Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum

Tissue & Cell 35 (2003) 353–361

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum J. Wistuba a,∗ , W. Völker b , J. Ehmcke a,1 , G. Clemen a a

Institute of Animal Evolution and Ecology, University of Münster, Hüfferstraße 1, D-48129 Münster, Germany Institute of Arterioskleriosis Research, University of Münster, Domagkstraße 3, D-48129 Münster, Germany

b

Received 13 December 2002; received in revised form 3 June 2003; accepted 11 June 2003

Abstract Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan–cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation. © 2003 Elsevier Ltd. All rights reserved. Keywords: Glycosaminoglycans; Cuprolinic blue; Extracellular matrix; Teeth; Ambystoma mexicanum

1. Introduction Glycosaminoglycans (GAGs) and proteoglycans (PGs) play an essential role in hydration, electrolyte regulation and structural organization of the extracellular matrix (ECM; see Scott, 1980; Völker, 2002). GAGs role in hard tissue mineralization has not been settled yet. They were reported to indicate both promoting and inhibition of mineralization. So they are assumed to be involved in binding of calcium ions and in the regulation of the mineralization processes (Marks and Popoff, 1988; Goldberg and Septier, 1992; Linde and Lundgren, 1995; see also Sasaki and Garant, 1996). GAGs are involved in several distinct morphogenetic pathways (Princivalle and de Agostini, 2002). Being part of PGs, they represent the major gly∗ Corresponding author. Present address: Institute of Reproductive Medicine, University of Münster, Domagkstraße11, D-48129 Münster, Germany. Tel.: +49-251-8356098; fax: +49-251-8356093. E-mail address: [email protected] (J. Wistuba). 1 Present address: Department of Neurology, University of Münster, Albert-Schweizer-Straße 33, D48149 Münster, Germany.

0040-8166/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0040-8166(03)00056-9

coconjugates in connective tissues and play an important role in the formation of cartilage, bone and teeth (Scott, 1980; see Linde and Lundgren, 1995; Sasaki et al., 1997; Princivalle and de Agostini, 2002; Blair et al., 2002). PGs are composed of a filamentous core protein (∼5%) to which various GAG side chains formed by repeated disaccharid units (∼95%) are covalently attached. Owing to the numerous sulphate ester groups in the GAG side chains, PGs are highly polyanionic. This is important for the binding of cations (Ca2+ , Mg2+ ) during the process of mineralization and for cytochemical staining with cationic dyes, such as cuprolinic blue (CB). In mammals sulphated GAGs (such as chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate) were reported to play a role in mineralization and collagen fibrillogenesis during dentin formation and reparative processes (Nishikawa et al., 2000). Decorin and biglycan, which are chondroitin sulphate-rich, and the keratan sulphate-rich lumican and fibromodulin are the predominant PGs in dentin and predentin of mammals (Embery et al., 2001). Using the CB staining, we examined whether GAGs are also involved in the mineralization of urodele teeth.

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Tooth development in Ambystoma mexicanum was demonstrated to be morphologically typical for urodeles and also to resemble general aspects of hard tissue formation in vertebrates (Clemen et al., 1980; Bolte et al., 1996; Wistuba et al., 2002, 2003). Two tooth types occur during ontogenetic development in urodeles. Teeth of the larvae are monocuspid, lack a dividing zone and possess enameloid in the crown, while the substantially different transformed teeth of the adult animals are bicuspid and present a dividing zone separating crown and pedicel (e.g. Bolte et al., 1996; Wistuba et al., 2002). Clemen et al. (1980) reported various mineral qualities found in the shaft dentine, the dividing zone and the pedicel of the transformed teeth. Differential developmental processes in the different tooth zones as well as in the different teeth types occurring during development were exhibited in former studies (Wistuba et al., 2002, 2003). However, studies dealing with the contribution of GAGs and PGs in urodeles and their role in tooth formation are very rare (Hillman and Greven, 1998; see also Kogaya, 1994) and data obtained with critical electrolyte concentrations (CECs) are missing, although these components of the ECM probably influence those processes considerably and could induce the observed differentiations in hard tissue formation as it was discussed in mammals (Goldberg and Septier, 1992; Linde and Lundgren, 1995). Therefore, we examined whether GAGs are present during the tooth formation in this urodele species and focus on the distribution of GAGs in the matrices of various zones of the transformed teeth of the adult, as well as in the teeth of the early-larvae of the Mexican Axolotl, A. mexicanum. The possible impact of the various GAGCB populations on the differential processes of mineralization is discussed.

