Intrafibrillar mineralization deficiency and osteogenesis imperfecta mouse bone fragility.

Osteogenesis imperfecta (OI), a brittle bone disease, is known to result in severe bone fragility. However, its ultrastructural origins are still poorly understood. In this study, we hypothesized that deficient intrafibrillar mineralization is a key contributor to the OI induced bone brittleness. To test this hypothesis, we explored the mechanical and ultrastructural changes in OI bone using the osteogenesis imperfecta murine (oim) model. Synchrotron X-ray scattering experiments indicated that oim bone had much less intrafibrillar mineralization than wild type bone, thus verifying that the loss of mineral crystals indeed primarily occurred in the intrafibrillar space of oim bone. It was also found that the mineral crystals were organized from preferentially in longitudinal axis in wild type bone to more randomly in oim bone. Moreover, it revealed that the deformation of mineral crystals was more coordinated with collagen fibrils in wild type than in oim bone, suggesting that the load transfer deteriorated between the two phases in oim bone. The micropillar test revealed that the compression work to fracture of oim bone (8.2 ± 0.9 MJ/m3) was significantly smaller (p < 0.05) than that of wild type bone (13.9 ± 2.7 MJ/m3), while the bone strength was not statistically different (p > 0.05) between the two genotype groups. In contrast, the uniaxial tensile test showed that the ultimate strength of wild type bone (50 ± 4.5 MPa) was significantly greater (p < 0.05) than that of oim bone (38 ± 5.3 MPa). Furthermore, the nanoscratch test showed that the toughness of oim bone was much less than that of wild type bone (6.6 ± 2.2 GJ/m3 vs. 12.6 ± 1.4 GJ/m3). Finally, in silico simulations using a finite element model of sub-lamellar bone confirmed the links between the reduced intrafibrillar mineralization and the observed changes in the mechanical behavior of OI bone. Taken together, these results provide important mechanistic insights into the underlying cause of poor mechanical quality of OI bone, thus pave the way toward future treatments of this brittle bone disease.

Contribution of extrafibrillar matrix to the mechanical behavior of bone using a novel cohesive finite element model.

The mechanical behavior of bone is determined at all hierarchical levels, including lamellae (the basic building block of bone) that are comprised of mineralized collagen fibrils and extrafibrillar matrix. The mechanical behavior of mineralized collagen fibrils has been investigated intensively using both experimental and computational approaches. Yet, the contribution of the extrafibrillar matrix to bone mechanical properties is poorly documented. In this study, we intended to address this issue using a novel cohesive finite element (FE) model, in conjunction with the experimental observations reported in the literature. In the FE model, the extrafibrillar matrix was considered as a nanocomposite of hydroxyapatite (HA) crystals bounded through a thin organic interface modeled as a cohesive interfacial zone. The parameters required by the cohesive FE model were defined based on the experimental data reported in the literature. This hybrid nanocomposite model was tested in two loading modes (i.e. tension and compression) and under two hydration conditions (i.e. wet and dry). The simulation results indicated that (1) the failure modes of the extrafibrillar matrix predicted using the cohesive FE model were closely coincided with those experimentally observed in tension and compression tests; (2) the pre-yield deformation (i.e. internal strain) of HA crystals with respect to the applied strain was consistent with that obtained from the synchrotron X-ray scattering measurements irrespective of the loading modes and hydration status; and (3) the mechanical behavior of the extrafibrillar matrix was dictated by the properties of the organic interface between the HA crystals. Taken together, we postulate that the extrafibrillar matrix plays a major role in the pre-yield deformation and the failure mode of bone, thus, giving rise to important insights in the ultrastructural origins of bone fragility.

Effect of water on nanomechanics of bone is different between tension and compression.

Water, an important constituent in bone, resides in different compartments in bone matrix and may impose significant effects on its bulk mechanical properties. However, a clear understanding of the mechanistic role of water in toughening bone is yet to emerge. To address this issue, this study used a progressive loading protocol, coupled with measurements of in situ mineral and collagen fibril deformations using synchrotron X-ray diffraction techniques. Using this unique approach, the contribution of water to the ultrastructural behavior of bone was examined by testing bone specimens in different loading modes (tension and compression) and hydration states (wet and dehydrated). The results indicated that the effect of water on the mechanical behavior of mineral and collagen phases at the ultrastructural level was loading-mode dependent and correlated with the bulk behavior of bone. Tensile loading elicited a transitional drop followed by an increase in load bearing by the mineral phase at the ultrastructural level, which was correlated with a strain hardening behavior of bone at the bulk level. Compression loading caused a continuous loss of load bearing by the mineral phase, which was reflected at the bulk level as a strain softening behavior. In addition, viscous strain relaxation and pre-strain reduction were observed in the mineral phase in the presence of water. Taken together, the results of this study suggest that water dictates the bulk behavior of bone by altering the interaction between mineral crystals and their surrounding matrix.

Water residing in small ultrastructural spaces plays a critical role in the mechanical behavior of bone.

Water may affect the mechanical behavior of bone by interacting with the mineral and organic phases through two major pathways: i.e. hydrogen bonding and polar interactions. In this study, dehydrated bone was soaked in several solvents (i.e. water, heavy water (D2O), ethylene glycol (EG), dimethylformamide (DMF), and carbon tetrachloride(CCl4)) that are chemically harmless to bone and different in polarity, hydrogen bonding capability and molecular size. The objective was to examine how replacing the original matrix water with the solvents would affect the mechanical behavior of bone. The mechanical properties of bone specimens soaked in these solvents were measured in tension in a progressive loading scheme. In addition, bone specimens without any treatments were tested as the baseline control whereas the dehydrated bone specimens served as the negative control. The experimental results indicated that 22.3±5.17vol% of original matrix water in bone could be replaced by CCl4, 71.8±3.77vol% by DMF, 85.5±5.15vol% by EG, and nearly 100% by D2O and H2O, respectively. CCl4 soaked specimens showed similar mechanical properties with the dehydrated ones. Despite of great differences in replacing water, only slight differences were observed in the mechanical behavior of EG and DMF soaked specimens compared with dehydrated bone samples. In contrast, D2O preserved the mechanical properties of bone comparable to water. The results of this study suggest that a limited portion of water (<15vol% of the original matrix water) plays a pivotal role in the mechanical behavior of bone and it most likely resides in small matrix spaces, into which the solvent molecules larger than 4.0Å cannot infiltrate.