We introduce a continuous modeling approach which combines elastic response of the trabecular bone structure with the concentration of signaling molecules within the bone and a mechanism for concentration dependent local bone formation and resorption. In an abstract setting bone can be considered as a shape changing structure. For similar problems in materials science phase field approximations have been established as an efficient computational tool. We adapt such an approach for trabecular bone remodeling. It allows for a smooth representation of the trabecular bone structure and drastically reduces computational costs if compared with traditional micro finite element approaches. We demonstrate the advantage of the approach within a minimal model. We quantitatively compare the results with established micro finite element approaches on simple geometries and consider the bone morphology within a bone segment obtained from micro-CT data of a sheep vertebra with realistic parameters.

Autoimmune diseases such as rheumatoid arthritis (RA) are associated with debilitating chronic inflammation, autoantibody production, articular bone erosions and systemic bone loss. The underlying mechanisms and cell types that initiate these diseases are not fully understood, and current therapies mainly address downstream mechanisms and do not fully halt disease progression in all patients. Moreover, previous studies have largely focused on the role of adaptive immunity in driving these diseases, and less attention has been given to the contribution of innate immune pathways such as DNA sensor signaling pathways in initiating and/or perpetuating autoimmunity and erosive inflammatory arthritis. Detection of microbial nucleic acids by DNA sensors such as endosomal toll-like receptors (TLRs) and cytosolic sensors is an early form of antiviral defense. Upon detection of nucleic acid, TLRs dependent on Unc93B and cytosolic sensors dependent on the adaptor stimulator of interferon genes (STING) orchestrate production of type 1 interferons and pro-inflammatory cytokines to resolve infection. Additionally, the cytosolic DNA sensor absent in melanoma 2 (AIM2), which is not dependent on STING, also recognizes microbial DNA and coordinates the cleavage of pro-IL-1β. Previous studies have largely focused on the role of these DNA sensors in macrophages and dendritic cells in the context of antiviral immunity. In recent years, however, the inappropriate recognition of host nucleic acids by these sensors has been associated with several autoimmune diseases including RA. This dissertation aims to delineate the mechanisms by which DNA sensors contribute to inflammatory arthritis and bone remodeling in the context of a murine model of autoimmunity. In DNase II deficient mice, excessive accrual of undegraded, endogenous DNA leads to robust production of type 1 interferons (IFNs) and proinflammatory cytokines. The high levels of type 1 IFNs result in anemia and embryonic lethality; therefore, the gene for the type 1 IFN receptor (IFNaR) has also been deleted so that the mice survive. DNase II-/- IFNaR-/- double knockout (DKO) mice develop erosive inflammatory arthritis, anti-nuclear antibodies, and splenomegaly not seen in the DNase II+/- IFNaR-/- (Het) control group. To evaluate whether cytosolic or endosomal DNA sensors contribute to the clinical manifestations of DKO mice, genes involved in TLR or cytosolic sensor signaling were deleted on the DKO background. Genetically altered mice include STING/DNaseII/IFNaR TKO (STING TKO), AIM2/DNase II/IFNaR TKO (AIM2 TKO), and Unc93b/DNase II/IFNaR TKO (Unc93 TKO) mice. Our hypothesis was that the STING, AIM2, and/or Unc93 pathways would contribute to the autoimmune manifestations in DNase II deficient mice. Rigorous examination of inflammation in these lines revealed important roles for both the STING and AIM2 pathways in arthritis. Despite the substantial effects of the STING and AIM2 pathways on arthritis, STING TKO and AIM2 TKO mice still exhibited prominent autoantibody production. Interestingly, inflammation persisted in Unc93 TKO mice while autoantibody production to nucleic acids was abrogated. Collectively, these data indicate that innate immune pathways contribute to the initiation/perpetuation of inflammatory arthritis and demonstrate that cytosolic and endosomal pathways play distinct roles in the manifestations of autoimmunity. Moreover, they reveal a previously undescribed role for AIM2 as a sensor of endogenous nucleic acids in inflammatory arthritis. Thus, therapeutics that target the STING and AIM2 pathways may be beneficial for the treatment of inflammatory joint diseases. While the role of hematopoietic cells in driving autoimmunity has been well established, the contribution of stromal elements to disease pathogenesis is less well understood. Therefore, we generated bone marrow chimeras to delineate the contribution of hematopoietic and non-hematopoietic cells to the various autoimmune manifestations in DKO mice. These studies revealed that both donor hematopoietic and host radioresistant cells are required for inflammation in the joint as well as for other features of autoimmunity in DKO mice, including splenomegaly, extramedullary hematopoiesis, and autoantibody production. This data demonstrates that stromal host cells play a major role in DNA-driven autoimmunity. Moreover, these results suggest that targeting not only hematopoietic but also stromal elements may be advantageous in the setting of inflammatory arthritis. In the final chapter of this thesis, a role for innate immune sensor pathways in bone is described. The majority of inflammatory arthritides have been shown to lead to systemic loss of bone. Surprisingly, however, we found that DKO mice accumulate trabecular bone in the long bones over time as well as ectopic bone in the spleens, both sites of robust DNA accrual. Moreover, deficiency of the STING pathway abrogated this bone accumulation. Collectively, these data demonstrate that DNA accrual promotes dysregulated bone remodeling through innate immune sensing pathways. These findings are the first to reveal a role for the STING pathway in bone and may unveil novel targets for the treatment of diseases associated with bone disorders.

