A biochemical and immuno-electron microscopical analysis of chondroitin sulphate-rich proteoglycans in human alveolar bone A. J. SMITH 1 , S. K. SINGHRAO 2 , G. R. NEWMAN 2 , R. J. WADDINGTON 3 and G. EMBERY 3 1 Department of Adult Dental Care, Glasgow Dental School, Glasgow, UK 2 Electron Microscopy Unit, University of Wales College of Medicine, Heath Park, Cardiff, UK, and 3 Department of Basic Dental Science, Cardiff Dental School, Heath Park, Cardiff, UK Received 5 January 1996 and in revised form 6 September 1996 Summary This study used biochemical and immunohistochemical methods to characterize the chondroitin sulphate-rich proteoglycans from human alveolar bone obtained from an oral source. Proteoglycans were extracted from bone by a sequential 4 M guanidine HCl extraction process, and purified by DEAE-ion exchange chromatography. SDS-PAGE and Western blot analysis, using CS-56 monoclonal antibody, demonstrated one major proteoglycan species with a core protein of 58 kDa, glycosaminoglycan chains of 45–66 kDa and a mean molecular weight of 205 kDa. This work confirmed the biochemistry of chondroitin sulphate-rich proteoglycans from a novel source of adult human alveolar bone, and pointed towards a proteoglycan with a high glutamate, glycine, aspartate, alanine, serine and leucine content. Sections of alveolar bone were embedded in LR White resin, labelled with CS-56 antibody and examined with the light and electron microscopes. At the light microscope level, labelling was restricted to the osteocyte lacunae and canaliculi. Ultrastructural observations showed that the labelling was localized to fine filamentous material in the walls of the osteocytes and canaliculi. Sparse labelling was associated with the collagen fibres immediately subjacent to the lamina limitans, but no labelling of the mineralized matrix was observed. These findings also indicated subtle differences in the distribution of chondroitin sulphate compared with previously reported work, which may indicate species or age differences in the samples used in this study. Ultrastructural analysis confirmed and extended observations of glycosaminoglycan localization at the osteocyte cell membrane of mature human alveolar bone. Introduction Non-collagenous proteins, including proteoglycans, constitute approximately 10% of the organic matrix of mammalian bone, and appear to be involved in secretion, assembly, maturation, mineralization and/ or maintenance of the extracellular matrix (Fisher, 1984; Boskey, 1989). The precise role of proteogly- cans and glycosaminoglycans in the mineralization process is not known. However, several lines of evidence suggest that these molecules may play a role in the transformation of non-mineralized tissues to mineralized tissues, and are therefore expected to play a vital role in the remodelling process of alveolar bone. Chondroitin 4-sulphate constitutes some 90% of the total glycosaminoglycan content within human alveolar bone (Waddington et al., 1989). The fact that chondroitin 4-sulphate proteoglycan is the major proteoglycan of bone is borne out by work from a wide variety of species, such as dog, ox, sheep, pig and man (Herring, 1968; Hjertquist & Vejlens, 1968; Vejlens, 1971; Waddington et al., 1989; Bartold, 1990; Waddington & Embery, 1991). Bone is now known to contain at least three distinct proteoglycan species, which include decorin (PG II) (Franzen & Heinegard, 1984a,b; Fisher et al., 1989), with one chondroitin sulphate chain, and biglycan (PG I), with two chondroitin sulphate chains. Both these proteoglycan species have similar core protein molecular weights (45 kDa each) but different amino acid compositions (Heinegard & Paulsson, 1984), and similar-size chondroitin sul- phate chains (40 kDa). Studies on mature human alveolar bone have confirmed these findings (Waddington & Embery, 1991). The third proteogly- Histochemical Journal 29, 1–9 (1997) 0018–2214 1997 Chapman & Hall