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. / Kinesiology / Accompanied by one .pdf file: Lamellar/Woven Database.

Schlüsselfaktoren der bemerkenswerten mechanischen Eigenschaften von Knochen sind seine komplexe hierarchische Struktur und chemische Zusammensetzung. Ziel dieser Dissertation war die simultane Untersuchung von Materialkomposition und 3D Struktur in Relation zu lokalen elastischen Eigenschaften von Knochengewebe unter Verwendung von neuen hochauflösenden experimentellen Konzepten. Im ersten Teil wurde polarisierte Raman Spektroskopie (PRS) eingesetzt um gesunden humanen kortikalen Knochen zu analysieren. Es konnte gezeigt werden, dass sich PRS eignet, um sowohl die chemische Zusammensetzung als auch die 3D Organisation der Kollagenfasern in einer Messung aufzuklären. Dominante Faserorientierungen ganzer Gewebedomänen konnten identifiziert und mit der Koexistenz zweier Faserorganisationsmuster verknüpft werden. Durch Kombination derPRS Experimente mit ko-lokalisierten Synchrotron-Phasenkontrast-Nano-Tomografie- undUltraschallmikroskopie-Messungen wurde eine komplementäre Untersuchung von Faserarchitektur, chemischer Komposition und elastischen Eigenschaften einzelner Knochenlamellen ermöglicht. Die multimodale Analyse ergab, dass die charakteristischen lamellären Ondulationen der Elastizität in erster Linie durch sich lokal ändernde Faserorientierungen bedingt werden und nicht durch Variationen der Materialzusammensetzung, Abweichungen der Mineralkristallpartikeleigenschaften oder durch Fluktuationen der Massendichte. Im letzten Teil wurde mittels akustischer Mikroskopie der Einfluss der Mutation des Neurofibromin 1 Genes auf die pathologische Entwicklung von mechanischen Knocheneigenschaften untersucht. Anhand zweier Knockout-Mausmodelle wurde festgestellt, dass nur eine Mutation in frühen mesenchymalen Vorläuferzellen die Steifigkeit der langen Röhrenknochen signifikant beeinträchtigt. Perspektivisch eignet sich der vorgestellte multimodale Ansatz für nicht-destruktive Charakterisierung eines breiten Spektrums biologischer und synthetischer Faserverbundwerkstoffe. / Key factors determining the remarkable mechanical performance of bone are its material composition and complex hierarchically structure. The aim of this thesis was the concurrent investigation of the chemical composition and 3D structure of bone tissue in relation to the local elastic properties by introducing novel high resolution experimental approaches. In the first part, polarized Raman spectroscopy (PRS) was applied to analyze healthy human cortical bone. In particular, it was demonstrated that PRS can be employed to simultaneously investigate the chemical composition and the 3D organization of collagen fibrils in a single experiment. Predominant fibril orientations in entire tissue domains were identified and linked to the coexistence of two fibril organization patterns. To further extend the analysis, PRS experiments were combined with synchrotron X-ray phase contrast nano tomography and scanning acoustic microscopy measurements in a site-matched study design. This multimodal approach enabled complementary imaging of the fibrillar architecture, tissue composition and resulting elastic properties of single bone lamellae. In line with earlier studies, crosscorrelation analysis strongly suggested that the characteristic elastic undulations of bone lamellae are the result of the twisting fibrillar orientation, rather than compositional variations, modulations of the mineral particle maturity, or mass density fluctuations. Finally, acoustic microscopy was applied to analyze the impact of the neurofibromin 1 gene mutation on the pathologic development of the mechanical properties of bone. Analysis of two knock-out mouse models revealed that only Nf1 ablation in early mesenchymal progenitor cells significantly impairs the elastic stiffness of long bones. In future studies, the presented multimodal methodology can be translated for non-destructive and high resolution characterization of a broad range of biological and synthetic fiber composite materials.