Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated.Key words: versican, dentine matrix, immunohistochemistry, TEM, FEISEM, fluorescence microscope.The human dentine organic matrix is composed by a large complex of macromolecules capable of self-assembly. The dentine matrix is represented predominantly by type I collagen and completed by non collagenous glycoproteins, elastin, hyaluronan and proteoglycans (PGs). While type I collagen is the backbone of the dentine with a predominant structural role, non-collagenous proteins, and in particular PGs, are believed to play fundamental functional roles during odontogenesis, mineralization and homeostasis of dentine.The process of odontogenesis appears to be controlled by a precise sequential expression of a pool of extracellular non-collagenous proteins that induces modifications within the extracellular environment of the predentine leading to the formation of the dentine matrix (Embery et al., 2001). Similarly, dentine mineralization involves a dynamic transition from the unmineralised predentine to the mineralised mature dentine, in which the role of specific regulative mineralisation proteins appears to be pivotal in the precipitation of the minerals and in the formation of apatite crystals (Embery et al., 2001). In particular, PGs has been shown to play crucial role in the mineralisation processes of dentine (Embery et al., 2001; Waddington et al., 2003).PGs are macro-molecules where, at least, one glycosaminoglycan side chain (GAGs) is covalently attached to the protein core of the molecules.Their size and structure can change and can be differentially found intracellulary, on the cell surface, or within the extracellular matrix.The majority of PGs have been identified by their antigenic and structural properties suggesting numerous biological functions (Embery et al., 2001). Biochemical, histochemical and immunohistochemical studies on PGs of dentine and predentine have yielded sufficient information to indicate that the predominant PGs belong to the small leucine-rich interstitial family (SLRP) (Fisher et al., 1983; Yoshiba et al., 1996). They include decorin and biglycan (Waddington et al., 2003; Orsini et al., 2007), which bear one or two chondroitin/dermatan sulphate GAGs, lumican, fibromodulin and osteoadherin that bear keratan sulphate GAGs chains (Iozzo et al., 1997, 1999; Neame et al., 2000). A second pool of PGs belongs to the large aggregation chondroitin/keratan sulphate family named hyaluronan-binding (HA), including aggrecan, versican, brevican and neurocan (Yamauchi et al., 1997).Versican was firstly isolated in chicken mesenchymal tissue, and it has been found to be expressed also in keratinocytes, smooth muscle cells of the vessels, brain and mesangial cells of the kidney. Similar PGs have been found in other connective tissues (Zimmermann et al., 1989; Shinomura et al., 1990; Zimmermann et al., 1994; Landolt et al.,1995) and recent studies have shown that, within the dental tissues, versican has been localised in gingival fibroblasts culture, dental pulp complex (Yamauchi et al., 1997; Bartold et al., 1995; Shibata et al., 2000; Shibata et al., 2002; Robey et al., 1993; Ababneh et al., 1999; Cheng et al., 1999), dentine Waddington et al., 2003), cementum (Ababneh et al., 1999; Cheng et al., 1999) and periodontal ligament (Sato et al., 2002).Within the dentine organic matrix versican can be detected either as fragments or as whole molecule. Waddington et al. (2003) reported that versican is mainly present as its degradation products (fragments), whereas the whole molecule has been isolated by Shibata et al. (1999; 2000) in rat dental pulp tissue.The aim of this study was to localise versican PG in human mature dentine by an immunohistochemical technique using a monoclonal antibody anti-versican (towards the whole molecule) and a polyclonal antibody anti-versican fragments, under high resolution field emission in-lens scanning electron microscope (FEI-SEM), electron transmission microscope (TEM) and fluorescence microscope (FM) and to confirm the morphological findings by a biochemical assay.