Immunogold detection of types I and II chondrocyte collagen fibrils: An in situ atomic force microscopic investigation

Chondrocyte tissue engineering is a major challenge in the field of cartilage repair. The phenotype of chondrocytes consists of cartilage specific proteoglycan and type II collagen. During serial passages, chondrocytes dedifferentiate into cells, presenting a fibroblast-like phenotype consisting predominately of type I collagen synthesis. Observation of native collagen fibers could be visualized by atomic force microscope. Here, we developed an original and useful atomic force microscopy-based immunogold technique allowing biochemical distinction between types I and II collagen fibers. Imaging of 40-nm gold particles staining collagen fibers was performed in tapping mode. Rat 1 fibroblasts and human chondrosarcoma cells were used as positive models for types I and II collagen, respectively. As demonstrated by our data, primary rat chondrocytes adhering for 48 h on a glass substrate synthesize type II collagen native fibers. This technique allows analyses of local areas of the extracellular matrix of fixed cells, providing complementary data about cartilage phenotype. This simple approach could be of major interest for the biologist community in routine laboratory investigations, to localize in situ, macromolecules of the extracellular matrix.