2. Materials and methods 2.1. Animals Ambystoma mexicanum was obtained from the departmental breeding station. The developmental stages and the types and zones of teeth were defined according to Wistuba et al. (2000, 2002, 2003). In brief, transformed (=metamorphosed) teeth of the maxillary were obtained from adult animals (older than 2 years) and the teeth of the larval type were obtained from the maxillary of early-larvae at the age of 6–8 weeks. Fig. 1 presents the examined stages of tooth formation and the analysed areas of the ECM.

2.2. Identification of GAGs Early-larvae (n = 3) and adult animals (n = 4) were anaesthestized in a solution of MS 222 (0.03%, Sigma) and sacrificed by decapitation. Afterwards small parts of the dentigerous bones of the upper jaw were removed, fixed for 2 h in glutaraldehyde (2.5% in 1.25 mol/l cacodylate buffer) and rinsed several times in 0.2 mol/l Na-acetate buffer (0.2 mol/l, pH 5.6) (in total 1 h). The molarity of the acetate buffer was higher than values reported in the literature (e.g. Scott, 1980; Hillman and Greven, 1998) in order to assure a complete removal of the cacodylate salt and to achieve the acidic pH in the tissues necessary for the CB staining. For electron microscopic investigation of the GAGs, tissues were stained with 1% CB (Polysciences) dissolved in acetate buffer (see Scott, 1980; Völker et al., 1986, 1987). Combination of CB staining with the CEC method allowed differentiation of GAG staining according to the degree of sulphation of their GAG side chains (Scott, 1985). Using increasing MgCl2 concentration, the affinity of the different populations of GAGs can be discriminated by loss of the capacity of CB staining (Scott, 1973). In highly sulphated GAGs such as chondroitin sulphate or keratan sulphate, higher concentrations of Mg2+ ions are needed to remove the GAGCB staining competetively (Scott, 1973, 1980; Goldberg and Septier, 1992). Various concentrations of MgCl2 (CEC: 0.0, 0.3, 0.6 or 2.0 mol/l) added to the buffers were used for CB staining and also for subsequent rinsing of the samples. Stainings lasted for about 3 h and the probes were washed three times (1 h in total). For controls, 2.0 mol/l MgCl2 , which completely supresses formation of GAGCB, was added to exclude false positive staining. This method allowed the identification of positively stained structures in the samples treated with the lower CECs. After washing, the samples were counterstained with 0.5% Na2 WO4 dissolved in 0.2 mol/l Na-acetate buffer for 1 h and thereafter in 0.5% Na2 WO4 in 30% ethanol. Samples were dehydrated in increasing concentrations of ethanol (3×30%, 50%, 70%, 96%, 2×100%). The samples were embedded in Spurr’s epoxy resin and ultrathin sections were cut with a diamond knife (Diatome) and observed in a transmission electron microscope (EM 900, Zeiss) at a voltage of 50 kV. The length of GAGCB were measured in selected areas of tissue on electron micrographs of the same order of magnification. To achieve sufficient numbers for statistical analysis, counts of the various GAGCB found in the different animals of one stage, but in identical compartments, were summed up. Subsequently the

䉴 Fig. 1. Schematic survey of investigated regions: (a) young replacement tooth of the early-larva; (b) young replacement tooth of the transformed tooth type; and (c) old replacement tooth of the transformed tooth type. The numbered boxes indicate the areas investigated for proteoglycans during the mineralization process. (1) Enamel during its formation, (2) enamel cap after secretion has finished; maturation is taking place, (3) mineralizing dentine of the shaft, (4) dividing zone, (5) mineralizing hard tissue of the pedicle. Abbreviations: d, dentine; db, dentigerous bone (maxillary, upper jaw); dl, dental lamina; dz, dividing zone; e, enamel; eo, enameloid; iee, inner enamel epithelium; oee, outer enamel epithelium; oe, oral epithelium; pe, pedicel; st, shaft (dentine shaft/crown). Stars: odontoblasts; circles: pulp cells building the pedicel. For the different stages of tooth formation in A. mexicanum, see also Wistuba et al. (2002).

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distribution of the GAGCB of various lengths in the different GAG populations found in the compartments examined and among the different regions investigated was statistically analysed by Student’s t-test and the Mann–Whitney U test.

