Localization of different alcian blue-proteoglycan particles in the intervertebral disc

A histochemical investigation was carried out on proteoglycans of bovine intervertebral disc. Samples obtained from the "annulus fibrosus" (A.F.) and "nucleus pulposus" (N.P.) were treated with Alcian blue (AB) diluted in solutions of MgCl2 at critical electrolyte concentrations (CEC); some samples were incubated in testicular hyaluronidase before AB treatment. At least four types of elongated AB-proteoglycan particles were recognized: a) in A.F. lamellae and N.P., 1 nm rod-like particles were arranged orthogonally to the collagen fibrils and spaced at a distance equivalent to the fibril D-period (Figs. 1-5); b) within the A.F. lamellae, other 16-20 nm particles formed a close network among the collagen fibrils (Figs. 1,2,3,5); c) in the A.F. interlamellar crevices, 30-50 nm leaf-like particles were present (Fig. 6); d) in the N.P.Z., 20-30 nm leaf-like particles formed a wide-mesh (Fig. 4). The alcianophylic particle sizes suggest they may correspond to proteoglycan monomers in the A.F. lamellae and mostly proteoglycan aggregates in A.F. interlamellar crevices and N.P.. Both alcianophylia degrees at MgCl2 CEC solutions and enzymatic susceptibility indicate the presence of chondroitin sulphate and keratan sulphate and that the large particles in the A.F. interlamellar crevices are the keratan sulphate richest proteoglycans. The features of the observed AB-proteoglycan particles are consistent with previous morphological data reported for other tissues as well as some biochemical data for the intervertebral disc and may be correlated to the composite mechanical properties of this tissue.