published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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Metastatic bone cancer causes severe pain that is primarily treated with opioids. A recently developed model of bone cancer pain was used to evaluate the effects of sustained morphine treatment. In cancer-treated mice, morphine enhanced spontaneous and evoked pain; these effects were dose-dependent and naloxone-sensitive. SP and CGRP positive DRG cells did not differ between sarcoma or control mice, but were increased following morphine in both groups. Morphine increased ATF-3 expression only in DRG cells of sarcoma mice. Morphine did not alter tumor growth in vitro or in vivo but increased sarcoma-induced bone destruction and incidence of spontaneous fracture in a dose- and naloxone-sensitive manner. Morphine increased osteoclast activity suggesting enhancement of sarcoma-induced osteolysis. Thus, morphine treatment may "add-on" additional mechanisms of pain beyond those induced by sarcoma. Despite the potential clinical significance, the exact mechanisms of opioid-induced hypersensitivity remain unknown. The vanilloid 1 receptor (TRPV1) is a molecular integrator of noxious stimuli. Sustained morphine elicited thermal and tactile hypersensitivity in WT, but not TRPV1 KO mice. Sustained morphine enhanced capsaicin-induced flinching and plasma extravasation in rats, indicating increased activity of these receptors in the periphery. Immunohistochemical studies indicate increase in TRPV1 expression in DRG and sciatic nerve, but not spinal cord, suggesting increased trafficking of TRPV1 channel to the periphery. Possible mechanisms of this enhanced expression and function of TRPV1 channels is activation of p38 MAPK. Sustained intrathecal infusion of p38 MAPK inhibitor prevents morphine-induced hypersensitivity and enhanced capsaicin-induced flinching, indicating the role of p38MAPK in the development of morphine-induced pain, possibly through sensitization of TRPV1 receptors. Acute administration of p38 MAPK inhibitor reversed morphine-induced pain suggesting the importance of p38 MAPK in the maintenance of morphine-induced hypersensitivity, likely through activation of TRPV1 channel. Sustained morphine also up-regulates NGF content in skin, which is then transported to DRG neurons where phosporilation of p38MAPK takes place. Single injection of anti-NGF peptibody blocked the development of morphine-induced hypersensitivity, enhanced capsaicin-induced flinching and plasma extravasation. Co-treatment with these compounds blocks the development of morphine-induced hyperalgesia and may optimize treatment of chronic pain states, like bone cancer pain.

This study focussed on the reduced platen compression (RPC) testing of the cancellous bone of the proximal tibia. The objective of this study was to improve the current testing methods with an emphasis on the location of the RPC specimens, and to correlate the mechanical properties of the rodent cancellous bone with the various density measures. Analytical studies were made to assess the effect of the size and shape of the platen based on the values from mechanical testing of the cancellous bone. RPC specimens are made from transverse slices of the proximal tibia metaphysis. Specimen location was determined using planar radiograph method at a distance of 1.75 mm distal to the growth plate. The distance from the top of the proximal tibia to the line at 1.75 mm distal to the growth plate was measured and this distance is termed as the first cut distance. Specimen thickness of 2 mm was then cut for the RPC test. Endocortical method was followed to determine the platen sizing for RPC testing. The cancellous bone was then tested in compression to failure. Correlations were performed between the mechanical properties of the cancellous bone and the density measures from pQCT and radiographic techniques. SigmaStat and TableCurve 2D were used to perform the correlations and estimate the P value for the correlation. Linear and power law fits were made for all the correlations. Based on this study, several improvements to the reduced platen compression test were recommended. An improved specimen location method was developed. However, it requires a corrective distance to account for the tissue that cannot be identified in the radiographic analysis. A new method for estimating the density of the cancellous bone that is directly tested by the platen was developed. Correlations between the density of the cancellous bone and the mechanical properties show that, there is a strong correlation between ultimate stress and aluminum layer intensity. Elastic modulus correlates best with the last batch tested in this study. Recommendations for future study include advanced technology like finite element analysis and custom shaped platens to enhance RPC testing.

Breast cancer has a high propensity to metastasize to bone. While the genetic and epigenetic changes associated with metastatic breast cancer progression are being identified, the changes that drive metastatic progression are poorly understood. Proteases, and in particular matrix metalloproteinases (MMPs), have been shown to play a pivotal role in certain aspects of tumor metastasis by modifying the affected microenvironment. Bone matrix-depositing mouse MC3T3 osteoblasts were co-cultured with metastatic human MDA-MB-23 1 (MDA23 1) cells or the bone-homing MDA-MB 231-1 833/TR (1 833/TR) variant in an effort to identify novel, osteoclast-independent, changes to the tumor/bone microenvironment. Co-culture-induced changes in the complete “protease and inhibitor” expression profile in the osteoblasts and the tumor cells were then determined using targeted murine and human specific microarray chips (CLIP-CHIP TM ). This analysis revealed an increase in the RNA expression of collagenase-3 (MMP 13) in the co-cultured osteoblasts that was confirmed by qPCR. Further, Western blotting indicated increased MIvIP13 protein secretion into the bone matrixltumor microenvironment by the co-cultured MC3T3 cells. The elevation in osteoblast-produced MMP13 was observed when the co- cultured tumor cells were in direct contact or separated by filters. Additionally, the elevation was also induced by conditioned medium derived from separate MDA23 1 or 1 833/TR cultures, which indicates that a soluble factor produced by the tumor cells is capable of inducing MMP 13. One soluble factor that appears to be produced by 1 833iTR cultures is oncostatin M. Oncostatin M is an interleukin-6 family cytokine that is known to upregulate MMP13 synthesis and secretion during chondrogenesis. Genome-wide Affymetrix® analysis revealed, and qPCR analysis confirmed, that oncostatin M receptor-specific subunit RNA was also significantly upregulated in co-cultured osteoblasts. Therefore, breast tumor cells may be capable of initiating protein degradative changes in the bone microenvironment that are independent of the much studied osteolytic degradation initiated by osteoclast activation.