3. Results 3.1. Electron microscopic characterization of different populations of GAGs In the presence of CEC of 0.0, 0.3 or 0.6 mol/l MgCl2 , GAGCB were found in all samples analysed (Table 1). GAGCB characterization of GAGCB was made related to the compartments of the ECM in which they were situated. The following compartments were defined: “collagenous matrix”, “soluble matrix”, “basal lamina bordering the ameloblasts”, (Figs. 2, 6 and 7) and “cell surface of odontoblasts” (not shown). When 2.0 mol/l MgCl2 was added, GAGCB were not detectable (Fig. 8). Both collagenous matrix and soluble matrix contained collagen fibres, but they showed differences concerning the degree of fibre organization. Only in the collagenous matrix compartment the fibres were arranged in bundles. The various compositions of the GAG populations found in the relevant compartments of the tooth-forming tissues in the early-larva and in the adult were analysed for significant differences in the distribution of the lengths of GAGCB (see Table 2). The collagenous matrix of the shaft of the bicuspid tooth of the adult animal revealed PGs of 38±15 nm in length (Table 2) when no MgCl2 was added (Fig. 6). These precipitates were associated periodically to the collagen fibres (Fig. 3). The mean value for the maximum length (mL) was about 58 nm, for the diameter (d) about 20 nm. These data are very similar to those found for the soluble matrix of the same tooth type (41±18 nm; mL 58 nm, d 19 nm; Table 2; Fig. 7). These data evaluated for the collagenous matrices of the shaft, the pedicel (43 ± 18 nm, mL 55 nm, d 19 nm) and the dividing zone (42 ± 18 nm, mL 59 nm, d 19 nm) were not significantly different (Table 2). In contrast, a prominent difference was found when the GAGs of the shaft of the early-larval tooth were measured.

They showed an average length of 65 ± 31 nm and a mean for the maximum length of about 94 nm. However, the diameter of about 19 nm was comparable to that of other precipitates (Table 2; Fig. 5). The average lengths of those precipitates associated with the basal lamina and the surface of the odontoblasts were smaller (Table 2; Figs. 2 and 6). Although their maximal length and diameter differed only a little from those compounds found in the matrix, the mean length was considerably lower (Table 2). The average length of GAGCB in the collagenous matrices evaluated decreased the higher the CEC added was. This was caused by the step-wise elimination of larger precipitates associated with the increasing MgCl2 concentration (0.3, 0.6, 0.9 mol/l) (Tables 1 and 2). For the collagenous dentine matrices of the shaft in the transformed tooth these differences were found to be significant (Table 1). Thus, populations were proved by the CEC method to include hybridized PGs with various GAG side chains (see Table 1). Comparison of the relative proportions of the GAGs in the various populations (CEC: 0.0 mol/l MgCl2 ) of the different compartments and tooth zones showed that the precipitate lengths were differently distributed in the collagenous matrices of the mineralizing pedicel, in the shaft and in the dividing zone (Tables 1 and 2). In the mineralizing dentine of the shaft of the transformed (metamorphosed) teeth, the soluble matrix was also different from the collagenous one (Tables 1 and 2). The soluble matrix of the shaft was not significantly different in composition of GAGCB populations from that of collagenous matrix of the dividing zone (Table 1). In addition, the GAG populations in the mineralizing collagenous matrices of the dentine in the shaft of the transformed (Table 2) teeth were found to differ significantly from those of the same compartment in the early-larval teeth (Table 2). 3.2. Morphology and localization of GAGCB in the ECM during formation of the mineral GAGs were located in the mineralizing collagenous matrix related to the dentine in the shaft, in the dividing zone and in the hard material of the pedicel of the transformed

