Identification and location of bone-forming cells within cartilage canals on their course into the secondary ossification centre

Abstract

Osteoblasts and osteocytes derive from the same precursors, and osteocytes are terminally differentiated osteoblasts. These two cell types are distinguishable by their morphology, localization and levels of expression of various bone cell-specific markers. In the present study on the chicken femur we investigated the properties of the mesenchymal cells within cartilage canals on their course into the secondary ossification centre (SOC). We examined several developmental stages after hatching by means of light microscopy, electron microscopy, immunohistochemistry and in situ hybridization. Cartilage canals appeared as extensions of the perichondrium into the developing distal epiphysis and they were arranged in a complex network. Within the epiphysis an SOC was formed and cartilage canals penetrated into it. In addition, they were successively incorporated into the SOC during its growth in the radial direction. Thus, the canals provided this centre with mesenchymal cells and vessels. It should be emphasized that regression of cartilage canals could never be observed in the growing bone. Outside the SOC the mesenchymal cells of the canals expressed type I collagen and periostin and thus these cells had the characteristics of preosteoblasts. Periostin was also expressed by numerous chondrocytes. Within the SOC the synthesis of periostin was down-regulated and the majority of osteoblasts were periostin negative. Furthermore, osteocytes did not secret this protein. Tissue-non-specific alkaline phosphatase (TNAP) staining was only detectable where matrix vesicles were present. These vesicles were found around the blind end of cartilage canals within the SOC where newly formed osteoid started to mineralize. The vesicles originated from osteoblasts as well as from late osteoblasts/preosteocytes and thus TNAP was only expressed by these cells. Our results provide evidence that the mesenchymal cells of cartilage canals express various bone cell-specific markers depending on their position. We suggest that these cells differentiate from preosteoblasts into osteocytes on their course into the SOC and consider that cartilage canals are essential for normal bone development within the epiphysis. Furthermore, we propose that the expression of periostin by preosteoblasts and several chondrocytes is required for adhesion of these cells to the extracellular matrix.

Fig. 1

Light micrographs. (A) Semithin cross-section through a cartilage canal (cc). This canal is located within the reserve zone (rz) of the epiphysis of a 40-day-old chicken (D40). The canal contains several blood vessels (v) and mesenchymal cells which are embedded in the canal extracellular matrix. The canal matrix does not stain with toluidine blue. Scale bar, 20 µm. (B) This histological longitudinal section through the femur shows an overview of the epiphysis (D17). A secondary ossification centre (soc) is present and one cartilage canal (cc) lies outside this centre. Scale bar, 100 µm. (C) Semithin cross-section through the epiphysis (D15). A cartilage canal (cc) penetrates into the SOC and mesenchymal cells and blood vessels migrate into it. Scale bar, 50 µm. (D) Semithin longitudinal section through a cartilage canal which terminates blind within the SOC (D17). Around the end of the canal the bone matrix stains deeper with toluidine blue (arrowheads). The mesenchymal cells (= osteoblasts) of the canal border on this extracellular layer. Scale bar, 50 µm. Inset: an osteoclast which resorbs the newly formed bone matrix (bm). Scale bar, 20 µm.

Fig. 2

Transmission electron micrographs. (A,B) Ultrathin sections through a cartilage canal (cc) outside the SOC within the reserve zone of the epiphysis (D28). The electron-translucent matrix (cm) of the canal contains numerous cross-banded collagen fibrils. At the edge of the cartilage canals adjacent to the cartilage matrix this type of collagen fibril is also detectable. Scale bars, 0.5 µm. (C) Ultrathin section through the blind end of a cartilage canal within the SOC (D15). Densely arranged osteoblasts (ob) border on the osteoid (o), which is formed around the canal (compare with Fig. 1D). The innermost layer of the osteoid (o) contains numerous electron-dense matrix vesicles. The cell that is completely trapped within this layer represents a late osteoblast (lob) or an early osteocyte. Scale bar, 2 µm. (D) Higher magnification of the matrix vesicles (mv) with their electron-dense content. Scale bar, 0.5 µm.

Fig. 3

Immunohistochemistry. (A) Cross-sections through a cartilage canal outside the SOC within the reserve zone (rz). Type I collagen is detectable within the canal and in addition a distinct layer around the canal is present (D17). (B) Section through the SOC. The bone matrix (bm) around the blind end of a cartilage canal is composed of type I collagen fibrils (D15). (C) Oblique section through a cartilage canal outside the SOC within the reserve zone (rz). No immunostaining for TNAP is found (D28). (D) Section trough the SOC. The innermost layer of the bone matrix around the cartilage canals labels positively for TNAP. In this area newly formed osteoid starts to calcify. Osteocytes do not express TNAP (D10). All scale bars, 20 µm.

Fig. 4

Immunohistochemistry/in situ hybridization. (A,B) Strong reaction for periostin is noted within a cartilage canal (cc). Furthermore, faint staining for periostin is visible close to the surface of chondrocytes of the reserve zone (rz), the proliferative zone (pz), the hypertrophic zone (hz), and within the adjacent extracellular matrix (D10). Insets in A and C: control sections where anti-periostin is omitted show no labelling. Scale bars, 20 µm. (D) Overview of the SOC. Some osteoblasts that border on the bone matrix around the cartilage canals exhibit strong expression for periostin (arrowheads) whereas others showed only weak labelling (arrow). However, the majority of osteoblasts (asterisks) do not express this protein at D7. Osteocytes do not synthesize periostin. Scale bar, 50 µm. (E) Cross-section through a cartilage canal within the reserve zone (rz). ISH shows expression of the periostin mRNA (dark blue staining). The signal is detectable within canal mesenchymal cells and chondrocytes. Hypertrophic chondrocytes show the same staining pattern (inset) (D10). (F) No signal is observed when sections were hybridized with the random control. Scale bars, 20 µm.

Fig. 5

Immunohistochemistry/in situ hybridization. Sections through the epiphysis (A–D) and the diaphysis (E,F). (A) The inner layer of the perichondrium (p) expresses high levels of type I collagen (D17). (B) Strong expression of periostin is observed within the perichondrium (p). Furthermore, the chondrocytes of the reserve zone show faint expression (D17). (C) Controls without application of anti-periostin do not reveal any immunoreaction. (D) Oblique section through the perichondrium (p). The mesenchymal cells (arrowheads) of this connective tissue express periostin mRNA (dark blue staining) (D10). (E) Section through the femoral diaphysis (D5). The cortical bone (cb) is covered by the periosteum (pe). High levels of periostin are visible in the cambial layer, whereas the outer fibrous layer shows less staining. Immunoreactivity for periostin was not detected in the osteocytes of the cortical bone. In addition, the cells of the endosteum did not express periostin. (F) Longitudinal section through the POC where trabecular bone (tb) is visible (D13). Only the innermost layer of the bone matrix shows a positive reaction for TNAP. Bars, 20 µm.