Indiana University-Purdue University Indianapolis (IUPUI) / Although the in vivo tibial loading model has been used to study the bone forma- tion response of mice to exercise, little emphasis has been placed on the translation of architectural and compositional modifications to changes in mechanical behaviour. The goals of the studies discussed below were to investigate the mechanical response in both healthy and osteolathrytic mice to this loading model and to determine the dose-depended effects of strain level on these properties. In two separately designed studies, strain levels ranging from 1700 to 2400 were applied to the right tibiae of 8 week old female C57BL/6 mice, while the left tibiae were used as non-loaded control. The first study consisted of loading both PBS- and BAPN-injected mice to 1750 microstrain which resulted in little bone formation but some tissue-level changes in mechanical analyses and an improvement in fatigue-resistance in terms of microdamage accumulation. The second study loaded healthy mice to three strain levels (1700, 2050, and 2400). Results indicated that the low end of the strain range did not engender a robust formation response, while the high end of the strain range resulted in a woven bone response in half of the animals in that group. Future studies will focus on the mid-strain level of 2050 which induced both significant architectural and mechanical improvements.

Advancements in computational power and micro-imaging has allowed the creation of finite element (FE) models on a microstructural level that can represent complex skeletal structures. These µFE models can analyze the structural integrity of individual trabeculae and may be used to model the impact of complex pathologies on skeletal stability. This thesis aims to: 1) optimize the histological identification of microdamage in healthy and mixed metastatic whole rat vertebrae, 2) quantify trabecular level stress and strain using µFE models and deformable registration generated from µCT data and 3) evaluate stress and strain in µFE models comparing undamaged regions with areas of mechanically induced microdamage. This novel technique allows the histological identification of microdamage in whole vertebrae with accurate alignment to 3D μCT data sets. In the μFE models, significantly higher stresses and strains were found in areas of damaged bone in both healthy and metastatically involved vertebrae.

Advancements in computational power and micro-imaging has allowed the creation of finite element (FE) models on a microstructural level that can represent complex skeletal structures. These µFE models can analyze the structural integrity of individual trabeculae and may be used to model the impact of complex pathologies on skeletal stability. This thesis aims to: 1) optimize the histological identification of microdamage in healthy and mixed metastatic whole rat vertebrae, 2) quantify trabecular level stress and strain using µFE models and deformable registration generated from µCT data and 3) evaluate stress and strain in µFE models comparing undamaged regions with areas of mechanically induced microdamage. This novel technique allows the histological identification of microdamage in whole vertebrae with accurate alignment to 3D μCT data sets. In the μFE models, significantly higher stresses and strains were found in areas of damaged bone in both healthy and metastatically involved vertebrae.

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Bisphosphonates (BPs) are used for the treatment of osteoporosis. This study evaluated changes in bone microdamage with BP treatment duration. Fifty-one iliac crest biopsies were obtained from Caucasian women, ages 41 to 87 years, who were previously diagnosed and treated for osteoporosis with oral BPs for 1-16 years duration. Patients diagnosed with any disease, drug, or substance abuse that may affect bone metabolism were excluded. Bone samples were sectioned, stained, and histologically examined using light and fluorescence microscopy. Bone area, number and length of microcracks were quantified. Following adjustment for age, BMD, BV/TV, trabecular thickness, and turnover, regression analysis revealed a relationship between microcrack density and treatment duration (p=0.018). No significant relationship was observed between microcrack length and treatment duration. This study provides novel data relating microdamage with varying BP treatment duration in human bone. Given information from other studies showing that microdamage amounts are related to changes in bone biomechanics, the BP treatment duration related changes in microdamage shown offer new information that may help optimize osteoporosis treatment.

