Kunstar A. - Recognizing different tissues in human fetal femur cartilage by label-free Raman microspectroscopy

Aliz Kunstar, Jeroen Leijten, Stefan van Leuveren, Janneke Hilderink, Cees Otto, Clemens A. van Blitterswijk, Marcel Karperien, and Aart A. van Apeldoorn

Journal of Biomedical Optics 2012


Traditionally, the composition of bone and cartilage is determined by standard histological methods. We
used Raman microscopy, which provides a molecular “fingerprint” of the investigated sample, to detect differences
between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area
scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate
and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral
datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of
individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and
was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/
ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward’s clustering method, and principal
component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of
all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms
and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous
zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and
clinical purposes