Indiana University-Purdue University Indianapolis (IUPUI) / The Keap1-Nrf2 pathway regulates a wide range of cytoprotective genes, and has been found to serve a protective and beneficial role in many body systems. There is limited information available, however, about its role in bone homeostasis. While Nrf2 activation has been suggested as an effective method of increasing bone mass and quality, there have been conflicting reports which associate Keap1 deficiency with detrimental phenotypes. As Keap1 deletion is a common method of Nrf2 activation, further study should address the impacts of various methods of regulating Nrf2 expression. Also, little research has been conducted on the specific pathways by which Nrf2 activation improves bone quality. In this study, the effects of alterations to Nrf2 activation levels were explored in two specific and varied scenarios. In the first experiment, moderate Nrf2 activation was achieved via partial deletion of its sequestering protein, Keap1, in an aging mouse model. The hypothesis tested here is that moderate Nrf2 activation improves bone quality by affecting bone metabolism and response to mechanical loading. The results of this first experiment suggest a subtle, sex-specific effect of moderate Nrf2 activation in aging mice which improves specific indices of bone quality to varying degrees, but does not affect loading-induced bone formation. It is likely that the overwhelming phenotypic impacts associated with aging or the systemic effects of global Keap1 deficiency may increase the difficulty in parsing out significant effects that can be attributed solely to Nrf2 activation. In the second experiment, a cell-specific knockout of Nrf2 in the osteocytes was achieved using a Cre/Lox breeding system. The hypothesis tested here is that osteocyte-specific deletion of Nrf2 impairs bone quality by affecting bone metabolism and response to mechanical loading. The results of this experiment suggest an important role of Nrf2 in osteocyte function which improves certain indices of bone quality, which impacts male and female bones in different 7 ways, but did not significantly impact loading-induced bone formation. Further studies should modify the method of Nrf2 activation in an effort to refine the animal model, allowing the effects of Nrf2 to be isolated from the potential systemic effects of Keap1 deletion. Future studies should also utilize other conditional knockout models to elucidate the effects of Nrf2 in other specific cell types.

No description available.

Marathon runners are prone to femoral stress fractures due to the high magnitudes and frequencies of lower extremity loads during training. Female runners tend to have a greater incidence of stress fracture compared to male runners. Sex-specific differences in body structure, joint pressure, and muscle activation patterns that influence bone remodeling may cause this observed difference in stress fracture occurrence. The goal of this thesis was to develop a finite element model of the femur during marathon training, then determine if marathon training affected bone properties of male and female runners differently. To achieve this goal, a finite element femur model was integrated with a bone remodeling algorithm. Sex-specific muscle and joint pressure loads corresponding to baseline activity and marathon training were applied to the finite element femur model. Axial strain, density, damage, and remodeling activity were quantified at regions predicted to be at high risk of stress fracture. The major results of this analysis predicted that marathon training increased bone damage at all regions of interest in both males and females, especially at the inferior neck. The model predicted that the superior neck, trochanter, and proximal diaphysis were more severely weakened in females than males after marathon training. While this model cannot directly quantify femoral stress fracture risk, it may be used to predict regions of bone weakness in male and female marathon runners. Future work may be done to improve accuracy of this model by using sex-specific femur geometry and bone remodeling parameters specific to male and female marathon runners. This model may be useful in future applications to study effectiveness of injury preventive methods, such as gait retraining, in reducing bone damage.

