Roles of Collagen and Water in the Fracture Resistance of Bone

A critical barrier to the development of new strategies for preventing costly fractures of the hip, spine, and proximal humerus is an incomplete understanding of the key age- and disease-related changes occurring in bone tissue. In particular, little is known about the pathogenic mechanisms by which the deterioration in the extracellular matrix (ECM) reduces the fracture resistance of bone or increases fracture risk. Non-enzymatic post-translational modifications (NE-PTMs) are potential contributors to poor ECM because they accumulate in matrix proteins as fracture risk increases. Therefore, the overall goals of this project are i) to determine which NE-PTMs of collagen I, the predominant ECM protein of bone, predict bone fracture resistance and are clinically relevant in osteoporosis and ii) to establish whether non-crosslinking or crosslinking NE-PTMs contribute to a decrease in fracture resistance of bone and whether they do so via alterations in the structure and hydration of collagen I. To achieve our goals, we will first generate specimens of cortical bone (dense) and trabecular bone (spongy) using cadaveric femurs collected from both female and male donors between 50 years and 100 years of age (Aim 1a). These specimens will be comprehensively analyzed to quantify: bone mineral density, bone volume fraction, ECM-bound water, secondary structure of collagen I, mature enzymatic & non-enzymatic collagen crosslinks, integrity of collagen I fibrils, the resistance to yielding (strength), the ability to deform after yielding (toughness), the resistance to crack growth (fracture toughness), and the resistance to damage accumulation (fatigue). From adjacent bone samples, we will also extract ECM proteins including collagen I and quantify NE-PTMs at specific amino acid residues that form its triple helix using mass spectrometry. By fitting the data to statistical models, we will determine whether the levels of certain NE-PTMs help explain differences among the donors in fracture resistance, collagen fibril integrity, ECM-bound water, and spectroscopic markers of helical structure. We will also generate bone specimens from proximal femurs acquired from cadavers without osteoarthritis (OA) and two types of orthopaedic surgical cases: total hip arthroplasty (THA) for OA and hemi- arthroplasty (HA) to fix a fragility fracture (Aim 1b). The specimens will be analyzed as in Aim 1a to determine whether NE-PTM levels are significantly higher while ECM-bound water and fracture resistance are significantly lower in HA (osteoporosis) vs. THA (OA) or the cadaveric controls. To identify a mechanism whereby NE-PTMs lowers fracture resistance of bone, we will treat bones ex vivo to accumulate specific types of NE-PTMs (Aim 2a). Following treatment, we will assess the bones to determine which specific NE-PTMs significantly affect the fracture resistance of bone in a manner similar to that of aging as determined in Aim 1. Lastly, we will perform molecular dynamics simulations and molecular modeling of collagen I to determine how relevant NE-PTMs affect the triple helix structure and hydration (Aim 2b). Successful completion of the project would shift the paradigm of bone health from a focus on bone mass to the inclusion of pathogenic contributions from bone ECM.