Dilatational band formation in bone

Abstract

Toughening in hierarchically structured materials like bone arises from the arrangement of constituent material elements and their interactions. Unlike microcracking, which entails micrometer-level separation, there is no known evidence of fracture at the level of bone's nanostructure. Here, we show that the initiation of fracture occurs in bone at the nanometer scale by dilatational bands. Through fatigue and indentation tests and laser confocal, scanning electron, and atomic force microscopies on human and bovine bone specimens, we established that dilatational bands of the order of 100 nm form as ellipsoidal voids in between fused mineral aggregates and two adjacent proteins, osteocalcin (OC) and osteopontin (OPN). Laser microdissection and ELISA of bone microdamage support our claim that OC and OPN colocalize with dilatational bands. Fracture tests on bones from OC and/or OPN knockout mice (OC(-/-), OPN(-/-), OC-OPN(-/-;-/-)) confirm that these two proteins regulate dilatational band formation and bone matrix toughness. On the basis of these observations, we propose molecular deformation and fracture mechanics models, illustrating the role of OC and OPN in dilatational band formation, and predict that the nanometer scale of tissue organization, associated with dilatational bands, affects fracture at higher scales and determines fracture toughness of bone.

Fig. 1.

Bone hierarchy and dilatational bands. Hierarchical model of bone toughness shows that dilatational bands and diffuse damage link to higher-level toughening mechanisms in bone. Loss or modification at the nanoscale translates through the different scales shown here and alters bone toughness.

Fig. 2.

Bone toughening mechanisms observed in damaged bone specimen. (A) The toughening mechanisms in bone: collagen fibril bridging, uncracked ligament, and microcracks in the fracture zone. (B) Under confocal laser microscopy (Zeiss; basic fuchsin, excitation = 543 nm, emission = 560 nm) extensive amounts of diffuse damage (DD) and linear microcracks (LM) were observed within uncracked ligaments and in the vicinity of the main fracture crack in a compact-tension specimen. (C) Diffuse damage area at high magnification showing submicroscopic cracks.

Fig. 3.

Formation of dilatational bands in damaged bone studied via atomic force and confocal microscopy. The images show the progression of dilatational band formation in bone. (A and B) The presence of bands in diffuse damage regions of fatigued longitudinal bone. (Scale bar, 10 μm.) Dilatational bands form in a direction normal to the loading direction (thick dark arrows). (C) A single dilatational band imaged by atomic force microscopy that corresponds to each of the circled entities in B. Encircled in red dotted lines are the fused mineral aggregates, each over 100 nm in size. Mineral aggregates connected by the osteocalcin and osteopontin complex (additional details in Fig. 6) separate (in the direction of arrows shown in D) to form dilatational bands (encircled). Growth of dilatational bands subsequently leads to the rupture and separation of mineralized fibrils (D) and formation of diffuse damage regions (E). (F) Three-dimensional rendering of AFM dilatational bands’ topography. Bands can be seen as depressions in the surface (marked by arrows in E and F).

Fig. 4.

Immunohistochemical staining of fatigued specimen and ELISA for osteocalcin and osteopontin quantification. (A) Associations of dilatational bands (Upper) and diffuse damage (Lower) with osteocalcin, osteopontin, and phosphoproteins. As seen in the micrographs, osteocalcin, osteopontin, and phosphorylated proteins colocalize with the bands (highlighted red by basic fuchsin, which stains for damage), indicating their possible role in band formation. (B) Mouse bones lacking OC (OC^−/−), OPN (OPN^−/−), and OC-OPN (OC-OPN^{−/−;−/−}) showed a significantly lower level of fracture toughness than their WT controls but were not different from each other. (C) Mouse bones lacking OC (OC^−/−), OPN (OPN^−/−), and OC-OPN (OC-OPN^{−/−;−/−}) showed a dramatic reduction in diffuse damage compared with their WT controls but were not different from each other. *P < 0.05; ***P < 0.001.

Fig. 5.

Hardness and toughness reduction in diffuse damage and linear microcrack regions. (A and B) Compared with the controls, the diffuse damage region displayed a 13% drop in hardness (A), whereas the 0- to 50-µm zone surrounding linear microcracks (B) lost only 7%. *P < 0.05.

Fig. 6.

Dilatational band formation in bone. Schematic shows dilatational band formation and subsequent collagen fibril rupture and slippage. The first stage (Left) illustrates two sites of dilatational band formation. Each site comprises two osteocalcin molecules (red) and an osteopontin molecule (green). The OC and OPN molecules are sandwiched between two fused mineral aggregates (i.e., aggregates composed of several individual mineral crystals). OC directly interacts with mineral (at point of contact with mineral aggregate). Possible long-range OPN interactions with mineral via Ca²⁺ ions are shown in the schematic as dashed white lines. The mineral aggregate is also surrounded by additional OC molecules (shown in red above the mineral aggregate) that may not partake in dilatational band formation and play only the role of crystal growth regulator. The formation of dilatational bands occurs before deformation of collagen. The application of a load (Center) causes the OC-OPN-OC protein complex to unfold. Dilatational bands (highlighted in yellow) form and extend until the maximum extension of 135 nm is reached. Continuous loading causes the OC and OPN to separate (Right). The separation of OC and OPN dictates the subsequent rupture and shear of collagen fibrils. The shear is shown by the difference in longitudinal displacement of the fibrils.