䉴 Fig. 2. Early-larva, basal lamina lining the inner enamel epithelium (iee) during the secretion of enamel. CEC: 0.0 mol/l MgCl2 . When the secretion of enamel starts, the basal lamina (bl) is present at the outer face of the enamel (∗) and shows GAGCB (arrowheads). Bar = 400 nm. Fig. 3. Adult, transformed tooth of the maxillary. CEC: 0.3 mol/l MgCl2 . Precipitates are associated regularly (arrowheads) with the banding pattern of the collagenous fibres. Bar = 300 nm. Fig. 4. Adult, enamel maturation. CEC: 0.6 mol/l MgCl2 . After secretion of enamel has ended, the precipitates associated with the basal lamina are lacking. Instead, small GAGCB are present (arrowheads). Close to the enamel (∗), they are numerous and their number decreases in the cytoplasm (iee) of the ameloblasts with growing distance from the mineral. Bar = 400 nm. Fig. 5. Early-larva, tooth of the maxillary. CEC: 0.0 mol/l MgCl2 . Elongated precipitates (arrows) are only found within the collagenous matrix (cm) of the shaft dentine. Bar = 300 nm. Fig. 6. Adult, transformed tooth; basal lamina of the inner enamel epithelium (iee). CEC: 0.0 mol/l MgCl2 . The basal lamina (bl) can be identified at the basal regions of the replacement teeth by the precipitates (arrowheads) typically associated with it. The collagenous matrix of the dentine (cm) and the inner enamel epithelium are separated by a basal lamina. Bar = 400 nm. Fig. 7. Adult, transformed tooth. CEC: 0.0 mol/l MgCl2 . Compartments of the extracellular matrix. The different organization of collagenous matrix (cm) and soluble matrix (sm) is clearly distinguishable. Bar = 300 nm. Fig. 8. Adult, transformed tooth of the upper jaw, interface between enamel (∗) and inner enamel epithelium (iee). No precipitates were found at a CEC of 2.0 mol/l MgCl2 . Bar = 630 nm. Fig. 9. Adult animal, transformed tooth. CEC: 0.0 mol/l MgCl2 . Some precipitates (arrows) are surrounded by crystals of the growing mineral. Bar = 180 nm.

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Table 1 Distribution of the relative proportions of precipitates (in percent) in the various compartments Compartment

Proportion of GAGCB precipitates CM EL shaft CM Ad DZ; CM Ad Pe; CM Ad shafta

CEC (mol/l MgCl2 )

0.0

CM Ad DZ CM EL; CM Ad Pe; CM Ad shafta 0.0

CM Ad Pe CM EL; CM Ad Dza 0.0

CM Ad shaft CM EL-CM Ad Pe; CM Ad shaft 0.3; 0.6; 0.9 CEC, SM Ad shafta 0.0

CM Ad shaft CM Ad

shafta

0.3

CM Ad shaft CM Ad

shafta

0.6

CM Ad shaft CM Ad

0.9

shafta

SM Ad shaft CM Ad shafta

0.0

6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120 126 132 138 144

0.73 (%) 2.19 1.46 5.84 6.57 6.57 5.11 9.49 7.3 4.38 5.84 7.3 2.92 7.3 6.57 5.11 3.65 6.57 0 0.73 0 3.65 0 0.73

0 (%) 7.69 6.67 11.79 8.72 6.67 15.9 13.34 11.29 7.18 2.05 3.59 2.05 2.56 0.51 0 0 0 0 0 0 0 0 0

0 (%) 6.69 3.33 11.67 8.34 15 5 18.34 8.34 11.67 6.69 3.33 0 0 0 1.67 0 0 0 0 0 0 0 0

0 (%) 4.57 6.85 16.56 10.28 24.55 9.14 10.28 2.86 8.57 1.71 3.43 0.57 0.57 0 0 0 0 0 0 0 0 0 0

CM: collagenous matrix; SM: soluble matrix; DZ: dividing zone; EL: early-larva; Pe: pedicle, Ad: adultus. a Statistically different from Student’s t-test (P < 0.05) and Mann–Whitney U test (P < 0.05).

1.35 (%) 8.43 12.73 17.87 13.74 22.98 8.43 10.11 2.39 2.39 0.34 0.65 0 0 0 0 0 0 0 0 0 0 0 0

1.17 (%) 7.02 15.21 28.67 15.21 18.14 5.27 5.27 2.34 1.76 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 (%) 0.95 8.57 14.28 14.28 23.8 7.62 13.33 6.66 8.57 0.95 0.95 0 0 0 0 0 0 0 0 0 0 0 0

0 (%) 8.66 3.25 11.36 11.9 12.98 8.66 15.69 5.95 11.36 2.7 4.87 1.08 1.08 0.54 0 0 0 0 0 0 0 0 0

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Categories of longitudinal size measured (nm)

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Table 2 GAGCB in the compartments examined Compartment

Length (mean ± S.D.) (nm)

n (measured PGCB)

Transformed tooth, adult animal Collagenous matrix, shaft CEC 0.0 0.3 0.6

38 ± 15, mmin : 20, mmax : 58 31 ± 12, mmin : 19, mmax : 49 28 ± 11, mmin : 20, mmax : 52

175 297 171

Soluble matrix CEC 0.0 0.3 0.6

41 ± 18, mmin : 19, mmax : 58 36 ± 14, mmin : 21, mmax : 50 26 ± 9, mmin : 19, mmax : 40

