This work was supported by National Science Foundation [Mechanics of Materials and Structures Program, grant number 1562656]; and the American Society of Mechanical Engineers [Haythornthwaite Young Investigator Award].

<ol>
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The authors have nothing to disclose.

Several steps of the protocol are particularly important for ensuring that forces and displacements are measured accurately. While some of these critical steps are universal to all three-point bending tests, others are unique to this mechanical testing device.
In step 1.2 of the protocol the LP mirror is cleaned and inspected for scratches, and in step 1.6 of the protocol the FODS gain is set. It is important for the gain and the LP mirror reflectance to be constant for steps 2, 4, and 5 of the protocol. For this reason, the two calibration steps (steps 2 and 4 of the protocol) should be performed immediately before the bending test (step 5 of the protocol).
In steps 1.9 and 3.7 of the protocol the stage is leveled with respect to the surface of the isolation table. These steps ensure that <img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg" /> is the component of force perpendicular to the spicule&#39;s longitudinal axis. The frame of the mechanical testing device is manufactured so that the cantilever, LP mirror, and surface of the FODS are all parallel to the surface of the isolation table. This means that the force sensor will measure the component of force and displacement normal to the isolation table surface. If the top of the stage is misaligned by an angle&#160;<img alt="Equation 53" src="/files/ftp_upload/56571/56571eq53.jpg" /> with respect to the surface of the isolation table, then the measured displacement of the LPT will be <img alt="Equation 55" src="/files/ftp_upload/56571/56571eq55.jpg" />, where <img alt="Equation 54" src="/files/ftp_upload/56571/56571eq54.jpg" /> is the actual displacement in the direction perpendicular to the spicule&#39;s longitudinal axis (see Figure 7). Since <img alt="Equation 56" src="/files/ftp_upload/56571/56571eq56.jpg" />, this results in an over prediction of applied forces and the under prediction of spicule displacements per equations (1) and (2).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56571/56571fig7.jpg" /> 
Figure 7: Effect of stage leveling on displacement measurements. (A) The stage is tilted at an angle, <img alt="Equation 53" src="/files/ftp_upload/56571/56571eq53.jpg" />, with respect to the surface of the isolation table and the bottom surface of the cantilever. (B) The displacement of the LP in the vertical direction, <img alt="Equation 50" src="/files/ftp_upload/56571/56571eq50.jpg" /> (see Figure 1 (D)), is measured by the FODS. The component of the LP displacement in the direction perpendicular to the spicule&#39;s axis is <img alt="Equation 54" src="/files/ftp_upload/56571/56571eq54.jpg" />. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56571/56571fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In steps 5.1-5.3 of the protocol the LPT is positioned mid way across the trench&#39;s span. Misalignment of the LPT with respect to the mid span will result in the specimen appearing stiffer than it actually is 31,32. That is, the spicule&#39;s displacement will be smaller than that which would be measured if the same force were applied at the mid span. This type of misalignment can be avoided by not removing the stage from the stage base plate or adjusting the x-axis stage position after the centering procedure is complete (steps 5.1-5.3 of the protocol).
One limitation of this method is that in order to reduce the relative measurement uncertainty of the force and displacement measurements, the cantilever stiffness should be selected so that the FODS output voltages span the full range of 1.8 to 4.5 V during the bending test. However, this voltage range corresponds to a cantilever displacement of approximately &#8776;250 &#181;m, which is roughly the same as the spicule displacement just before it fails (see Figure 6 (C)). This means that the cantilever and the spicule have similar stiffnesses. While this is not problematic for measuring the elastic response and strength properties of the spicules, it does preclude the accurate measurement of the spicules&#39; toughness properties. This is because in order to ensure accurate measurement of toughness properties, a crack in the spicule must propagate in a controlled manner 33. Typically, this is only possible if the testing device is much stiffer than the specimen 33. In order to increase the stiffness of the testing device, a more sensitive displacement sensor could be used in place of the FODS.
While the bending test protocol is demonstrated on E. aspergillum spicules, the mechanical testing device can be used to perform three-point bending tests on other LBBSs and synthetic materials as well. This mechanical testing device is most appropriate for specimens whose cross-sectional diameters range from 0.01 to 1 mm and for trench spans ranging from 1 to 10 mm. For larger diameters, the sample stage should be redesigned so that the specimen cannot roll across the stage. This is not an issue for smaller fibers, like the spicules, because the roughness of the stage&#39;s surface is enough to prevent the specimen from rolling. The radii of the trench edges and LPT should also be made larger to avoid introducing local damage at the points where the specimen is supported 31,32. Furthermore, the stage leveling plate should be fastened to the stage base plate (see Figure 4A) using &#188;&#34;-20 socket head cap screws after step 3.7 of the protocol to prevent stage tilting if forces exceed &#8776;1 N.
For accurate force and displacement measurement, the cantilever&#39;s stiffness should always be much smaller than the frame&#39;s stiffness (&#8776;107 N/m). This requirement limits the maximum force that can be applied by this device to &#8776;25 N. Consequently, it is important to estimate the maximum force a specimen can withstand before performing a bending test to determine if this device can be used to perform the test.
This work provides the protocol, technical drawings (see Supplementary File 1) and software (see Supplementary Code files) for reproducing and using our mechanical testing device. This will hopefully provide a platform for accurately measuring the flexural behavior of many different LBBSs. These measurements are a prerequisite for developing a deeper understanding the relationship between a LBBS&#39;s architecture and its mechanical properties.

