Keywords: cartilage mineralization, glycosaminoglycans, histochemistry, immunohistochemisty, laryngeal cartilages
 |  |
| ||||
Copyright © Anatomical Society of Great Britain and Ireland 2004 Gender-specific distribution of glycosaminoglycans during cartilage mineralization of human thyroid cartilage Correspondence Dr Horst Claassen, Institut für Anatomie und Zellbiologie der Martin-Luther-Universität Halle-Wittenberg, Grosse Steinstrasse 52, D-06097 Halle, Germany. T: +49 345557 1708; F: +49 345557 1700, E: Email: horst.claassen/at/medizin.uni-halle.de Accepted September 20, 2004. | |||||||
Abstract The role of glycosaminoglycans (GAG) in the process of cartilage mineralization, especially in the hypertrophic zone of growth plates, is not yet fully understood. Human thyroid cartilage can serve as a model to observe matrix changes associated with cartilage mineralization because the processes follow a distinct route, progress very slowly and show sexual differences. Histochemical staining for low sulphated GAG (chondroitin-4- and -6-sulphates) was decreased in the interterritorial matrix of thyroid cartilage starting at the beginning of the fifth decade, but not in the pericellular or territorial matrix of chondrocytes. Because cartilage mineralization progressed in the interterritorial matrix it seems likely that a decreasing content of chondroitin-4- and -6-sulphates is involved in the mineralization process. This hypothesis is supported by the observation that immunostaining for chondroitin-4- and -6-sulphates was weaker in mineralized cartilage areas than in unmineralized areas, whereas there was no difference in staining for keratan sulphate. In all life decades, female thyroid cartilages contained more chondrocytes with a territorial rim of chondroitin-4- and -6-sulphates probably preventing cartilage mineralization compared with age-matched male specimens. Taken together, the characteristic distribution pattern of chondroitin-4- and -6-sulphates being more concentrated in female than in male thyroid cartilages provided evidence that these macromolecules decrease in cartilage mineralization. Keywords: cartilage mineralization, glycosaminoglycans, histochemistry, immunohistochemisty, laryngeal cartilages | |||||||
Introduction Cartilage mineralization is one of the main events in endochondral ossification of growth plates due to the fact that only longitudinal but not transverse septs mineralize to form a solid base for the deposition of bone tissue. In addition to collagens, proteoglycans (PG) and glycosaminoglycans (GAG) are two of the main components of cartilage extracellular matrix (ECM). Despite numerous histological and transmission electron microscopical investigations that have focused on the impact of PG and GAG on cartilage mineralization in growth plates, the mechanism of mineralization is still not fully understood (Poole & Rosenberg, 1987; Matsui et al. 1991; Byers et al. 1992, 1997; Farquharson et al. 1994; Hagiwara et al. 1995; Ehrlich & Armstrong, 1997; Mwale et al. 2001; for a review see Boskey et al. 2002). The process progresses too rapidly to observe details of cartilage mineralization. By contrast, human laryngeal cartilages, especially thyroid cartilage, allow us to study particular aspects of cartilage mineralization because they mineralize slowly and in a gender-specific manner (Chievitz, 1882; Tillmann & Wustrow, 1982). Many radiological studies have shown that cartilage mineralization and ossification start at the upper and dorsal rim of thyroid cartilage, therafter spreading across the thyroid plate along a particular route (Scheier, 1902; Fraenkel, 1908; Roncallo, 1948; Heywang, 1953; Keen & Wainwright, 1958; Hately et al. 1965; Bugyi, 1968; Heinemann, 1969; Wastrak, 1970; Harrison & Denny, 1983; von Glass & Pesch, 1983). Most authors have reported sexual differences during the processes of cartilage mineralization and bone deposition (Scheier, 1902; Fraenkel, 1908; Roncallo, 1948; Heywang, 1953; Keen & Wainwright, 1958; Wastrak, 1970). Thyroid cartilage of men ossifies nearly completely by the age of 70–90 years. In women the process of cartilage mineralization and ossification is diminished, beginning in the fourth or fifth decade and the ventral half of the thyroid cartilage remains unmineralized and unossified until advanced age. The aim of the present study was to gain insights into the role of GAG in cartilage mineralization of human thyroid cartilage. Therefore, low (chondroitin-4- and -6-sulphates) and high (keratan sulphate) sulphated GAG were localized histochemically in the ECM of thyroid cartilages of males and females aged between the first and ninth decade. In addition, chondroitin-4- and 6-sulphates or keratan sulphate were localized immunohistochemically in mineralized and unmineralized cartilage areas. | |||||||
Materials and methods Sources of material and reparation Larynges were obtained from autopsies and laryngectomies as follows: two male children (5 months and 3 years of age), two female children (2 months and 9 years of age), four male adolescents (13, 16, 17 and 18 years of age), one female adolescent (16 years of age), 12 male adults (21, 22, 22, 23, 30, 44, 53, 59, 63, 67, 74 and 80 years of age) and 13 female adults (22, 25, 30, 34, 41, 44, 51, 63, 70, 73, 74, 78 and 81 years of age). In all cases the thyroid cartilages were removed from the bisected larynges and X-rayed to determine the extent of cartilage mineralization and ossification. In radiographs of thyroid cartilage, spotted and opaque cartilage mineralization can be distinguished clearly from ossification characterized by a filigree trabecular architecture. Finally, each cartilage was dissected into eight defined transverse or frontal segments (see Fig. 1). Cartilage segments 4 and B near the Adam's apple were of particular interest because their mineralization is extremely different in men and women.
