Raman imaging as a new analytical method to monitor osteogenic differentiation in forming bone tissue

Charlotte De Bleye, Pharmacy, University of Liege, Liege, Belgium (cdebleye@uliege.be)
Marie Fontaine, Novadip Biosciences, Mont-saint-guibert, Belgium
Pierre-Yves Sacre, Pharmacy, University Of Liege, Liege, Belgium
Philippe Hubert, Pharmacy, University Of Liege, Liege, Belgium
Nicolas Theys, Novadip Biosciences, Mont-saint-guibert, Belgium
Eric Ziemons, Pharmacy, University Of Liege, Liege, Belgium

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.

[1] H.A. Declercq et al., Biomaterials, 34 2013, 1004-1017.
[2] S. Veriter et al., PLoS ONE, 10 2015, 1-18.
[3] K. Esmonde-White et al. Boca Raton: Taylors & Francis Group, 2014, 59-71.

---

Abstract Reference & Short Personal Biography of Presenting Author

Charlotte De Bleye was graduated in Pharmaceutical Sciences at the University of Liege in 2011. Thereafter, she had a grant holder form the F.R.S.-FNRS and started a Ph.D. thesis dealing with the investigation of quantitative performances of surface-enhanced Raman scattering (SERS) in the laboratory of Pharmaceutical Analytical Chemistry (LCAP) of Professor Philippe Hubert. She received her Ph.D. in Biomedical and Pharmaceutical Sciences in 2016. Nowadays, she is still working in the LCAP and her research is focused on SERS and Raman imaging on pharmaceutical and biological materials.