We investigate the cellular and molecular mechanisms governing bone growth and development. One goal of our work is to improve medical treatment of growth disorders and childhood metabolic bone diseases. 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 also seek to uncover general principles of developmental biology.
Longitudinal bone growth: cellular and molecular mechanisms
Baron, Barnes, Marino, Nilsson, Schrier, Hegde, Emons, Gafni, Nwosu1
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. The 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.
With age, growth plate chondrocyte proliferation slows, causing longitudinal bone growth to slow and eventually stop. Also with increasing age, the growth plate undergoes structural changes. These functional and structural changes appear not to be attributable to a systemic mechanism but instead to a mechanism intrinsic to the growth plate. Our term for this intrinsic mechanism is programmed growth plate senescence. We have found evidence that it is a function not of time per se but rather of the number of replications that the growth plate chondrocytes have undergone. In particular, our studies suggest that stem-like cells, located in the resting zone of the growth plate, have a finite proliferative capacity that is gradually exhausted, thus producing growth deceleration and other senescent changes. However, if resting zone chondrocytes are placed in cell culture, they can be induced to proliferate substantially, even if the donor animal was older, near the end of longitudinal bone growth. This finding raises the possibility that the loss of proliferative potential is context-dependent and reversible. We have also shown that growth plate senescence is associated with a loss of DNA methylation in resting zone chondrocytes, suggesting that growth plate senescence may involve epigenetic changes.
Another possible mechanism that could contribute to growth plate senescence involves telomere shortening. Telomeres are hexameric repeat sequences that flank eukaryotic chromosomes. The telomere hypothesis of cellular aging proposes that replication of normal somatic cells leads to progressive telomere shortening, which induces replicative senescence. To test the hypothesis that telomere shortening in resting zone chondrocytes leads to replicative senescence, we compared the telomere restriction fragment (TRF) length in resting zone chondrocytes of Mus casteneus at 1, 4, 8, and 56 weeks of age. We found that TRF length did not diminish measurably with age, suggesting that telomere shortening in resting zone chondrocytes is not the mechanism that limits proliferation of growth plate chondrocytes in vivo.
Cadet ER, Gafni RI, McCarthy EF, McCray DR, Bacher JD, Barnes KM, Baron J. Mechanisms responsible for longitudinal growth of the cortex: coalescence of trabecular bone into cortical bone. J Bone Joint Surg Am 2003;85:1739-1748.
Nilsson O, Baron, J. Fundamental limits on longitudinal bone growth: growth plate senescence and epiphyseal fusion. Trends Endocrinol Metab 2004;15:370-374.
Nilsson O, Falk J, Ritzen EM, Baron J, Savendahl L. Raloxifene acts as an estrogen agonist on the rabbit growth plate. Endocrinology 2003;144:1481-1485.
Nilsson O, Mitchum RD, Schrier L, Barnes KM, Troendle JF, Baron J. Growth plate senescence is associated with loss of DNA methylation. J Endocrinol 2005;186:241-249.
Nwosu BU, Nilsson O, Mitchum RD, Coco M, Barnes KM, Baron J. Lack of telomere shortening with age in mouse resting zone chondrocytes. Horm Res 2005;63:125-128.
Human growth and postnatal development: clinical studies
Marino, Gafni, Leschek,2 Barnes, Emons, Nwosu,1 Baron
After a period of growth inhibition, the linear growth rate in children usually exceeds the normal range. This phenomenon, known as catch-up growth, has been observed in humans and other mammals following a wide variety of growth-inhibiting conditions. We have evidence that catch-up growth is not attributable to a hormonal or other systemic mechanism but rather to a local mechanism intrinsic to the growth plate. In particular, our findings suggest that catch-up growth occurs because growth-inhibiting conditions slow growth plate chondrocyte proliferation, thus conserving the proliferative capacity of the chondrocytes and consequently slowing senescence. As a result, following transient growth inhibition, growth plates retain a greater proliferative capacity, are less senescent, and hence show a greater growth rate than expected for age, resulting in catch-up growth.
We recently conducted a clinical study to determine whether the pattern of catch-up growth in children is consistent with the delayed senescence hypothesis. If catch-up growth is solely the result of a delay in growth plate senescence, then the linear growth rate of a child experiencing catch-up growth should be equal to the growth rate of a normal younger child. Thus, the growth curve during catch-up growth should represent a simple time-shift of the normal growth curve of a younger child. To test this theory, we analyzed catch-up growth in children with celiac disease placed on a gluten-free diet. At onset of the gluten-free diet, the average height age and bone age were delayed by 0.7 years. Mathematical analysis showed that the growth rate over the ensuing 36 months increased for chronological age but that the growth pattern was normal for a child 0.7 years younger. The findings are consistent with the hypothesis that catch-up growth results from delayed senescence of the growth plate.
In a second clinical study, we examined some of the possible causes of idiopathic short stature. It has been suggested that many children with unexplained short stature might in fact have subtle abnormalities of the growth hormone (GH)–insulin-like growth factor-I (IGF-I) axis that are not detected by traditional diagnostic criteria. Traditionally, the diagnosis of GH deficiency is often made on the basis of peak serum GH concentrations of less than 7 microgram/L after two GH stimulation tests. A peak GH (pGH) of 7 to10 microgram/L is now often considered indicative of partial GH deficiency. However, we found that children with pGH of 7 to 10 microgram L did not show characteristics intermediate between those with pGH of less than 7 microgram/L and those with pGH greater than or equal to10 microgram/L. Instead, they resembled those with pGH of greater than or equal to 10 microgram/L. Furthermore, among the children with pGH greater than or equal to 7 microgram/L, we found no correlation between pGH and any markers of GH deficiency. Thus, we found no evidence that children with pGH of 7 to 10 microgram/L have partial GH deficiency. We also studied children with low circulating IGF-I levels but normal pGH levels to determine whether, as previously suggested in the literature, they have partial GH insensitivity. The children did not show quantitative characteristics suggesting a decrease in GH action. Therefore, our data do not support the hypothesis that the children have partial GH insensitivity.
Emons JAM, Boersma B, Baron J, Wit JM. Catch-up growth: testing the hypothesis of delayed growth plate senescence in humans. J Pediatr 2005;147:843-846.
Gafni RI, Baron J. Overdiagnosis of osteoporosis in children due to misinterpretation of dual-energy x-ray absorptiometry (DEXA). J Pediatr 2004;144:253-257.
Leschek EW, Rose SR, Yanovski JA, Troendle JF, Quigley CA, Chipman JJ, Crowe BJ, Ross JL, Cassorla FG, Cutler GB, Baron J. Effect of growth hormone treatment on adult height in peripubertal children with idiopathic short stature: a randomized, double-blind, placebo-controlled trial. J Clin Endocrinol Metab 2004;89:3140-3148.
Nwosu BU, Coco M, Jones J, Barnes KM, Yanovski JA, Baron J. Short stature with normal growth hormone stimulation testing: lack of evidence for partial growth hormone deficiency or insensitivity. Horm Res 2004;62:97-102.
Weise M, Flor A, Barnes KM, Cutler GB, Baron J. Determinants of growth during gonadotropin-releasing hormone analog therapy for precocious puberty. J Clin Endocrinol Metab 2004;89:103-107.
1Benjamin Nwosu, MD, former Special Volunteer
2Ellen Leschek, MD, former Special Volunteer
COLLABORATORS
James Troendle, PhD, Biometry and Statistics Branch, NICHD, Bethesda, MD
Jan-Maarten Wit, MD, Leids Universitair Medisch Centrum, Leiden, Netherlands
For further information, contact jbaron@mail.nih.gov.