In order to prepare cryostat sections, the segments from the right side of the thyroid cartilage were submerged in Tris-buffered-saline (TBS), pH 7.3, complemented with 10% saccharose for 2 h. Complementation with saccharose is required for cryoprotection of cartilage during the freezing process. The specimens were then cooled in liquid nitrogen. Sections of 10 µm thickness were cut at −21 °C with a cryostat (Frigocut 2800 N, Reichert-Jung Co.) and were mounted on gelatine-coated slides. To prepare paraffin sections the segments from the left side of the thyroid cartilage were fixed in 4% paraformaldehyde buffered with phosphate-buffered saline (PBS), pH 7.3, for at least 24 h. Specimens were then decalcified with ethylenediaminetetra-acetic acid (EDTA), pH 7.3, for about 1 week at 37 °C. Decalcification was monitored by X-rays. After washing, the specimens were dehydrated in several portions of ethanol and embedded in paraffin. Sections of 7 µm thickness were cut with a microtome (Leitz Co.) and mounted on slides. Antibodies For immunolocalization of chondroitin-4-sulphate, chondroitin-6-sulphate and keratan sulphate the following antibodies, kindly provided by Professor Bruce Caterson (School of Molecular and Medical Biosciences, Connective Tissue Biology Laboratory, Cardiff, UK), were applied:
Immunohistochemistry Different types of GAG were demonstrated immunohistochemically in cryostat sections. After fixation for 5 min with cold acetone, sections were rinsed with TBS, pH 7.4, and incubated with chondroitinase ABC (Sigma C-2905, 1 U/100 µL), diluted 1 : 75, pH 8.0, at 37 °C for 30 min in a humid chamber. Chondroitinase ABC was used for digesting chondroitin sulphates to produce the stub of unsaturated disaccharides of chondroitin-4-sulphate and chondroitin-6-sulphate or to unmask the epitope of keratan sulphate masked by chondroitin sulphate (Caterson et al. 1983, 1985). After washing in TBS for 3 × 5 min, endogenous peroxidases were blocked with 5% H2O2-methanol for 10 min. After additional washing in TBS for 3 × 5 min, sections were incubated with antibodies against chondroitin-4- and -6-sulphates (diluted 1 : 100 with TBS) or against keratan sulphate (diluted 1 : 75 with TBS) for 60 min at room temperature in a humid chamber. The following tissues were used as controls, because they were certain to contain the antigen: a specimen of carotid artery for immunodetection of chondroitin-4- and -6-sulphates and a specimen of intervertebral disc for immunodetection of keratan sulphate. A section was covered with non-immune serum replacing the primary antibodies as negative control. After washing, the sections were incubated with peroxidase-conjugated rabbit anti-mouse IgG (Dako P0260), diluted 1 : 100 with 1 : 10 prediluted human serum, for 30 min at room temperature in a humid chamber. After additional washing, immunoreaction was developed with a DAB-kit (Vector SK-4100) for 10 min. Sections were dehydrated in several portions of ethanol and xylol and covered with DePeX.Histochemistry Low (chondroitin-4- and -6-sulphates) and high (keratan sulphate) sulphated GAG were demonstrated histochemically (Scott & Dorling, 1965) with alcian blue containing 0.3 m or 0.8 m magnesium chloride, respectively, in cryostat and paraffin sections. For pre-incubation 0.3 m MgCl2 or 0.8 m MgCl2 were dissolved in 100 mL 0.025 m sodium acetate buffer, pH 5.8 (solutions 1a/b). For incubation 0.05% alcian blue was dissolved in 100 mL 0.025 m sodium acetate buffer, pH 5.8, containing 0.3 m or 0.8 m MgCl2 (solutions 2a/b). Cryostat sections or deparaffinized paraffin sections were pre-incubated for 60 min in solutions 1a/b without alcian blue. Afterwards, specimens were incubated for at least 12 h in solutions 2a/b. Sections were washed in solutions 1a/b and aqua dest. After dehydration in several portions of ethanol, specimens were covered with DePeX.The degree of staining and the distribution of chondroitin-4- and -6-sulphates or keratan sulphate in the territorial and interterritorial matrix were recorded while viewing parallel sections. The degree of staining was graded as negative, weak, intermediate or strong. | |||||||
Results Histochemical staining for low (chondroitin-4- and -6-sulphates) and high (keratan sulphate) sulphated GAG The extent of mineralization and the grade of staining for low (chondroitin-4- and -6-sulphates) and high (keratan sulphate) sulphated GAG in the ECM of thyroid cartilages of both sexes from the first up to the ninth decade are given in Table 1.