185 98 57

Basal lamina, enamel epithelium CEC 0.0 0.3

36 ± 16, mmin : 18, mmax : 56 38 ± 15, mmin : 18, mmax : 54

71 41

Odontoblasts surface CEC 0.0

30 ± 13, mmin : 18, mmax : 56

93

Collagenous matrix, pedicel CEC 0.0 0.3

43 ± 18, mmin : 19, mmax : 55 38 ± 14, mmin : 20, mmax : 53

60 179

Collagenous matrix, dividing zone CEC 0.0

42 ± 18, mmin : 19, mmax : 59

195

65 ± 31, mmin : 19, mmax : 94

137

Early-larval tooth Collagenous matrix, shaft CEC 0.0

m: mean length of all measured precipitates; mmin : mean of the smallest measured precipitates (diameter), mmax : mean of the largest measured precipitates (long-shape).

teeth of the adult axolotl. Additionally, the matrix of the shaft of early-larval teeth was analysed (Fig. 1). The GAGCB were analysed with regard to topographic aspects, their variations in length and associations with collagen fibres. The basal lamina bordering the inner enamel epithelium along the basal parts of the transformed replacement teeth clearly revealed a layer composed of GAGCB (Fig. 2). The collagenous matrix adjacent to the dentine and the inner enamel epithelium was organized more diffusely than in areas close to the basal lamina (Fig. 6). In early stages of the secretion and mineralization of enamel, GAGCB remained along the basal lamina at the apex of replacement teeth (Fig. 2). However, they disappeared during the ongoing formation of the enamel cap. The basal lamina was broadened and its matrix components seemed to be resorbed by cells of the inner enamel epithelium at the apex of the tooth. The basal lamina disappeared during differentiation of tissue and its mineralization (Fig. 4). In this stage, GAGCB appeared smaller (about 7 ± 2 nm) than in the stages observed before (Fig. 4). This indicated degradation of GAGs. Close to sites of mineral deposition, small-sized GAGCB were concentrated. The density was the lowest close to the enamel cap (Fig. 4). After application of a CEC of 2.0 mol/l MgCl2 , the small-sized GAGCB were not detected in electron micrographs. Furthermore, GAGCB closely associated with crystals (Fig. 9) were observed and another population of GAGCB was found directly associated with the collagen

fibres. These precipitates were regularly arranged along the fibrils at intervals of about 60 nm (Fig. 3). This periodicity corresponded with the banding-pattern of the collagen fibres.

4. Discussion In the present study, GAGs were localized and characterized in tooth-forming tissue of the Mexican Axolotl by staining with CB. GAGCB and the basal lamina were absent from the ameloblast cells of the inner enamel epithelium secreting the enamel (see also Wistuba et al., 2002). However, in intracellular vesicles of these ameloblasts cells, precipitates smaller than 7 nm in size were seen during the period when enamel was produced at the apex of the tooth. It is very likely that these precipitates represent remainders of the GAG side chains. The staining of these precipitates can be suppressed by raising CEC (2 mol/l MgCl2 ) excluding false positive staining of structures. The small precipitates are probably produced during maturation of the enamel when compounds are withdrawn from the matrix and resorbed by the ameloblasts (see Wistuba et al., 2002, 2003). This indicates the presence of polysulphated GAG as a major constituent of PGs in the ECM of these areas and confirms former observations that the ameloblasts of A. mexicanum also may have resorptive functions (Wistuba

