Oxidative damage of bone collagen as a cause of reduced fracture resistance in human cortical bone

Abstract

Bone fragility and fracture remain a prominent issue in the populace especially for the older adult population with associated significant health, social and economic implications. The X-ray based clinical tools used to assess bone fracture risk only “see” the mineral and are unable to predict fracture risk accurately in certain individuals especially for those with type 2 diabetes (T2D) and chronic kidney diseases (CKD). This suggest that factors not measured by these X-ray based clinical tools may be important contributors to predicting bone fracture risk. One such factor is bone collagen which forms about 41% by volume of the bone tissue and has been shown to be vital to the fracture resistance of bone and possibly an important determinant for bone fracture risk assessment. Ex vivo human bone studies have shown that bone collagen network connectivity degrades with age, and this is associated with a reduction in the fracture resistance of cortical bone. Collagen network connectivity is a term used to describe how well linked together the collagen molecules are in the collagen network of the bone tissue. However, the exact mechanisms that lead to this degradation of connectivity remain inconclusive. Interestingly, elevated levels of oxidative stress has been implicated in the development and progression of osteoporosis, T2D and CKD, diseases with higher incidences of fracture. Oxidative stress is known to cause damage to macromolecules, referred to as oxidative damage, which includes long lived proteins such as collagen. Consequently, the overarching goal of this thesis was to investigate oxidative damage as a cause of collagen network connectivity degradation leading to reduced fracture resistance in cortical bone. To achieve this goal, this thesis work consisted of two large studies. The first study sought to establish the most accurate and precise way to measure cortical bone fracture toughness. Therefore, current methods for cortical bone fracture testing were critically evaluated. Specifically, the unloading compliance (UC) method developed for metals that has been widely used in cortical bone J-integral resistance curve fracture testing was assessed for its accuracy and precision. The UC method employs multiple progressive load partial unload cycles and uses the unloading compliance of the unloading curve to estimate the current crack extension. Fracture toughness measures from the UC method were compared to those generated using a continuously monotonic load and crack extension measured by an optical method. Results showed that the UC method on average underestimated crack extension by 73% and this consequently resulted in inaccurate and less precise fracture toughness measures from the UC method. This indicates optical based approaches provide more accurate and precise results and highlights a need for rigorous and standardized methods for cortical bone fracture resistance testing. Results from this study were used as a guide to conduct fracture toughness testing of cortical bone specimens in the second study. In the second study, oxidative damage was investigated as a cause of collagen network degradation associated with reduced human cortical fracture toughness. For this, fracture toughness testing of ex vivo human femoral cortical bone specimens from a large heterogenous group of 81 donors with and without a history of T2D and/or CKD were performed. In addition, various biomarkers of oxidative damage alongside collagen network quality measures, cortical porosity as well as mineralization profiles were measured in these specimens. Relationships between the oxidative damage biomarkers, collagen network quality measures and cortical bone fracture toughness measures were investigated using statistical methods. In addition, differences between these measures for the donors with T2D and/or CKD and those without (controls) were also investigated. Results show that advanced glycation end-products (AGEs), one of the biomarkers of oxidative damage were higher in quantity in the T2D and/or CKD donor group compared to controls. However, fracture toughness measures were not different between the two groups suggesting the impact of oxidative damage on bone collagen happens on a spectrum and is general to both groups. Further, in general, the oxidative damage biomarkers correlated with measures of immature crosslinking and collagen chain fragmentation which in turn correlated with other measures of collagen network quality. Multiple linear regression models that were developed showed that interaction terms between collagen network connectivity and immature crosslinking, collagen chain fragmentation as well as the lysine-based AGE adducts were important to explaining fracture toughness measures. From all this, it was proposed that oxidative damage indirectly impacts collagen network connectivity through disruption of immature crosslink formation and collagen chain fragmentation. This in turns negatively impacts the collagen network connectivity leading to reduced fracture resistance of human cortical bone. This work provides a better understanding of how oxidative stress may contribute to loss of fracture resistance in human cortical bone and this new knowledge could be critical towards the development of better means to detect, prevent and treat bone fragility.