Immunohistochemical and biochemical analyses of 20,000-25,000-year-old fossil cartilage.

A cracked, irregular pellicle adhering to fossilized bone excavated from the Enlène cave (Ariège) and estimated to date from 20,000-25,000 years BP was examined to verify its cartilaginous nature, suggested previously on the basis of optical and electron microscopic investigations. Immunolabeling of the organic component revealed the presence of type II and IX collagens, associated with residual glycosaminoglycans, in the external zone of the pellicle. The cartilaginous nature of the pellicle was also demonstrated by biochemical identification of type II collagen as the major protein in the demineralized sample: the amino acid compositions of insoluble and soluble fractions were similar to that of pure type II collagen; cyanogen-bromide-generated peptides, prepared after reduction of the sample, had an electrophoretic pattern similar to that of cyanogen bromide peptides derived from type II collagen. The amino acid sequences of four tryptic peptides were identical to the corresponding human type II sequences. It was impossible to isolate intact alpha chains. All of the solubilized fractions were composed of a wide range of low-molecular-mass peptides demonstrating significant degradation of the collagen molecules that was not reflected in the well-preserved fibrillar structure observed at the ultrastructural level. The mineral fraction, characterized by X-ray diffraction, consisted of apatite (as in sub-chondral bone) associated with contaminating poorly crystallized components originating from the cave sediment. Energy dispersive spectrometry showed that the cartilaginous zone contained three times less phosphorus and calcium than the underlying bone. These results confirm the cartilaginous nature of the sample and the preservation of tissue-specific components, and suggest that the process of fossilization is closely related to a mechanism of phosphatization.