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A major health concern for subjects with diabetes is weaker bones and increased fracture risk. Current clinical assessment of the bone strength is performed by measuring Bone Mineral Density (BMD), where low BMD-values are associated with an increased risk of fracture. However, subjects with Type 2 Diabetes (T2D) have been shown to have normal or higher BMD-levels compared to healthy controls, which does not reflect the recognized bone fragility among diabetics. Thus, there is need for more research about diabetes-related bone fragility to find other factors of impaired bone health. One potential biomarker that has recently been studied is Bone Marrow Fat (BMF). The data in this project consisted of whole-body water-fat Magnetic Resonance Imaging (MRI) volumes from the UK Biobank Imaging study (UKBB). Each subject in this data has a water volume and a fat volume, allowing for a quantitative assessment of water and fat content in the body. To analyze and perform quantitative measurements of the bones specifically, a Deep Learning (DL) model was trained, validated, and tested for performing fully automated and objective skeleton segmentation, where six different bones were segmented: spine, femur, pelvis, scapula, clavicle and humerus. The model was trained and validated on 120 subjects with 6-fold cross-validation and tested on eight subjects. All ground-truth segmentations of the training and test data were generated using two semi-automatic pipelines. The model was evaluated for each bone separately as well as the overall skeleton segmentation and achieved varying accuracy, performing better on larger bones than on smaller ones. The final trained model was applied on a larger dataset of 9562 subjects (16% type 2 diabetics) and the BMF, as well as bone marrow volume (BMV) and cortical bone volume (CBV), were measured in the segmented bones of each subject. The results of the quantified biomarkers were compared between T2D and healthy subjects. The comparison revealed possible differences between healthy and diabetic subjects, suggesting a potential for new findings related to diabetes and associated bone fragility.

Introduction: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a platelet count less than 100 x 109/L. Platelet-bound autoantibodies are detected in the peripheral blood of only 40-50% of ITP patients. The subset of ITP patients who do not have detectable autoantibodies may truly be seropositive, but autoantibodies may not be detected due to limitations of the assays. Studies have also suggested that autoantibodies might be sequestered in the bone marrow where autoantibodies may impair platelet production. In addition, detecting autoreactive antibody-secreting B cells using the Enzyme-linked Immunospot (ELISPOT) assay was shown to be highly sensitive. In this study, the bone marrow and the peripheral blood compartments of ITP patients were tested for the presence of anti-GPIIbIIIa and anti-GPIbIX IgG autoantibodies and autoreactive B cells. Methods: Bone marrow aspirate and peripheral blood samples were collected from ITP patients (n=12), non-immune thrombocytopenic patient controls (n=3), and healthy controls (n=5). Mononuclear cells were isolated and tested for the presence of anti-GPIIbIIIa and anti-GPIbIX IgG autoreactive B cells before stimulation and after stimulation with R848 and IL-2 using the ELISPOT assay. Anti-GPIIbIIIa and anti-GPIbIX IgG autoantibodies were detected in the bone marrow and the peripheral blood using the direct antigen capture assay. Results: In our study, we detected autoantibodies and autoreactive B cells of known specificity in the bone marrow of a subset of ITP patients. Detecting anti-GPIIbIIIa and anti-GPIbIX autoantibodies in the bone marrow or the peripheral blood had a sensitivity of 42% and testing both compartments increased the sensitivity to 58%, while maintaining 100% specificity. Autoreactive B cells were detected at low frequencies with low specificity in the bone marrow and the peripheral blood of a subset of ITP patients. The majority of the ITP patients without detectable autoantibodies in the peripheral blood did not have autoantibodies in the bone marrow, and autoreactive B cells were not detected in either compartment. Conclusion: Examining both the bone marrow and the peripheral blood compartments for autoantibodies or autoreactive B cells increased the sensitivity. Furthermore, detecting autoantibodies using the antigen capture assay is more sensitive and specific than detecting autoreactive B cells using the ELISPOT assay. / Thesis / Master of Science (MSc)

The knee joint is a diarthrodial joint that rotates in the flexion-extension axis to provide individuals mobility. A limitation in the passive range of motion (ROM) is detrimental for function and this limitation is termed a joint contracture. A commonly shared characteristic between conditions that lead to contracture formation is prolonged periods of immobilization. However, the etiology of immobility-induced joint contractures is not well described and requires quantitative data on anatomical structures limiting knee mobility to design new interventions aimed at restoring function. In turn, our research group has developed an experimental animal model to study the temporal pathophysiology of knee immobilization and reversibility through unassisted remobilization. With durations of immobilization ranging from 1 to 32 weeks and remobilization up to 48 weeks, our experimental design provides a comprehensive temporal overview on the various stages of contracture formation: initiation, progression, and severity. A combination of muscles and articular structures are involved in the pathophysiology of knee flexion contractures, but the posterior joint capsule is of particular interest. Through histomorphological analysis, we provided quantitative data on the contribution of the reduced posterior capsule length in the limitation of knee extension and increased joint stiffness. Moreover, elucidation of synoviocyte profiles within the synovium of the capsule provided insights to potential mechanisms of capsule shortening. Our novel measurable outcome of mechanical joint stiffness revealed distinct temporal differences with ROM measurements after joint immobilization and remobilization, suggesting that alterations in the biomechanical properties of articular tissue structures are also contributing to the limitation in function. Malleability of the dynamic reciprocal relationship between trabecular bone loss and accumulation of marrow adipose tissue (predominately through adipocyte hyperplasia) after knee immobilization underscores the sensitivity of the bone marrow microenvironment in response to mechanical stimuli and lack thereof. Remobilization of the knee joint is limited in its capacity to reverse detriments induced by extended periods of joint immobilization. Findings from this work point to the temporal changes detected in different musculoskeletal tissues during knee immobilization and emphasizes the contribution of the joint capsule in limiting joint mobility.

