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

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

Os bisfosfonatos (BF) são amplamente utilizados no tratamento de doenças osteolíticas como metástases ósseas e osteoporose. A osteonecrose dos maxilares associada ao uso de BF (OMAB) é caracterizada pela presença de osso exposto ou que pode ser sondado através de uma fístula que persiste por mais de oito semanas em pacientes com história de terapia de BF e sem história de radioterapia na região de cabeça e pescoço e/ou sem doença metastática nos maxilares. A incidência de OMAB aumenta com a potência, duração do tratamento e dose de BF recebida. Até o presente momento, a fisiopatologia da OMAB não está clara, dificultando a prevenção e o tratamento. O objetivo deste estudo foi avaliar o efeito da administração de altas doses Ácido Zoledrônico (AZ) por período prolongado no osso esponjoso da mandíbula e da metáfise proximal do fêmur de ratos Wistar. Para relacionar as descobertas à fisiopatologia da OMAB, o regime de administração de BF de um modelo animal relevante desta lesão foi reproduzido. Seis animais receberam AZ (0,6 mg / kg) e seis receberam solução salina no mesmo volume (Controles). Os compostos foram administrados por via intraperitoneal em cinco doses a cada 28 dias. A eutanásia dos animais ocorreu após 150 dias de início da terapia. As hemimandíbulas e fêmures direitos foram escaneados usando Micro-tomografia computadorizada (Micro-CT) de alta resolução (14 m). Para a primeira análise realizada neste estudo, os dados morfométricos do osso esponjoso foram calculados na região do segundo e primeiro molar na mandíbula e na metáfise do fêmur usando CTAnalyzer (Bruker, Bélgica). Para a segunda análise, cinco amostras de hemimandíbulas de cada grupo foram cortadas em lâminas histológicas (5 m) e coradas com Hematoxilina e Eosina. Para comparar os parâmetros morfométricos na Micro-CT e histologia, as imagens de Micro-CT foram espacialmente alinhadas à histologia. Os dados morfométricos do osso alveolar foram calculados usando o software CTAnalyzer (Bruker, Bélgica) na região entre as raízes mesial e distal do primeiro molar. A densidade da área vascular (área vascular/área total; VA/TA) e os dados histomorfométricos ósseos foram estimados usando Axiovision na mesma região (entre as raízes mesial e distal do primeiro molar). Foi adotada significância estatística de 5% ( = 0,05). Os animais tratados com AZ apresentaram aumento significativo na porcentagem de volume ósseo (p <0,05) com trabéculas mais espessas, osso mais compacto com menor separação trabecular na mandíbula e no fêmur. Na mandíbula, o aumento da densidade óssea e diminuição da separação trabecular foram fortemente correlacionados com a diminuição da área vascular observada no grupo AZ (p <0,05). Em conclusão, o tratamento de longa duração com altas doses de AZ foi significativamente associado ao aumento na densidade óssea e à diminuição dos espaços medulares, canais nutritivos e vasculatura do osso alveolar. A análise com Micro-CT revelou alterações semelhantes na estrutura óssea tanto na mandíbula quanto no fêmur do grupo AZ. / Bisphosphonates (BFs) are widely used in the treatment of osteolytic diseases such as bone metastases and osteoporosis. The osteonecrosis of the jaws related to BF (ONB) is characterized by the presence of exposed bone or bone that can be probed through a fistula that persists for more than eight weeks in patients with a history of BF therapy and without history of head and neck radiotherapy and / or without metastatic disease in the jaws. The incidence of ONB increases with potency, duration of treatment and dose of BF received. Thus far, the pathophysiology of ONB is unclear, hampering prevention and treatment. The aim of this study was to objectively assess the effect of long-term high-dose Zoledronic Acid (ZA) on cancellous bone in the jaw and femur of Wistar rats. In order to link our findings to the physiopathology of ONB, the therapeutic regiment of a relevant ONB animal model was reproduced. Twelve Wistar rats were randomly divided in two groups: six received Zoledronic acid (ZA; 0.6 mg / kg) and six (Controls) received saline solution in the same volume. The compounds were administrated intraperitoneally in five doses each 28 days. The rats were killed after 150 days of the therapy onset. Mandibles and femurs were scanned using a high-resolution (14m) micro-computerized tomography (Micro-CT). For the first analysis carried in this study, cancellous bone morphometric data were calculates in the region of the second and first molar in the mandible and in the proximal femur using CTAnalyzer (Bruker, Belgium). For the second analysis five samples were cut into histological slices (5m) and stained with Hematoxylin and Eosin. In order to compare the same morphological structures in Micro-CT and histology, the Micro-CT images were aligned to histology. Alveolar bone morphometric data (Micro-CT) was calculated using CTAnalyzer (Bruker, Belgium) in the region between the mesial and distal roots of the first molar. Blood vessels density and bone histomorphometric data were calculated using Axiovision (Carl Zeiss, Germany) in the same region used for Micro-CT evaluation. Statistical significance of 5% (=0.05) was adopted. ZA treated rats presented significant increase in the percentage of bone volume (p<0.05) with thicker trabeculae and more compact bone with smaller marrow spaces in the mandible and femur. In the mandible, the increase in bone density and decrease of marrow spaces size was strongly correlated with the decrease in the vascular area noticed in the ZA group (p<0.05). In conclusion, long-term high-dose ZA treatment was significant associated with the increase of bone density and the diminution of medullary spaces and nutritive canals size as well as decrease in vascularity of the alveolar bone. Micro-CT investigation showed similar changes in bone structure in the mandible and femur in the ZA group.