Endochondral ossification of costal cartilage is arrested after chondrocytes have reached hypertrophic stage of late differentiation

Safarali Bahrami, Ulrich Plate, Rita Dreier, Alfred DuChesne, Günter-Heinrich Willital and Peter Bruckner

Department of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany
Department of Medical Physics and Biophysics, University of Münster, Münster, Germany
Department of Forensic Medicine, University of Münster, Münster, Germany
Department of Pediatric Surgery, University of Münster, Münster, Germany
Institut für Physiologische Chemie & Pathobiochemie, Westfälische Wilhelms-Universität, Waldeyerstrasse 15, 48149 Münster, Germany

– Introduction
– Discussion
– References

Late cartilage differentiation during endochondral bone formation is a multistep process. Chondrocytes transit through a differentiation cascade under the direction of environmental signals that either stimulate or repress progression from one step to the next. In human costal cartilage, chondrocytes reach very advanced stages of late differentiation and express collagen X. However, remodeling of the tissue into bone is strongly repressed. The second hypertrophy marker, alkaline phosphatase, is not expressed before puberty. Upon sexual maturity, both alkaline phosphatase and collagen X activity levels are increased and slow ossification takes place. Thus, the expression of the two hypertrophy markers is widely separated in time in costal cartilage. Progression of endochondral ossification in this tissue beyond the stage of hypertrophic cartilage appears to be associated with the expression of alkaline phosphatase activity. Costal chondrocytes in culture are stimulated by parathyroid hormone in a PTH/PTHrP receptor-mediated manner to express the fully differentiated hypertrophic phenotype. In addition, the hormone stimulates hypertrophic development even more powerfully through its carboxyterminal domain, presumably by interaction with receptors distinct from PTH/PTHrP receptors. Therefore, PTH can support late cartilage differentiation at very advanced stages, whereas the same signal negatively controls the process at earlier stages.

1. Introduction

2. Materials and methods

3. Results

4. Discussion

Acknowledgements

References

• Abstract
• Discussion
• References

In several tissues, hyaline cartilage persists throughout life and serves structural and biomechani-cartilaginous templates are eventually invaded by blood vessels before they are remodeled into trabecular bone.
At the cartilaginous stages, the cells transit through an ordered sequence of proliferation and late differentiation steps, culminating in chondrocyte hypertrophy and matrix mineralization. In this process, the extracellular matrix is extensively remodeled! which results from the activation of stage-specific genes (Cancedda et al., 1995). Aggrecan and collagens II and IX are expressed
throughout, albeit at different levels. By contrast, collagen VI and matrilin-1 (cartilage matrix protein, CMP) are markers of early and late proliferative stages, respectively (Quarto et al., 1993; Chen et al., 1995;Muratoglu et al., 1995; Szüts et al., 1998).

Collagen X and alkaline phos-phatase are characteristic products of hypertrophic chondrocytes (Schmid and Conrad, 1982).

Chondrocyte maturation requires extracellular stimuli that include thyroid hormones and insulin, or insulinlike growth factors. However, it is now established that there are also powerful negative control elements (Kato and Iwamoto, 1990; Tschan et al., 1993; Böhme et al., 1995; Colvin et al., 1996; Deng et al., 1996; Vortkamp et al., 1996) that either delay or entirely repress
endochondral ossification. In the cranial section of chick embryonic sterna (Böhme et al., 1995) or articular cartilage (D’Angelo and Pacifici, 1997), late chondrocyte differentiation proceeds spontaneously unless prevented at early or late proliferative stages by soluble and/or stationary environmental factors that interfere at distinct checkpoints of the maturation cascade (Vortkamp et al., 1996; Szüts et al., 1998; Pathiet al., 1999). This complexity is required to achieve appropriate progression rates and final extents of chondrocyte maturity. For example, severe skeletal abnormalities are the consequences of gain- or loss-offunction mutations in genesontrolling chondrocyte proliferation and differentiation. Such genes include those of fibroblast growth factors and their receptors (Rousseau et al., 1994; Neilson and Friesel, 1995;
Colvin et al., 1996; Deng et al., 1996; Burke et al., 1998).During adolescence, human ribs contain two cartilaginous regions, i.e. costal cartilage joining ribs to the sternum, and costal growth plates responsible for the longitudinal growth of the ribs. The two cartilaginous regions are separated from each other by bone tissue.

