We have investigated the early cellular events that take place during the change in lineage commitment from hypertrophic chondrocytes to osteoblast-like cells. We have induced this osteogenic differentiation by cutting through the hypertrophic cartilage of embryonic chick femurs and culturing the explants. Immunocytochemical characterization, [3H]thymidine pulse-chase labeling, in situ nick translation or end labeling of DNA breaks were combined with ultrastructural studies to characterize the changing pattern of differentiation. The first responses to the cutting, seen after 2 d, were upregulation of alkaline phosphatase activity, synthesis of type I collagen and single-stranded DNA breaks, probably indicating a metastable state. Associated with the change from chondrogenic to osteogenic commitment was an asymmetric cell division with diverging fates of the two daughter cells, where one daughter cell remained viable and the other one died. The available evidence suggests that the viable daughter cell then divided and generated osteogenic cells, while the other daughter cell died by apoptosis. The results suggest a new concept of how changes in lineage commitment of differentiated cells may occur. The concepts also reconcile previously opposing views of the fate of the hypertrophic chondrocyte.
AbstractWe have investigated the early cellular events that take place during the phenotypic switch from hypertrophic chondrocytes to bone-forming cells in a) chondrocytes located inside intact lacunae after embryonic chick femurs had been cut through the hypertrophic cartilage and cultured for 1–15 days; and b) at the cartilage/marrow interface of femurs after short-term culture. Ultrastructural studies were combined with in situ methods localizing proliferating and apoptotic cells, and 3D-reconstructions of confocal images of the cartilage/marrow edge. The crucial event in the phenotypic switch was an asymmetric cell division which resulted in one daughter cell which underwent apoptosis and another viable daughter cell which subsequently differentiated to an osteogenic cell, i.e to a smaller basophilic cell that was positive for alkaline phosphatase, type I collagen, osteonectin, osteopontin, bone sialoprotein and osteocalcin and that, after 12–15 days in culture, could synthesize a mineralized bone matrix within intact lacunae. The present results suggest a mechanism whereby differentiated cells can change their phenotype. At least one mitotic division seems to be required to fix the commitment to the new phenotype.Key Words: Transdifferentiationhypertrophicfateosteogenesisendochondral
Chondromodulin-I (ChM-I) is a bifunctional autocrine regulator of cartilage, initially isolated from fetal bovine epiphyseal cartilage 1 . ChM-I stimulates matrix synthesis of chondrocytes, but inhibits the growth of endothelial cells 1, 2 thus ChM-I may be one of the anti-angiogenic molecules present in cartilage. In fetal bovine bone, ChM-I was expressed by all epiphyseal and growth plate chondrocytes except hypertrophic chondrocytes and was present in the matrix throughout the epiphysis and the growth plate, except the hypertrophic zone 2, 3 , consistent with its proposed role as anti-angiogenic factor. To examine the possible role of chondromodulin-I in relation to angiogenesis at the vascular front, we studied the immunolocalisation in femoral growth plates from young and mature rats (2–16 weeks) as well as aged rats (62–80 weeks), using a rabbit polyclonal antibody raised against the entire region of mature human recombinant ChM-I. In 2-week old rats, ChM-I was synthesised by all epiphyseal chondrocytes and strong immunostaining was found in the matrix. In the growth plates, ChM-I staining was present in chondrocytes and matrix of the reserve, proliferating and maturing zones with loss of staining in the hypertrophic zone. However, ChM-I was also present where cartilage canals had penetrated into the chondroepiphysis. In 4–16 week old rats, there was a progressive change in the localisation of ChM-I. Hypertrophic chondrocytes also became positive for ChM-I, while cellular staining gradually disappeared from the other zones. By 12–16 weeks, very strong immunostaining was present almost exclusively on the inner perimeter of the lacunae of hypertrophic chondrocytes. As lacunae were opened at the vascular front, ChM-I initially remained on the cartilage-side of the lacunae, and then disappeared completely. In aged rats, very little ChM-I was present in the cells and matrix of the growth plates, except where remodelling had occurred or chondrocytes had become re-activated. The rate of longitudinal growth in rats is high between 1–5 weeks, then declines until skeletal maturity at approximately 12 weeks, after which a very slow rate of growth continues until 26 weeks. In young rats, the loss of ChM-I in the hypertrophic zone was as expected for an anti-angiogenic factor, i.e. loss was required before vascular invasion could take place. However, the same did not apply to cartilage canal formation, since there was no loss of ChM-I around cartilage canals. The change in the localisation of ChM-I in mature rats, in particular the very intense immunolocalisation around hypertrophic chondrocytes, might be related to the reduced rate of growth. It is possible that rapid vascular invasion must be slowed down in these growth plates and that ChM-I prevented vascular invasion until degraded by proteases, such as MMP-9. The relative absence of ChM-I in the stationary growth plates of aged rats suggests that other anti-angiogenic factors prevent vascular invasion in these growth plates.
Chondrocytes of the growth plate are generally assumed to undergo apoptosis, but the mechanisms which induce this cell death are not known. The Fas receptor is a mediator of the apoptotic signal in some systems. We studied its expression in situ in growth plates of rabbits aged from five to 20 weeks. In addition, we investigated the immunolocalisation in the growth plates of the bone proteins, osteonectin and osteocalcin, and the changes in their expression with age. The Fas-positive chondrocytes were found mostly in the hypertrophic zone, as were the osteonectin-positive and osteocalcin-positive cells. The percentage of Fas-positive cells increased with age whereas little change was found in the number of osteonectin-positive and osteocalcin-positive chondrocytes. Many of the Fas-positive chondrocytes were also TUNEL-positive. This strongly suggests that apoptosis in the growth plate is mediated through the Fas system. Double immunostaining for osteocalcin and Fas showed that not all hypertrophic chondrocytes were of the same cell type. Some chondrocytes stained for osteocalcin only, others for Fas only, while some were positive for both.
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