Bone is the second most transplanted tissue in the body, with approximately 2.2 million bone graft procedures performed annually worldwide. Currently, autogenous bone is the gold standard for bone grafting due to its ability to achieve functional healing; however, it is limited in supply and results in secondary injury at the donor site. Tissue engineering has emerged as a promising means for the development of new bone graft substitutes in order to overcome the limitations of the current grafts. In this research project, the specific approach for bone tissue engineering involves seeding osteoprogenitor cells within a biomaterial scaffold then culturing this construct in a biodynamic bioreactor. The bioreactor imparts osteoinductive mechanical stimuli on the cells to stimulate the synthesis of an extracellular matrix rich in osteogenic and angiogenic factors that are envisioned to guide bone healing in vivo. Fluid flow, which exerts a hydrodynamic shear stress on adherent cells, has been identified as one of the strongest stimuli on bone cell behavior. It has been shown to enhance the deposition of osteoblastic matrix proteins in vitro, and is particularly important for the delivery of oxygen and nutrients to cells within large scaffolds suitable for bone tissue regeneration. In particular, dynamic flow profiles have been shown to be more efficient at initiating mechanotransductive signaling and enhancing gene expression of osteoblastic cells in vitro relative to steady flow. However, the molecular signaling mechanisms by which bone cells convert hydrodynamic shear stress into biochemical signals and express osteoblastic matrix proteins are not fully understood. Therefore, the overall goal of this research project was to determine the effect of dynamic fluid flow on mechanotransductive signaling and expression of bioactive factors and bone matrix proteins. In the first study, an intermittent flow regimen, in which 5 min rest periods were inserted during fluid flow, was examined. Results showed that signaling molecules, mitogen activated protein kinases (MAPKs) and prostaglandin E2, were modulated with the flow regimen, but that expression of bone matrix proteins, collagen 1α1, osteopontin, bone sialoprotein (BSP), and osteocalcin (OC), were similar under continuous and intermittent flow. Thus, this study suggested that variation of the flow regimen modulates mechanotranductive signaling. In the second study, four flow conditions were examined: continuous flow, 0.074 Hz, 0.044 Hz, and 0.015 Hz pulsatile flow. This study demonstrated that pulsatile flow enhances expression of BSP and OC over steady flow. Similarly, bone morphogenetic protein (BMP)-2 and -7 were enhanced with pulsatile flow, while BMP-4 was suppressed with all flow conditions, suggesting that the mechanism by which fluid flow enhances bone matrix proteins may involve the induction of BMP-2 and -7, but not BMP-4. In the third study, the molecular mechanism by which fluid flow simulates expression of BMPs was examined. Results from this study suggest that this mechanism may involve activation of MAPKs, but BMP-2, -4, and -7 are regulated through multiple different signaling pathways. Overall, the results from this research demonstrate that dynamic flow modulates mechanotransductive signaling and expression of osteoblastic matrix proteins by osteoblast cells. In particular, BMPs, important for formation in vivo, were shown to be induced by fluid flow. Therefore, this work may be beneficial in understanding and developing 3D perfusion culture systems for the creation of a clinically effective engineering bone tissue. / Ph. D.

Rupture of the anterior cruciate ligament (ACL) is one of the most common ligamentous injuries of the knee. Post rupture, the ACL does not heal on itself due to poor vasculature and hence surgical intervention is required to treat the ACL. Current surgical management of ACL rupture consists of reconstruction with autografts or allografts. However, the limitations associated with these grafts have prompted interest in tissue engineered solutions that combine cells, scaffolds and stimuli to facilitate ACL regeneration. This thesis describes a ligament tissue engineering strategy that involves incorporating biomolecules within fibers-based electrospun meshes which mimics the extra-cellular matrix microarchitecture of ligament. However, challenges exist with incorporation of biomolecules. Therefore, the goal of this research project was to develop two techniques to incorporate biomolecules within electrospun meshes: (1) co-axially electrospinning fibers that support surface-grafting of biomolecules, and (2) co-axially electrospinning fibers decorated with biomolecule-loaded microspheres. In the first approach, chitosan was co-axially electrospun on the shell side of poly caprolactone (PCL) and arginine-glycine-aspartate (RGD) was attached to the electrospun meshes. Bone marrow stromal cells (BMSCs) attached, spread and proliferated on these meshes. In the second approach, fluorescein isothiocyanate labelled bovine serum albumin (FITC-BSA) loaded chitosan-alginate (CS-AL) microspheres were fabricated. The effects of cation to alginate ratio, type of alginate and concentration of CaCl2 on microsphere size, FITC-BSA loading and release were systematically evaluated. The CS-AL microspheres were then incorporated into the sheath phase of co-axially electrospun meshes to achieve microsphere-decorated fiber composite meshes. The results from these model study suggest that both approaches are tractable for incorporating biomolecules within fibers-based electrospun meshes. Both these approaches provide platform for future studies that can focus on ligament-relevant biomolecules such as FGF-2 and GDF-5. / Master of Science

