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In this study, Atomic Force Microscopy (AFM) was used to examine nano-scale structure difference in superficial and intermediate layers of bovine cartilage under dehydrated and hydrated conditions. The nanostructure of cartilage was quantitatively described using collagen fibril D-periodic spacing, fibril diameter, and surface roughness. Cartilage collagen fiber distribution was inhomogeneous in dehydrated condition but more homogeneous in hydrated condition. AFM images showed that the cartilage in dehydrated condition has higher roughness than that in hydrated condition. This study indicates that water is an important factor in maintaining the structure of extracellular matrix of cartilage. The loss of water can directly result in a rougher structure both on surface and bulk of articular cartilage. The morphology change of hydration-dehydration processes in cartilage is reversible.