Date Published: August 1, 2018
Author(s): L. Xi, P. De Falco, E. Barbieri, A. Karunaratne, L. Bentley, C.T. Esapa, N.J. Terrill, S.D.M. Brown, R.D. Cox, G.R. Davis, N.M. Pugno, R.V. Thakker, H.S. Gupta.
Glucocorticoid-induced osteoporosis (GIOP) is a major secondary form of osteoporosis, with the fracture risk significantly elevated – at similar levels of bone mineral density – in patients taking glucocorticoids compared with non-users. The adverse bone structural changes at multiple hierarchical levels in GIOP, and their mechanistic consequences leading to reduced load-bearing capacity, are not clearly understood. Here we combine experimental X-ray nanoscale mechanical imaging with analytical modelling of the bone matrix mechanics to determine mechanisms causing bone material quality deterioration during development of GIOP. In situ synchrotron small-angle X-ray diffraction combined with tensile testing was used to measure nanoscale deformation mechanisms in a murine model of GIOP, due to a corticotrophin-releasing hormone promoter mutation, at multiple ages (8-, 12-, 24- and 36 weeks), complemented by quantitative micro-computed tomography and backscattered electron imaging to determine mineral concentrations. We develop a two-level hierarchical model of the bone matrix (mineralized fibril and lamella) to predict fibrillar mechanical response as a function of architectural parameters of the mineralized matrix. The fibrillar elastic modulus of GIOP-bone is lower than healthy bone throughout development, and nearly constant in time, in contrast to the progressively increasing stiffness in healthy bone. The lower mineral platelet aspect ratio value for GIOP compared to healthy bone in the multiscale model can explain the fibrillar deformation. Consistent with this result, independent measurement of mineral platelet lengths from wide-angle X-ray diffraction finds a shorter mineral platelet length in GIOP. Our results show how lowered mineralization combined with altered mineral nanostructure in GIOP leads to lowered mechanical competence.
Increased fragility in musculoskeletal disorders like osteoporosis are believed to arise due to alterations in bone structure at multiple length-scales from the organ down to the supramolecular-level, where collagen molecules and elongated mineral nanoparticles form stiff fibrils. However, the nature of these molecular-level alterations are not known. Here we used X-ray scattering to determine both how bone fibrils deform in secondary osteoporosis, as well as how the fibril orientation and mineral nanoparticle structure changes. We found that osteoporotic fibrils become less stiff both because the mineral nanoparticles became shorter and less efficient at transferring load from collagen, and because the fibrils are more randomly oriented. These results will help in the design of new composite musculoskeletal implants for bone repair.
The reduced mechanical and structural competence of bone in musculoskeletal disorders (e.g. osteoporosis and osteoarthritis) arise from both alterations in the mineralization dynamics (via altered cellular activity) as well as intrinsic changes in the mineralized collagen matrix (as in disorders like osteogenesis imperfecta), but the mechanisms linking matrix alterations to reduced functionality are not always clear , , , . Glucocorticoid-induced osteoporosis (GIOP) is one of such disorders, affects 1–3% of the general population and is characterised by a rapid increase in fracture risk in patients taking anti-inflammatory steroidal medications (glucocorticoids or GCs) , , , . About 30 to 50 percent of patients with chronic glucocorticoid treatment suffer from osteoporotic fractures . In cancellous bone, GCs can cause suppression of bone formation and enhanced and prolonged resorption through direct effects on osteoblasts, osteoclasts and osteocytes , . As a result, in GIOP, bone resorption (osteoclastic activity) is not matched by bone formation (osteoblastic activity), which results in reduced bone mass. Most notably, however, the increase in fracture risk in GIOP is larger than predicted from changes in bone mass alone (relative to healthy bone) , implying that changes in the bone matrix and microarchitecture play an important role. For example, increased vertebral fracture risk in GIOP-patients is associated with trabecular thinning (microarchitecture), and material-level changes have been observed as well in cancellous bone , , . Alterations in bone metabolism – mainly affecting cancellous bone – in terms of cellular changes have been recognized in GIOP , . Therefore, understanding the structural mechanisms in bone tissue – at multiple hierarchical levels – which cause the reduction in mechanical properties in GIOP would thus be of considerable clinical relevance. Such an understanding would also shed light on the mechanical relevance of bone matrix quality , ,  in a prototypical example of secondary osteoporosis, where the current gold standard method of assessment of osteoporotic fracture risk – bone mineral density or BMD , ,  – is inadequate for fracture risk predictions , , .
In summary, our main findings are:•Consistently reduced fibrillar modulus in Crh−120/+ mouse bone in comparison to Crh+/+ mouse bone, with minimal increase in fibrillar modulus with age in Crh−120/+ mouse bone, in contrast to an over 2-fold increase in Crh+/+ mouse bone fibril modulus (Fig. 3).•A heterogeneous and lower mineralization density distribution at the microscale, accompanied by a more porous 3D microarchitecture in Crh−120/+ mouse bone (Fig. 4).•A laminate fibril array model – based on the rotated plywood lamella , , and deriving its parameters from experimental structural data – explains the reduced effective fibril modulus (measured experimentally) in Crh−120/+ mouse bone in comparison to Crh+/+ mouse bone (Fig. 5).•In terms of the model parameters, the main difference between Crh−120/+ and Crh+/+ is a reduced mineral platelet aspect ratio AR (15.0 (Crh+/+) to 9.6 (Crh−120/+)) at the scale of the mineralized collagen fibril (Fig. 2, Fig. 5).•Reduced length of mineral platelets in Crh−120/+ bone, as measured from the Debye-Scherrer width, consistent with the reduced AR predicted by the model (Fig. 6).•A greater degree of fibrillar disorganization (measured via the ρ-parameter) in Crh−120/+ bone compared to Crh+/+ (Fig. 6).