This work was funded by the EPSRC CDT for Formulation Engineering in the School of Chemical Engineering at the University of Birmingham, UK, Grant reference EP/L015153/1, and the Royal Centre for Defence Medicine.

1. Sample Preparation
<ol>
	<li>Prepare the sample and load it into the syringe.
	<ol>
		<li>To simulate a pre-loaded syringe, prepare the sample in advance, load it into the syringe, and attach the needle. Store as required, until testing. This may be suitable for hydrogels and materials that do not change with time. 
		NOTE: For example, to prepare 2% alginate solutions, dissolve 2 g of alginic acid sodium salt in 100 mL of deionized water, by stirring at room temperature. Aspirate the solution into 5 mL syringes, and store for 24 h at room temperature.</li>
		<li>Alternatively, to simulate an injection formulated directly prior to the application, prepare the sample in the same way it would be made in the clinic, allowing for any setting times. Load into the syringe and attach the needle. This may be suitable for cements, and materials whose properties change with time. 
		NOTE: For example, to prepare calcium sulfate cement, manually mix 4 g of calcium sulfate hemihydrate into 5 mL of deionized water with a spatula for 1 min. Remove the plunger from the syringe and load the cement into the syringe barrel with the spatula. Begin the mechanical testing after 4 min. 
		CAUTION: Needles pose a safety risk, use blunt needles if possible. If the material contains cells or other biological materials, extra care should be taken to prevent sharps injuries.</li>
	</ol>
	</li>
</ol>
2. Set up the mechanical tester
<ol>
	<li>Attach flat platens (for compression testing) to the mechanical tester.</li>
	<li>Manually equip the mechanical tester with a load cell with a maximum load of 200 N. 
	NOTE: A larger load cell may be used, provided it has sufficient precision at the 1 &#8211; 200 N range. Samples that are more viscous and not intended to be injected by hand may require a larger load cell.</li>
	<li>Separate the plates, using the manual control buttons, to allow for sufficient space for the needle, syringe and plunger (around 30 cm will be sufficient).</li>
	<li>Create a testing protocol.
	<ol>
		<li>Open the test wizard and set the test type to uniaxial compression.</li>
		<li>Set the pre-load. This is the measured force value at which testing will begin. 0.5 N is sufficient.</li>
		<li>Set the speed to pre-load to 5 mm/min. This is the speed the crosshead will move down until it encounters the pre-load.</li>
		<li>Set the loading to displacement control and select an appropriate test speed. 1 mm/s is an appropriate speed for a standard 5 mL syringe.</li>
		<li>Set an upper force limit at which to stop the test, e.g., 200 N. This is primarily for safety reasons. The test may also be stopped automatically at a given displacement, e.g., the length of the syringe.</li>
	</ol>
	</li>
</ol>
3. Set up the clamping system
<ol>
	<li>Attach two sets of clamps to two stands, with grips large enough to securely ensconce the chosen syringe.</li>
	<li>Place the grips between the crosshead and baseplate, with enough space below the grips for the syringe and needle.</li>
	<li>Line up the centers of the two grips, and line these up with the center of the crosshead. 
	NOTE: Alignment of the clamp grips with each other and the center of the crosshead may take some time and iteration to achieve, but is important to acquire high quality data.</li>
	<li>Ensure the clamps are secured firmly so that there is no movement in the clamps when a downward force is applied.</li>
	<li>Place a dish onto the bottom plate to collect the extruded material.</li>
</ol>
4. Run the injectability protocol
<ol>
	<li>Insert the syringe into the clamp grips and close them. The grips should hold the syringe in place, but allow it to move up and down without resistance.</li>
	<li>Ensure the syringe and plunger are perpendicular to the crosshead. This ensures that only uniaxial compression of the material will be measured. 
	NOTE: An empty syringe should be used to check steps&#160;4.1&#160;and 4.2.</li>
	<li>Lower the top plate to a position just above the plunger, using the manual movement buttons. 
	NOTE: It may be possible to select a &#8216;Start position&#8217; in the mechanical tester protocol, such that the original position above the plunger is reached automatically and is consistent throughout testing.</li>
	<li>Zero the measured force by clicking &#8216;Zero Force&#8217;.</li>
	<li>Run the testing protocol by pressing &#8216;Run&#8217;. 
	CAUTION: The experimenter should always be present to observe each trial, and ready to activate the emergency stop in case of a mishap.</li>
	<li>Raise the plates to a sufficient height, using the manual movement buttons, such that the syringe can be removed.</li>
	<li>Repeat step 4 for each sample. 
	NOTE: At this point, the syringe and extruded sample can be discarded if no further analysis is required, but may be kept in order&#160;to examine filter pressing, self-healing, the effects on cells, etc.</li>
</ol>
5. Data collection
<ol>
	<li>Save the data from each trial in a format from which a table of force and displacement values can be generated (.txt, .xls, .xlsx).</li>
	<li>Plot the results from each trial, with displacement on the x-axis and force on the y-axis.</li>
	<li>Read the maximum force (if it exists) and plateau force from the graphs.</li>
</ol>