Indiana University-Purdue University Indianapolis (IUPUI) / It has been proposed that the accumulation of microdamage in bone of aging individuals may play a causative and synergistic role in increased fracture incidence. If microdamage production were somehow increased, or reparative remodeling was somehow decreased, the scales may tip towards pathologic fracture. It is known that bisphosphonates increase microdamage accumulation in ribs, lumbar vertebrae, and ilium. The specific aim of this study was to histomorphometrically quantify the microdamage effect of the bisphosphonates alendronate and risedronate therapy on alveolar bone surrounding the first mandibular molar in the dog to determine if this response differs from that in non-bisphosphonate treated dogs. Thirty-four dogs were randomly assigned in two test, and one control groups. Test groups received pharmacologically equivalent doses of either alendronate (11 dogs) or risedronate (11 dogs). The control group (12 dogs) received subcutaneous injections of saline solution. The mandibular right first molar was analyzed for this study. Histomorphometric measurements were made using a x150 Nikon Optiphot-2 fluorescence microscope equipped with brightfield sources (Nikon, Inc.) using the semi-automatic Bioqant digitizing system (R & M Biometrics). There was no significant differences in cortical bone area across treatment groups for any of the regions, nor were any expected. Overall, there was almost twice as much crotical bone found in the Middle (Combined) regions compared with the Coronal (Combined) regions. The precent cortical area was universally high across all treatment groups averaging in the mid-90% range. The Apical region averaged 96.05%, followed by the Coronal region with 95.04% and the Middle region with 93.80%. The number of labeled osteons per cortical area in the alendronate and risedronate groups both tended to be lower relative to the control group (0.92/mm2 and 0.93/mm2 vs. 1.26mm2, respectively), but were not significantly different. On average, the coronal regions had nearly three times the LOn/CtAr as the Middle and Apical regions (1.90/mm2 vs. 0.63/mm2 and 0.57/mm2 respectively). Only in one region was MAR statistically higher in the Coronal (1mm) region, relative to all other regions compared. The Middle region demonstrated a low MAR. The WTh was significantly higher in the risedronate and alendronate groups than that of the control group for the Coronal region. This illustrates that with respect to the bisphosphonates, there is more formation and less resorption. In one region of a significantly lower WTh for the alendronate group relative to the risedronate group was noted. This implies a more potent inhibition in the risedronate treated groups. The WTh for the entire Coronal was statistically lower than every Middle measurement, but was not different than observed in the Apical region. This would tend to signify that in the Coronal, the turnover rate is more of a rapid nature, and therefore the osteons are not as large, while in the Apical, there were so many missing values due to the low rate of turnover, the numbers are skewed to the low end. In the Coronal (Combined) region, the risedronate (108.79 days) group exhibiting a significantly higher FP than the alendronate (62.88 days) and the control (56.13 days) groups. This would imply an increased potency of risedronate over alendronate. Regionally, the FP was significantly lower in the Coronal, relative to Middle or Apical. This is consistent with a more rapid turnover in the Coronal regions observed earlier. The Acf for alendronate (6.41/mm2 per day) and risedronate (5.69/mm2 per day) both tended to be lower by approximately 40% when compared with the control group (10.11/mm2 per day). Overall, the Acf for the Coronal region was 14.15/mm2 per day vs. 2.98/mm2 per day for the Middle and 9.13/mm2 per day for the Apical regions. This shows a significantly increased amount of turnover events taking place not just in the Coronal region, but in the region immediately adjacent to the tooth in the Coronal region. In no region did bone formation differ significantly when treated with bisphosphonates. The Coronal (1mm) region was statistically greater than every region it was measured against, individual and combined. Based on this observation, the second hypothesis that within the first molar alveolar site, bisphosphonate therapy with alendronate, and risedronate would inhibit remodeling more in the coronal region than in the middle and apical region, is rejected. When measuring microdamage accumulation (CrDn), only in the Middle (1mm) region was a significant difference across treatment groups notes. There were no other statistical differences across groups for any other regions. This observation demonstrates that bisphosphonate treatment does not increase the accumulation of microcracks in the dentate alveolar bone. Therefore, the first hypothesis that within the dentate mandible, bisphosphonate therapy with alendronate and risedronate would increase microdamage accumulation around the first molar compound to control, is rejected. When CrDn was compared by region, significant differences were noted. As expected, the Coronal (1mm) region demonstrated a significantly increased CrDN compared with the Apical and Middle regions. Coupled with the information that the BFR is increased in the Coronal and Middle (1-3mm) regions would argue for a reparative function of remodeling in the Coronal and outer Middle regions, which is in response to microdamage accumulation. Significant differences were observed in the Middle (1mm) and Middle (Combined) regions, with the alendronate group demonstrating an increased CrSDn relative to control. There was no statistical difference across treatment groups for any of the regions studied. When compared by regions, the Coronal (1mm) was statistically higher than all regions it was measured against. The Middle regions demonstrated elevated CrSDn relative to the apical region, which displayed the lowest CrSDn values of all regions. One final measure of microdamage is mean crack length. There were no statistically significant differences across any groups for any regions. The only significant differences, when observed across regions, was in reference to the Middle (1mm) region, which was significantly larger than the Coronal (1mm), Coronal (Combined), and the Middle (1-3mm) region. Otherwise, there is no observable trend, and no significant difference between regions. In conclusion, this study found that there was no an increase in microdamage in the dentate mandible of the dogs with bisphosphonate therapy, thereby rejecting the first hypothesis. While there were isolated regions of remodeling inhibition, the hypothesis that bisphosphonate therapy would inhibit remodeling more in the coronal region than in the middle and apical region is rejected. Therefore, based on the findings of this study, we conclude that bisphosphonates do inhibit remodeling in the dentate alveolus generally, but inhibition is not localized to any particular region. Finally, the administration of bisphosphonates do not result in an increase in microdamage accumulation in the dentate alveolus of dogs.