In thyroid cartilages from males and females of the first to the fourth decade the whole ECM reacted with alcian blue containing 0.3 m MgCl2, indicating a high concentration of chondroitin-4- and -6-sulphates (Fig. 2A,C,E,G). In particular, the territorial matrix of chondrocytes reacted with alcian blue containing 0.8 m MgCl2, indicating a concentration of keratan sulphate in this area (Fig. 2B,D,F,H). By contrast, the histochemical reaction for keratan sulphate in the interterritorial matrix was only faint or negative.
In male and female thyroid cartilages belonging to the fifth to the eighth decade chondroitin-4- and -6-sulphates were concentrated in the territorial matrix. In the interterritorial matrix chondroitin-4- and -6-sulphates were less concentrated but with an increasing trend. The decrease in staining was more pronounced in men (Fig. 2I,M,Q) than in women (Fig. 2K,S). The interterritorial spaces were more extended in men than in women and, in addition, contained asbestoid fibres (Fig. 2I,M,Q). In women interterritorial spaces were narrower and contained hardly any asbestoid fibres as compared with age-matched men (Fig. 2K,S). Keratan sulphate in both sexes was particularly distributed in the territorial matrix, whereas the histochemical reaction was mainly negative in the interterritorial spaces of the matrix (Fig. 2J,L,N,R,T). A transition zone between unmineralized and mineralized cartilage can be observed in thyroid cartilage segment 4 of a 63-year-old woman (Fig. 2O,P). The unmineralized cartilage was stained more strongly for chondroitin-4- and -6-sulphates than the mineralized cartilage (Fig. 2O). The histochemical reaction for keratan sulphate was weak in the unmineralized cartilage and negative in the mineralized cartilage (Fig. 2P). In thyroid cartilages from men and women of the ninth decade the distribution of chondroitin-4- and -6-sulphates was limited to the pericellular (Fig. 2U) or territorial matrix (Fig. 2W). In the interterritorial matrix staining for these macromolecules was negative in men (Fig. 2U) and weak in women (Fig. 2W). High sulphated GAG were detected in the pericellular matrix only, in both men and women (Fig. 2V,X). Furthermore, a check of all specimens revealed that thyroid cartilage segments of women (Fig. 2G,K,O,S,W) contained more chondrocytes with a territorial rim of chondroitin-4- and -6-sulphates per field of vision than those of age-matched men (Fig. 2E,I,M,Q,U). Immunohistochemical detection of chondroitin-4- and -6-sulphate or keratan sulphate In the thyroid cartilage matrix of a 5-month-old boy immunostaining for chondroitin-4- and -6-sulphates or keratan sulphate was positive (Fig. 3A–C). An even distribution of these three types of GAG was observed in specimens of boys and girls of the first decade.