By studying the architectures of load bearing biological structures (LBBSs), such as shell and bone, engineers have developed new composite materials that are both strong and tough 1. It has been shown that the remarkable mechanical properties of LBBSs and their bio-inspired counterparts are related to their intricate internal architectures 2. However, the relationships between LBBS architectures and mechanical properties are not fully understood. Measuring a LBBS&#39;s mechanical response is the first step toward understanding how its architecture enhances its mechanical properties.
However, it is important that the type of test used to measure a LBBS&#39;s mechanical response is consistent with its mechanical function. For example, since feathers must support aerodynamic loads, the primary function of a feather rachis is to provide flexural stiffness 3. Therefore, a bending test is preferred to a uniaxial tension test for measuring its mechanical response. In fact, many LBBSs&#8212;such as feather rachises 3, grass stems 4, and spicules 5,6,7,8&#8212;primarily deform by bending. This is because these LBBSs are slender&#8212;i.e., their length is much greater than their width or depth. However, performing bending tests on these LBBSs is challenging because the forces and displacements that they can withstand before failing range from 10-2 to 102 N and 10-4 to 10-3 m, respectively 3,4,5,7,8. Consequently, the device used to perform these mechanical tests should have force and displacement resolutions of &#8776;10-5 N and &#8776;10-7 m (i.e., 0.1% of the sensor&#39;s maximum measureable force and displacement), respectively.
Commercially available, large scale, mechanical testing systems typically cannot measure forces and displacements with this resolution. While atomic force microscope-based 9,10 or microelectromechanical systems-based 11 testing devices have adequate resolution, the maximum force (respective displacement) they can measure is smaller than the maximum force (respective displacement) that the LBBS can withstand. Therefore, to perform bending tests on these LBBSs, engineers and scientists must rely on custom-built mechanical testing devices 5,7,12,13. The primary advantage of these custom-built devices is that they can accommodate large ranges of forces and displacements. However, the construction and operation of these devices is not well documented in the literature.
A protocol is described 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. Technical drawings, including all dimensions, of the components of the mechanical testing device are provided in the Supplementary Material. The primary advantage of this mechanical testing device is that the force and displacement ranges can be easily adjusted to suit different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope 9. In this device, a specimen is placed across a trench cut in a stainless steel plate (see Figure 1A-C). The span of the trench is measured from optical micrographs to be 1278 &#177; 3 &#181;m (mean &#177; standard deviation; n = 10). The trench edges support the specimen during a bending test (see Figure 1C, and D). This sample stage is attached to a three-axis translation stage and positioned beneath an aluminum wedge so that the wedge is located midway across the trench&#39;s span (see Figure 1C). By moving the stage in the <img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg" /> direction (see Figure 1A, and C), the specimen is pushed into the wedge causing the specimen to bend.
We refer to the wedge as the load point tip (LPT) and the component of the device that contains the wedge as the load point (LP). The LP is attached to the end of a cantilever whose displacement is measured by a fiber optic displacement sensor (FODS). The FODS emits infrared light, which is reflected off of a mirror located on the top surface of the LP (see Figure 1B) and received by an optical fiber in the FODS. A &#8776;5 mm square piece of a polished silicon wafer is used as the LP mirror and is affixed to the LP using epoxy. The FODS measures displacements by comparing the intensities of the emitted and reflected light. The cantilever stiffness and displacement are used to compute the force,&#160;<img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg" />, experienced by the wedge due to its interaction with the specimen. The cantilever displacement is also used to compute the displacement of the specimen&#39;s cross-section beneath the wedge, <img alt="Equation 3" src="/files/ftp_upload/56571/56571eq3.jpg" />. Cantilever-based force sensors have been used in a number of micro- and macro-scale mechanical testing studies 10,11,12,13,14. The specific design presented here is adapted from a mechanical testing device used for performing adhesive contact experiments 14. A similar design has also been used in a commercially available micro-tribometer 15,16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56571/56571fig1.jpg" /> 
