Self-Repair of a Biological Fiber Guided by an Ordered Elastic Framework

Incorporating sacrificial cross-links into polymers represents an exciting new avenue for the development of self-healing materials, but it is unclear to what extent their spatial arrangement is important for this functionality. In this respect, self-healing biological materials, such as mussel byssal threads, can provide important chemical and structural insights. In this study, we employ in situ small-angle X-ray scattering (SAXS) measurements during mechanical deformation to show that byssal threads consist of a partially crystalline protein framework capable of large reversible deformations via unfolding of tightly folded protein domains. The long-range structural order is destroyed by stretching the fiber but reappears rapidly after removal of load. Full mechanical recovery, however, proceeds more slowly, suggesting the presence of strong and slowly reversible sacrificial cross-links. One likely role of the highly ordered elastic framework is to bring sacrificial binding sites back into register upon stress release, facilitating bond reformation and self-repair

Microstructural and micro-scale mechanical properties of <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− cortical bone.

<p>(A). Light microscopy of osteocyte lacunae in <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− samples, respectively (scale bar: 20 µm) (B). Laser scanning confocal microscopy using Rhodamine-B as a contrasting agent showing osteocytic and canalicular networks in <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− samples, light areas are intensely stained with Rhodamine-B, scale bar: 20 µm. (C) Backscatter scanning electron microscopy revealing the microstructure at the surface and no significant differences in density, scale bar: 10 µm. (D) Nanoindentation (n = 80) measurements of the indentation moduli and hardness of <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− bone samples (E) Micro-tensile (n = 20) measurements of tensile strength and elastic moduli in wildtype and fetuin-A deficient bone samples (F) Representative <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− fracture surfaces showing evidence of brittle failure, scale bar: 20 µm, inset: higher magnification of the fracture surface, scale bar: 2 µm.</p

Normal trabecular bone mass, but increased cortical bone strength in fetuin-A deficient mice.

<p>(<i>A</i>) Von Kossa/van Gieson-stained undecalcified sections of the spine from 4 months old wildtype and fetuin-A deficient mice. (B) Histomorphometric quantification of the trabecular bone volume (BV/TV, bone volume per tissue volume) and the trabecular number (Tb.N.). (C) Histomorphometric quantification of the osteoblast number (N.Ob./B.Pm, number of osteoblasts per bone perimeter) and the osteoclast number (N.Oc./B.Pm, number of osteoclasts per bone perimeter). (D) Contact radiographs of the hindlegs from 4 months old <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− mice. The femoral length is given below. (E) Cross-sectional µCT scanning of the femora. (F) Quantification of the cortical thickness and the force to failure in three-point-bending assays. All values represent mean±SD (n = 8 per group). Asterisks indicate statistically significant differences (p<0.05).</p

Growth plate morphology and mineralization in <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− mice.

<p>(A) Wildtype mice had dark blue stained cartilage cores within metaphyseal trabeculae of the growth plates, whereas <i>Ahsg</i>−/− had completed the cartilage-to-bone transition without the remains of cartilage cores within the trabeculae. Moreover, <i>Ahsg</i> −/− mice showed pronounced discontinuities in the chondrocyte column organization in comparison to the wildtype mice (Toluidine blue staining). (B) <i>Ahsg</i> −/− mice frequently showed thickened calcified bridge formations across their growth plates, which was confirmed by backscattered electron microscopy. The orange and black areas correspond to mineralized and non-mineralized tissue, respectively. (C) The mineral content in <i>Ahsg</i>−/− mice was significantly increased in both tibial and femoral growth plates in comparison to wildtype mice as judged by quantitative backscattered electron imaging. (D) In contrast, the mineralization (mean Ca Wt%) of the femoral and tibial cortices was similar in <i>Ahsg</i>−/− and <i>Ahsg</i>+/+ mice.</p

Mineral and organic components in <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− femoral cortical bone.

<p>(A) A typical Raman spectrum of representative <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− samples with peaks at 910–990 cm⁻¹ and 1600–1700 cm⁻¹ representing PO₄³⁻ (mineral) and Amide I (organic matrix) groups, respectively. inset: Normalized intensity measurements at polarization angles of −45, 0, 45, 90 were made to address the orientation artifacts of Raman intensity of type I collagen for both <i>Ahsg</i>+/+ and <i>Ahsg</i>−/−. The dashed lines are fits which estimate parameters characteristic of sample orientation and mineralization. The solid lines indicate mean intensity values of mineralization in the samples. (B) The mineral content normalized with the organic matrix can be observed by Raman ratios between <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− samples. Further complementing these observations, measurements of the mineral component were made with X-ray attenuation, absorption, as well as small-angle X-ray scattering. (C) Comparing fibrillar versus tissue strains in <i>Ahsg</i>+/+ (green circles) and <i>Ahsg</i>−/− (red squares) bone. Samples were measured with in-situ synchrotron small angle X-ray scattering to determine the amount of strain contributed by the collagenous fibrils within the <i>Ahsg</i>+/+ and <i>Ahsg</i>−/− samples. Dashed lines represent orientation guides.</p