With the beginning of the second decade, the immunohistochemical distribution of chondroitin-4-, chondroitin-6- and keratan sulphates in the neighbourhood of mineralized cartilage areas formed a pattern that did not change until advanced age. In the thyroid cartilage of a 30-year-old man, territorial matrix of chondrocytes near the mineralization front reacted stronger with antibodies against chondroitin-4-, chondroitin-6- and keratan sulphates than did the interterritorial matrix (Fig. 3D–F). A transition zone between unmineralized and mineralized cartilage was observed in the thyroid cartilage of a 59-year-old man. The mineralized cartilage was investigated after decalcification with EDTA. The immunoreaction for chondroitin-4-, chondroitin-6- and keratan sulphates was stronger in the unmineralized matrix than in the mineralized matrix (Fig. 3G–I). Small unmineralized cartilage islands reacted strongly with antibodies against the three types of GAG, whereas the surrounding mineralized matrix showed a faint immunoreaction only (Fig. 3G–I). | |||||||
Discussion Due to a slow progressing and gender-specific cartilage mineralization, human thyroid cartilage can serve as a model to study the distribution of GAG during the process of cartilage mineralization. The time window to observe matrix being prepared for mineralization is small in rapidly calcifying growth plates. Histochemical and immunohistochemical localization of GAG in thyroid cartilages of different sex and age was performed to observe the distribution of these macromolecules while the matrix is being prepared for mineralization. The present results show a decreased reaction of the interterritorial matrix of thyroid cartilage with alcian blue containing 0.3 m MgCl2 beginning with the fifth decade. Because mineralization progressed preferentially in the interterritorial matrix, a loss of chondroitin-4- and -6-sulphates seemed to be involved in cartilage mineralization. Negatively charged carboxyl- and sulphate-groups of GAG were able to bind positively charged calcium, resulting in a prevention of cartilage mineralization (Buckwalter, 1983). However, the impact of PG and GAG on cartilage mineralization is a controversial issue, and, as mentioned by Howell & Pita (1976), depends on more than one system. Although some investigators believe that PG (and their component GAG) need to be lost prior to the onset of calcification and have demonstrated the proteoglycan degradative changes that occur in the growth plate (Lohmander & Hjerpe, 1975; Howell & Pita, 1976; Chen et al. 1984; Dziewiatkowski & Majznerski, 1985; Farquharson et al. 1994; Hagiwara et al. 1995; Boskey et al. 1997; Ehrlich & Armstrong, 1997), others (Poole & Rosenberg, 1987; Hunter, 1991; Matsui et al. 1991; Mwale et al. 2001) suggest that there is a persistence of PG when calcification starts, and that these PG cause calcification. However, because many of these studies were completed before the recognition that there was another class of PG, the small non-aggregating PG, it could not be determined whether the persistence of GAG staining was due to the presence of large or small PG. In vitro studies have shown that PG suppressed the formation and growth of apatite crystals (Chen et al. 1984; Dziewiatkowski & Majznerski, 1985). Accordingly, a loss of PG was recorded in rib cartilage of guinea-pigs with the onset of mineralization (Lohmander & Hjerpe, 1975). The hypertrophic zone of avian growth plates was characterized by a lower alcian blue staining of sulphated GAG compared with the proliferative zone (Farquharson et al. 1994). Furthermore, a decreased content of chondroitin-6-sulphate was observed in the calcifying zone of rat growth plates by immunoelectron microscopic analysis (Hagiwara et al. 1995). More recently, Boskey et al. (1997) have shown in differentiating chick limb-bud mesenchymal cells that extensive mineral deposition occurs when proteoglycan synthesis is blocked by treatment with 10−10 m retinoic acid. In addition, PG-degrading enzymes have been demonstrated in the hypertrophic zone of growth plates, where calcification occurs (Ehrlich & Armstrong, 1997). By contrast, immunoelectron microscopic (Poole & Rosenberg, 1987) or biochemical analytical methods (Matsui et al. 1991) have revealed a concentration of PG at the sites of mineralization in the hypertrophic zone of growth plates. Hunter (1991) also suggested that PG may promote hydroxyl apatite formation under in vivo conditions of infinite calcium availability. Moreover, in cultured growth plate chondrocytes the progression of mineralization was accompanied by a resorption of type II collagen, but was not accompanied by a preservation of PG (Mwale et al. 2001). In addition to a loss of alcian blue staining for chondroitin-4- and -6-sulphates in the interterritorial matrix of thyroid cartilage, asbestoid fibres were found in these areas. They were more pronounced in male than in female thyroid cartilages. Asbestoid fibres are regions of thyroid cartilage, where initial invasion of blood capillaries running in cartilage canals took place (Claassen & Kirsch, 1995). In addition, studies have shown that initial cartilage mineralization occurred close to cartilage canals in thyroid cartilage (Claassen et al. 1996). The loss of alcian blue staining in the interterritorial spaces well defined by asbestoid fibres and cartilage canals, structures involved in thyroid cartilage mineralization, added further evidence for a decreased level of GAG in areas of tissue mineralization. In accordance with the concept of