Low Estrogen Model and Percent Lamellar Bone Pre and Post Puberty

Abstract

INTRODUCTION: Pubertal growth is an important time during development for bone accrual and attainment of peak bone mass. Suboptimal bone gain has been observed in females with reproductive abnormalities such as primary and secondary amenorrhea and these conditions are very prevalent in female athletes. Amenorrhea is associated with decreased estradiol levels. Previous research has shown that in prepubertal animals a low estrogen environment significantly decreased mechanical strength, but there was no significant loss in bone area and actually an increase in moment of inertia. The decrease in mechanical properties may be related to the microstructure of the bone. Two types of bone are involved in growth-- woven bone, which is added for structural support in the short term, and lamellar bone , which is highly organized and has a greater contribution to overall strength. We will test the hypotheses that suppressed estradiol will result in bones with no change in cortical area and decreased strength properties but will have a larger composition of non lamellar bone as opposed to lamellar bone. PURPOSE: The goal of this study was to determine the relative amounts of woven and lamellar tissue in a bone and the relationship with the bone's mechanical strength in two models of low estrogen-- pre- and post-pubertal onset. METHODS: Fifty-Five female Sprague-Dawley rats were randomly assigned into four groups: a control group (n=14) and three experimental groups injected with gonadotropin releasing-hormone antagonist (GnRH-a)-- the Dose 1 group was injected with 1.25 mg/kg/dose daily (n=14), the Dose 2 was injected with 2.5 mg/kg/dose daily (n=14), and the Dose 3 group was injected with 5.0 mg/kg/dose, 5 days per week (n=13). All groups were sacrificed at Day 49. Additionally, twenty-nine Sprague Dawley rats were randomly assigned into three groups. The baseline day 65 group (BL 65) was sacrificed on day 65 (n=9). There was an aged match control group that was sacrificed on day 90 (n=12). Finally, there was an AMEN experiment group injected with 2.5 mg/kg/dose daily that was sacrificed on day 90 (n=9). All experimental groups for both protocols received injections of gonadotropin releasing hormone antagonists (GnRH-a) (Zentaris GmbH) intraperitoneally. Left femora were mechanically tested under 3-point bending. The right femora were dehydrated, embedded in polymethylmethacrylate, cut and ground to 100 µm thickness. Bones were analyzed under polarized light using Stereo Investigator Software (MBF Bioscience, VT). The proportion of the cortex with primary lamellar vs. non-lamellar/other primary tissue type was measured and expressed as percent of the total cortical bone area. Outcome measures included lamellar endocortical area, lamellar periosteal area, cortical area, endocortical area, % lamellar area and % non-lamellar area. RESULTS: There was a significant decrease (p<.05) in the distribution of lamellar versus non-lamellar cortical tissue type in the experimental group in the model of delayed puberty. Additionally, the pre-pubertal bones had a lower percentage of lamellar periosteal and endocortical area. The post-pubertal group showed no significant differences between the control and experimental group in any of the outcome measures. CONCLUSION: There were significant differences in relative bone distribution throughout the femoral cortex. Relative decreases in lamellar tissue distribution, especially on the periosteal surface, will result in decreased mechanical strength due to increased percentage of woven bone in pre-pubertal models.