Figure 1: Overview of the custom-built mechanical testing device. (A) A computer aided design rendering of the device. The stage components are highlighted in green. The force sensing subassembly (cantilever, load point (LP)) is highlighted in red. (B) A magnified view of (A). The LP mirror is shown in blue on the top surface of the LP beneath the FODS and is labeled LPM. (C) The coordinate system used to describe the motion of the translation stage. By leveling the stage in step 1.9 of the protocol, the&#160;<img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg" /> direction is made to coincide with the vector normal to the surface of the LP mirror. (D) A schematic of the three-point bending configuration showing the deformation of the spicule and the measured displacements <img alt="Equation 49" src="/files/ftp_upload/56571/56571eq49.jpg" />, and <img alt="Equation 50" src="/files/ftp_upload/56571/56571eq50.jpg" />. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56571/56571fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum6,7. This sponge&#39;s skeleton is an assembly of filaments, called spicules (see Figure 2A). The spicules are &#8776;50 &#181;m thick and are composed primarily of silica 6. Biosilica-based spicules are found in sponges belonging to the classes Demospongiae, Homoscleromorpha, and Hexactinellida. Sponges, such as E. aspergillum, that belong to the class Hexactinellida are also known as &#34;glass sponges.&#34; While the spicules of glass sponges are composed primarily of silica, it has been shown that the silica often contains an organic matrix composed of either collagen 17,18 or chitin 19,20,21. This organic matrix plays an important role in silica biomineralization 18,20. Furthermore, in some spicules the organic matrix also serves as a template for the biomineralization of calcium 22. In addition to being distributed within the silica, the organic matrix can also form distinct layers that partition the spicule&#39;s silica into concentric, cylindrical lamellae 6,23. It has been shown that this concentric, lamellar architecture can affect the spicules&#39; deformation behavior 6,7,8,24,25,26. Consequently, the spicules&#39; mechanical properties are determined by a combination of their chemistry (i.e., the chemical structure of the silica-protein composite) and their architecture 27. Both the chemical structure and architecture of glass sponge spicules are still under investigation 24,28,29.
Most of the spicules in E. aspergillum are cemented together to form a stiff skeletal cage. However, at the base of the skeleton there is a tuft of very long (&#8776;10 cm) spicules known as the anchor spicules (see Figure 2A). We describe the protocol for performing three-point bending tests on small sections of the anchor spicules.
In step 1 of the protocol, the procedure for assembling and aligning the components of the custom-built mechanical testing device is described. Steps 2 and 4 of the protocol provide instructions for generating calibration data used to compute forces and displacements in the bending test. The steps taken to prepare a section of a spicule and mount it to the test fixture are described in step 3. The procedure for conducting the bending test on the spicule section is described in step 5. Finally, in the Representative Results section the calibration data obtained in steps 2 and 4 are used along with the bending test data obtained in step 5 to compute <img alt="Equation 2" src="/files/ftp_upload/56571/56571eq2.jpg" /> and <img alt="Equation 3" src="/files/ftp_upload/56571/56571eq3.jpg" />.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56571/56571fig2.jpg" /> 
Figure 2: Procedure for sectioning and inspecting E. aspergillum spicules. (A) The skeleton of E. aspergillum. The tuft of free-standing anchor spicules is shown at the base of the skeleton. The scale bar is ~25 mm. (B) A single anchor spicule is held in place on a microscope slide using a #00000 red sable brush and sectioned using a razor blade. The scale bar is ~12 mm. (C) A section of an E. aspergillum spicule placed across the trench on the sample stage. The trench edges and trench ridge are highlighted in teal and orange, respectively. The spicule is pushed against the trench ridge to ensure that its axis is perpendicular to the trench edges. (D) A micrograph of a spicule that passes the inspection procedure described in step 3.4 of the protocol, which describes how to determine if a spicule section is damaged and should be discarded. (E) A micrograph of a spicule containing many cracks and missing large sections of silica layers that would fail the inspection procedure described in step 3.4 of the protocol. Scale bars =&#160; 250 &#181;m (C), 100 &#181;m (D), and 100 &#181;m&#160;(E). <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56571/56571fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>TMC 36&quot; x 48&quot; isolation table with 4&quot; CleanTop breadboard</td><td>TMC</td><td>63-563</td><td>Isolation Table</td></tr><tr><td>Diffeential Screw Adjuster</td><td>Thorlabs</td><td>DAS110</td><td>For stage leveling plate</td></tr><tr><td>1&quot; Travel Micrometer Head with 0.001&quot; Graduations</td><td>Thorlabs</td><td>150-801ME</td><td>For stage leveling plate</td></tr><tr><td>Right-Angle Bracket for PT Series Translation Stages, 1/4&quot;-20 Mounting Holes</td><td>Thorlabs</td><td>PT102</td><td>For microscope mount</td></tr><tr><td>1&quot; Dovetail Translation Stage, 1/4&quot;-20 Taps</td><td>Thorlabs</td><td>DT25</td><td>For microscope mount</td></tr><tr><td>1&quot; Translation Stage with 1/4&quot;-170 Adjustment Screw, 1/4&quot;-20 Taps</td><td>Thorlabs</td><td>PT1B</td><td>For microscope mount</td></tr><tr><td>12&quot; Length, Dovetail Optical Rail</td><td>Edmund