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The aim of this research was to test and to compare the homogenous cancellous bone glycerin-stored with the autogenous cancellous bone graft in the atlantoaxial arthrodesis. Fifteen dogs, weighing between 8 and 12 kg, were randomly distributed in three groups denominated one (30 days), two (60 days) and three (90 days). The atlantoaxial joint was immobilized through the ventral access using Steinmann pins associated the acrylic resin. A defect was created in the right hemi-joint atlantoaxial and put 100mg of the homogenous implant and, in the left hemi-joint, 100mg of the autogenous graft. Was analyzed after 30, 60 and 90 days, survey radiographs in the joint atlantoaxial, histopathological and bone density by computed tomography of the fragments collected through biopsies. The right and left hemi-joint atlantoaxial has moderate formation in the 30 days pos-operative. In the 60 days, has bone formation with partial fusion more in the autogenous side (left) and the 90 days, the bone formation with partial fusion was high in the homogenous side (right). Computed tomography evaluation in the 30 and 60 days post-operative has high bone density in the fragments collected in the right hemi-joint atlantoaxial (homogenous implant). In the 90 days, the bone density in the fragments collected in the right and left hemi-joint were similar with tendency in cortical bone formation. The evaluation histological of the bone fragments in the left hemi-joint (cancellous bone graft) and right hemi-joint (homogenous implant) atlantoaxial were similar in the 30 and 60 days post-operative. In the 90 days, bone tissue mature and organized was observed. No has significant difference the right hemi-joint atlantoaxial (homogenous implant) with the left hemi-joint (cancellous bone graft) when comparison the grade fusion joint and bone density the fragments collected by biopsies. The use of the homogenous cancellous bone implant glycerin-stored in dogs submitted atlantoaxial arthrodesis with Steinmamm pins associated acrylic resin is a viability method for treatment in the atlantoaxial instability. / O objetivo desta pesquisa foi testar e comparar o implante homógeno de osso esponjoso conservado em glicerina a 98% com o enxerto autógeno em cães submetidos à artrodese atlantoaxial. Foram utilizados 15 cães, pesando entre 8 e 12 kg, distribuídos aleatoriamente em três grupos iguais de acordo com o período pósoperatório (PO) denominados de I (30dias), II (60 dias) e III (90 dias). A articulação atlantoaxial foi submetida à artrodese através do acesso ventral utilizando pinos de Steinmann associados à resina acrílica autopolimerizável. Foi criado um defeito na hemiarticulação atlantoaxial direita para colocação de 100mg de osso esponjoso homógeno e, na esquerda, 100 mg de osso esponjoso autógeno. Decorridos o período 30, 60 e 90 dias de PO, foram realizadas análise radiográfica da articulação atlantoaxial, histológica e da densidade óssea por tomografia computadorizada dos fragmentos ósseos coletados por biópsia. A análise radiográfica revelou aos 30 dias que as hemiarticulações atlantoaxiais esquerda e direita apresentavam formação óssea moderada. Já aos 60 dias de PO, houve uma tendência à formação óssea com grau de fusão parcial mais no lado autógeno (esquerdo) e aos 90 dias, a formação óssea com grau de fusão parcial predominou no lado homógeno (direito). O exame por tomografia computadorizada (TC) aos 30 e 60 dias de PO revelou maior densidade óssea nos fragmentos coletados na hemiarticulação atlantoaxial direita (implante homógeno). Aos 90 dias, a densidade óssea dos fragmentos coletado das hemiarticulações direita e esquerda foram semelhantes com tendência à formação de osso cortical. Os achados histológicos dos fragmentos coletados da hemi-articulação direita (enxerto autógeno) e esquerda (implante homógeno) foram similares aos 30 e 60 dias de PO, sendo que aos 90 dias de PO havia presença de um tecido ósseo maduro e organizado. Não foi encontrada diferença estatística entre o enxerto autógeno e implante homógeno em relação ao grau de fusão articular e densidade óssea dos fragmentos. O uso de implante homógeno conservado em glicerina a 98% em cães submetidos à artrodese atlantoaxial com pinos de Steinmann e resina acrílica autopolimerizável não apresenta diferença quando comparado ao enxerto autógeno e é um método viável para tratamento de instabilidade atlantoaxial.