Cartilage degenerative diseases like osteoarthritis affect the organization of the biological extracellular matrix
(ECM) surrounding chondrocytes. This ECM is mainly composed by collagen giving rise to a strong Second Harmonic
Generation (SHG) Signal, due to its high non linear susceptibility.
Mechanical stress leads to perturbation of the collagen network comparable to modification occurring in
disease. To be sure that SHG signal comes specifically from the collagen network, the enzymatical action of Collagenase
was followed. We clearly noted the decrease of the collagen specific signal according to incubation time due to
To characterize structural modification on the arrangement of collagen fibers in the ECM, we used image
analysis based on co-occurrence matrix (Haralick). Textural features give information like homogeneity ('Angular
Second Moment') or size of textural elements ('Inverse Difference Moment', 'Correlation'). Samples submitted to
compression are characterized by higher 'Correlation', associated with a decrease of 'IDM' and 'ASM'. Those
evolutions suggest the presence of long linear structures, an effect of packing of collagen fibrils and the apparition of
nodes where the density of collagen is important versus areas showing a lack of molecules.
Collagen I, II and VI are biomarkers characterising disease states since its presence is increased in pathological
cartilage (osteoarthritis). Fluorescence Lifetime Imaging Microscopy (FLIM) associated to Spectral and SHG analysis
confirmed the presence of Collagen I and II in the extracellular and Collagen VI in the pericellular matrix of
SHG, FLIM and Spectral Imaging combined with multiphoton excitation enable tissue imaging at deep
penetration. We pointed out a local modification of the ECM of cartilage without any labelling (SHG) under mechanical
stress. Thus the association of all these techniques represents a potential diagnosis tool for disorganization of collagen.