Optics</td><td>54-401</td><td>For microscope mount</td></tr><tr><td>2.5&quot; Width, Dovetail Carrier</td><td>Edmund Optics</td><td>54-404</td><td>For microscope mount</td></tr><tr><td>0.5&quot; Width, Dovetail Carrier</td><td>Edmund Optics</td><td>54-403</td><td>For microscope mount</td></tr><tr><td>InfiniTube Mounting C-Clamp with &amp;frac14;-20</td><td>Edmund Optics</td><td>57-788</td><td>Microscope component</td></tr><tr><td>Standard (with no In-Line Attachment), InfiniTube</td><td>Edmund Optics</td><td>56-125</td><td>Microscope component</td></tr><tr><td>Standard In-Line Attachment (Optimized at 2X-10X), InfiniTube</td><td>Edmund Optics</td><td>56-126</td><td>Microscope component</td></tr><tr><td>Mitutoyo/Achrovid Objective Adapter (M26 to M27)</td><td>Edmund Optics</td><td>53-787</td><td>Microscope component</td></tr><tr><td>5X Infinity Achrovid Microscope Objective</td><td>Edmund Optics</td><td>55-790</td><td>Microscope component</td></tr><tr><td>0.316&quot; ID, Fiber Optic Adapter SX-6</td><td>Edmund Optics</td><td>38-944</td><td>Microscope component</td></tr><tr><td>&amp;frac14;&quot; x 36&quot;, Flexible Fiber Optic Light Guide</td><td>Edmund Optics</td><td>42-347</td><td>Microscope component</td></tr><tr><td>115V, MI-150 Fiber Optic Illuminator w/IR Filter and Holder</td><td>Edmund Optics</td><td>55-718</td><td>Microscope component</td></tr><tr><td>Allied Vision Manta G-223 2/3&quot; Color CMOS Camera</td><td>Edmund Optics</td><td>88-452</td><td>Microscope component</td></tr><tr><td>Power Supply for Manta/ Guppy Pro/ Stingray/ Pike</td><td>Edmund Optics</td><td>68-586</td><td>Microscope component</td></tr><tr><td>1/4&quot; Travel Single Axis Translation Stage</td><td>Thorlabs</td><td>MS1S</td><td>FODS micrometer</td></tr><tr><td>Analog Reflectance Dependent Fiber Optic Displacement Sensor</td><td>Philtec</td><td>D20</td><td>FODS</td></tr><tr><td>30V, 3A DC Power Supply</td><td>Agilent</td><td>U8001A</td><td>Power supply for DAQ and FODS</td></tr><tr><td>14-Bit, 48 kS/s Low-Cost Multifunction DAQ</td><td>National Instruments</td><td>USB-6009</td><td>DAQ for FODS</td></tr><tr><td>Three Axis Motorized Translation Stage</td><td>Thorlabs</td><td>Thorlabs T25 XYZ-E/M</td><td>Translation stage</td></tr><tr><td>T-Cube DC Servo Motor Controller</td><td>Thorlabs</td><td>TDC001</td><td>Motor controller for stage</td></tr><tr><td>T-Cube Power Supply</td><td>Thorlabs</td><td>TPS001</td><td>Power supply for motor controller</td></tr><tr><td>National Instruments LabVIEW (2013 SP1)</td><td>National Instruments</td><td></td><td>Used for running software</td></tr><tr><td>National Instruments LabVIEW Vision Acquisition Software (2016)</td><td>National Instruments</td><td></td><td>Used for running software</td></tr><tr><td>Nikon Eclipse Ci-POL Main Body</td><td>MVI</td><td>MDA96000</td><td>Polarized light microscope</td></tr><tr><td>Nikon Pi Intermediate Tube with Analyzer Slider</td><td>MVI</td><td>MDB45305</td><td>Polarized light microscope</td></tr><tr><td>Nikon Dia-Polarizer</td><td>MVI</td><td>MDN11920</td><td>Polarized light microscope</td></tr><tr><td>Power Cord - 7&#039;6&quot;</td><td>MVI</td><td>79035</td><td>Polarized light microscope</td></tr><tr><td>Nikon P-Amh Mechanical Stage</td><td>MVI</td><td>MDC45000</td><td>Polarized light microscope</td></tr><tr><td>Nikon Lwd Achromat Condenser</td><td>MVI</td><td>MBL16100</td><td>Polarized light microscope</td></tr><tr><td>Nikon LV-NBD5BD-CH Manual Quint Nosepiece ESD</td><td>MVI</td><td>MBP60125</td><td>Polarized light microscope</td></tr><tr><td>Nikon C-TF Trinocular Tube F</td><td>MVI</td><td>MBB93100</td><td>Polarized light microscope</td></tr><tr><td>Nikon CFI 10X Eyepiece FN 22mm NC</td><td>MVI</td><td>MAK10110</td><td>Polarized light microscope</td></tr><tr><td>Nikon TU Plan Flour BD 10x Objective</td><td>MVI</td><td>MUE42100</td><td>Polarized light microscope</td></tr><tr><td>Venus Flower Basket Sponge</td><td>Denis Brand</td><td>N/A</td><td>Sponge skeleton</td></tr><tr><td>3.5X Headband Flip-Up Magnifier</td><td>McMaster Carr</td><td>1490T5</td><td>Used for spicule sectioning</td></tr><tr><td>&amp;Oslash;1&quot; Silicon Wafer, Type P / &amp;lt;100&gt;</td><td>Ted Pella</td><td>16011</td><td>Used for load point mirror</td></tr><tr><td>Low Lint Tapered Tip Cotton Swab</td><td>McMaster Carr</td><td>71035T31</td><td>Used for cleaning LP mirror</td></tr><tr><td>Rubber grip precision knife</td><td>McMaster Carr</td><td>35575A68</td><td>Used for sectioning spicules</td></tr><tr><td>Microscope Slides, frosted end, 75 x 25 x 1mm</td><td>Ted Pella</td><td>260409</td><td>Used for sectioning spicules</td></tr><tr><td>Sable Brushes, #00000, 0.08mm W x 4.0mm L</td><td>Ted Pella</td><td>11806</td><td>Used for handling spicules</td></tr><tr><td>PELCO Pro High Precision Tweezers, extra fine tips, superior finish</td><td>Ted Pella</td><td>5367-5NM</td><td>Used for handling spicules</td></tr><tr><td>Dual Axis Linear Scale Micrometer</td><td>Edmund Optics</td><td>58-608</td><td>Used for calibrating the microscopes</td></tr><tr><td>FLEX-A-TOP FT-38 CAS</td><td>ESD Plastic Containers</td><td>FT-38-CAS</td><td>Used for storing spicules</td></tr><tr><td>Plastic Vial Bullseye Level</td><td>McMaster Carr</td><td>2147A11</td><td>Used for leveling the stage</td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo</td><td>MS105DU</td><td>Used to mass calibration weights</td></tr></tbody></table>