<ol>
	<li>Webber, M. J., Appel, E. A., Meijer, E. W., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+biomaterials.">Supramolecular biomaterials.</a> Nature Materials. 15, 13-26 (2015).</li><li>Mathew, A. P., Uthaman, S., Cho, K. -. H., Cho, C. -. S., Park, I. -. 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The authors have nothing to disclose.

Mechanical testing is perhaps the simplest and most reliable way to quantify injectability. A key advantage of this protocol is that no special equipment is required, other than the mechanical tester, which is common in materials laboratories. This protocol is highly versatile; any material, needle gauge and syringe size can be used, provided the syringe can be accommodated by the clamps. This has been verified in this protocol for syringes up to 10 mL. Further, the material can be prepared exactly as it would for the real-world application25. Finally, this procedure is very fast, taking only up to a few minutes per sample, allowing tens of samples to be processed per hour.
For samples that give typical curves, two values can be extracted: the maximum force and the plateau force curves. The maximum force is arguably more objective and can be extracted computationally from the data table for each sample. Conversely, the plateau force may be more representative, as this will be the force experienced for the greatest amount of time and, as an average, is less affected by curves with large fluctuations. These fluctuations may be caused by air bubbles or particles in the material causing intermittent changes as they are extruded, or by low instrument precision for small force measurements. However, it is notable that, for many samples, there is no maximum force peak, and so the maximum and plateau value are the same. Objective comparisons between injection forces can be made so long as a consistent value is used.
The data obtained can be used in several ways. The injectability force values may be compared to ease of injection, to establish which formulations, syringe and needle sizes are viable for translation8. Alternatively, comparing between samples allows for the quantification of changes to formulations on injectability. For example, in cements, changing the viscosity of the liquid phase, the particle size distribution, and adding additives such as citrate to alter the colloidal properties, can have large changes in injectability9. These tests may also inform formulation protocol for cements, for example mixing time, time to loading and time to application, for optimum injection and post-injection performance. In addition, this method may be used to test the initial feasibility of novel bioinks for 3D printing.
This protocol can be modified in several ways. The clamp system may be replaced with a bespoke 3D printed construct to hold the syringe, which may make it easier to ensure the syringe and plunger are perpendicular to the crosshead, and the syringe held securely. The needle can be replaced with a cannula or any device that extrudes material by compression of a plunger and can be of any size and geometry. In order to increase the fidelity of the results, the tip of the needle can be placed into a tissue or hydrogel, in order to more accurately simulate clinical injection. However, this adds further complexities to the protocol, as tissue/gel composition and needle depth must be kept constant. Further, this protocol utilizes displacement-controlled extrusion, to measure the force required to inject at the specified speed. Alternatively, the injection force can be specified, and the amount of extrusion can be measured against time. This may be useful for materials with time dependent properties, such as cements. For example, by using a correlation between injection force and ease of injectability to select a force8, this protocol may be used to establish whether the entire volume of cement can be injected with this speed prior to setting. Finally, this protocol can easily be combined with other experiments, in order to test the effect of injection on the material properties and examine phenomena such as filter pressing and self-healing, or the effect of injection on cells.
The main limitation of this protocol is that a universal mechanical tester is required. While these are common in materials testing labs, they are expensive to purchase if the user cannot access one. Further, the mechanical tester provides uniaxial compression at either a set force or rate of displacement, whereas the applied force and injection speed may vary over the course of injection by hand. This protocol is also unsuitable for replicating some real world injections, such as injections into complex tissues in theatre, or injecting at different angles. To quantify the force of injection in the clinic, force and displacement transducers may be a better method.

