Moderate energy restriction in young, exercising women attenuates the positive effects of exercise on bone density. Studies have shown that in the absence of adequate levels of circulating estrogen, there may not be enough functional estrogen receptor-a (ER-a) to respond adequately to loading. The experiment described in this document is significant because this model has not been explored under conditions of energy restriction (EnR) which are known to reduce circulating estrogen levels; it has been tested only in ovariectomized animals. The central hypothesis of this research is that reductions in estrogen due to EnR limit the ability of bone to respond to mechanical loading (LOAD) through a down-regulation of ER-a. Study one determined which nutrient’s (calcium or energy) restriction (-40 percent) had the greatest negative effects on the skeletal integrity of exercising female rats and whether exercise (EX) could mitigate these deleterious changes. EnR caused detrimental effects in many of the structural properties of bone; however EX attenuated losses in cancellous bone. Study two ascertained whether EX maintained cancellous bone mass in female rats subjected to graded EnR (-20 or -40 percent) and whether changes in endocrine factors were related. EX preserved cancellous bone volume and osteoblast activity under both levels of EnR, in addition to total body lean mass and bone mineral content. A similar maintenance of serum insulin-like growth factor and estradiol occurred in the EX EnR(40 percent) group suggesting that these changes may be related to the protective effects of EX. Study three determined the effects of 40 percent EnR on bone formation rate to LOAD in young adult female rats and tracked alterations in ovarian function (estradiol). Although higher than non-loaded animals, the response of bone to LOAD in EnR animals was dampened in comparison to energy-replete animals. The experiments described in this document are significant because these are the first experiments to explore the relationship between EnR and estrogen levels on cancellous bone response to LOAD. This is particularly important for physically active, energy restricted women because cancellous bone in these women will not experience the same effects of loading which can increase their risk for developing osteoporosis.

This study looks at improving reduced platen compression (RPC) specimen preparation procedures by developing a better method for locating the ideal RPC specimen on each bone. These improvements are aimed at decreasing the amount of time required to complete an RPC analysis and improving the quality of the obtained results. High-resolution micro-CT scans are used to gain a better understanding of rat long bone anatomy by quantifying the location, shape, and orientation of the growth plate, primary spongiosa, and secondary spongiosa. Micro-CT analysis shows that there are easily identifiable external landmarks on the anterior side of both tibias and femurs that identify the end of the growth plate and the point at which the top of an ideal RPC specimen should be located. The landmarks are the most proximal tip of the patellar surface for the femur and the base of the tibial tuberosity for the tibia. This study also analyzes the effect of variations in the actual RPC specimen location from the ideal location and the effect of different platen sizes on test results using BMD as a surrogate for mechanical properties. The analysis shows that the BMD increases as the target RPC specimen location approaches the growth plate and decreases on moving away from the growth plate. The study also indicates that consistency is necessary when obtaining RPC specimens to avoid error due to variation from the specified landmark. Additionally, the BMD decreased as the diameter of the platen is reduced. Choosing platen size then becomes a trade off between testing the greatest amount of cancellous bone possible and potentially higher load sharing by the cortical shell with larger platen sizes as well as the risk of compressing cortical bone during the test.

The aim of this master’s thesis is to determine the effective material properties of cancellous bone. In the first part of this work, finite element models are used for numerical homogenization of trabecular structures. It is shown that the applied boundary conditions have a strong influence on the effective material properties. To this end, different boundary condition are opposed and discused. In the second part, the Levenberg-Marquardt method is used to identify the preferred direction. Furthermore, it is shown that cancellous bone can be modelled as a transverse isotropic material. Finally, the homogenized continua are compared with the microstructural models of cancellous bone. / Ziel der Masterarbeit ist die Bestimmung der effektiven Materialparameter von spongiösem Knochen (lat. spongia „Schwamm“). Die numerische Homogenisierung von Trabekelstrukturen erfolgt mit Hilfe der Finite-Elemente-Methode. Es wird gezeigt, dass die verwendeten Randbedingungen einen starken Einfluss auf die effektiven Materialparameter ausüben. Die verschiedenen Randbedingungen werden dazu gegenübergestellt und diskutiert. Im zweiten Teil erfolgt mit Hilfe des Levenberg-Marquardt-Verfahrens die Identifizierung von ausgezeichneten Richtungen.Weiterhin wird gezeigt, dass die Spongiosa näherungsweise als transversalisotropes Material modelliert werden kann. Am Ende erfolgt der Vergleich des homogenen Ersatzkontinuums mit dem Mikrostrukturmodell der Spongiosa.

