Raman imaging as a new analytical tool for the quality control of the monitoring of osteogenic differentiation in forming 3D bone tissue

Tissue engineering and stem cells technology are emerging fields in modern medicine, aiming to
produce living tissues in vitro to replace, regenerate or repair in vivo damaged tissues or organs.
In this study, adipose-derived stem cells (ASCs) are used to produce 3D bone tissue grafts [1,2].
The safety and the feasibility of use of these grafts has been demonstrated by several clinical
applications as it is largely described by Vériter et al. [2]. Here, Raman imaging is investigated as
a non-destructive and non-invasive method to monitor the synthesis of extracellular matrix by the
cells and its progressive mineralization during formation of an osteogenic tissue.
Raman imaging is a vibrational technique allowing to acquire spectral fingerprints of molecules
while visualizing their spatial distribution within the sample. The fast data acquisition time of this
technique further allows time-resolved analyses.
Here, Raman imaging is used for the first time to monitor extracellular matrix formation and
mineralization by human cells in a live 3D structure. Our attention was focused on Raman bands
related to this matrix, namely phosphate, phenylalanine and hydroxyproline, which are very
distinctive and intense [3]. Several batches of ASCs were cultured in a bone tissue differentiation
medium then sampled and analyzed using Raman imaging at different time points. From the
Raman spectra, mineral to organic matrix ratios (MTMR) were calculated from phosphate and
hydroxyproline signal intensities to evaluate the formation of mineral deposits accompanying
extra-cellular matrix synthesis which is indicative of an ongoing osteogenic differentiation
process [3]. Is was observed that these ratios peaked between day 35 and 49 but also the spatial
distribution of individual signal intensities vary in the forming 3D structures whilst maintaining a
same MTMR ratios at the end of the culture process. A study was conducted to evaluate the
influence of the position of the analyzed samples in the forming tissue in vitro to define a protocol
to acquire the Raman data for future analyses. Finally, the repeatability and the specificity of this
Raman imaging method were evaluated.
To conclude, Raman imaging allows a time-resolved and non-invasive monitoring in vitro of the
mineralization of extracellular matrix during osteogenic differentiation.