Introduction - Materials and methods.
Versican is a large interstitial chondroitin sulphate proteoglycan initially isolated from human fibroblast cultures (Zimmermann & Ruoslahti 1989) and similar proteoglycans have been found in various connective tissues (Shinomura et al. 1990, Zimmermann et al. 1994, Landolt et al. 1995). Versican interferes in vitro with the attachment of cells to various extracellular matrix components (Yamagata et al. 1989). In vivo , versican seems to act as a barrier to migratory neural crest cells and outgrowing axons (Landolt et al. 1995), and also may participate in the control of keratinocyte and dermal fibroblast proliferation (Zimmermann et al. 1994). Versican has a peptide domain homologous to hyaluronan (HA)- binding domain of aggrecan and is thought to be able to bind HA via this domain to form large aggregate structure (LeBaron et al. 1992). HA is a large glycosaminoglycan, without core protein and is believed to be involved in tissue formation and cell differentiation (Toole 1991).
Versican (Bartold et al. 1995, Yamauchi et al. 1997) and HA (Linde 1973, Mangkornkarn & Steiner 1992, Nieminen et al. 1993) have been also identified in the dental pulp and recently a proteoglycan aggregate consisting of versican, HA and link protein was isolated from the rat incisor dental pulp (Shibata et al. 2000). A previous histochemical study showed that both HA and versican were localized in the radicular dental pulp of the rat molar, but that there was considerable regional variation in the staining intensities for both molecules (Shibata et al. 1999). These results lead to the hypothesis that the distribution of both molecules can vary with physiological conditions in the dental pulp. As a first trial to confirm this, we determined to investigate the developmental changes in histochemical localization of both molecules, since rat molars undertake a series of developmental changes including completion of crown formation, root formation and eruption during the postnatal period.
A number of structural differences have been described between the coronal and radicular parts of the dental pulp, including those in the vascular system (Nakamura 1986), the nervous system (Maeda et al. 1987) and the number of immunocompetent cells (Okiji et al. 1992). These studies indicate that there are different physiological properties such as in defence mechanisms between the two regions of dental pulps. Therefore, in line with the above hypothesis, we also sought to assess regional differences in the distribution of HA and versican.
Materials and methods.
All animals were housed in facilities approved by the Tokyo Medical and Dental University. The animal-use protocol form conforming to NIH guidelines as stated in the ÔÇśPrinciples of Laboratory Animal CareÔÇÖ (NIH Guidelines 1985) was reviewed and approved by the Screening Committee for Animal Research of the Tokyo Medical and Dental University prior to the study.
Two pregnant Sprague-Dawley rats were purchased from Sankyo laboratories (Tokyo, Japan) and 30 newborn rats were raised during the experimental period. Postnatal rats, 1, 7, 14, 21, 28, 35, 42 and 49 days old, were anaesthetized with ether and killed by cervical dislocation. Three to four rats in each age group were used. The mandibles were dissected and immersed in 4% paraformaldehyde solution (0.1 mol L ´Çş´Çá1 phosphate buffer, pH 7.4, at room temperature) for 1 day, decalcified in 10% EDTA for 2 weeks at room temperature and embedded in paraffin. Five to six teeth samples of the mandibular first molars in each age group were investigated. Sagittal and horizontal sections (4 ´üş´Çám) were mounted on glass slides coated with poly-L-lysine.
The immunohistochemistry for versican and histochemical staining for HA has been described in detail (Shibata et al. 1999). Briefly, sections digested with trypsin (Wako Chemicals, Tokyo, Japan) were treated with monoclonal antibodies 12C5 (antiversican HAbinding region, the Developmental Studies Hybridoma Bank, Iowa City, IA, USA) and CS-56 (antichondroitin sulphate chain, Seikagaku Corporation, Tokyo, Japan), or with biotin-labelled HA-binding protein (Seikagaku Corporation, Tokyo, Japan). These probes have been well characterized (Asher et al. 1991, Asari et al. 1992, Shibata et al. 2000). We earlier confirmed that 12C5 reacts with the chondroitinase ABC digested core protein of the large proteoglycan from the rat dental pulp (Shibata et al. 2000). Its molecular weight (400ÔÇô500 Kda) is equivalent to the versican core protein. Furthermore, antichondroitin sulphate monoclonal antibodies, including CS-56, are known to recognize versican, but not decorin (Sorrell et al. 1999). The streptoavidin-biotin method was then applied to the sections using a HISTOFINE SAB kit (Nichirei, Tokyo, Japan). Biotin-labelled antimouse IgG + IgM + IgA in this kit was used as the secondary antibody for immunostaining. Finally, sections were treated with AEC (3-amino-9-ethylcarbazole) (Nichirei, Tokyo, Japan) to reveal any reaction. Negative control sections for versican immunohistochemistry were incubated with normal mouse IgG instead of the primary antibodies. Negative controls for HA staining were prepared by treating sections with Streptomyces hyalurolyticus hyaluronidase (Seikagaku Corporation, Tokyo, Japan) after trypsin digestion. All sections were examined after counterstaining with haematoxylin.