an exponential function during the initial growth stage and that saturation occurs between days 11 and 14. Further, the growth rate of the extracellular matrix was found to increase toward the surface of the chitosan scaffold. Our work demonstrates the use of multiphoton microscopy for performing long-term monitoring and quantification of the tissue engineering process.
我们使用多光子自发荧光和二次谐波产生显微镜的组合成像方式来研究在壳聚糖支架中培养的人骨髓间充质干细胞的软骨生成过程。将分离的人骨髓间充质干细胞接种到壳聚糖支架上，通过添加转化生长因子-β3 来诱导其发生软骨生成。连续培养后，在相同支架位置的工程组织在长达 49 天的不同时间点进行成像。通过对软骨生成过程的获取图像，我们通过监测来自工程组织的多光子自发荧光和二次谐波产生信号的变化来定量组织形态发生。我们发现，在初始生长阶段，细胞外基质的生成可以用指数函数来建模，并且在第 11 天到第 14 天之间达到饱和。此外，还发现细胞外基质的生长速度朝着壳聚糖支架的表面增加。我们的工作证明了多光子显微镜在进行组织工程过程的长期监测和定量方面的应用。
