3.1. In costal cartilage, collagen X is expressed much
earlier than alkaline phosphatase

The expression of collagen X in normal human costal cartilage tissue from donors of different ages
was examined by extracting the tissues with neutral buffers containing high concentrations of guanidinium hydrochloride, i.e. under conditions denaturing soluble collagens.
The crude extraction mixtures were subjected to polyacrylamide gel elcctrophoresis in SDS and collagen X was probed by immunoblotting with an antiserum specifically reacting with human collagen X (Kirsch and von der Mark, 1991).

Equal amounts of total protein from each donor were loaded

Fig. 1. Collagen X is a component of costal cartilage matrix already in childhood. Collagens were extracted with 6 M guanidinium hydrochloride from costal cartilage of normal donors of several
ages. Equal amounts of total protein were run on a 4.5-15% polyacrylamide gradient gel. An immunoblot is shown and the donor age is indicated below the lanes.

Fig. 2. Collagen X immunostaining of human rib cartilage from a 3-year-old donor. Costal cartilage and adjacent connective tissue were recovered from a central location remote from the bony parts of both the sternum and ribs, as well as the costal growth plate.
Staining was with a rabbit antiserum to human collagen X, detected by an antibody peroxidase anti-peroxidase kit. P, perichondrium; Sand I, surface- and inner zone, respectively. Note: staining occurred in the extracellular cartilage matrix in the vicinity of all chondrocytes,
but appeared somewhat stronger in locations rich in cells close to the perichondrial boundaries.
Tissues surrounding cartilage were negative (upper left corner).

onto the gels and collagen X was identified as an immunoreactive band with an apparent molecular mass of approximately 60 kDa. Collagen X was clearly detectable in tissues from 3-year-old donors and reached maximal levels in 7-year-old children, i.e. well before puberty (Fig. 1).

Collagen X-producing chondrocytes occur throughout the costal cartilage and deposit the protein predominantly into the extracellular matrix in their vicinity (Fig. 2). Thus, regardless of the donor age, costal cartilage contains chondrocytes having reached the most advanced stages of late differentiation.

Gels examined by staining with coomassie blue displayed strong bands of al(II)-chains
of collagen II, but failed to produce evidence for a2(I)-chains of collagen I (not shown).
Rib cartilage was also extracted with neutral saltbuffer without guanidinium chloride and alkaline
phosphatase activity was determined in the extracts.
Similar levels of enzyme activity were recovered from rib cartilage of subjects older than 17 years, but not pre-pubertal children (Fig. 3). Alkaline phosphatase activity was not demonstrable by enzyme histology in rib cartilage of a 3-year-old child (not shown). Therefore, unlike in all other cartilaginous tissues undergoing late differentiation, a clear separation occurs in
rib cartilage in the expression of the two markers of chondrocyte hypertrophy. The occurrence of alkaline phosphatase activity, but not collagen X, coincides with tissue mineralization and ossification.

Only in costal cartilage from post-pubertal donors, are matrix vesicles containing apatite mineral (Fig. 4) observed by electron microscopy on unstained sections with zero-loss filtering.

The mineral of the matrix vesicles was further investigated by electron diffraction. Bragg
reflections or reflection rings were recorded (Fig. 4, inset) and corresponded to a lattice spacing of 0.344 and 0.272 nm, respectively, which are characteristic for biological apatite consisting of a mixture of hydroxyl apatite and dahlite.

3.2. Proliferation and maturation of costal chondrocytes in culture

The capacity of costal chondrocytes to advance to late stages of differentiation was further investigated in long-term suspension culture in agarose gels. Under these conditions, chondrocytes from the cranial portion of chick embryo sterna proliferated and became
overtly hypertrophic within 1-2 weeks if the media contained 100 ng / ml of IGF-1 or insulin, or 50ng / ml of thyroxine or FBS (Böhme et al., 1995).
Costal chondrocytes from a 7-year-old proband were exposed to media containing FBS, or other factors without serum, and proliferation was assessed by direct counting of the cells in several representative microscopic fields revisited throughout the cultures. 710 a1 (X)

The cells only slightly proliferated (approx. 1.1-fold after 3 weeks) after stimulation by 10% FBS. Cell division was apparent in a few, but not all cells (arrows in Fig. 5, panels Q T). Lesser amounts of serum, as well as insulin, IGF-1, thyroxine, or PTH at maximal concentrations compatible with cell survival did not stimulate cell division (Fig. 5, panels E-P, and results not shown). These results indicated that most costal chondrocytes do not divide frequently during childhood.
However, cells cultured with 10% FBS expressed hypertrophy markers (Figs. 6 and 7) and increased their size (arrowheads in Fig. 5, panels Q-T). Exposure to the carboxyterminal fragment (residues 53-84) and, to a lesser extent, the aminoterminal portion (residues 1-34)
of parathyroid hormone, also raised synthesis of collagen X well above the detection limit (Fig. 6), but the cells did not increase their size (Fig. 5, panels I-P).

By contrast, collagen X was not synthesized in the presence of other signals, including insulin, IGF-1, and thyroid hormone (not shown), whereas the cell size was increased by treatment with IGF-1 (Fig. 5, panels M-P) or insulin (not shown). Therefore, collagen X-synthesis was not uniformly associated with a large cellular volume as seen in cartilage undergoing late
differentiation during endochondral ossification, such as in growth plates. Alkaline phosphatase activity was generated by the cells only under the influence of the carboxyterminal fragment of PTH for 48 h or 10% FBS.

All other factors in the defined culture medium were ineffective (Fig. 7). Thus, similarly to authentic tissue, the two hypertrophy markers were not simultaneously expressed by cultured costal chondrocytes under the direction of the aminoterminal domain of PTH (residues
1-34). This difference was not apparent in cells under the control of 10% FBS or, to a lesser extent, the carboxyterminal domain of PTH (residues 53-84). Analogously to native tissues, however, mineralized matrix vesicles similar to those shown in Fig. 4 only appeared in cultures expressing both hypertrophy markers (not shown).

Fig. 3. Alkaline phosphatase activity occurs only in post-pubertal costal cartilage. Proteins were extracted under non-denaturing conditions from costal cartilage fragments from donors of different ages. Alkaline phosphatase activity was determined by hydrolysis of pnitrophenyl-

phosphate in aliquots of the extracts containing equal amounts of total protein and values are given in arbitrary relative

Fig. 4. Matrix vesicles containing biological apatite mineral occur in post-pubertal costal cartilage. Electron micrograph of an unstained section of costal cartilage-matrix from a 17-year-old male donor. The tissue fragment was derived from a central location remote from
overtly osseous tissues. Inset: diffraction pattern of the mineral within the matrix vesicle shown in main panel. Several crystalline reflections (arrowhead) and a diffraction ring (arrow) are visible, corresponding to lattice spacings of 0.344 and 0.272 nm, respectively. Both patterns are characteristic for biological apatite mineral. Bar, 0.1 mm.

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