Sawtooth patterns in flexural force curves of structural biological materials are not signatures of toughness enhancement: Part I.

Layered architectures are prevalent in tough biological composites, such as nacre and bone. Another example of a biological composite with layered architecture is the skeletal elements-called spicules-from the sponge Euplectella aspergillum. Based on the similarities between the architectures, it has been speculated that the spicules are also tough. Such speculation is in part supported by a sequence of sudden force drops (sawtooth patterns) that are observed in the spicules' force-displacement curves from flexural tests, which are thought to reflect the operation of fracture toughness enhancing mechanisms. In this study, we performed three-point bending tests on the spicules, which also yielded the aforementioned sawtooth patterns. However, based on the analysis of the micrographs obtained during the tests, we found that the sawtooth patterns were in fact a consequence of slip events in the flexural tests. This is put into perspective by our recent study, in which we showed that the spicules' layered architecture contributes minimally to their toughness, and that the toughness enhancement in them is meager in comparison to what is observed in bone and nacre [Monn MA, Vijaykumar K, Kochiyama S, Kesari H (2020): Nat Commun 11:373]. Our past and current results underline the importance of inferring a material's fracture toughness through direct measurements, rather than relying on visual similarities in architectures or force-displacement curve patterns. Our results also suggest that since the spicules do not possess remarkable toughness, re-examining the mechanical function of the spicule's intricate architecture could lead to the discovery of new engineering design principles.

Lamellar architectures in stiff biomaterials may not always be templates for enhancing toughness in composites.

The layered architecture of stiff biological materials often endows them with surprisingly high fracture toughness in spite of their brittle ceramic constituents. Understanding the link between organic-inorganic layered architectures and toughness could help to identify new ways to improve the toughness of biomimetic engineering composites. We study the cylindrically layered architecture found in the spicules of the marine sponge Euplectella aspergillum. We cut micrometer-size notches in the spicules and measure their initiation toughness and average crack growth resistance using flexural tests. We find that while the spicule's architecture provides toughness enhancements, these enhancements are relatively small compared to prototypically tough biological materials, like nacre. We investigate these modest toughness enhancements using computational fracture mechanics simulations.

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules.

Many load bearing biological structures (LBBSs)-such as feather rachises and spicules-are small (<1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions. We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device's operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device's force range can be adjusted by using cantilevers of different stiffnesses. The device's capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements-known as spicules-are silica fibers that are approximately 50 µm in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a ≈1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

Enhanced bending failure strain in biological glass fibers due to internal lamellar architecture.

The remarkable mechanical properties of biological structures, like tooth and bone, are often a consequence of their architecture. The tree ring-like layers that comprise the skeletal elements of the marine sponge Euplectella aspergillum are a quintessential example of the intricate architectures prevalent in biological structures. These skeletal elements, known as spicules, are hair-like fibers that consist of a concentric array of silica cylinders separated by thin, organic layers. Thousands of spicules act like roots to anchor the sponge to the sea floor. While spicules have been the subject of several structure-property investigations, those studies have mostly focused on the relationship between the spicule's layered architecture and toughness properties. In contrast, we hypothesize that the spicule's layered architecture enhances its bending failure strain, thereby allowing it to provide a better anchorage to the sea floor. We test our hypothesis by performing three-point bending tests on E. aspergillum spicules, measuring their bending failure strains, and comparing them to those of spicules from a related sponge, Tethya aurantia. The T. aurantia spicules have a similar chemical composition to E. aspergillum spicules but have no architecture. Thus, any difference between the bending failure strains of the two types of spicules can be attributed to the E. aspergillum spicules' layered architecture. We found that the bending failure strains of the E. aspergillum spicules were roughly 2.4 times larger than those of the T. aurantia spicules.

A new structure-property connection in the skeletal elements of the marine sponge Tethya aurantia that guards against buckling instability.

