Department of Mechanical Engineering, McGill University, Montreal, Quebec, Canada.
Faculty of Dentistry, Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
Bone. 2018 May;110:304-311. doi: 10.1016/j.bone.2018.02.022. Epub 2018 Feb 24.
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.
骨骼中最突出的结构成分是胶原蛋白和矿物质。然而，骨骼还含有大量的非胶原蛋白（尤其是 SIBLING 蛋白家族），其中一些可能作为复合混合胶原蛋白/矿物质支架的粘性/粘性“粘结剂”，无论是在骨骼的整体相中，还是在其界面处。一种这样的非胶原蛋白 - 骨桥蛋白（OPN） - 似乎对骨骼的可变形性和断裂韧性至关重要。在本研究中，我们使用了重建的合成矿物质-OPN-矿物质界面和生物源性（天然牙本质）矿物质/胶原蛋白-OPN-矿物质/胶原蛋白界面，来测量矿化基质上 OPN 的断裂韧性。我们使用该系统来测试这样一个假设，即存在于骨骼中的酶组织转谷氨酰胺酶 2（TG2）交联 OPN 可增强界面粘附力，从而提高骨骼的断裂韧性。为此，我们制备了合成纯羟磷灰石矿物质和独角鲸牙本质的双悬臂梁底物，并在不同的 OPN/交联条件下将它们直接彼此相对，使用微型加载台测试断裂韧性。测量了不同 OPN 配方（单体与聚合物）、交联状态和基质组成的 OPN 界面的断裂功。非交联的 OPN 在纯羟磷灰石上几乎没有提供粘附力，而 OPN 交联（通过化学交联剂戊二醛和 TG2 酶）则通过单体和聚合物 OPN 为羟磷灰石和牙本质提供了强的界面粘附力。在底物梁上预涂单体 OPN 进一步提高了样品的粘附性能，这可能是因为允许这种新生 OPN 形式与矿物质/基质成分有效结合，这种预附着为底物之间的进一步交联提供了蛋白质层。