Osteoporosis is a disease characterized by reduced bone mass and reduced bone quality. This deterioration manifests itself in osteoporotic fractures at skeletal sites containing large proportions of cancellous bone (ie. forearm, hip, spine). Given the costs associated with these fractures, improvements in our ability to model and predict the behaviour of cancellous bone would be of great financial and social benefit to society. This thesis makes contributions in three areas within the much larger goal of developing a comprehensive model for describing the mechanical behaviour of cancellous bone. Since the accuracy of model predictions can only be as good as the test data against which it is compared, the effect of experimental artifacts introduced by specimen geometry is examined for cored samples. The apparent elastic modulus of cancellous bone is found to be relatively insensitive to specimen (or gauge) length, such that it can be reduced below the recommended 2:1 aspect ratio without introducing detectable artifact. Conversely, apparent modulus is found to be much more sensitive to specimen diameter. The role of water is also examined. Dehydration at room temperature was found to increase the apparent elastic modulus by roughly 14%. This net increase results from the competing effects of an increased tissue modulus and a decreased bone volume fraction due to shrinkage. Finally, preliminary work is presented which attempts to relate micro-CT voxel intensity and locally measured nanoindentation moduli, in order to provide an experimental basis for assigning heterogeneous material properties to finite element method (FEM) models. / Thesis (Ph.D, Mechanical and Materials Engineering) -- Queen's University, 2009-04-24 14:28:17.772

Extended periods of mechanical unloading are known to be detrimental to bone health. Astronauts who spend months in microgravity aboard the International Space Station (ISS) are at particular risk. It is anticipated that NASA will not drastically increase the size of the astronaut corps, and this will mean increased likelihood of repeat missions for more astronauts. Thus, it is important to better understand the effects that prolonged, multiple bouts of unloading have on bone. This study utilized the hindlimb unloaded (HU) rat model to examine bone loss and recovery for single and double unloading bouts. Adult male Sprague-Dawley rats (6 months old) were randomized into the following groups: baseline (sacrificed at 6 months), 1HU7 (unloaded for 1 month, weight-bearing recovery for 3 months), 2HU10 (unloaded for 1 month, recovered for 2 months, unloaded for another month, and then recovered 2 months), 1HU10 (normal cage activity until 1 month HU ending at month 10, 2 month recovery followed), and aging controls (remained ambulatory throughout experiment). Every month (28 days), animals were terminated and the left femurs were excised, resulting in n=15 per group for each time point. Mineral and geometric properties were measured using peripheral quantitative computed tomography (pQCT) at the distal femur metaphysis, and quasi-static reduced platen compression (RPC) was used to estimate the mechanical properties of cancellous bone. Strength indices based on pQCT parameters were calculated as predictors of mechanical properties. Bone mass properties decreased due to HU and recovered within 2-3 months post-HU. A combination of increased periosteal apposition and endocortical resorption also occurred during HU. The initial HU bout suppressed normal age-related increases in mechanical properties and recovered within 1-2 months. Cancellous compressive strength index (CSI) most closely matched changes in mechanical properties. A second HU bout after two months recovery had a less detrimental effect on pQCT parameters but a greater negative impact on mechanical properties, when compared to pre-HU values. The opposite is true for mechanical properties if loss is characterized relative to aging controls. Recovery after the second HU period did not appear to be significantly affected by a previous bout of HU.

The management of significant bone defects, delayed and non-union of fractures can be extremely challenging. Development of specific treatment is hindered by an absence of information regarding the molecular events which regulate these processes. In this thesis, a bilateral cancellous bone defect model of the femur and tibia was developed in a rodent and the spatiotemporal profile of TGF-β, BMP 2 and 7, Smads 1, 4 and 5 characterised. Next, the capability of acid solution to augment healing was tested in both a bone defect and in a closed femoral fracture model. Finally, a long term oestrogen deficiency (OVX) rat model of postmenopausal osteoporosis was characterised and the spatiotemporal profiles of IGF-1, IGFR-1, MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, BMP-2, BMP-4, BMP-7, TGF-β, Smad4, Smad7, VEGF, Flt-1, Ihh and FGF-2 were compared in femoral osteotomies between OVX and Sham groups. The bilateral cancellous defect model was successfully created with a number of advantages with which to recommend its use in future studies. TGF-β, BMP 2 and 7, Smads 1, 4 and 5 had characteristic spatiotemporal profiles during cancellous bone defect healing suggesting that they have a regulatory role. The results of the acid study were inconclusive and problems with substance delivery and maintenance at the desired site need to be addressed in the future to fully test this hypothesis. No significant differences were detected on histology or three-point mechanical testing between the fracture calluses of acid and control groups. In the final study, OVX rats after six months had significantly increased weight and decreased bone mineral density compared to their sham counterparts. A histological delay in osteotomy healing was observed in the OVX group but no significant differences on tensile testing were seen between OVX and Sham groups up to six weeks. Immunohistochemistry revealed that delayed healing may be due to the down-regulation of IGF-1, BMP-2, 4, and 7 and the up-regulation of MMP-3 in OVX compared to Sham groups. In conclusion, the results of this thesis give some insight into the molecular biology of bone defects and osteoporotic fractures. This information may also be useful in the development of specific treatments aimed at augmenting healing in bone defects and osteoporotic fractures.