We identify a new structure-property connection in the skeletal elements of the marine sponge Tethya aurantia. The skeletal elements, known as spicules, are millimeter-long, axisymmetric, silica rods that are tapered along their lengths. Mechanical designs in other structural biomaterials, such as nacre and bone, have been studied primarily for their benefits to toughness properties. The structure-property connection we identify, however, falls in the entirely new category of buckling resistance. We use computational mechanics calculations and information about the spicules' arrangement within the sponge to develop a structural mechanics model for the spicules. We use our structural mechanics model along with measurements of the spicules' shape to estimate the load they can transmit before buckling. Compared to a cylinder with the same length and volume, we predict that the spicules' shape enhances this critical load by up to 30%. We also find that the spicules' shape is close to the shape of the column that is optimized to transmit the largest load before buckling. In man-made structures, many strategies are used to prevent buckling. We find, however, that the spicules use a completely new strategy. We hope our discussion will generate a greater appreciation for nature's ability to produce beneficial designs.

New functional insights into the internal architecture of the laminated anchor spicules of Euplectella aspergillum.

To adapt to a wide range of physically demanding environmental conditions, biological systems have evolved a diverse variety of robust skeletal architectures. One such example, Euplectella aspergillum, is a sediment-dwelling marine sponge that is anchored into the sea floor by a flexible holdfast apparatus consisting of thousands of anchor spicules (long, hair-like glassy fibers). Each spicule is covered with recurved barbs and has an internal architecture consisting of a solid core of silica surrounded by an assembly of coaxial silica cylinders, each of which is separated by a thin organic layer. The thickness of each silica cylinder progressively decreases from the spicule's core to its periphery, which we hypothesize is an adaptation for redistributing internal stresses, thus increasing the overall strength of each spicule. To evaluate this hypothesis, we created a spicule structural mechanics model, in which we fixed the radii of the silica cylinders such that the force transmitted from the surface barbs to the remainder of the skeletal system was maximized. Compared with measurements of these parameters in the native sponge spicules, our modeling results correlate remarkably well, highlighting the beneficial nature of this elastically heterogeneous lamellar design strategy. The structural principles obtained from this study thus provide potential design insights for the fabrication of high-strength beams for load-bearing applications through the modification of their internal architecture, rather than their external geometry.

Performance of a commercially available in-clinic ELISA for detection of antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii and Dirofilaria immitis antigen in dogs.

OBJECTIVE: To evaluate the performance of an in-clinic ELISA designed for detection of heartworm antigen and antibodies against 5 tick-borne pathogens. DESIGN: Validation study. SAMPLE: 1,601 serum or matched serum, plasma, and blood samples from dogs. PROCEDURES: Samples were tested for Dirofilaria immitis (heartworm) antigen and antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii by means of an in-clinic ELISA. Evaluation of assay sensitivity and specificity, agreement of results among sample types, and cross-reactivity of E canis antigens in the assay with anti-Ehrlichia chaffeensis antibodies in stored samples from experimentally infected dogs were performed at a reference laboratory. Field tests of the in-clinic ELISA were performed at 6 veterinary facilities. Results were compared with confirmatory test results. RESULTS: Sensitivity and specificity of the in-clinic ELISA were > 89% for detection of antibodies against A phagocytophilum (93.2% and 99.2%, respectively), A platys (89.2% and 99.2%, respectively), B burgdorferi (96.7% and 98.8%, respectively), E canis (97.8% and 92.3%, respectively), and E ewingii (96.5% and 93.9%, respectively). Sensitivity of the assay for detection of D immitis was 98.9%, with 99.3% specificity. The in-clinic ELISA identified exposure to > 1 vector-borne pathogen in 354 of 1,195 samples. Cross-reactivity of E canis antigens with anti-E chaffeensis antibodies was confirmed. Results of field evaluations confirmed that the in-clinic ELISA could be reliably used under typical clinical conditions to identify dogs exposed to the pathogens of interest. CONCLUSIONS AND CLINICAL RELEVANCE: The in-clinic ELISA provided a comprehensive in-house serologic screening test for all vector-borne pathogens evaluated.