The discrimination of type I and type II collagen and the label-free imaging of engineered cartilage tissue.

Using excitation polarization-resolved second harmonic generation (SHG) microscopy, we measured SHG intensity as a function of the excitation polarization angle for type I and type II collagens. We determined the second order susceptibility (χ((2))) tensor ratios of type I and II collagens at each pixel, and displayed the results as images. We found that the χ((2)) tensor ratios can be used to distinguish the two types of collagen. In particular, we obtained χ(zzz)/χ(zxx) = 1.40 ± 0.04 and χ(xzx)/χ(zxx) = 0.53 ± 0.10 for type I collagen from rat tail tendon, and χ(zzz)/χ(zxx) = 1.14 ± 0.09 and χ(xzx)/χ(zxx) = 0.29 ± 0.11 for type II collagen from rat trachea cartilage. We also applied this methodology on the label-free imaging of engineered cartilage tissue which produces type I and II collagen simultaneously. By displaying the χ((2)) tensor ratios in the image format, the variation in the χ((2)) tensor ratios can be used as a contrast mechanism for distinguishing type I and II collagens.

Multiphoton imaging and quantitative analysis of collagen production by chondrogenic human mesenchymal stem cells cultured in chitosan scaffold.

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.