| Rheumatoid arthritis (RA) is a chronic, systemic and immune-mediated inflammatory disease that mainly affects the synovial membrane of multiple small joints. As a consequence, RA results in cartilage and bone damage, leading to functional impairment and an increase in morbidity and mortality. Early diagnosis and adequate treatment are critical to prevent RA progression, as joint destruction can occur immediately after its onset. The most characteristic feature of RA is synovial hyperplasia, which is mediated by several immune cells, such as T-cells, B-cells, neutrophils, macrophages and by a complex cytokine network, especially interleukin (IL)-1beta, tumor necrosis factor (TNF) and IL-6. RA inflammatory environment induces osteoclastogenesis, promoting disturbances in skeletal bone remodeling, which ultimately leads to the development of secondary osteoporosis and consequent bone fragility. An opportunity for a more effective treatment intervention was identified in early RA, when permanent damage can be prevented and a higher number of patients can achieve remission. Early treatment intervention might also interfere systemically with bone biology preventing bone micro and nano architectural damage. The development of therapeutic strategies able to control both inflammation and bone degradation, with a high rate of disease remission, low incidence of side effects and low production costs is still an unmet medical need in RA. Our hypothesis is that the impact of inflammation on bone micro and nano properties (intrinsic bone tissue properties, independent of the overall bone architecture and directly dependent on the way bone cells, collagen and calcium crystals interact) occurs almost immediately, upon first symptoms, and that these effects can be prevented by early intervention with drugs able to control inflammation and capable of interfering also with bone metabolism. This thesis characterizes the early events of bone damage in RA and explores the effect of novel treatment interventions in this context. Accordingly, in the first part of this thesis, we used an adjuvant induced arthritis (AIA) rat model and observed a synovial sublining layer infiltration, increased lining layer cells, bone erosions and cartilage surface damage present since the early stages of arthritis, as well as increased levels of IL-6. This inflammatory environment promotes osteoclastogenesis, which is related to the observed local bone erosion and may interfere systemically with bone skeletal remodeling. Indeed, AIA animals showed an increased bone turnover, as depicted by increased CTX-I (Carboxy-terminal telopeptide of type I collagen) and P1NP (amino terminal propeptides of type I collagen) levels since the early stages of arthritis. Bone histology was consistent with this early onset spur of bone remodeling. Arthritic animals showed concentric lamellas in secondary osteons (SO), which are the consequence of intense bone remodeling. On the contrary, healthy animals presented more parallel-lamellae (PL) structures than SO structures and these PL structures are 10% harder than SO structures, representing the mature bone structure (normal bone remodeling). Thus, arthritic bone tissue was composed of a larger number of younger, less mineralized and less hard structures, explaining the reduced hardness that we have observed by nanoindentation. Moreover, an increased area occupied by osteocyte lacunae was detected early on in the arthritis process. This apparent change of osteocyte morphology might be related to bone necrosis, leading to mineral loss, decreased hardness and possibly mechanical weakness. In addition, we have also demonstrated that arthritis induces mineral and collagen loss in trabecular bone since the early phase of arthritis development. At a higher organizational level data, micro computed tomography (micro-CT) revealed in arthritic animals a lower fraction of cortical and trabecular bone volume with reduced trabecular thickness together with a higher trabecular separation, in comparison with controls. Results also demonstrated cortical differences in polar moment of inertia, suggesting mechanical weakness in arthritic groups since the early phase of arthritis. Furthermore, cortical and trabecular porosity were increased in the arthritic groups compared to healthy controls. We also confirmed these observations by classic histomorphometry, which demonstrated a decreased structural integrity in arthritic animals. Coherent with these structural defects, our results also showed that in very early arthritis bone has low mechanical competence. Altogether, these results revealed that inflammation promotes bone nano and micro structural disturbances, leading to bone fragility since the early stages of arthritis. In addition, we also provided the basis for using the AIA animal model of arthritis as an adequate model for studying the impact of inflammation on bone and for assessing candidate compounds for the control of arthritis and its associated bone damage. The quest for new RA treatments, more effective at inflammation and bone damage control, safer and less expensive is still a major need. Previously, we had demonstrated that celastrol, acts by downregulating IL1beta and TNF production, was a promising RA therapeutic candidate. Herein we have demonstrated that celastrol was able to reduce the number of synovial B and T-cells as well as fibroblasts and CD68 macrophages. Accordingly, we showed that celastrol protects cartilage and bone from inflammation-induced focal damage. At a systemic level, we observed a reduction in bone turnover together with preservation of bone structural and mechanical properties. Moreover, celastrol therapy showed superior effects if administrated in an early phase of arthritis development, which highlights the importance of an early treatment to limit inflammation-induced bone damage. Tofacitinib was also tested in order to assess the effects on micro and nano structural and mechanical properties of bone in an AIA rat model of arthritis. Tofacitinib is a selective inhibitor of janus kinase 1 (JAK1) and janus kinase 3 (JAK 3). Results showed significant reduced arthritis manifestations, synovial tissue inflammation and bone erosions, accompanied by a reduced bone turnover rate and a predominance of parallel structures on bone tissue. At tissue level, measurements performed by nanoindentation showed that tofacitinib increased bone cortical and trabecular hardness. However, micro-CT and 3-point bending tests revealed that tofacitinib did not revert the effects of arthritis on cortical and trabecular bone structure and mechanical properties. This effect on bone might be related to the mechanism of action of tofacitinib which has complex and conflictual molecular interactions with bone. We suggest that these interactions have an overall negative effect not totally compensated by the benefits resulting from the control of inflammation. On the other hand, tofacitinib may require more exposure time to have an impact on bone quality. Overall, the results of the present thesis support the hypothesis that the impact of inflammation on bone micro and nano properties occurs almost immediately, upon the appearance of first symptoms. Moreover, these observed effects can be prevented by very early intervention with drugs able to control inflammation and capable of interfering with bone metabolism. |