a millimeter scale flexural testing system for measuring the mechanical properties of marine sponge spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;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&#39;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&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;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.

The most basic outputs of any mechanical test are the magnitude of the force applied to the specimen and the displacement at the location where the force is applied. In the case of a three-point bending test, the goal is to obtain the magnitude of the force applied by the LPT, <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />, and the displacement of the specimen&#39;s cross-section beneath the LPT in the <img alt="Equation 4" src="/files/ftp_upload/56571/56571eq4.jpg" /> direction, <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" />. However, for the mechanical testing device described here, several post-processing steps must be performed to transform the output data obtained from steps 2, 4 and 5 of the protocol into this desired <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />- <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> data. The data files obtained from the three-point bending test are: 1) the voltage-displacement interpolation file; 2) the force calibration file; and 3) the bending test file. A summary of the measured and derived quantities is shown in Table 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Symbol</td>
			<td colspan="4">Definition</td>
		</tr>
		<tr>
			<td>Nh</td>
			<td colspan="4">Number of voltages values in the voltage-displacement interpolation output file</td>
		</tr>
		<tr>
			<td>Vh</td>
			<td colspan="4">Measured voltage values in step 4 of the Protocol</td>
		</tr>
		<tr>
			<td>&#963;Vh</td>
			<td colspan="4">Standard deviation of Vh</td>
		</tr>
		<tr>
			<td>zsh</td>
			<td colspan="4">Measured stage position in step 4 of the Protocol</td>
		</tr>
		<tr>
			<td>Nc</td>
			<td colspan="4">Number of force measurements in the force calibration output file</td>
		</tr>
		<tr>
			<td>Fc</td>
			<td colspan="4">Force applied by calibration weights in step 2 of the Protocol</td>
		</tr>
		<tr>
			<td>Vc</td>
			<td colspan="4">Measured voltage values in step 2 of the Protocol</td>
		</tr>
		<tr>
			<td>&#963;Vc</td>
			<td colspan="4">Standard deviation of Vc</td>
		</tr>
		<tr>
			<td>zlc</td>
			<td colspan="4">Position of the LP in step 2 of the Protocol computed using Vh and Vc</td>
		</tr>
		<tr>
			<td>wlc</td>
			<td colspan="4">Displacement of the LP in step 2 of the Protocol computed from zlc</td>
		</tr>
		<tr>
			<td>Nt</td>
			<td colspan="4">Number of force and displacement measurements in the bending test output file</td>
		</tr>
		<tr>
			<td>zst</td>
			<td colspan="4">Position of the stage in step 5 of the Protocol</td>
		</tr>
		<tr>
			<td>wst</td>
			<td colspan="4">Displacement of the stage in step 5 of the Protocol</td>
		</tr>
		<tr>
			<td>Vt</td>
			<td colspan="4">Measured voltage values in step 5 of the Protocol</td>
		</tr>
		<tr>
			<td>&#963;Vt</td>
			<td colspan="4">Standard deviation of Vt</td>
		</tr>
		<tr>
			<td>zlt</td>
			<td colspan="4">Position of the LP in step 5 of the Protocol computed using Vh and Vt</td>
		</tr>
		<tr>
			<td>wlt</td>
			<td colspan="4">Displacement of the LP in step 5 of the Protocol computed from zlt</td>
		</tr>
		<tr>
			<td>F</td>
			<td colspan="4">Force applied by the LP in step 5 of the Protocol computed from zlt</td>
		</tr>
		<tr>
			<td>w0</td>
			<td colspan="4">Displacement of the spicule&#8217;s cross-section under the LP in step 5 of the Protocol</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of symbols used for quantities measured in steps 2, 4 and 5 of the Protocol and computed in the Representative Results section.
The purpose of the voltage-displacement interpolation file is to relate measured FODS output voltages to LPT displacements. This is done by rigidly coupling the LPT to the translation stage so that as the stage is moved in the <img alt="Equation 1" src="/files/ftp_upload/56571/56571eq1.jpg" /> direction, the change in the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" />-axis stage position is equal to the LPT displacement (step 4 of the protocol). The voltage-displacement interpolation file contains a set of points <img alt="Equation 15" src="/files/ftp_upload/56571/56571eq15.jpg" />, where&#160;<img alt="Equation 16" src="/files/ftp_upload/56571/56571eq16.jpg" /> is the average FODS output voltage taken over 100 measurements at a sampling rate of 1000 Hz, <img alt="Equation 17" src="/files/ftp_upload/56571/56571eq17.jpg" /> is the associated standard deviation of the 100 voltage measurements, <img alt="Equation 18" src="/files/ftp_upload/56571/56571eq18.jpg" /> is the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" />-axis stage position and&#160;<img alt="Equation 19" src="/files/ftp_upload/56571/56571eq19.jpg" /> is the number of stage displacement steps (see Figure 6 (B)).
The force calibration file allows the cantilever stiffness to be measured so that LP displacements can be used to compute the magnitude of the force applied by the LP. The force calibration file contains a set of points <img alt="Equation 20" src="/files/ftp_upload/56571/56571eq20.jpg" />, where&#160;<img alt="Equation 21" src="/files/ftp_upload/56571/56571eq21.jpg" /> is the average FODS output voltage taken over 100 measurements at a sampling rate of 1000 Hz,&#160;<img alt="Equation 22" src="/files/ftp_upload/56571/56571eq22.jpg" /> is the associated standard deviation of the 100 voltage measurements,&#160;<img alt="Equation 23" src="/files/ftp_upload/56571/56571eq23.jpg" /> is the force exerted by the weights on the LPT, and&#160;<img alt="Equation 24" src="/files/ftp_upload/56571/56571eq24.jpg" /> is the number of calibration weights used. Notice that there are two more points than there are calibration weights because the first point is measured for zero applied force and the second point for the force exerted by the wire hook alone.