Introduction: The process by which children's bones grow has not been fully charcterised. The current dogma is that girls fill in their medullary canal area by forming bone at the endosteum. It has been argued that the sex difference in how bone strength is conferred -- favouring boys -- may contribute to the relative protection that aging men have over aging women with respect to fracture incidence and the prevalence of osteoporosis. Primary Objectives: 1)To compare bone surface changes at the periosteal and endosteal surface of the tibial midshaft in boys and girls. 2)To compare how bone density at the tibial midshaft is accrued in boys and girls. 3) To compare sex differences in bone strength accrual. Methods: Design and Participants: Participants were obtained from a 20-month randomized, controlled school-based physical activity intervention. As we found no difference in the effect of the intervention on pQCT bone outcome variables, both groups were combined for our current study. A total of 183 participants (93 boys, 89 girls) received a pQCT scan at baseline. Results: Sex-specific comparisons of the pQCT bone outcome variables showed significantly greater rates of change (slope) for boys for the total area (ToA), cortical area (CoA), medullary canal area (MedA) and strength-strain index (SSI) measures, p<0.001. No significant differences were observed for CoD, p=0.904. The magnitude of these differences is 60.8% for ToA, 55.7% for CoA, 75.6% for MedA, 1.3% for CoD, and 54.7% for SSI. Examination of differences between the sexes (intercept) revealed significant differences with greater gains observed for boys for all measures p<0.001 except for CoD where girls exhibited greater gains p<0.001. Conclusion: Girls showed a similar pattern of cortical bone growth at the tibial midshaft- periosteal apposition dominated over endosteal resorption. Boys' increased changes and pattern of growth were of a greater magnitude at both surfaces compared to girls. This resulted in a greater increase in strength as measured by SSI in boys which can partly be explained by their larger size. Girls exhibited greater increases in CoD; however, no significant difference in the change in CoD was observed between the two.

The Er:YAG laser and the USPL are the most promising when considering the previous study results and their physical characteristics. This investigation compared the healing of various laser ablation units versus conventional mechanical cutting to explore the future applications for bone surgery and the effects when combined with rhBMP-7. A full-thickness circular defect was created on the parietal bones of mice for all the groups. Hard tissue healing was assessed using a microcomputerized tomography. Wound closure analyses suggested that the femtosecond laser created wounds displayed slightly healing delay in closure over the healing period when compared to mechanical instrumentation. The Er:YAG laser showed a healing rate similar to that of the mechanically ablated groups. In summary, femtosecond and Er:YAG lasers are two modalities suitable for bone ablation comparable to mechanical instrumentation.

Background: Hyperbaric Oxygen Therapy (HBOT) is used to promote soft and hard tissue repair at sites of injury or disease. It is not known whether it has any effect on uninjured tissues. Objective: To evaluate the effect of HBO on vertebral bones distant from a calvarial surgical site. Materials and Methods: 22 male New Zealand white rabbits were divided into two groups (n=11). All animals underwent surgery to produce bilateral calvarial critical sized defects. Group 1 received HBOT while Group 2 served as controls breathing room air (NBO). The Marx HBO protocol was used (90min, 100% oxygen at 2.4 ATA, 5 days a week for 4 weeks). Subjects were sacrificed at 6 weeks. The vertebrae were analyzed by micro-CT (μCT) and histology. Results: There were no statistical significant differences between the two groups. Conclusion: we concluded that there were no harmful effects of HBOT on non-injured vertebrae at 6 weeks.

No description available.

Background: Hyperbaric Oxygen Therapy (HBOT) is used to promote soft and hard tissue repair at sites of injury or disease. It is not known whether it has any effect on uninjured tissues. Objective: To evaluate the effect of HBO on vertebral bones distant from a calvarial surgical site. Materials and Methods: 22 male New Zealand white rabbits were divided into two groups (n=11). All animals underwent surgery to produce bilateral calvarial critical sized defects. Group 1 received HBOT while Group 2 served as controls breathing room air (NBO). The Marx HBO protocol was used (90min, 100% oxygen at 2.4 ATA, 5 days a week for 4 weeks). Subjects were sacrificed at 6 weeks. The vertebrae were analyzed by micro-CT (μCT) and histology. Results: There were no statistical significant differences between the two groups. Conclusion: we concluded that there were no harmful effects of HBOT on non-injured vertebrae at 6 weeks.