Identification of mechanisms of delayed puberty on bone strength deficits during development

Osteoporosis which is frequently referred to as a pediatric disease with geriatric consequences (Golden, 2000) can result from a lack of optimal bone accrual during the development (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy, 2001). Pubertal timing is a key factor that contributes to optimal bone accrual and strength (Bonjour et al., 1994; 21 Warren et al., 2002). Bone mass doubles during the onset of puberty and young adulthood (Katzman et al., 1991) with more than 90% of peak bone mass being accrued at the end of second decade of life (Schneider & Wade, 2000). The rate of periosteal expansion is elevated during the pubertal period (Specker et al., 1987; Bradney et al., 2000) and this expansion parallels longitudinal growth (Parfitt, 1994). Irrespective of other changes, periosteal expansion lowers fracture risk by improving the strength of long bones by increasing the moment of inertia (Orwoll, 2003). Therefore, a delay in puberty may actually increase the time available for periosteal development and positively affect bone strength. Previous animal studies have shown decreases in strength, endocortical bone formation and increases in periosteal bone formation with delayed puberty. Clinical studies report negative effects of delayed puberty on bone mass accrual suggesting that delayed puberty is a multifactorial problem affecting bone strength development.;The purpose of this study was to determine the effect of delayed puberty on mechanical strength and endocortical bone marrow cells in two models: female rats treated with gonadatropin releasing hormone antagonists (GnRH-a) and energy restriction (30%).;Thirty-two female Sprague Dawley rats (21 to 22 days-of-age) were received from (Charles Rivers Laboratories, Wilmington, MA, USA) and housed individually at the Temple University Central Animal Facility (Temple University Weiss Hall). Animals were randomly assigned to one of three groups; control (n=10), GnRH-a (n=10) and energy restriction (ER) (n=12). The GnRH-a group was injected with gonadotropin releasing antagonist injections (GnRH-a) (Antide, Bachem, Torrance, Ca. USA) at a dose of 2.5 mg/kg/BW. The ER group received a 30% energy restricted diet (0pen Source diet (D07100606)(Research Diets, New Brunswick, NJ). All animals were sacrificed on Day 51. One-way analysis of variance testing (ANOVA) with a significance level of 0.05 was used to assess group differences.;Following the two protocols the uterine weight in the GnRH-a group was 80.6% lower than control; no change in the ER group. Ovarian weight was significantly lower in the GnRH-a group (83.3%) and in the ER group (33.3%) as compared to controls. A 22.7% lower muscle weight was found in the ER group but was equal to control and GnRH-a when normalized by body weight (BW). The retro-peritoneal fat pad weight was significantly decreased by 64.95% in the ER group as compared to controls. Energy restriction did not result in any deficit in bone strength when normalized by body weight however the GnRH-a group had a 26.2% lower bone strength compared to control. Histomorphometric changes were not significantly different between groups, but the ratio for periosteal versus endocortical bone formation rates for the control group was 1.38, GnRH-a was significantly higher with a ratio of 5.54 and for ER was 3.02 indicating that periosteal BFR are almost twice endocortical BFR in the experimental groups. There was a significant decrease in the trabecular percent bone volume (BV/TV) of the lumbar vertebra in the GnRH-a group (20.2%) compared to control. However BV/TV was significantly higher in the ER (18.4%) compared to the control group. Proliferation was suppressed to 59.6% of control in the GnRH-a group but only 85.5% of control in the ER group. The alkaline phosphatase activity was 31.2% lower in the GnRH-a group and 63.9% lower in the ER group. The relative quantification (RQ) of RUNX2 gene expression was lowest in control followed by GnRH-a and highest in ER group although no statistical significance was observed between any groups.;Thus our data infers that 30% energy restriction does not negatively impact bone health. Thirty percent food restriction with no deficits in micronutrients or hormone suppression may just suppress growth as indicated by the maintenance of bone strength per body weight and equivalent muscle mass per body weight in the ER group compared to control. The GnRH-a injections resulted in decreased bone strength and trabecular bone volume. Female Athlete Triad or Anorexia Nervosa are the two clinical conditions hypothesized to result from a combination of ER and estrogen deficient environment. Studies replacing estrogen in hypothalamic amenorrhea or IGF-1 in anorexia alone have failed to improve bone mineral density (BMD), but a combination of IGF-1 and estrogen has been successful in improving BMD. This suggests that estrogen dependant and independent mechanisms work in combination to protect bone. Our study investigated both mechanisms separately and indicates that ER at 30% may be protective for bone health. Since estrogen deficiency may be the extreme end of the spectrum affecting trabecular bone, treatment therapies may have to be based on age, magnitude and severity of energy restriction and presence or absence of menstrual status.