No description available.

No description available.

No description available.

The human skeleton is a dynamic organ that serves multiple functions to maintain normal physiology and health. It protects vital organs, provides support for movement, houses marrow and maintains calcium homeostasis. The skeleton is maintained by the work of two cells with opposing functions: osteoblasts, cells that synthesize organic bone matrix and osteoclasts that degrade and resorb it. These cells interact with one another in a tightly regulated process known as the bone remodeling cycle. This cycle maintains the health of bone by removing and replacing weak or damaged bone and responding to stress loads by remodeling portions of the skeleton that require reinforcement. Osteoblasts differentiate from mesenchymal stem cells and respond to hormonal stimuli by synthesizing and secreting cytokines necessary for osteoclast differentiation. Osteoblasts may become embedded within mineralized matrix, becoming osteocytes, cells that can sense changes in mechanical loading and facilitate localization of the remodeling cycle. Osteoclasts differentiate from hematopoietic stem cells (HSC) when the cell surface receptors, c-FMS and RANK, are activated by ligands produced by osteoblasts, M-CSF and RANKL respectively. In addition to c-FMS and RANK stimulation, another calcium-mediated, co-stimulatory pathway must be activated to ensure proper osteoclast differentiation. This pathway is activated by two immunoreceptors, OSCAR and TREM-2 that interact with adaptor proteins termed FcRγ and DAP12 respectively. These adaptor proteins harbor immunoreceptor tyrosine-based activation motifs (ITAM), which exist on their cytoplasmic tail. Once the immunoreceptors are triggered, specific tyrosines within the ITAM motifs become phosphorylated and act as docking points for the tyrosine kinase, Syk. Once bound, Syk autophosphorylates and acts on its downstream targets. Syk dephosphorylation is, therefore, necessary to attenuate this signal to prevent over activation of osteoclasts. Recently, a novel tyrosine phosphatase, T-cell Ubiquitin ligand -2 (TULA-2) has been shown to dephosphorylate specific phosphotyrosine residues on Syk in various systems and has shown an increased specificity to dephosphorylate tyrosine 352. The goal of this project is to determine how TULA-2 mediated dephosphorylation of Syk regulates osteoclast differentiation and function. TULA-2 is a member of the TULA family of proteins, TULA and TULA-2. In spite of a significant homology and similar domain organization between TULA and TULA-2, only TULA-2 has significant phosphatase activity. Furthermore, whereas TULA is expressed only in lymphocytes, TULA-2 is expressed in most tissues albeit a higher level of expression is seen in cells of hematopoietic origin. In vivo analysis including Micro-computed tomography (Micro CT) and histomorphometry indicated that mice that lack both TULA and TULA-2 (DKO) have decreased bone mass compared to wild-type (WT) counterparts. An in vitro cell differentiation assay revealed that a larger population of osteoclast-like cells (OCL) could be cultivated from bone marrow isolated from DKO mice compared to OCL derived from WT bone marrow. An in vitro resorption pit assay revealed that DKO osteoclasts could resorb bone at a faster rate than WT counterparts. Additionally, over-expression of phosphatase-dead TULA-2 in WT osteoclasts increased the ability of the cells to resorb bone. At the molecular level, activation of the co-stimulatory pathway revealed increased tyrosine phosphorylation of Syk 352 in DKO pre-osteoclasts when compared to phosphorylation of Syk isolated from WT pre-osteoclasts. Cumulatively, the above data indicates that the absence of TULA-2 results in an increased signaling response leading to a larger population of hyperactive osteoclasts, which contributes to decreased bone mass in mice. These data suggest that the phosphatase activity of TULA-2 is required for negative regulation of bone resorption. / Cell Biology