New markers, prevalence and burden of osteoporosis and fragility fractures

In this PhD thesis, we aimed to determine the prevalence and burden of osteoporosis and fragility fractures in Portugal to provide evidence in support of new health strategies to improve clinical care and reduce or prevent disability and mortality among elderly. We also aimed to better identify senior women at high risk for a fragility fracture through the use of novel noninvasive biomarkers. To achieve this goal, we evaluated cellular (osteoblast) and tissue (bone mechanical properties) mechanism dysfunction to identify potential serum markers of bone fragility. We hypothesized that bone fragility in the elderly is associated with dysregulation of mineralization because of osteoblast terminal differentiation and disturbances in Wnt regulators [dickkopf-related protein (DKK)1 and DKK2, sclerostin (SOST) and secreted frizzled related protein-1 (sFRP-1)]. Moreover, we hypothesized that serum levels of Wnt regulators are associated with bone fragility and fractures and can constitute new markers for osteoporosis treatment decision. Finally, to create awareness and reduce new fragility fractures in Portugal, we aimed to develop national clinical consensus recommendations for osteoporosis diagnosis and treatment. The studies presented in this thesis crossed two main areas of clinical research, patientoriented mechanistic research, and epidemiological research. We used two samples of participants/patients. One sample was composed of patients undergoing hip replacement surgery (for osteoarthritis or a fragility fracture) in the Orthopaedic Department of Hospital de Santa Maria, Centro Hospitalar Lisboa Norte from 2008 to 2012. It was used to analyse clinical determinants of bone fragility and fractures in the elderly, particularly the associations between osteoblast dysfunction, bone mechanical properties, and fragility fractures. The other sample was a population based on a nationwide sample evaluated in the EpiReumaPt (2011-2013) study. Using the EpiReumaPt sample, we determined the prevalence and individual burden of osteoporosis in Portugal. Moreover, we analysed the prevalence, burden, and risk factors of fragility fractures in a particularly vulnerable stratum, senior women. The EpiReumaPt population was then followed in two more waves of evaluation (2011-2016) under the scope of the Epidemiology of Chronic Diseases (EpiDoC) cohort. Using data from the EpiDoC cohort, we analysed the association between serum markers of Wnt inhibitors (osteoblast regulators) and fragility fractures. Finally, a review of the literature was performed to develop national clinical recommendations regarding fracture risk assessment and osteoporosis treatment. In the first study of this thesis, using the EpiReumaPt sample, we found that 10.2% of Portuguese adults have osteoporosis. The prevalence is higher in women (17.0%) than in men (2.6%) and increases with age. Almost half (40.0%) of Portuguese adults 75 years and older have osteoporosis, and an osteoporosis diagnosis was associated with substantial physical function impairment but not with anxiety or depression symptoms. The second study, also using EpiReumaPt data, showed that self-reported fragility fractures were highly prevalent among senior women (20.7%). This high prevalence was in stark contrast with the low rate of osteoporosis treatment (13.9%). Non-hip and nonvertebral fractures (i.e., lower leg, wrist, humerus, rib, clavicle, and elbow fractures) accounted for the majority of fragility fractures, and clinical risk factors independently associated with prevalent fragility fractures were increased age, obesity, and lower distal bone mineral density (BMD). The challenge to better identify seniors (people aged ≥65 years old) at high risk for a fragility fracture led us to search for novel noninvasive biomarkers of bone fragility and fractures. To achieve this goal, we conducted patient-oriented mechanistic research, evaluating associations between cellular mechanism dysfunction and bone fragility among patients undergoing hip replacement surgery. In the third research work of this thesis, we demonstrated that when adjusted for differences in age, sex, and body mass index, the only macrostructural bone characteristic that remained significantly different between patients with hip fragility fractures and those with osteoarthritis was trabecular stiffness (which is linked to mineralization disturbances). Stiffness was lower in patients with fragility fractures. We also found that smoking habits and female sex were independently associated with lower stiffness in patients with a fragility fracture. In the fourth study of this thesis, we evaluated markers of osteoblast differentiation, as these are the cells responsible for the production of mineralized tissue. We found that osteocalcin (OCL) relative bone expression and the OCL/type 1 collagen, alpha 1 chain (COL1A1) expression ratio in bone (a marker of osteoblast terminal differentiation) were significantly lower in patients with hip fractures than in those with osteoarthritis. Consistent with these results, in a subset of patients, fewer osteoblasts stained for OCL in patients with a fragility fracture than in those with osteoarthritis. We also demonstrated that in patients with hip fractures, a low bone OCL/COL1A1 expression ratio was associated with worse trabecular mechanical behaviour. This work reinforced the importance of osteoblast dysfunction in bone intrinsic properties and fractures among the elderly. In our fifth study, we examined whether the Wnt inhibitors DKK1, DKK2, SOST, WIF-1, and sFRP-1 (regulators of osteoblast differentiation and bone formation) were associated with BMD or fragility fractures in a population-based cohort. Using EpiDoC cohort data, we found that low serum levels of DKK2 predicted low-impact fractures, independent of BMD, and clinical risk factors for fracture. For every 1 standard deviation decrease in DKK2, fracture risk increased by approximately 1.5-fold. Serum levels of DKK2 were not associated with vertebral or hip BMD. Our results suggest a possible interaction among BMD, FRAX score without BMD, and serum DKK2 levels in the assessment of fracture risk, which requires further investigation in a larger study with longer follow-up. The final work of this PhD thesis involved performing a literature review and establishing national consensus recommendations regarding fracture risk assessment and osteoporosis clinical management and treatment to change clinical practice and reduce the incidence of fragility fractures in Portugal. In conclusion, this thesis provided rigorous epidemiological data regarding the prevalence of osteoporosis and fragility fractures in Portugal. It contributed to refining fracture risk assessment through the identification of new serum markers (among regulators of osteoblast-mediated bone formation) of bone fragility and fractures. Mechanistic research regarding bone biomechanics and osteoblast dysfunction showed that fragility fractures are associated with reduced bone stiffness, reflecting mineralization disturbances. Furthermore, reduced osteoblast terminal differentiation was associated with poor bone mechanics and fractures. Finally, national consensus recommendations were created to improve fracture risk assessment of individuals, as well clinical management and treatment of osteoporosis, with the goal of reducing fragility fractures in Portugal