Exposure to spores and hyphae of Alternaria alternata, an airborne ubiquitous fungus, is clinically associated with allergic airway disorders including allergic rhinitis, asthma, and chronic rhinosinusitis. Dendritic cells are known as the type of antigen presenting cells most often associated with allergic inflammation. In this study, we used mouse bone marrow-derived dendritic cells (BMDCs) as a model to study the ability of A. alternata spores and different components of the spore cell wall to stimulate innate immune responses. We found that BMDCs were highly sensitive to A. alternata spores, chitin and the major allergen Alt a 1. Following stimulation with these molecules, the expression of MHC II and other co-stimulators, like CD40, CD86, and OX40L, were highly up regulated. In order to determine how different cell wall components affect the T cells, we conducted co-culture experiments of BMDCs and lymphocytes in this study. Both spores and Alt a1 did not induce IL-4 in mixed lymphocytes reactions. Interestingly, we found that Alt a 1 induced the switching of the CD4+ T cell population to the Th17 type, with a major increase in IL-17A secretion. This study reveals that A. alternata components may balance the innate immune responses between Th2 and Th17 pathways, and/or contributes to the development and exacerbation of more serve neutrophilic forms of asthma. / Master of Science

No / Improving the patient¿s experience of a bone marrow biopsy ¿ an RCT Aims. To compare nitrous oxide 50%/oxygen 50% (N2O/O2 ¿ entonox) plus local anaesthetic (LA) with placebo (oxygen) plus LA in the management of pain experienced by patients undergoing a bone marrow biopsy. . Bone marrow biopsies are a common procedure for many haematological conditions. Despite the use of a LA, pain during the procedure has frequently been reported by patients. Previous research in pain management of other invasive diagnostic procedures (e.g. sigmoidoscopy) has reported N2O/O2 as an effective alternative to LA. Design. Double-blind randomized controlled trial. Methods. Forty-eight patients requiring a bone marrow biopsy were randomized to receive either N2O/O2 or oxygen in addition to their LA. Participants were asked to complete a pain score and comment on their experience of the procedure. Results. Although the overall pain scores were moderate, there was a wide range of scores. N2O/O2 resulted in significantly less pain for men, but not for women. All patients who had had previous biopsies reported significantly more pain, regardless of the gas used. There were no significant adverse effects in either group. Conclusion. N2O/O2 is a safe, effective, easy-to-use analgesic which merits further investigation in potentially painful diagnostic (and other) interventions.

Long-term exposure to pharmacological doses of glucocorticoids has been associated with the development of osteopenia and avascular necrosis. Bone loss may be partially attributed to a steroid-induced decrease in the osteoblastic differentiation of multipotent progenitor cells found in the bone marrow. In order to determine if there is a change in the osteogenic potential of the bone marrow stroma following glucocorticoid treatment, Sprague-Dawley rats were administered methylprednisolone for up to six weeks, then sacrificed at 0, 2, 4, or 6 weeks during treatment or 4 weeks after cessation of treatment. Femurs were collected and analyzed for evidence of steroid-induced osteopenia and bone marrow adipogenesis. Although glucocorticoid treatment did inhibit bone growth, differences in ultimate shear stress and mineral content were not detected. The volume of marrow fat increased with increasing duration of treatment, but returned to near control levels after cessation of treatment. Marrow stromal cells were isolated from tibias, cultured in the presence of osteogenic supplements, and analyzed for their capacity to differentiate into osteoblast-like cells in vitro. Glucocorticoid treatment diminished the absolute number of isolated stromal cells, but did not inhibit the relative levels of bone-like mineral deposition or osteocalcin expression and secretion. Although pharmacological glucocorticoid levels induce bone loss in vivo, physiologically equivalent concentrations have been shown to enhance the formation of bone-like tissue in vitro. However, glucocorticoids have also been reported to inhibit proliferation and type I collagen synthesis in marrow stromal cell cultures. In order to assess the effects of intermittent dexamethasone treatment on the progression of osteogenesis in rat marrow stromal cell culture, this synthetic glucocorticoid was removed from the culture medium after a variable period of initial supplementation. Cell layers were analyzed for total cell number, collagen synthesis, phenotypic marker expression, and matrix mineralization. Prolonged supplementation with dexamethasone decreased proliferation, but did not significantly affect collagen synthesis. Furthermore, increased treatment duration was found to increase bone sialoprotein expression and mineral deposition. The duration of glucocorticoid treatment may be a key factor for controlling the extent of differentiation in vitro. / Master of Science