Costal cartilage undergoes incomplete ossification, which is not initiated before the onset of puberty. In addition, the process is very slow in comparison with bone formation during development or in growth plates, including the costal growth plate. Unlike articular cartilage, rib cartilage is vascularized immediately after birth but, nevertheless, is not mineralized before early
adulthood. Thus, the tissue is a permanent hyaline cartilage, in which endochondral ossification cal functions. For example, joint cartilage permanently conditions the surface of long bones to permit loadbearing and smooth articulation. Costal cartilage flexibly joins the bony part of the ribs to the sternum, and cartilaginous rings maintain the lumen of the trachea, while providing essential elasticity.

In skeletal elements undergoing endochondral ossification, however, hyaline cartilage is a transient tissue. During development, growth, and repair of bones, avascular is arrested only after chondrocyte differentiation has advanced to very late stages. This study was undertaken
to define the checkpoint of this negative control and to identify extracellular signals required to overcome the differentiation barrier. We have studied the occurrence of cartilage maturation markers and mineral deposition in situ and have investigated the capacity of costal chondrocytes to express a fully hypertrophic phenotype in vitro.

• Abstract
• Introduction
• References

Endochondral ossification consists of a highly complex sequence of proliferation, differentiation, and tissue remodeling events and includes several distinct steps of late cartilage differentiation. Each of the steps is subject to positive and negative control by environmental signals, which interfere at distinct checkpoints. Several anatomically distinct hyaline cartilage tissues can undergo late differentiation and can sequentially express markers for each stage. However, this inherent capacity may not become manifest under normal conditions, because it is suppressed by a powerful environmental control mechanism.

Under pathological conditions, however, this arrest may be released. For example, osteoarthritis may well result from inappropriate hypertrophy of articular cartilage (von der Mark et al., 1992).

The consequence is degeneration and loss or remodeling of the tissue that would be normal in other circumstances, such as in endochondral ossification of callus tissue during the repair of bone fractures. Our results clearly show that hypertrophic development takes place in costal cartilage well before the tissue is mineralized and ossified. This implies a novel checkpoint of negative control downstream of the expression of the overtly hyper-trophic cartilage
phenotype.
Collagen X is a well-recognized marker of terminal chondrocyte differentiation.
The protein has been found in many tissues undergoing endochondral ossification, including rib growth plates (Remington et al., 1983, 1984; Grant et al., 1985). In costal cartilage, however, the in situ occurrence of collagen X has escaped previous detection by immunohistology (Claassen et al., 1996), even though this tissue undergoes ossification very slowly. However, we have previously found evidence for the protein made in vitro by costal cartilage cells derived from funnel-chest patients (Erdmann et al., 1996).

As shown here, the protein was recovered from rib cartilage of very young children and was identified by immunoblotting and immunohistochemistry.

Its amounts increased to maximal levels well before puberty, i.e. before the onset of alkaline pbosphatase expression and slow mineralization and ossification. This clear separation in time of the expression of the two hypertrophy markers has not been found previously in any system undergoing late chondrocyte differentiation.

This necessitates the existence of independent control mechanisms for the activation of the genes for collagen X and alkaline phosphatase.

The function of collagen X still is not entirely understood. The protein is a prominent component of hypertrophic cartilage and is specific for this tissue, but its suprastructural organization is still incompletely understood. Collagen VIII, a structural homo-logue of collagen X, associates into hexagonal networks in corneal Descemet’s membrane (Sawada et al,, 1990), and collagen X can form similar suprastruc-tures in vitro (Kwan et al., 1991). In addition, collagen X may associate with cartilage fibrils, thus modulating the suprastructure of hypertrophic cartilage matrix (Chen et al., 1992).

