Jeffrey Baron, MD, Head, Section on Growth and Development
Kevin Barnes, PhD, Senior Research Assistant
Gabriella Finkielstain, MD, Postdoctoral Fellow
Ola Nilsson, MD, Postdoctoral Fellow
Elizabeth Parker, MD, Postdoctoral Fellow
Jacob Lazarus, Predoctoral Fellow
Anenisia Andrade, MD, Special Volunteer
Rachel Gafni, MD, Special Volunteer

We investigate the cellular and molecular mechanisms governing skeletal growth and development. One of our goals is to improve medical treatment of growth disorders and childhood metabolic bone diseases. In addition, we seek to uncover general principles of developmental biology, given that the cellular processes underlying bone growth, such as cell proliferation, terminal differentiation, angiogenesis, and cell migration, are also essential for the development of other tissues. In recent years, we have focused on the mechanisms that cause longitudinal bone growth to decelerate dramatically with age. This decline occurs because of an intrinsic program in the growth plate, which we have termed growth plate senescence. The program is extraordinarily potent, capable of reducing human growth from 100 cm per year in utero to 5 cm per year by mid-childhood and eventually to 0 cm per year in late adolescence. The phenomenon is also of interest because the concept of growth plate senescence appears to have broad explanatory powers, providing simple explanations for previously mysterious clinical phenomena. For example, the concept of senescence appears to be pivotal to understanding catch-up growth, the timing of epiphyseal fusion, and the effects of estrogen on the growth plate.

Longitudinal bone growth: cellular and molecular mechanisms

Andrade, Lazarus, Parker, Nilsson, Barnes, Baron

Longitudinal bone growth occurs at the growth plate, a thin layer of cartilage that lies near the ends of long bones and vertebrae. The growth plate consists of three principal layers: the resting zone, the proliferative zone, and the hypertrophic zone. Studies in our laboratory indicate that the resting zone contains stem-like cells that are capable of generating new clones of proliferative chondrocytes. These proliferative cells undergo clonal expansion followed by cellular hypertrophy. The resulting new cartilage is then remodeled into bone tissue. The net effect is that new bone tissue is progressively created at the bottom of the growth plate, resulting in bone elongation.

With age, growth plate chondrocyte proliferation decelerates, causing longitudinal bone growth to slow and eventually stop. This functional change in the growth plate is accompanied by structural changes; with age, the number of resting, proliferative, and hypertrophic chondrocytes declines as does the size of individual hypertrophic cells. The chondrocyte columns also become more widely spaced. This so-called growth plate senescence appears to be caused by a mechanism intrinsic to the growth plate. Our previous studies suggested that the growth plate retains information about growth history and influences subsequent growth plate structure and function.

The cell most likely to carry information about growth history is the resting-zone chondrocyte because it is the only cell involved in the growth process that remains in the growth plate over long periods of time. We recently obtained evidence that resting-zone chondrocytes serve as a pool of stem-like cells that generate the columnar clones of proliferative zone chondrocytes. We also found evidence suggesting that growth plate senescence is not a function of time per se but rather of the cumulative number of divisions that the chondrocytes have undergone. Therefore, taken together, previous findings suggest the following model: (1) stem-like cells in the resting zone have a finite proliferative capacity that is gradually exhausted; and (2) as proliferative capacity is exhausted, the proliferation rate of the proliferative-zone chondrocytes (which are derived from the stem-like cells) declines, causing longitudinal bone growth to slow with age and then eventually cease. Consistent with this hypothesis, we found that the proliferation rate in resting-zone chondrocytes (assessed by continuous bromodeoxyuridine labeling) declines 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 chondrocyte proliferative capacity. Consistent with this hypothesis, we found that dexamethasone treatment lowered the proliferation rate of resting-zone chondrocytes and slowed the numeric depletion of these cells.

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, thereby conserving the proliferative capacity of the growth plate.

We have also investigated in detail a gene that might be involved in growth plate senescence. The cyclin-dependent kinase inhibitor p27/Kip1 negatively regulates proliferation of several cell types. We therefore assessed the role of p27 in the regulation of growth plate chondrocyte proliferation. We detected p27 mRNA expression by real-time PCR in both proliferative/resting and hypertrophic zones of the mouse growth plate. To determine whether such expression is physiologically important, we studied skeletal growth in seven-week-old mice lacking a functional p27 gene. The body length of these mice was modestly higher than that of wild-type littermates. In the proximal tibiae, proliferation of growth-plate chondrocytes was higher, but tibial length was not significantly greater than in controls. We observed no measurable effect of p27 ablation on growth plate morphology, including the number of proliferative or hypertrophic chondrocytes. Treatment with dexamethasone in vivo inhibited longitudinal bone growth similarly in p27-deficient mice and controls, indicating that p27 is not required for the inhibitory effects of glucocorticoid on growth plate function. The femoral diaphysis of mice deficient in p27 was wider, suggesting that p27 acts normally to inhibit periosteal bone growth. In conclusion, our findings suggest that p27 acts a negative regulator of growth plate chondrocyte proliferation.

Human growth and postnatal development: clinical studies

Gafni, Barnes, Baron

The primary mechanism that initiates puberty is unknown. One possible clue is that pubertal maturation often parallels skeletal maturation. Conditions that delay skeletal maturation also tend to delay the onset of puberty, whereas conditions that accelerate skeletal maturation tend to hasten the onset of puberty. To examine this relationship, we previously studied boys with congenital adrenal hyperplasia and familial male-limited precocious puberty, two conditions that accelerate maturational tempo, and boys with idiopathic short stature in which maturational tempo is sometimes slowed. In all three conditions, the onset of central puberty generally occurred at an abnormal chronological age but a normal bone age. Boys with the greatest skeletal advancement began central puberty at the earliest age while boys with the greatest skeletal delay began puberty at the latest age. Furthermore, the magnitude of the skeletal advancement or delay matched the magnitude of the pubertal advancement or delay. We observed this synchrony between skeletal maturation and hypothalamic-pituitary-gonadal axis maturation among patients within each condition and between conditions. In contrast, the maturation of the hypothalamic-pituitary-gonadal axis does not remain synchronous with other maturational processes, including increases in weight, height, or body mass index. We concluded that, in boys with abnormal developmental tempo, maturation of the skeleton and hypothalamic-pituitary-gonadal axis remains synchronous.

The synchrony raises the possibility that skeletal maturation influences pubertal onset. To determine whether such concordance is also present in normal children, we analyzed data from 30 normal boys participating in a longitudinal study. Height, weight, and serum testosterone concentrations were assessed every six months and bone age every year. Pubertal onset was defined by a serum testosterone value ≥ 30 ng/dL. The variability in bone age at onset of puberty was no lower than the variability in chronological age. In addition, we observed no significant correlation between skeletal advancement and pubertal advancement. Similarly, we noted no significant correlation between pubertal advancement and height-age advancement, weight-age advancement, or BMI-age advancement. These findings do not support the hypothesis that skeletal maturation directly influences the age of pubertal onset in normal boys. An explanation of the findings in normal boys and of the earlier findings in boys with abnormal maturation will require a more complex model.

COLLABORATORS

James Troendle, PhD, Biometry and Statistics Branch, NICHD, Bethesda, MD
Jan-Maarten Wit, MD, Leids Universitair Medisch Centrum, Leiden, The Netherlands

For further information, contact jeffrey_baron@nih.gov.