Imaging articular cartilage tissue using atomic force microscopy (AFM)

Cartilage is a complex avascular tissue composed of cells ("chondrocytes") embedded in an extracellular matrix (ECM) consisting of 70%-80% water. The primary components of the ECM are negatively charged aggrecans and collagen II fibrils, which possess a characteristic, ordered three-dimensional structure. The components interact to ensure that the cartilage is able to absorb shock and can function to protect the bone ends. Atomic force microscopy (AFM) can be used to examine structure-function relationships of cartilage at both micrometer and nanometer scales. When imaged at the micrometer scale with microspheres, only the ECM and chondrocytes can be distinguished. Correspondingly, mechanical testing of cartilage at the micrometer scale results in unimodal distribution of the stiffness because the bulk elastic property of the ECM is probed. In contrast, bare AFM tips are able to reveal the molecular components of the ECM at the nanometer scale. Mechanical testing at the nanometer scale reveals a bimodal distribution of the stiffness and reflects the distinct stiffness of the collagen network and the proteoglycan moiety. In this protocol, the corresponding AFM image and force map are used to reveal the distinct morphology of the collagen fibers and proteoglycan gel. Although, in principle, these experiments can be performed using any AFM, an AFM with tube scanners that have manual screws for tilting the sample is preferable because cartilage has macroscopically rough surface features. By manually tilting the probe over the sample, an optimal angle for tip approach can be achieved.