Studies on gene defects further support the role of collagen X as a structural component. Mutations in the human collagen X-gene can cause Schmid metaphyseal chondrodysplasia (Dhar-mavaram et al., 1994; Mclntosh et al., 1994; Wallis et al., 1994) and mice with mutated or absent collagen X show disturbed matrix compartmentalization during endochondral ossification.

This results in abnormal formation of marrow cavities in long bones, which, in turn, impairs ematopoiesis (Kwan et al., 1997; Chan and Jacenko, 1998).
In addition, collagen X may regulate mineral deposition in hypertrophic cartilage. The protein reportedly modulates the calcium flux in matrix vesicles through binding to extracellular annexin V (Kirsch et al., 1997).

Our observations, however, imply that collagen X expression is not sufficient for cartilage mineralization and ossification. Only after the appearance of alkaline phosphatase activity can mineral deposition take place.
The studies on costal cartilage presented here have also afforded new insights into environmental
signals controlling late cartilage differentiation.
PTH may positively or negatively control chondrocyte hypertrophy, depending on the stage at which it interferes. The aminoterminal fragment, comprising residues 1-34 of the hormone, achieves negative signaling through cell-surface PTH/PTHrP receptors that also recognize the homologous region PTH-related protein (PTHrP) (Iwamoto et al., 1995; O’Keefe et al., 1997;
Fitzpatrick and Bilezikian, 1999; Karaplis and Goltzman, 1999).

Hedgehog-induced, perichondrially derived PTHrP prevents progression of growth-plate chondrocytes from a late proliferative stage towards a hypertrophycompetent stage (Vortkamp et al., 1996). As shown here, costal chondrocytes have advanced in situ to post-proliferative stages of terminal  if ferentiation and respond inversely towards this signal. Under the direction of the aminoterminal portion of PTH, the cells increased expression of collagen X, but not alkaline phosphatase. By contrast, the carboxyterminal portion of the hormone, acting through receptoKs) distinct from the PTH/PTHrP receptors, stimulated the production of both hypertrophy markers.

A comparable response has been reported previously for osteocarcinoma cells in culture
(Murray et al., 1991). Studies on the distinct responses to the PTH segments and their intracellular signaling cascade are in progress.

Fig. 5. Mo st co stal cho ndro c ytes in culture are p a st their proliferative p ha se. P ha se co ntra st-micro grap hs are shown of represe ntative fields revisited thro ugho ut the entire c ulture p eriod , as indic ated at the top of the figure. The med ia were sup plemented by spe cific signals or FBS, as indica ted on the le ft ma rgin. Panels A-D were d erived from a serum-free co ntrol culture (DMEM).

Note: isolated cells exhibit proliferation capa city in re spo nse to FBS (arrows in p anels Q-R). The ce lls uniformly incre ase their size thro ugho ut t h e cultures und er the d ire ction of IGF -1 or 10% FBS (arrowhe ads in panels E -H and Q-T).

Fig. 6. Hypertrophy of costal chondrocytes in agarose culture, as revealed by collagen X synthesis, is stimulated by parathyroid hormone. Costal chondrocytes were cultured in agarose gels at a density of 1.5 ´ 106 cells / ml for 2 weeks. The medium was DMEM and, during the last 48 h, contained 10 -8 M PTH (1-34) (lane 2), 10 -8 M PTH (53-84) (lane 3), or 10% FBS (lane 4). Serum-free control, lane 1. Collagens were extracted by limited digestion with pepsin and were subjected to electrophoresis on a 4.5-15% polyacrylamide gradient gel. Immunoblots with a polyclona! serum specific for human collagen X are shown.

Fig. 7. Hypertrophy of costal chondrocytes in agarose culture, as revealed by alkaline phosphatase activity, is stimulated by the carboxyterminal region of parathyroid hormone. Chondrocytes were cultured for the times indicated and alkaline phosphatase activity in culture media was determined by hydrolysis of p-nitrophenyl phosphate (arbitrary units).

The culture conditions were: full circles, 10% of fetal bovine serum; full squares, 10 -8 M PTH (53 84); and open symbols, 10 -8 M PTH (1-34), 100 ng / ml of IGF-1, 50 ng / ml of thyroxine, or serum-free control.

• Abstract
• Introduction
• Discussion

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