Finally, the bending test file is used to compute&#160;<img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> and <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />. It contains a set of points <img alt="Equation 25" src="/files/ftp_upload/56571/56571eq25.jpg" />, where <img alt="Equation 26" src="/files/ftp_upload/56571/56571eq26.jpg" /> is the average FODS output voltage taken over 100 measurements at a sampling rate of 1000 Hz, <img alt="Equation 27" src="/files/ftp_upload/56571/56571eq27.jpg" /> is the associated standard deviation of the 100 voltage measurements, <img alt="Equation 28" src="/files/ftp_upload/56571/56571eq28.jpg" /> is the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" />-axis stage position and&#160;<img alt="Equation 29" src="/files/ftp_upload/56571/56571eq29.jpg" /> is the number of stage displacement steps during the bending test.
First, the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT&#39;s position during the force calibration, <img alt="Equation 30" src="/files/ftp_upload/56571/56571eq30.jpg" />, is found by using the set&#160;<img alt="Equation 31" src="/files/ftp_upload/56571/56571eq31.jpg" /> to map <img alt="Equation 21" src="/files/ftp_upload/56571/56571eq21.jpg" />values to <img alt="Equation 32" src="/files/ftp_upload/56571/56571eq32.jpg" /> values via linear interpolation. The <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT displacement is given by <img alt="Equation 33" src="/files/ftp_upload/56571/56571eq33.jpg" />, <img alt="Equation 34" src="/files/ftp_upload/56571/56571eq34.jpg" />. Since the LPT displacements are small compared to the length of the cantilever, the relationship between <img alt="Equation 23" src="/files/ftp_upload/56571/56571eq23.jpg" /> and <img alt="Equation 35" src="/files/ftp_upload/56571/56571eq35.jpg" /> appears to be linear. Therefore, the cantilever stiffness can be computed by fitting a line to the <img alt="Equation 36" src="/files/ftp_upload/56571/56571eq36.jpg" /> data and computing the slope, <img alt="Equation 37" src="/files/ftp_upload/56571/56571eq37.jpg" />. A representative set of points <img alt="Equation 36" src="/files/ftp_upload/56571/56571eq36.jpg" />and its corresponding fitted line are shown in Figure 6A. The stiffness of the cantilever used in the bending experiments was 90.6 &#177; 0.3 N/m.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56571/56571fig6.jpg" /> 
Figure 6: Representative results of the three-point bending test. (A) Force and displacement data (gray) obtained in step 2 of the protocol along with the linear fit (blue) used for estimating the stiffness of the cantilever. (B) Representative example of the data contained within the voltage-displacement interpolation output file. For a measured FODS output voltage, <img alt="Equation 51" src="/files/ftp_upload/56571/56571eq51.jpg" />, the position of the stage, <img alt="Equation 52" src="/files/ftp_upload/56571/56571eq52.jpg" />, can be obtained via linear interpolation. This is used to measure the cantilever displacement, <img alt="Equation 50" src="/files/ftp_upload/56571/56571eq50.jpg" />, during the bending test. (C) Representative force-displacement responses of 3 different E. aspergillum anchor spicules from successful three-point bending tests. (D) A force-displacement response from an unsuccessful three-point bending test. The nonlinearity of the curve suggests that the spicule was not properly seated on the sample stage and slid or reoriented after initial contact was made with the LPT. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56571/56571fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, the <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT&#39;s position during the bending test, <img alt="Equation 38" src="/files/ftp_upload/56571/56571eq38.jpg" />, is found by using the set <img alt="Equation 31" src="/files/ftp_upload/56571/56571eq31.jpg" /> to map <img alt="Equation 26" src="/files/ftp_upload/56571/56571eq26.jpg" /> values to <img alt="Equation 39" src="/files/ftp_upload/56571/56571eq39.jpg" /> values via linear interpolation. The <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the LPT displacement during the bending test is given by <img alt="Equation 40" src="/files/ftp_upload/56571/56571eq40.jpg" />,&#160;<img alt="Equation 41" src="/files/ftp_upload/56571/56571eq41.jpg" />. The <img alt="Equation 5" src="/files/ftp_upload/56571/56571eq5.jpg" /> component of the stage displacement during the bending test is given by <img alt="Equation 42" src="/files/ftp_upload/56571/56571eq42.jpg" />.
Since the LPT and the spicule are in contact during the entirety of the bending test, the spicule displacement,&#160;<img alt="Equation 43" src="/files/ftp_upload/56571/56571eq43.jpg" /> is given by
<img alt="Equation 44" src="/files/ftp_upload/56571/56571eq44.jpg" /> (1)
and the force applied by the LPT, <img alt="Equation 45" src="/files/ftp_upload/56571/56571eq45.jpg" />, is
<img alt="Equation 46" src="/files/ftp_upload/56571/56571eq46.jpg" /> (2)
It is important to note that since the set <img alt="Equation 31" src="/files/ftp_upload/56571/56571eq31.jpg" /> is used to obtain both <img alt="Equation 32" src="/files/ftp_upload/56571/56571eq32.jpg" /> and <img alt="Equation 39" src="/files/ftp_upload/56571/56571eq39.jpg" /> values via interpolation, the values of the <img alt="Equation 47" src="/files/ftp_upload/56571/56571eq47.jpg" /> and <img alt="Equation 26" src="/files/ftp_upload/56571/56571eq26.jpg" /> must be within the range of <img alt="Equation 16" src="/files/ftp_upload/56571/56571eq16.jpg" />. This is ensured by setting appropriate values for the starting voltage and high voltage stop values in steps 2, 4 and 5 of the protocol.
Figure 6C shows force-displacement curves for three representative spicules. For slender, linear elastic structures loaded in three-point bending, <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" /> is expected to increase linearly with <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> for small values of <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> 30. Nonlinearity of the <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" />- <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> curve for small <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> (e.g., see Figure 6D) typically suggests that the spicule may not be seated correctly on the sample stage. In this case, the test should be stopped and the spicule repositioned on the sample stage (step 3.6 of the protocol).
In order to ensure sufficient accuracy of the <img alt="Equation 13" src="/files/ftp_upload/56571/56571eq13.jpg" /> and <img alt="Equation 14" src="/files/ftp_upload/56571/56571eq14.jpg" /> measurements, the total voltage change over the course of the bending test, <img alt="Equation 48" src="/files/ftp_upload/56571/56571eq48.jpg" />, should be at least 1 V. If the total voltage change is less than 1 V, a more compliant cantilever should be selected.