Biomaterials are often studied and used as scaffolds for cell-based tissue regeneration and depots for targeted, sustained delivery of therapeutics1. Within this field, injectable biomaterials are growing in popularity as they are minimally invasive, which reduces the risk of infection, pain and scarring associated with implantation2. Further, because they are usually applied as fluids, they conform perfectly to tissue defects, and drugs and cells may be mixed into them immediately prior to the application3,4,5. As such, while injectable biomaterials may be manufactured as pre-loaded syringes, they are often prepared by clinicians directly prior to application. For example, cements begin to set once the powder and liquid phases are mixed, and so cannot be stored for long periods before use6. The characterization of these materials is thus time dependent and inextricably linked to their preparation.
Common injectable biomaterials include calcium cements, polymethyl methacrylate, bioglasses, and various polymeric hydrogels3,7. Unlike traditional injections of drugs, which have the same rheological properties as water, these injectable biomaterials are typically more viscous, non-Newtonian, may have some elastic character, and may also change over time. Therefore, the injectability of these materials cannot be assumed but must be assessed experimentally. By quantifying the force required for injection and correlating it to the ease of injection, early decisions about which biomaterial formulations, syringe, and needle sizes to take forward may be made early in the developmental process8. Such experiments may also quantify the effects of changing formulations on injectability9.
There are several methods to assess the properties of injectable materials. Rotational rheology is often utilized to assess viscosity, non-Newtonian behavior, post-shear recovery, setting time, and other properties of these materials10,11,12. Whilst this type of test is useful to establish fundamental properties of the materials, these properties do not correlate directly to injectability. For a Newtonian fluid and cylindrical syringe and needle, the injection force can be&#160; estimated from a form of the Hagen&#8211;Poiseuille equation13:
<img alt="Equation 1" src="/files/ftp_upload/61417/61417eq1.jpg" />
Where F is the force required for injection (N), Rs is the internal syringe radius (m), Rn is the internal needle radius (m), L is the needle length (m), Q is fluid flow rate (m3 s-1), &#951; is the dynamic viscosity (Pa.s) and Ff is the friction force between the plunger and barrel wall (N). Thus, if the viscosity is measured via rotational rheology, the dimensions of the syringe and needle are known and the flow rate estimated, the injection force can be estimated. However, this equation does not account for the conical end of the syringe or any other geometries, such as off-center outlets, and Ff must be estimated or found experimentally by mechanical testing. Further, biomaterials are typically not Newtonian, but exhibit complex rheological properties. For a simple shear thinning fluid, the equation becomes14:
<img alt="Equation 2" src="/files/ftp_upload/61417/61417eq2.jpg" />
Where n is the power index (-) and K is the consistency index (Pa.sn) from the Ostwald de Waele expression: <img alt="Equation 3" src="/files/ftp_upload/61417/61417eq3.jpg" />, where <img alt="Equation 4" src="/files/ftp_upload/61417/61417eq4.jpg" /> is the shear rate (s-1). The complexity vastly increases for materials whose rheological properties cannot be characterized by two values, and particularly for time-dependent materials such as setting cements. Additionally, if the material properties are shear dependent, then the material must be tested at the shear rate expected in the needle, which may far exceed the range of a rotational rheometer15.
Another quantitative method for measuring injectability involves attaching pressure and displacement sensors to a syringe while performing an injection, either by hand or using a syringe pump. This equipment is relatively inexpensive, however, requires users to generate scripts and calibration curves to convert into force data16. Further, a syringe pump may not possess sufficient torque to compress the plunger at a precise rate if high forces are required to extrude viscous or semi-solid materials. Alternatively, utilizing these sensors when injecting by hand may be useful as they can be used in a real clinical scenario, during clinical procedures17. However, this will take much longer and may introduce user bias, and will, therefore, need larger numbers of repetitions with different users to obtain reliable results. This may, thus, be more appropriate for materials that are further down the translational pipeline, or products already in clinical use.
In this protocol, a mechanical tester is used to compress the plunger at a set rate, and measure the force required to do so. This type of mechanical tester is common in materials laboratories and has been used to quantify injectability for various biomaterials18,19,20,21,22,23,24. This test can be used with any size and geometry of syringe and needle, containing any material. Further, in the case of biomaterials that are made immediately prior to the use, the exact formulation procedure that would be used in the clinic or surgery can be followed prior to testing. A further advantage of this procedure is that it is relatively fast; once the mechanical tester is set up, tens of samples can be studied in an hour, depending on extrusion speed and syringe volume. This is in contrast to rotational rheology, which typically takes at least 5 &#8211; 10 minutes per test, plus loading, equilibration and cleaning time. Using a mechanical tester produces a reliable extrusion rate equally over the plunger, which is particularly advantageous for viscous formulations or those with time dependent properties. Following testing, minimal post-processing of data is required to pull out important values for objective comparisons.

