We investigate the cellular and molecular mechanisms governing bone growth and development. One of our goals is to improve medical treatment of growth disorders and childhood metabolic bone diseases. In addition, given that the cellular processes underlying bone growth, such as cell proliferation, terminal differentiation, angiogenesis, and cell migration, are also essential for development in other tissues, we seek to uncover general principles of developmental biology.

Developmental Regulation of Growth Plate Chondrogenesis
Baron, Barnes, Nilsson, Gafni, Flor, Nwosu, Mitchum
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. We have demonstrated that the resting zone contains stem-like cells that are capable of generating new clones of proliferative chondrocytes. We have also shown that the resting zone directs the spatial orientation of the proliferative clones, causing them to form columns parallel to the long axis of the bone. These proliferative cells undergo clonal expansion followed by cellular hypertrophy. The hypertrophic 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.
Overall body proportions are determined by the size of the skeleton, which, in turn, is determined by the rate and duration of longitudinal bone growth. The rate falls progressively with age. In humans, fetal growth exceeds 100 centimeters per year. By birth, the growth rate has decreased to 50 centimeters per year, and by mid-childhood, five centimeters per year. A similar progressive decline in bone growth occurs in other mammals. The differences among mammals in final skeletal size (e.g., between a mouse and an elephant) are determined largely by the rapidity of the decline. The decline in growth rate with increasing age is due primarily to a decrease in the rate of growth plate chondrocyte proliferation.

Our in vivo studies suggest that the decrease in chondrocyte proliferation occurs because stem-like cells of the growth plate have a finite proliferative capacity, which is gradually exhausted. Early in life, when the stem-like cells have a great proliferative capacity, the growth plate chondrocytes replicate rapidly, causing rapid longitudinal bone growth. Later in life, when the stem-like cells have expended much of their proliferative capacity, the derivative clones of chondrocytes replicate more slowly, resulting in slower longitudinal bone growth. The cellular and molecular mechanisms that limit proliferation of growth plate chondrocytes are currently under investigation.

Growth-inhibiting conditions, such as glucocorticoid excess, slow the proliferation of growth plate chondrocytes and thus conserve the proliferative capacity of these cells. If the growth-inhibiting condition resolves, the chondrocytes would therefore retain a greater proliferative capacity than normal and thus would proliferate more rapidly and for a longer period of time. Therefore, the conservation of proliferative capacity provides an explanation for catch-up growth, which has been observed in both humans and other mammals. We have demonstrated this mechanistic explanation in vivo.

Eventually, in a process known as epiphyseal fusion, growth ceases and the growth plate is replaced by bone. Our findings suggest that epiphyseal fusion is triggered when the proliferative capacity of the growth plate chondrocytes is finally exhausted. We have found evidence that estrogen accelerates the proliferative exhaustion of the growth plate chondrocytes. As a result, estrogen leads to early termination of linear growth and early epiphyseal fusion. Consistent with this hypothesis, we have found that both alpha and beta estrogen receptors are expressed in growth plate chondrocytes throughout postnatal development in rats and rabbits.

We are currently conducting a clinical study to determine whether estrogen accelerates proliferative exhaustion in human growth plate chondrocytes. We are analyzing growth data from girls with precocious puberty treated with a gonadotropin-releasing hormone analog (GnRHa). In these girls, the precocious puberty caused the growth plates to be exposed to high levels of estrogen. Our animal studies suggest that such estrogen exposure would cause accelerated proliferative exhaustion of growth plate chondrocytes. Treatment with GnRHa causes normalization of estrogen levels. However, if the previous estrogen exposure exhausts the proliferative capacity of the growth plate chondrocytes, we would expect the growth rate to be low during the GnRHa treatment. Preliminary analysis of the growth data is consistent with this prediction. Furthermore, the severity of the growth impairment appears to be correlated with the severity of the estrogen exposure before treatment. Thus, the preliminary analysis suggests that estrogen does accelerate proliferative exhaustion of growth plate chondrocytes in humans as it does in other mammals.

Developmental Regulation of Skeletal Strength
Baron, Barnes, Gafni
The process of bone growth not only determines body size but also affects the structural integrity of the skeleton. Thus, understanding skeletal growth may provide insights into the origins of osteoporosis. However, most basic and clinical studies of bone formation and osteoporosis are performed in adult individuals, focusing on the process of bone remodeling. Far less attention has been paid to the juvenile skeleton, in which bone is not only remodeled but also created through growth.
It is often assumed that decreased bone mineral acquisition during childhood will cause a permanent decrease in bone mineral density, which will increase the risk of fractures in late adulthood. To the contrary, we found evidence that bone mineral acquisition early in life has little or no effect on adult bone mass because many areas of the juvenile skeleton are replaced in toto through skeletal growth. In the growing long bone, new trabeculae are formed adjacent to the growth plate. Older trabeculae are resorbed as they approach the medullary cavity. Therefore, trabeculae formed early in childhood are completely replaced by new trabeculae later in childhood. Similarly, cortical bone is resorbed at its endosteal (interior) surface and extended at its periosteal (exterior) surface. As a result, cortical bone formed in early childhood is completely replaced over time. We have demonstrated these phenomena in vivo and have shown that they can cause recovery even from severe osteoporosis in a growing animal. Thus, our data suggest that bone mineral acquisition in early life has little effect on adult bone density. If this concept generalizes to humans, then interventions to maximize peak bone mass would be more effective if directed at adolescents rather than at young children.

Clinical Studies
Gafni, Leschek, Baron
We are conducting a clinical trial of alendronate for the treatment of idiopathic juvenile osteoporosis. Children with idiopathic juvenile osteoporosis are randomly assigned to receive either alendronate or placebo. Alendronate, a bisphosphonate that can be administered orally, inhibits bone resorption. It has proven effective in the treatment of postmenopausal osteoporosis and glucocorticoid-induced osteoporosis in adults. The purpose of our study is to determine whether the medication will provide a safe and effective treatment for childhood osteoporosis.

We are also completing a randomized double-blind placebo-controlled trial of growth hormone therapy in children with non-growth-hormone–deficient extreme short stature. Currently, thousands of children with extreme non-growth-hormone–deficient short stature receive growth hormone therapy despite the absence of conclusive data regarding long-term efficacy and safety. Non-randomized long-term studies have yielded conflicting results as to whether growth hormone therapy increases the adult height of children without growth hormone deficiency. This public health issue was foreseen in 1983; the Conference on Uses and Possible Abuses of Biosynthetic Human Growth Hormone, convened by the NICHD, concluded that “there is an urgent need for therapeutic trials to determine the effect of growth hormone in short children who do not have a growth hormone deficiency.” Similarly, in 1987, the FDA Endocrinologic and Metabolic Drugs Advisory Committee called for well-controlled studies on the long-term safety and efficacy of growth hormone for children who are not growth hormone–deficient. In response, NICHD initiated the current study. The final data are currently under analysis.