Investigation of articular cartilage structural and biomechanical properties by atomic-force microscopy

Abstract

Purpose: Although Osteoarthritis (OA) is a painful disease, which leads to a low quality of life for the patient as well as to a high socioeconomic burden, a functional cure remains elusive. Gaining new insights into the onset and progression of the disease is crucial but limited by the lack of biological tools able to display detailed structural and biomechanical properties of healthy and pathological cartilage. However, the cartilaginous ultrastructure and its mechanical properties are key factors for the overall functionality of the joint and should be considered as unique biomarkers of cartilage health status. The direct correlation of OA parameters, analyzed by histology and the OARSI scoring system, with cartilage structural and biomechanical properties assessed by atomic-force microscopy (AFM), will give new insights into the mechanisms underlying OA pathogenesis, into the impact of individual ECM molecules, and into disease progression. Methods: Paraffin embedded hind limbs, collected from euthanized wildtype mice (C57BL/6J), were dewaxed, re-hydrated and sectioned. AFM measurements were carried out using a BioScope Catalyst (Bruker) in combination with an inverse optical microscope (Zeiss LSM 150 Meta). For contact mode imaging and indentation-type AFM silicon nitride cantilevers with a nominal spring constant of 100 mN/m and integrated pyramidal tips with nominal radius of 20 nm were used. Images were recorded in air with a resolution of 512 × 512 pixels at a line rate of 1 Hz. After recording an overview image of the articular cartilage (AC), the scan area was reduced to obtain structural details within the ECM of the superficial layer. After AFM imaging, PBS at pH 7.4 was added to the sample for indentation measurements. Young's modulus was extracted from the approach force-distance curves using a modified Hertz model for a pyramidal indenter. Results: First measurements show that, with the help of AFM, we are able to image cartilage overall structure, collagen fibril orientation and D-band structure, as well as to measure tissue biomechanical properties on deparaffinized tissue sections. Indentation measurements demonstrate differences in the biomechanical properties of the outermost superficial layer to its adjacent middle zone as well as to the pericellular matrix of AC chondrocytes. Conclusions: Although cartilage biomechanical properties will change due to tissue fixation and paraffinization, our measurements on deparaffinized tissue sections are comparable to other AFM studies in the literature, which show similar trends in cartilage biomechanical properties, such as the reduced stiffness of the pericellular matrix of chondrocytes, in cartilage explants or cryo-sections. Our next step is to perform AFM measurements on different murine OA model (e.g. DMM and ageing models) using depraffinized tissue sections. The comparison of cartilage OARSI scoring and other staining methods such as IHC for OA markers, to the ultrastructure and biomechanical properties of cartilage using sections from the same animal and joint, will allow us to directly compare these parameters and gain new insights into the onset and progression of OA.