<table border="0" cellpadding="0" cellspacing="0" style="width:637px;" width="638">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alginic Acid Sodium Salt</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:119px;">A2033-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Blunt Needles</td>
			<td style="width:136px;">Needlez</td>
			<td style="width:119px;">NB19G1.5</td>
			<td>Any size may be used, depending on application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium Sulphate Hemihydrate</td>
			<td style="width:136px;">Acros Organics</td>
			<td style="width:119px;">22441.296</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Clamp stand</td>
			<td style="width:136px;">Eisco</td>
			<td style="width:119px;">MTST5</td>
			<td>Two required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Clamps</td>
			<td style="width:136px;">R&#38;L Enterprises</td>
			<td style="width:119px;">41</td>
			<td>Two required, should have flat tops</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringes</td>
			<td style="width:136px;">BD</td>
			<td style="width:119px;">307731</td>
			<td>Any size can be used, depending on application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Universal Mechanical Tester</td>
			<td style="width:136px;">Zwick Roell</td>
			<td style="width:119px;">Z030</td>
			<td></td>
		</tr>
	</tbody>
</table>

the quantification of injectability by mechanical testing

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

The set-up of the mechanical tester and clamping system is shown in Figure 1A. This protocol generates a table and graph of force versus displacement for each tested sample. A typical force displacement curve consists of three sections (Figure 1B): an initial gradient, as the plunger overcomes friction from the barrel and the material is accelerated, a force maximum, and a plateau, as the material is extruded at a steady state.
However, a distinct maximum only exists where the plateau force is lower than the force required to accelerate the plunger. As such, peaks are only seen for inviscid samples passing through wide needles. For viscous samples passing through a more narrow orifice, the force needed to inject the sample at constant speed is greater than the force required to overcome friction in the barrel and accelerate the material, and no distinct peak is seen (Figure 1C). For highly viscous samples or very narrow needles, the force required to extrude the material may be so great that the syringe buckles and fails, often with very little extrusion of the material (Figure 1D). If the material being injected contains particles or is undergoing setting, such as cement, filter pressing (preferential expulsion of the liquid phase) or bulk setting may occur, leading to incomplete injection (Figure 1E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61417/61417fig1.jpg" /> 
Figure 1: Sample curves generated by this protocol. (A) Set up of the mechanical tester for this protocol. (B) Typical force-extrusion curve. (C) Force-extrusion curve with no distinct maximum peak. (D) Force-extrusion curve for syringe failure. (E) Force-extrusion curve for a setting cement. This figure is adapted from Robinson et al.8. <a href="https://www.jove.com/files/ftp_upload/61417/61417fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The Quantification of Injectability by Mechanical Testing

Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more ...