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

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

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8.9K Views.  Brown University. Presenteren we een protocol voor het verrichten van drie-punt buigende proeven op sub millimeter schaal vezels met behulp van een custom-built mechanische beproevingstoestel. Het apparaat kan meten krachten variërend van 20 µN tot 10 N en is daarom geschikt voor een verscheidenheid van grootte van de vezels.

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

A Testing Platform for Durability Studies of Polymers and Fiber-reinforced Polymer Composites under Concurrent Hygrothermo-mechanical Stimuli

The durability of polymers and fiber-reinforced polymer composites under service condition is a critical aspect to be addressed for their robust designs and condition-based maintenance. These materials are adopted in a wide range of engineering applications, from aircraft and ship structures, to bridges, wind turbine blades, biomaterials and biomedical implants. Polymers are viscoelastic materials, and their response may be highly nonlinear and thus make it challenging to predict and monitor their in-service performance. The laboratory-scale testing platform presented herein assists the investigation of the influence of concurrent mechanical loadings and environmental conditions on these materials. The platform was designed to be low-cost and user-friendly. Its chemically resistant materials make the platform adaptable to studies of chemical degradation due to in-service exposure to fluids. An example of experiment was conducted at RT on closed-cell polyurethane foam samples loaded with a weight corresponding to ~50% of their ultimate static and dry load. Results show that the testing apparatus is appropriate for these studies. Results also highlight the larger vulnerability of the polymer under concurrent loading, based on the higher mid-point displacements and lower residual failure loads. Recommendations are made for additional improvements to the testing apparatus.

The durability of polymers and fiber-reinforced polymer composites in service is a critical aspect for their designs and condition-based maintenance. We present a novel low-cost laboratory testing platform for the investigation of the influence of concurrent mechanical and environmental loadings, and may help design more efficient yet safer composite structures.

The durability of polymers and fiber-reinforced polymer composites under service condition is a critical aspect to be addressed for their robust designs ...

Engineering

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Micro/Nano-scale Strain Distribution Measurement from Sampling Moir&#233; Fringes

This work describes the measurement procedure and principles of a sampling moir&#233; technique for full-field micro/nano-scale deformation measurements. The developed technique can be performed in two ways: using the reconstructed multiplication moir&#233; method or the spatial phase-shifting sampling moir&#233; method. When the specimen grid pitch is around 2 pixels, 2-pixel sampling moir&#233; fringes are generated to reconstruct a multiplication moir&#233; pattern for a deformation measurement. Both the displacement and strain sensitivities are twice as high as in the traditional scanning moir&#233; method in the same wide field of view. When the specimen grid pitch is around or greater than 3 pixels, multi-pixel sampling moir&#233; fringes are generated, and a spatial phase-shifting technique is combined for a full-field deformation measurement. The strain measurement accuracy is significantly improved, and automatic batch measurement is easily achievable. Both methods can measure the two-dimensional (2D) strain distributions from a single-shot grid image without rotating the specimen or scanning lines, as in traditional moir&#233; techniques. As examples, the 2D displacement and strain distributions, including the shear strains of two carbon fiber-reinforced plastic specimens, were measured in three-point bending tests. The proposed technique is expected to play an important role in the non-destructive quantitative evaluations of mechanical properties, crack occurrences, and residual stresses of a variety of materials.

A sampling moir&#233; technique featuring 2-pixel and multi-pixel sampling methods for high-accuracy strain distribution measurements at the micro/nano-scale is presented here.

This work describes the measurement procedure and principles of a sampling moir&#233; technique for full-field micro/nano-scale deformation measurements. ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

A Coupled Experiment-finite Element Modeling Methodology for Assessing High Strain Rate Mechanical Response of Soft Biomaterials

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. brain, liver, tendon, fat, etc.) when exposed to high strain rates. This study utilized a Split-Hopkinson Pressure Bar (SHPB) to generate strain rates of 100-1,500 sec-1. The SHPB employed a striker bar consisting of a viscoelastic material (polycarbonate). A sample of the biomaterial was obtained shortly postmortem and prepared for SHPB testing. The specimen was interposed between the incident and transmitted bars, and the pneumatic components of the SHPB were activated to drive the striker bar toward the incident bar. The resulting impact generated a compressive stress wave (i.e. incident wave) that traveled through the incident bar. When the compressive stress wave reached the end of the incident bar, a portion continued forward through the sample and transmitted bar (i.e. transmitted wave) while another portion reversed through the incident bar as a tensile wave (i.e. reflected wave). These waves were measured using strain gages mounted on the incident and transmitted bars. The true stress-strain behavior of the sample was determined from equations based on wave propagation and dynamic force equilibrium. The experimental stress-strain response was three dimensional in nature because the specimen bulged. As such, the hydrostatic stress (first invariant) was used to generate the stress-strain response. In order to extract the uniaxial (one-dimensional) mechanical response of the tissue, an iterative coupled optimization was performed using experimental results and Finite Element Analysis (FEA), which contained an Internal State Variable (ISV) material model used for the tissue. The ISV material model used in the FE simulations of the experimental setup was iteratively calibrated (i.e. optimized) to the experimental data such that the experiment and FEA strain gage values and first invariant of stresses were in good agreement.