Background and objectives: In the treatment of bone-related diseases, the biomechanical testing data of cadaveric bone plays a fundamental role in the design and application of orthopedic prosthesis and artificial bone materials, as well as the building of FEMs. Of these, the sample preparation is directly related to the accuracy of test results, including the removal of bone marrow, and the treatment of chemical solutions. In addition, it is also essential to evaluate the bone quality of bone specimens being tested prior to mechanical testing. Based on these issues, in this thesis, our studies aimed to systematically summarize the methods currently available for assessing bone quality in human bone tissue, as well as to investigate the efficiency of different chemical solvents on the removal of marrow from cancellous bone and their effects on the mechanical properties. Methods: In the first study (i), a systematic review of the literature was conducted using PubMed and Web of Science databases with the goal of finding all articles describing the methods of assessing bone quality in human bone specimens. The search terms were “bone quality [Title/Abstract]” AND “human bone specimens”. In the second study (ii), cylindrical cancellous bone samples (Ø8 × 2 mm) were obtained from fresh-frozen human cadaver femoral condyles and assigned to two groups, treated with chemical solvent soaking (solvent group) and ultrasonic cleaning (ultrasound group), respectively. Each group was divided into several subgroups based on different treatments. The differences in material density (Δρb), apparent density (Δρapp), and porosity (ΔP) before and after treatment were used as evaluation indications. In the third study (iii), cylindrical cancellous bone samples (Ø6 × 12 mm) were harvested from fresh-frozen human bones, including spinal vertebrae and femoral heads. All samples were divided into two groups, i.e., immersed in physiological saline (saline-immersed group) and treated with a combination of acetone and physiological saline (acetone and saline-treated group). Before and after treatment, all samples were subjected to non-destructive compression tests (0.45% strain), and finally destructive tests with a strain of 50%. Actual density (ρact), initial modulus (E0), maximum stress (σmax), energy absorption (W), and plateau stress (σp) were calculated as evaluation indicators. Results: Firstly, based on previous studies, our systematic review (i) found that advanced techniques are playing an increasingly important role based on their multiple advantages. Non- invasive imaging modalities and mechanical testing techniques, as well as the assessment of bone composition, need to complement each other to provide comprehensive and ideal information on the bone quality of human bone specimens. Secondly, our second study (ii) showed that a combination of 99% ethanol and detergent solution (v/ v = 1:20), as well as a mixture of 99% ethanol and acetone (v/v = 1:1), seem to be the optimal defatting methods for 2 mm thick cancellous bone slices because of their advantages of effectiveness, easy- availability, low-cost and relative-safety. Moreover, chemical soaking for 24h indicated better marrow removal than ultrasonic cleaning with 99% ethanol or acetone for 20 or 40min. Finally, for the larger samples, which were investigated in the third study (iii), according to the results, a combination of acetone and ultrasonic bath for 4h failed to completely remove bone marrow from cancellous bone samples (Ø6×12mm). In this study, it was also observed that short-term storage in physiological saline is an appropriate choice and has no effect on the E0 of cancellous bone. However, treatment of cancellous bone with acetone resulted in changes in mechanical properties that could not be reversed by subsequent immersion in physiological saline. Conclusions: The results of this work provide a reference for bone quality assessment, sample preservation, and mechanical testing of cancellous bone, which is important for obtaining accurate biomechanical data. It may serve as a basis for future clinical applications and in vitro model building.

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Objectives: To compare optical values in the osteotomy gap created after a Tibial Tuberosity Advancement (TTA) treated with autogenous cancellous bone graft (ACBG), extracorporeal shock wave therapy (ESWT), a combination of ACBG and ESWT, and absence of both ACBG and ESWT using densitometry. Methods: Dogs presenting for surgical repair of a cranial cruciate ligament rupture were randomly assigned to one of four groups; TTA with ACBG (TTA-G), TTA with ACBG and ESWT (TTA-GS), TTA with ESWT (TTA-S), and TTA with no additional therapy (TTA-O). Mediolateral radiographs at 0, 4 and 8 weeks after surgery were evaluated to compare healing of the osteotomy gap via densitometry. An analysis of variance (ANOVA) statistical analysis was used to compare the densitometric values between groups. Results: At 4 weeks after surgery, a significant difference in osteotomy gap density was noted between TTA-GS (8.4 millimeters of Aluminum equivalent [mmAleq]) and TTA-S (6.1mmAleq), and between TTA-GS (8.4 mmAleq) and TTA-O (6.4 mmAleq). There were no significant differences noted between groups at the 8 week recheck. Clinical Significance: There were no significant differences in the osteotomy gap density at 8 weeks after surgery regardless of the treatment modality used. The combination of ACBG and ESWT may lead to increased density of the osteotomy gap in the first 4 weeks after surgery. Densitometry using an aluminum step wedge is a feasible method for comparison of bone healing after TTA in dogs. / Master of Science