a mineralization-preventing effect of GAG, our results show that immunostaining for chondroitin-4- and -6-sulphate was weaker in the EDTA-decalcified mineralized cartilage than in the unmineralized cartilage, indicating a diminished concentration of these low sulphated GAG in mineralized cartilage. By contrast, unmineralized and mineralized cartilage reacted similarly with antibodies against keratan sulphate. Furthermore, a loss of sulphated GAG was observed inside the vocal ligament tendon, a structure which attaches the vocal ligament to thyroid cartilage at the anterior commissure (Paulsen et al. 2000). This occurred when ossification of the laryngeal skeleton had started involving hyaline and fibrocartilage at the anterior commissure. Chondroitin-4-sulphate was also demonstrated immunohistochemically in the territorial matrix of human nasal septal cartilage (Üstünel et al. 2003a). Because parts of the hyaline septal cartilage mineralizes with advanced age (our unpublished observations), it is of note that an age-dependent decrease in GAG was reported for this cartilage (Rotter et al. 2002). By contrast, in articular cartilage, immunohistochemical staining for chondroitin-4-sulphate was strongest in the tangential zone where mineralization did not occur (Üstünel et al. 2003b). Altogether, a large body of evidence has accumulated emphasizing a correlation between a decreasing content in chondroitin-4- and -6-sulphates with their sulphated side chains and the beginning of mineralization in thyroid cartilage and growth plate cartilage. The results of Embery et al. (2001), who reviewed the role of PG in dentinogenesis, also confirm a decreasing gradient of chondroitin sulphate toward the mineralization front. In contrast to growth plates where cartilage mineralization is physiological, it is not yet clear if physiological age-related changes or degenerative changes lead to cartilage mineralization in human thyroid cartilage. Physiologically aged articular cartilage differs from the arthritic cartilage in its keratan sulphate content. In aged cartilage the concentration of keratan sulphate is increased (Mathew & Glagov, 1966; Thonar et al. 1986), whereas it is decreased in arthritic cartilage compared with unchanged cartilage of age-matched persons (Bayliss, 1986, 1992). Keratan sulphate as high sulphated GAG, mainly concentrated in the territorial matrix of thyroid cartilage, can be localized until the ninth decade. As no decrease in the keratan sulphate staining was observed, it seems likely that physiological, age-related changes were involved in the mineralization process of human thyroid cartilage. Furthermore, mineralization of thyroid cartilage does not lead to a pathological dysfunction of the organ as it would in articular cartilage. Mineralization of thyroid cartilage differs in males and females. In contrast to male thyroid cartilages, where cartilage mineralization continued until advanced age followed by nearly complete ossification, the ventral half of the female thyroid cartilage remained unmineralized and did not ossify (Claassen, 1995). Surprisingly, the ventral half of male thyroid cartilages (cartilage segment 4, see Fig. 1) contained fewer chondrocytes with a territorial rim of chondroitin-4- and -6-sulphates, probably preventing mineralization, compared with the ventral half of age-matched female thyroid cartilage. The absence of cartilage mineralization in female thyroid cartilage may partly be due to the dense distribution pattern of chondroitin-4- and -6-sulphates in its ECM. Recently, four different PG were extracted from human laryngeal cartilage: aggrecan as the major PG and versican, decorin and biglycan in minor amounts (Skandalis et al. 2004). Because the biglycan gene has been mapped to the Xq27–q28 region (Traupe et al. 1992), it seems possible that this chondroitin sulphate-rich PG is accumulated especially in female thyroid cartilage, preventing cartilage mineralization. However, other matrix components, such as matrix GLA protein, a regulator of hypertrophic cartilage mineralization (Yagami et al. 1999), may be involved in the processes described above. As mentioned, cartilage mineralization and ossification are accompanied by vascularization. It has been shown recently that vascular endothelial growth factor (VEGF, an important angiogenic peptide) concentrations measured by ELISA increased with age in male but decreased in female thyroid cartilages (Pufe et al. 2004). In conclusion, in addition to type X collagen as a main matrix protein involved in mineralization of human thyroid cartilage (Claassen & Kirsch, 1994), chondroitin-4- and -6-sulphates as low sulphated GAG also seem to participate in the mineralization of this tissue. We were able to provide an explanation for the observed gender-specific cartilage mineralization. A lack of chondrocytes with a territorial rim of negatively charged chondroitin-4- and -6-sulphates capable of binding calcium seems to be one reason for a more extensive cartilage mineralization in male thyroid cartilage. However, further experiments are needed to determine if the GAG lost had belonged to large or small PG. | |||||||
Acknowledgments We wish to thanks the following from the Department of Anatomy, Christian-Albrechts-University, Kiel, Germany, for their valuable help: Mrs A. Haupt, former assistant medical technician in the laboratory of Professor Dr B. Tillmann, for expert technical assistance, Mrs H. Waluk for the photographic work and Mr C. C. Franke for drawing the section diagram. | |||||||
References
| |||||||
