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 downregulated 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.

Bone development in the femoral epiphysis of mice: The role of cartilage canals and the fate of resting chondrocytes

“…In contrast, this labeling pattern has not been noted within the normal as well as clubfoot calcaneus (Gilbert et al, 2001). However, in the chicken femur cartilage canal, mesenchymal cells unambiguously express various bone-relevant markers and differentiate from preosteoblasts into osteocytes on their course into the SOC (Blumer et al, 2006). In avian, the canals are more numerous than in mice and are arranged in a complex network.…”

“…Longitudinal sections (6 m) from paraffin-embedded tissue were deparaffinized and heat-induced target retrieval was carried out as described before and in a recent study (Blumer et al, 2006). This was followed by prehybridization in a ready-to-use HybriBuffer ISH (Biognostik, Göttingen, Germany) for 3 hr at 30°C.…”

“…In chicken, however, evidence has been provided that cartilage canal cells fully belong to the bone lineage and they seem to differentiate from preosteoblasts into osteocytes on their course into the SOC. Based on these observations, cartilage canals are considered essential for endochondral bone development in the avian epiphysis (Blumer et al, 2005(Blumer et al, , 2006. Interestingly, it was noted that in several mammals cartilage canals can regress as the animal gets older (Wilsman and Van Sickle, 1972;Cole and Wezeman, 1985;Ytrehus et al, 2004a,b).…”

“…These canals run through the uncalcified hyaline cartilage prior to the establishment of a secondary ossification centre (SOC) in long bones (e.g., femur, tibia) of avian (Lutfi, 1970;Blumer et al, 2004aBlumer et al, ,b, 2005Blumer et al, , 2006Eslaminejad et al, 2006) and numerous mammals (Stockwell, 1971;Cole and Wezeman, 1985;Cole and Cole, 1989;Visco et al, 1990;Burkus et al, 1993;Ganey et al, 1995;Roach et al, 1998;Rivas and Shapiro, 2002;Doschak et al, 2003;Á lvarez et al, 2005a,b). Similarly, in human short bones without epiphysis (e.g., tarsal bones, vertebrae), the canals are present before the initial site of an ossification nucleus is evident (Agrawal et al, 1986;Chandraraj and Briggs, 1988;Fritsch and Eggers, 1999;Fritsch et al, 2001).…”

Histological Sequences of Long Bone Development in the New Zealand White Rabbits

“…In another study, the cartilage canals were shown to contain mesenchymal cells, osteogenic cells and blood vessels. These contents suggested to provide both the vasculature and nutrition to the growing chondrocytes as well as mesenchymal cells, precursors of osteoblasts, that may be needed to the formation of the SOC and osteoclasts need for degradation of cartilage remnants, which present in the margins and tip of cartilage canals (Blumer et al, 2004;Blumer et al, 2005;Blumer et al, 2006;Blumer et al, 2007;Blumer et al, 2008). In addition, it was presented that some of the mesenchymal cells differentiated into osteoblasts that participate in the formation of medullary bone during endochondral ossification, while other mesenchymal cells differentiated into chondrocytes (Ytrehus et al, 2004).…”