Interest of Second Harmonic Generation imaging to study collageneous matrix modification in osteoarthritis disease

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 enzymatic degradation. 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 chondrocytes. 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.