Bone toughness at the molecular scale: A model for fracture toughness using crosslinked osteopontin on synthetic and biogenic mineral substrates

The most prominent structural components in bone are collagen and mineral. However, bone additionally contains a substantial amount of noncollagenous proteins (most notably of the SIBLING protein family), some of which may act as cohesive/adhesive “binders” for the composite hybrid collagen/mineral scaffolding, whether in the bulk phase of bone, or at its interfaces. One such noncollagenous protein - osteopontin (OPN) - appears to be critical to the deformability and fracture toughness of bone. In the present study, we used a reconstructed synthetic mineral-OPN-mineral interface, and a biogenic (natural tooth dentin) mineral/collagen-OPN-mineral/collagen interface, to measure the fracture toughness of OPN on mineralized substrates. We used this system to test the hypothesis that OPN crosslinking by the enzyme tissue transglutaminase 2 (TG2) that is found in bone enhances interfacial adhesion to increase the fracture toughness of bone. For this, we prepared double-cantilever beam substrates of synthetic pure hydroxyapatite mineral, and of narwhal dentin, and directly apposed them to one another under different intervening OPN/crosslinking conditions, and fracture toughness was tested using a miniaturized loading stage. The work-of-fracture of the OPN interface was measured for different OPN formulations (monomer vs. polymer), crosslinking states, and substrate composition. Noncrosslinked OPN provided negligible adhesion on pure hydroxyapatite, whereas OPN crosslinking (by the chemical crosslinker glutaraldehyde, and TG2 enzyme) provided strong interfacial adhesion for both hydroxyapatite and dentin using monomeric and polymeric OPN. Pre-coating of the substrate beams with monomeric OPN further improved the adhesive performance of the samples, likely by allowing effective binding of this nascent OPN form to mineral/matrix components, with this pre-attachment providing a protein layer for additional crosslinking between the substrates.