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et al., 2002, 2003) as was shown for the enamel-producing cells in mammals (Nanci et al., 1996; Sasaki et al., 1997). Basal lamina and surface of odontoblasts possessed smaller GAGs. The capability of GAGs to bind cations plays an important regulatory role in the mineralization of the apatite positioned close to the cell surface, although the role of GAGs is still a matter of discussion. Thus, the odontoblasts may have the function either to accumulate or to dissolve cations in the ECM (see Linde and Lundgren, 1995). GAGs found in the basal lamina, similar to those found in A. mexicanum, have been extensively investigated in mammals (e.g. Marks and Popoff, 1988; Princivalle and de Agostini, 2002; Blair et al., 2002). Such PGs are situated in associated and integrated components (e.g. Vischer et al., 1997), that form the basal lamina of vertebrates such as fibronectin, collagen IV, laminin and nidogen. Thus, the GAGCB related to the basal lamina lining the ameloblasts found in A. mexicanum are possibly heparan sulphate-rich integrated or weakly bound PGs. The GAGs found associated with basal laminae are supposed to play a role in the inhibition of cell growth or to function as co-receptors (Völker, 1992; Vischer et al., 1997). The precipitate lengths found within the different compartments of the transformed tooth revealed similar values for collagenous and soluble matrix. The precipitates of the collagenous matrices reached an average length of 40 nm and a mean maximum length of about 60 nm. The GAGs which are localized in the collagenous matrices of the transformed teeth are situated at periodic intervals along the collagen fibres, almost identical to the period of the banding pattern of the fibres and partly associated with the fibres. The distances of about 60 nm between the precipitates are very similar to those found for mammalian PGs related to collagen fibres (Scott, 1980). Therefore, we suggest that similar to findings in mammalian connective tissues the periodically arranged precipitates found in A. mexicanum, also similar in length, are probably dermatan sulphate-rich PGs, which were shown to be also involved in collagen fibrillogenesis and dentine formation in mammals (Nishikawa et al., 2000). Hence, they are decorin-like components of the matrix, involved in the organization of the collagen nerwork and associated with the fibres which are formed by collagen type II or type I/III (Scott, 1980; Embery et al., 2001). There is evidence that these PGs play an important role in linking of collagen fibres (e.g. Svensson et al., 2001) and that they are also responsible for the degree of organization existing in this special part of the ECM (Völker, 2002), functionally involved in the subsequent processes of mineralization (see Goldberg and Septier, 1992; Nakamura et al., 2001; Embery et al., 2001). The glycoconjugates present in the different collagenous matrices of the tooth zones are probably the same in all areas. However, a certain proportion of the populations of GAGs, characterized by the different distributions of precipitates, differed significantly between the matrices of these zones. Possibly the different degrees of mineralization in the various parts of the urodele tooth (Clemen et al., 1980;

Bolte et al., 1996) are initiated and regulated by the compositions of the GAG populations giving a substantial signal for the formation of mineral. In mammalian connective tissues, these components reveal an organising function as well as an impact on the way of crystalization (Goldberg and Septier, 1992; Nakamura et al., 2001; Embery et al., 2001). The distribution of the GAGs within one area might also reflect differences in the general organization of the ECM. There is no significant difference in the PG populations between the soluble matrix of areas mineralizing later and the collagenous matrix of the dividing zone, which develops into the weakest mineralized part of the transformed tooth after its formation is finished (Clemen et al., 1980; Bolte et al., 1996). The soluble matrix shows a lower degree of organization and a weaker mineralization than the collagenous matrix, but in the area of the dividing zone the GAG populations in the collagenous compartment seem to be composed similarly and the tendency to form hard material also seems to decrease, but considerably more than in the soluble matrices which mineralize later during tooth formation. The characterization of the matrix components shown suggests there might first be chondroitin sulphate-rich PGs present in both of these compartments. Chondroitin sulphate gradients were demonstrated to decrease toward the mineralizing front in mammalian predentin, while keratan sulphate-rich PG increased (Embery et al., 2001). Thus, GAGs of the soluble matrix may be changed by an increase of lower sulphated side chains and might become keratan sulphate rich. In bone formation processes, osteoblasts were suggested to synthesize keratan sulphate actively in their Golgi apparatus. The keratan sulphate was found in close contact to the calcified nodules of the hard tissue indicating a contribution of this GAG in trapping calcium ions through its negative charge (Nakamura et al., 2001). This change to a lower amount of sulphates in the side chains was suggested to support mineralization by regulating the balance of ions needed (Goldberg and Septier, 1992; Embery et al., 2001; Völker, 2002), but chondroitin sulphate- and keratan sulphate-rich PGs can not be separated by their size. Larger GAGCB were only seen in the dentine matrix of the shaft of the early-larval tooth, which is therefore different from all collagenous matrices of the adult animals teeth. These larger precipitates in the larva are distributed less densely. They probably represent two types of PGs, of which the larger ones are likely keratan sulphate rich. It is not known whether this PG composition in the matrix of the early-larval tooth modulates the way of mineralization or influences the quality of the hard material formed. Short, as well as very large and thin, precipitates in the matrix of the growing teeth of larvae were also shown in Salamandra salamandra (Hillman and Greven, 1998). There has not been any use of CECs higher than 0.1 mol/l MgCl2 in former studies, thus we have demonstrated for the first time the hard tissues of a urodele to contain PGs with hybridized GAG side-chain compositions. It is suggested that these extracellular components are similar to those of more advanced

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