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Epidemiological studies suggest that poor growth during fetal life and infancy is associated with decreased bone mass in adulthood. However, theses observations have not, to date, been corroborated in animal models. To address this issue we evaluated the influence of maternal protein restriction on bone mass and growth plate morphology among the adult offspring, using a rat model. Maternal protein restriction resulted in a reduction in bone area and BMC, but not BMD, among the offspring in late adulthood. The widened epiphyseal growth plate in the protein-restricted offspring is compatible with the programming of cartilage and bone growth by maternal nutrition in early life.

Chondrocytes at the lower zone of the growth plate must be eliminated to facilitate longitudinal growth; this is generally assumed to involve apoptosis. We attempted to provide definitive electron-microscopic evidence of apoptosis in chondrocytes of physes and chondroepiphyses in the rabbit. We were, however, unable to find a single chondrocyte with the ultrastructure of 'classical' apoptosis in vivo, although such a cell was found in vitro. Instead, condensed chondrocytes had a convoluted nucleus with patchy chromatin condensations while the cytoplasm was dark with excessive amounts of endoplasmic reticulum. These cells were termed 'dark chondrocytes'. A detailed study of their ultrastructure combined with localisation methods in situ suggested a different mechanism of programmed cell death. In addition, another type of death was identified among the immature chondrocytes of the chondroepiphysis. These cells had the same nucleus as dark chondrocytes, but the lumen of the endoplasmic reticulum had expanded to fill the entire non-nuclear space, and all cytoplasm and organelles had been reduced to dark, worm-like inclusions. Since these cells appeared to be 'in limbo', they were termed 'paralysed' cells. It is proposed that 'dark chondrocytes' and 'paralysed cells' are examples of physiological cell death which does not involve apoptosis. It is possible that the confinement of chondrocytes within their lacunae, which would prevent phagocytosis of apoptotic bodies, necessitates different mechanisms of elimination.

Growth plates taken from five- to 20-week-old Japanese white rabbits were immunostained for c-Myc protein. This was localised both in the proliferating zone and upper hypertrophic zone at five weeks, whereas after ten weeks it was found mostly in the lower hypertrophic zone. The proliferating chondrocytes tended to show nuclear staining and the hypertrophic cells cytoplasmic staining, although the terminal hypertrophic chondrocytes sometimes expressed the protein in their nuclei. In the younger rabbits, c-Myc co-localised with proliferating cell nuclear antigen, whereas in the hypertrophic zone of older rabbits, it was present in some chondrocytes the nuclei of which also contained DNA breaks. Our study suggests that, in the rabbit growth plate, c-Myc is associated with different cellular processes, depending on the age and the developmental stage of the chondrocytes.

The efficient elimination of apoptotic cells depends on heterophagocytosis by other cells, which is difficult or impossible when the dying cells are embedded in an extracellular matrix. This situation is exemplified by the epiphyseal chondrocytes during the development of the chondroepiphyses of long bones. A detailed ultrastructural study identified an unusual type of epiphyseal chondrocyte which contained a very dark nucleus with irregular patches of condensed chromatin and a crenated nuclear membrane. The cytosol consisted of excessively expanded endoplasmic reticulum lumen, containing "islands" of cytoplasm and organelles. Since these cells appeared to be "in limbo," neither viable nor dead, they are referred to as "paralyzed" cells. By studying cells of intermediate morphologies, we were able to demonstrate the sequence of events leading to cell paralysis. It is proposed that the paralysis represents an intermediate state in the physiological cell death of epiphyseal chondrocytes in which destruction is orderly and avoids a inflammatory, potentially locally destructive, reaction. The cell is rendered paralyzed in terms of function but impotent in respect of damaging consequences. Paralysis is compared and contrasted with apoptosis, autophagocytosis, and necrosis and may represent another mode of programmed cell death in situations where cells are immature and/or where phagocytosis by neighboring cells is difficult.

The epiphyses of femurs from 7.5-15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, 'dark chondrocytes' representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours. This connection is briefly analysed in view of some current concepts of carcinogenesis.

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.

Growth plate chondrocytes of embryonic chick femurs were examined by electron microscopy, cytophotometry and autoradiography. Apart from the well-described 'light' chondrocyte, a different 'dark' type of chondrocyte was present, comprising 10 - 35% of the cell population. They were found at all stages of chondrocyte differentiation and in all ages of the femurs studied. Well developed rough endoplasmatic reticulum and Golgi complex, many secretory vesicles, energetically active mitochondria and a lot of glycogen, indicating high activity of the cytoplasm, were combined with low RNA synthesis, gentle margination and scattered compaction of the chromatin. DNA cytometry revealed that most of dark cells were diploid, but 15 - 30% were tetraploid, with the absence of an S-phase. Substantial loss of DNA was found in about 10% of dark chondrocytes. The TUNEL reaction demonstrated a limited number of DNA strand breaks. Advanced dark cells possessed the nuclear features of both apoptosis and necrosis. Besides chromomeric-chromonemic compaction, a chromatin arrangement similar to that of prometaphase and metaphase, as well as amitotic nuclear segregation, all of them degenerative, were found. Our interpretation is that the dark chondrocytes undergo an aberrant type of cell death which may be combined with aberrant cell cycle. Cell death of dark chondrocytes is preceded by a pre-mortal burst of secretion.

A detailed histological study of the growth plates from 9- to 20-day-old embryonic chick long bones was carried out with the aim of clarifying the long-debated question of the fate of the hypertrophic chondrocytes. Since resorption in chick bones does not occur synchronously across the plate as it does in mammals, specialized regions develop and the fate of the chondrocyte depends on its location within the growth plate. Where resorption took place, as at the sites of primary vascular invasion or at the main cartilage/marrow interface, chondrocytes underwent apoptosis before the lacunae were opened. In addition, spontaneous apoptosis of chondrocytes occurred at apparently random sites throughout all stages of chondrocyte differentiation. In older chick bones, a thick layer of endochondral bone matrix covered the cartilage edge. This consisted of type I collagen and the typical noncollagenous bone proteins but, in addition, contained tartrate-resistant acid phosphatase in the mineralized matrix. Where such matrix temporarily protected the subjacent cartilage from resorption, chondrocytes differentiated to bone-forming cells and deposited bone matrix inside their lacunae. At sites of first endochondral bone formation, some chondrocytes underwent an asymmetric cell division resulting in one daughter cell which underwent apoptosis, while the other cell remained viable and re-entered the cell cycle. This provided further support for the notion that chondrocytes as well as marrow stromal cells give rise to endochondral osteoblasts.

We 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.