Intrinsic mechanical behavior of femoral cortical bone in young, osteoporotic and bisphosphonate-treated individuals in low- and high energy fracture conditions

Abstract

Bisphosphonates are a common treatment to reduce osteoporotic fractures. This treatment induces osseous structural and compositional changes accompanied by positive effects on osteoblasts and osteocytes. Here, we test the hypothesis that restored osseous cell behavior, which resembles characteristics of younger, healthy cortical bone, leads to improved bone quality. Microarchitecture and mechanical properties of young, treatment-naïve osteoporosis, and bisphosphonate-treated cases were investigated in femoral cortices. Tissue strength was measured using three-point bending. Collagen fibril-level deformation was assessed in non-traumatic and traumatic fracture states using synchrotron small-angle x-ray scattering (SAXS) at low and high strain rates. The lower modulus, strength and fibril deformation measured at low strain rates reflects susceptibility for osteoporotic low-energy fragility fractures. Independent of age, disease and treatment status, SAXS revealed reduced fibril plasticity at high strain rates, characteristic of traumatic fracture. The significantly reduced mechanical integrity in osteoporosis may originate from porosity and alterations to the intra/extrafibrillar structure, while the fibril deformation under treatment indicates improved nano-scale characteristics. In conclusion, losses in strength and fibril deformation at low strain rates correlate with the occurrence of fragility fractures in osteoporosis, while improvements in structural and mechanical properties following bisphosphonate treatment may foster resistance to fracture during physiological strain rates.

Figure 1. Structure of human cortical bone.

Human bone comes in the form of either a porous trabecular framework or a dense cortical structure. Here, we focus on the cortical tissue, which can be found in the mid-diaphysis of the femur. In cortical bone, the microstructure consists of osteons (170–250 μm diameter), which are the units of bone produced during remodeling. The osteons contain a central vascular canal called the Haversian canal (60–90 μm diameter) that is concentrically surrounded by lamellae (2–9 μm thickness). The lamellae have a twisted plywood arrangement, where neighboring lamellae have different fibril orientations. Here, osteocyte cells reside in lacunae (15–25 μm diameter) that interconnect through canaliculi (100–400 nm in diameter). The lamellae are composed of collagen fibrils (80–100 nm diameter). The fibrils are surrounded by polycrystalline extrafibrillar mineral platelets. In addition to mineral, the extrafibrillar as well as the intrafibrillar matrix contains molecular components, such as non-collageneous proteins or cross-links, promoting the formation of sacrificial bonds. Within the fibrils, type I collagen molecules (1.5 nm diameter, 300 nm length) and hydroxyapatite nanocrystals (50 nm width, 25 nm height, 1.5–4 nm thickness) form a composite structure, where arrays of collagen molecules staggered at 67 nm are embedded with nano-platelets of hydroxyapatite mineral. Adapted from Zimmermann et al..

Figure 2. Microstructural characteristics.

The porosity of the bending samples was measured with micro-computed tomography scans. (a) The young samples had a lower porosity than the osteoporosis (OP) and bisphosphonate-treated (BP) samples. The changes in porosity were significant between the young and osteoporosis cases (p = 0.01). Representative changes in porosity among the (b) young, (c) osteoporosis, and (d) bisphosphonate-treated cases are visible in three-dimensional images from the micro-computed tomography scans. Scale bars are 250 μm. (e–g) Backscattered scanning electron microscopy and (h–j) Toluidine-blue-stained histological sections show the differences in microstructural features for each case. (k) Indeed, the number of Haversian canals in the osteoporotic bone is 29% greater than the young (p = 0.004) and 24% greater than the bisphosphonate-treated cases (p = 0.010). (i) The number of mineralized lacunae was found to be significantly higher in the osteoporosis group in comparison the young (p < 0.001) and bisphosphonate-treated groups (p = 0.009). (m) The size of the Haversian area reflecting the 2D porosity was 2.7-fold greater in the osteoporotic tissue than the young (p < 0.001) and 2.3-fold greater in the bisphosphonate-treated group than in the young group (p = 0.002).

Figure 3. Cortical bone strength with bisphosphonates.

Human cortical bone samples from the mid-diaphysis of the femur were tested in three-point bending to measure the strength. (a) The stress-strain curves are shown for the young, osteoporosis (OP) and bisphosphonate-treated (BP) cases. Here, the shaded area contains all of the stress-strain curves for each group. (b) The bending modulus of the OP (p = 0.02) and BP (p = 0.04) groups were both significantly lower than in the young cases, which may be due to the lower mineralization values previously reported for the same cases. (c) The yield stress and (d) maximum bending stress were also both significantly lower in the OP cases (p = 0.02).

Figure 4. Small angle x-ray scattering (SAXS) of bisphosphonate-treated human cortical bone.

Human cortical bone samples from the mid-diaphysis of the femur were mechanically tested in tension at a low strain rate, while simultaneously using synchrotron SAXS to discern the fibril-level deformation in the young, osteoporosis, and bisphosphonate-treated osteoporosis cases. (a) The strain in the fibril during the mechanical tension test is measured as a function of the strain applied to the whole sample (i.e., the tissue strain). Essentially, the material behavior indicates elastic stretching of the fibril during the initial linear portion of the curve. After the linear region, inelastic deformation begins to occur, which can result in more heterogeneous behavior. However, a plateau in the curve, where a steady-state fibril strain is reached, may indicate that fibrils are slipping past one another while the tissue continues to stretch (i.e., fibrillar sliding). The osteoporosis cases have a more pronounced plateau in the fibril strain (p < 0.001), indicating lower fibril deformation than the young cases, which has been similarly seen in aging and could lead to earlier failure. In contrast, the bisphosphonate-treated cases exhibit more fibrillar deformation, which corresponds to the behavior of the young cases, and may explain the improvements in strength. (b) A profile view of the tensile samples shows the roughness of the fracture surfaces for each case. At low strain rates, the samples all exhibit rough fracture surfaces, which is characteristic of the generation of plasticity at low strain rates. Scale bar is 250 μm.

Figure 5. Small angle x-ray scattering (SAXS) at high strain rates.

Human femoral cortical bone samples were also mechanically tested in tension at a high strain rate, while using synchrotron SAXS to discern the fibril-level deformation. The fibril strain can be measured as a function of the strain applied to the whole sample (i.e., the tissue strain). (a) At high strain rates, the fibril versus tissue strain is very linear, which indicates that the plasticity sliding mechanisms are “locking up” and deformation is dominated by elastic stretching of the fibril. Similar behavior has been found for young bone at high strain rates. Indeed, plasticity in human cortical bone most likely derives from the viscoelasticity of the fibril structure, which enables fibrillar sliding and sacrificial bonding mechanisms to absorb energy. Higher strain rates “lock up” the plasticity mechanisms at the fibril level. (b) The profile of the tensile samples shows that the fracture surfaces have a lower roughness at high strain rates, which corresponds with the lower levels of plasticity measured during the SAXS tests. Scale bar is 250 μm.