Investigation of activated remodelling in the healing of experimental stress fractures and the influence of anti-inflammatory treatments Lisa Jane Kidd Abstract Targeted and focal remodelling are important processes in bone homeostasis and pathology. However, the factors that initiate and direct remodelling to repair microcracks, or respond to excess loading are still poorly understood. The rat ulna-loading (RUL) model has been widely used to examine modelling and remodelling responses to axial cyclic loading. However the model has not yet been fully characterised. Stress fractures are common amongst athletes, dancers and military recruits, but there is almost no information available on the mechanism of healing of these fractures. Although cyclooxygenase-2 (COX-2) is a key mediator of bone resorption and bone formation, very little information is available on the effect of non-steroidal antiinflammatories (NSAIDs) on stress fracture healing. Remodelling may play a role in the pathogenesis of stress fractures, and there is growing interest in the potential use of bisphosphonates to prevent them. Nonetheless, the effect of bisphosphonates on stress fracture healing is not known. PMX53 is a C5a receptor antagonist developed as a novel anti-inflammatory agent. It is effective against inflammatory arthritis, but has not been tested in any fracture models. The aims of this study were to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by RUL, and to use this model to determine the effects of selective and non-selective NSAIDs, a bisphosphonate and PMX53 on stress fracture healing. To characterise the RUL model, fatigue fractures were created by loading ulnae until displacement was observed to increase by between 4% and 50%. Ulnae were bulk-stained in basic fuchsin and processed for undecalcified histology. For all remaining experiments, loading was stopped when the displacement had increased by 10%. For detailed histology and histomorphometry, ulnae were decalcified, paraffin embedded and stained with toluidine blue, saffranin-O or for tartrate resistant acid phosphatase (TRAP). Ulnae were examined at 1, 2, 4, 6, 8, and 10 weeks after loading. The effects of DFU (a selective COX-2 inhibitor, 2 mg/kg po), ibuprofen (a non-selective NSAID, 30 mg/kg po) and PMX53 (10 mg/kg po) were examined at 2, 4 and 6 weeks after loading. Effects of risedronate (a bisphosphonate) were examined at a high (1.0 mg/kg po) and low dose (0.1 mg/kg po) at 2, 6 and 10 weeks after loading. RUL did not create isolated intracortical microcracks, but curvilinear fatigue fractures that occurred at a standard position in the medial cortex of the distal ulna diaphysis. These stress fractures induced rapid periosteal woven bone formation and direct intracortical remodelling along the fracture line that originated at the periosteum and progressed towards the medullary cavity. Basic multicellular units (BMUs) could be followed through serial sections extending along the fracture line towards the centre of the bone. Quantitative, real-time PCR was performed at 4 hours, 24 hours, 4 days, 7 days and 14 days after fatigue fracture. Following each period, bones were dissected and mRNA was extracted using standard protocols. Gene expression was compared between loaded and unloaded ulnae and to an unloaded control group. Four hours after loading, there was a marked, 220-fold increase (P<0.0001) in expression of Interleukin-6 (IL-6). There were also prominent peak increases in mRNA expression for Osteoprotegerin (OPG), cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF) (all P<0.0001). At 24 hours there was a peak increase in mRNA expression for IL-11 (73-fold increase, P<0.0001). At 4 days there was a significant increase in mRNA expression for Bcl-2, COX-1, bone morphogenic protein (BMP)-2, insulin-like growth factor (IGF)-1, osteopontin (OPN), and stromal cell derived factor SDF-1. At 7 days there was a significant increase in mRNA expression of Receptor activator of nuclear factor kappa β ligand (RANKL) and OPN. The dramatic, early up-regulation of IL-6 and IL-11 suggests they play a central role in initiating signalling events for stress fracture healing. Treatment with PMX53 did not affect any measures of woven bone formation or stress fracture remodelling. There were no treatment effects of Ibuprofen or DFU on the area of woven bone. DFU treatment resulted in a significant reduction in the area of porosity (resorption) and BMU area along the fracture line at 2 weeks after fracture. Ibuprofen treatment resulted in a significant reduction in length and area of BMUs and new bone formation along the fracture line at 6 weeks (p < 0.05). This is the first report to demonstrate a negative effect on stress fracture healing of both a selective COX-2 inhibitor and a non-selective NSAID. These data confirm the importance of cyclooxygenase in bone resorption and formation during remodelling. Bisphosphonates are potent inhibitors of osteoclastic bone resorption Two, 6 and 10 weeks after loading, measures of resorption and new bone formation were significantly reduced along the fracture line by high dose risedronate treatment, but not by the low dose. Only the porosity along the fracture line 2 weeks after loading was significantly reduced by the low dose risedronate. The low dose more closely resembles the clinical dose used to treat patients. Woven bone formation and consolidation were not affected by the low or high doses of risedronate. In conclusion, fatigue fractures in the rat ulna are highly reproducible, induce exuberant periosteal woven bone formation, and heal by direct remodelling along the fracture line. Remodelling is associated with gene expression for molecules typically associated with bone resorption and formation, angiogenesis and cell signalling. Remodelling of the stress fracture line was adversely affected by treatment with selective and non-selective COX inhibitors, by high dose treatment with risedronate, but not by PMX53, a C5a antagonist.

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