The overall body size of vertebrates is primarily determined by longitudinal bone growth at the growth plate. With age, the growth plate undergoes programmed senescence, causing longitudinal bone growth to slow and eventually cease. Indirect evidence suggests that growth plate senescence occurs because stem-like cells in the growth plate resting zone have a finite proliferative capacity that is gradually exhausted. Similar limits on replication have been observed when many types of animal cells are placed in cell culture, an effect known as the Hayflick phenomenon. However, we found that the number of population doublings of rabbit resting zone chondrocytes in culture did not depend on the age of the animal from which the cells were harvested, suggesting that the mechanisms limiting replicative capacity of growth plate chondrocytes in vivo are distinct from those in vitro. We also observed that the level of DNA methylation in resting zone chondrocytes decreased with age in vivo. This loss of methylation appeared to occur specifically with the slow proliferation of resting zone chondrocytes in vivo and was not observed with the rapid proliferation of proliferative zone chondrocytes in vivo (i.e. the level of DNA methylation did not change from the resting zone to the hypertrophic zone), with proliferation of chondrocytes in vitro, or with growth of the liver in vivo. Thus, the overall level of DNA methylation decreases during growth plate senescence. This finding is consistent with the hypothesis that the mechanism limiting replication of growth plate chondrocytes in vivo involves loss of DNA methylation and, thus, loss of DNA methylation might be a fundamental biological mechanism that limits longitudinal bone growth in mammals, thereby determining the overall adult size of the organism.

With age, the growth plate undergoes senescent changes that cause linear bone growth to slow and finally cease. Based on previous indirect evidence, we hypothesized that this senescent decline occurs because growth plate stem-like cells, located in the resting zone, have a finite proliferative capacity that is gradually depleted. Consistent with this hypothesis, we found that the proliferation rate in rabbit resting zone chondrocytes (assessed by continuous 5-bromo-2′-deoxy-uridine labeling) decreases with age, as does the number of resting zone chondrocytes per area of growth plate.

Glucocorticoid excess slows growth plate senescence. To explain this effect, we hypothesized that glucocorticoid inhibits resting zone chondrocyte proliferation, thus conserving their proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment decreased the proliferation rate of rabbit resting zone chondrocytes and slowed the numerical depletion of these cells. Estrogen is known to accelerate growth plate senescence. However, we found that estradiol cypionate treatment slowed resting zone chondrocyte proliferation.

Our findings support the hypotheses that growth plate senescence is caused by qualitative and quantitative depletion of stem-like cells in the resting zone and that growth-inhibiting conditions, such as glucocorticoid excess, slow senescence by slowing resting zone chondrocyte proliferation and slowing the numerical depletion of these cells, thereby conserving the proliferative capacity of the growth plate. We speculate that estrogen might accelerate senescence by a proliferation-independent mechanism, or by increasing the loss of proliferative capacity per cell cycle.