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JoVE Journal

Bioengineering

7.6K Views.  University of Birmingham. Presented here is a protocol for quantitatively evaluating the injectability of a material through a syringe-needle system using a standard mechanical testing rig.

Video: The Quantification of Injectability by Mechanical Testing

Mechanical Testing of Mouse Carotid Arteries: from Newborn to Adult

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall1. When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation2. In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments.
	Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental3 and aging studies4 can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression5 and disease treatment6. Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process7. One drawback of the mouse model, especially for examining young ages, is the size of the arteries. 
	We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.

Passive mechanical testing of mouse carotid arteries is described, with special consideration for adapting to different specimen ages. The procedures include determining the in vivo length of the artery, mounting it in a pressure myograph, recording data, measuring the unloaded dimensions and analyzing the resulting data.

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances (EPS). The EPS serves as a physical and protective scaffold that houses the bacterial cells and consists of a variety of materials that includes proteins, exopolysaccharides and DNA. The composition of the EPS may change, which remodels the mechanic properties of the biofilm to further develop or support alternative biofilm structures, such as streamers, as a response to environmental cues. Despite this, there are little quantitative descriptions on how EPS components contribute to the mechanical properties and function of biofilms. Rheology, the study of the flow of matter, is of particular relevance to biofilms as many biofilms grow in flow conditions and are constantly exposed to shear stress. It also provides measurement and insight on the spreading of the biofilm on a surface. Here, particle-tracking microrheology is used to examine the viscoelasticity and effective crosslinking roles of different matrix components in various parts of the biofilm during development. This approach allows researchers to measure mechanic properties of biofilms at the micro-scale, which might provide useful information for controlling and engineering biofilms.

In Situ Mapping of the Mechanical Properties of Biofilms by Particle-tracking Microrheology

Particle-tracking microrheology investigates the viscoelasticity of materials. Here, the technique is used to determine the viscoelasticity, creep compliance and effective crosslinking roles of different matrix components of a bacterial biofilm. The matrix consists of polymeric substances secreted by the bacteria and its components determine biofilm structure and mechanical properties.

Bacterial cells are able to form surface-attached biofilm communities known as biofilms by encasing themselves in extracellular polymeric substances ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

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.

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 ...

Outer-Boundary Assisted Segmentation and Quantification of Trabecular Bones by an Imagej Plugin

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different bone loss conditions. However, the standard approach for trabecular analysis of micro-CT images is slice-by-slice semi-automatic hand-contouring, which is labor intensive and error prone. Described here is an efficient method for automatic segmentation of trabecular bones according to the bone's outer boundaries, where trabecular bones can be identified and segmented automatically with accuracy with less operator bias when appropriate segmentation parameters are set. To profile satisfactory segmentation parameters, an image stack of segmentation results is displayed, where all possible combinations of the segmentation parameters are changed one by one in sequence, and segmentation results with associated parameters can easily be visually checked. As a quality-control feature of the plugin, simulated standard objects are quantified where the measured quantities can be compared with theoretical values. Layer-by-layer quantification of trabecular properties and trabecular thicknesses are reported by such a plugin, and the distributions of such properties within the selected regions can be profiled easily. Although layer-by-layer quantification retains more information about trabecular bones and facilitates further statistical analysis of structural changes, such measures are unavailable from the output of current commercial software, where only a single quantified value for each parameter is reported for each sample. Therefore, the described workflows are better approaches for analyzing trabecular bones with accuracy and efficiency.

We present a workflow for segmenting and quantifying trabecular bones for 2D and 3D images based on the bone&#39;s outer boundary using an ImageJ plugin. This approach is more efficient and accurate than the current manual hand-contouring approach, and provides layer-by-layer quantifications, which are not available in current commercial software.

Micro-computed tomography (micro-CT) is routinely used to assess bone quantity and trabecular microstructural properties in small animals under different ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...