The current study prescribes a coupled experiment-finite element simulation methodology to obtain the uniaxial dynamic mechanical response of soft biomaterials (brain, liver, tendon, fat, etc.). The multiaxial experimental results that arose because of specimen bulging obtained from Split-Hopkinson Pressure Bar testing were rendered to a uniaxial true stress-strain behavior when simulated through iterative optimization of the finite element analysis of the biomaterial.

This study offers a combined experimental and finite element (FE) simulation approach for examining the mechanical behavior of soft biomaterials (e.g. ...

Cutting Procedures, Tensile Testing, and Ageing of Flexible Unidirectional Composite Laminates

Many body armor designs incorporate unidirectional (UD) laminates. UD laminates are constructed of thin (&#60;0.05 mm) layers of high-performance yarns, where the yarns in each layer are oriented parallel to each other and held in place using binder resins and thin polymer films. The armor is constructed by stacking the unidirectional layers in different orientations. To date, only very preliminary work has been performed to characterize the ageing of the binder resins used in unidirectional laminates and the effects on their performance. For example, during the development of the conditioning protocol used in the National Institute of Justice Standard-0101.06, UD laminates showed visual signs of delamination and reductions in V50, which is the velocity at which half of the projectiles are expected to perforate the armor, after ageing. A better understanding of the material property changes in UD laminates is necessary to comprehend the long-term performance of armors constructed from these materials. There are no current standards recommended for mechanically interrogating unidirectional (UD) laminate materials. This study explores methods and best practices for accurately testing the mechanical properties of these materials and proposes a new test methodology for these materials. Best practices for ageing these materials are also described.

The goal of the study was to develop protocols to prepare consistent specimens for accurate mechanical testing of high-strength aramid or ultra-high-molar-mass polyethylene-based flexible unidirectional composite laminate materials and to describe protocols for performing artificial ageing on these materials.

Many body armor designs incorporate unidirectional (UD) laminates. UD laminates are constructed of thin (&#60;0.05 mm) layers of high-performance yarns, ...

Fabrication and Mechanical Property Assessment of Fiber Composite Laminates

Measuring the Mechanical Properties of Glass Fiber Reinforcement Polymer Composite Laminates Obtained by Different Fabrication Processes

The traditional wet hand lay-up process (WL) has been widely applied in the manufacturing of fiber composite laminates. However, due insufficiency in the forming pressure, the mass fraction of fiber is reduced and lots of air bubbles are trapped inside, resulting in low-quality laminates (low stiffness and strength). The wet hand lay-up/vacuum bag (WLVB) process for the fabrication of composite laminates is based on the traditional wet hand lay-up process, using a vacuum bag to remove air bubbles and provide pressure, and then carrying out the heating and curing process. 
Compared with the traditional hand lay-up process, laminates manufactured by the WLVB process show superior mechanical properties, including better strength and stiffness, higher fiber volume fraction, and lower void volume fraction, which are all benefits for composite laminates. This process is completely manual, and it is greatly influenced by the skills of the preparation personnel. Therefore, the products are prone to defects such as voids and uneven thickness, leading to unstable qualities and mechanical properties of the laminate. Hence, it is necessary to finely describe the WLVB process, finely control steps, and quantify material ratios, in order to ensure the mechanical properties of laminates. 
This paper describes the meticulous process of the WLVB process for preparing woven plain patterned glass fiber reinforcement composite laminates (GFRPs). The fiber volume content of laminates was calculated using the formula method, and the calculated results showed that the fiber volume content of WL laminates was 42.04%, while that of WLVB laminates was 57.82%, increasing by 15.78%. The mechanical properties of the laminates were characterized using tensile and impact tests. The experimental results revealed that with the WLVB process, the strength and modulus of the laminates were enhanced by 17.4% and 16.35%, respectively, and the specific absorbed energy was increased by 19.48%.

Author Spotlight: Enhancing Fiber Composite Laminate Quality with the Wet Hand Lay-Up/Vacuum Bag Process 

This paper describes a fabrication process for fiber-reinforced polymer matrix composite laminates obtained using the wet hand lay-up/vacuum bag method.

The traditional wet hand lay-up process (WL) has been widely applied in the manufacturing of fiber composite laminates. However, due insufficiency in the ...

Fragility Assessment of Bovine Cortical Bone Using Scratch Tests

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the fragility of bone, making it fracture-prone. Owing to the large socio-economic impact of bone fracture in our society, there is a need for novel ways to assess the mechanical performance of each hierarchical level of bone. Although stiffness and strength can be probed at all scales &#8211; nano-, micro-, meso-, and macroscopic &#8211; fracture assessment has so far been confined to macroscopic testing. This limitation restricts our understanding of bone fracture and constrains the scope of laboratory and clinical studies. In this research, we investigate the fracture resistance of bone from the microscopic to the mesoscopic length scales using micro scratch tests combined with nonlinear fracture mechanics. The tests are performed in the short longitudinal orientation on bovine cortical bone specimens. A meticulous experimental protocol is developed and a large number (102) of tests are conducted to assess the fracture toughness of cortical bone specimens while accounting for the heterogeneity associated with bone microstructure.

This study assesses the fracture toughness of bovine cortical bone at the sub-meso levels using microscopic scratch tests. This is an original, objective, rigorous, and reproducible method proposed to probe fracture toughness below the macroscopic scale. Potential applications are studying changes in bone fragility due to diseases like osteoporosis.

Bone is a complex hierarchical material with five distinct levels of organization. Factors like aging and diseases like osteoporosis increase the ...

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Substrates with stiffness in the kilopascal-range are useful to study the response of cells to physiologically relevant micro-environment stiffness. Using just a widefield fluorescence microscope, the Young's modulus of soft silicone gels can be determined using an indentation with a suitable sphere.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been ...

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

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