Name: biaxial mechanical characterizations of atrioventricular heart valves
Uploaded: 2019-04-09T13:00:00
Description: Watch this Scientific Journal Video about Biaxial Mechanical Characterizations of Atrioventricular Heart Valves at JoVE.com

This work was supported by the American Heart Association Scientist Development Grant 16SDG27760143. The authors would also like to acknowledge the Mentored Research Fellowship from the University of Oklahoma&#39;s Office of Undergraduate Research for supporting both Colton Ross and Devin Laurence.

All methods described were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Oklahoma. All animal tissues were acquired from a United States Department of Agriculture (USDA)-approved slaughterhouse (Country Home Meat Co., Edmond, OK).
1. Tissue acquisition and cleaning
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	<li>Retrieve the animal hearts on the same day as the animal is slaughtered and store the hearts in an ice chest to ensure the tissue freshness. Transport the hearts to the laborato...

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A., Amini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+biaxial+mechanical+response+of+porcine+tricuspid+valve+leaflets.">On the biaxial mechanical response of porcine tricuspid valve leaflets.</a> Journal of Biomechanical Engineering. 138 (10), 104504 (2016).</li><li>Grashow, J. S., Yoganathan, A. P., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biaixal+stress-stretch+behavior+of+the+mitral+valve+anterior+leaflet+at+physiologic+strain+rates.">Biaixal stress-stretch behavior of the mitral valve anterior leaflet at physiologic strain rates.</a> Annals of Biomedical Engineering. 34 (2), 315-325 (2006).</li><li>Huang, H. -. Y. S., Lu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biaxial+mechanical+properties+of+bovine+jugular+venous+valve+leaflet+tissues.">Biaxial mechanical properties of bovine jugular venous valve leaflet tissues.</a> Biomechanics and Modeling in Mechanobiology. , 1-13 (2017).</li><li>Stella, J. A., Liao, J., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+biaxial+mechanical+behavior+of+the+aortic+heart+valve+leaflet.">Time-dependent biaxial mechanical behavior of the aortic heart valve leaflet.</a> Journal of Biomechanics. 40 (14), 3169-3177 (2007).</li><li>Sacks, M. S., David Merryman, W., Schmidt, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+biomechanics+of+heart+valve+function.">On the biomechanics of heart valve function.</a> Journal of Biomechanics. 42 (12), 1804-1824 (2009).</li><li>Sacks, M. S., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+function:+a+biomechanical+perspective.">Heart valve function: a biomechanical perspective.</a> Philosophical Transactions of the Royal Society of London B: Biological Sciences. 362 (1484), 1369-1391 (2007).</li><li>Jett, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+the+anisotropic+mechanical+properties+and+anatomical+structure+of+porcine+atrioventricular+heart+valves.">An investigation of the anisotropic mechanical properties and anatomical structure of porcine atrioventricular heart valves.</a> Journal of the Mechanical Behavior of Biomedical Materials. 87, 155-171 (2018).</li><li>Ruifrok, A. C., Johnston, D. 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Critical steps for this biaxial mechanical testing include (i) the proper orientation of the leaflet, (ii)&#160;proper biaxial tester setup for negligible shear, and (iii) a careful application of the fiducial markers. The orientation of the leaflet is crucial to the obtained mechanical characterization of the leaflet tissue as the material is anisotropic in nature. Thus, the radial and circumferential directions need to be known for properly aligning the tissue specimens with the testing X- and Y-directions. It is also ...

Proper heart function relies on appropriate mechanical behaviors of the heart valve leaflets. In situations where heart valve leaflet mechanics are compromised, heart valve disease occurs, which may lead to other heart-related issues. Understanding heart valve disease requires a thorough understanding of the leaflets&#39; proper mechanical behaviors for use in computational models and therapeutic development, and as such, a testing scheme must be developed to accurately retrieve the healthy leaflets&#39; mechanical properties. In previous literature, this mechanical characterization has been conducted using biaxial mechanical testing procedures.
Biaxial mechanical testing procedures for soft tissues vary throughout the literature, with different testing frameworks utilized to retrieve different characteristics1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19. Testing methods have been extended for investigations of the mechanical characteristics of heart valve leaflets. In general, biaxial mechanical testing involves loading the heart valve tissue with simultaneous forces in the two principal directions, but how this testing is performed varies based on the biomechanical properties to be observed. Some of these testing protocols include (i) strain-rate, (ii) creep, (iii) stress-relaxation, and (iv) force-controlled testing.
First, strain-rate testing has been utilized to determine the time-dependent behaviors of the tissue leaflets18,20. In this testing protocol, leaflets are loaded to a maximum membrane tension at different half-cycle times (i.e., 1, 0.5, 0.1, and 0.05 s) to determine if there is a significant difference in peak stretch or hysteresis between loading times. However, these tests have demonstrated a negligible difference in the observed stretch with varying strain-rates. Second, in creep testing, the tissue is loaded to the peak membrane tension and held at peak membrane tension. This testing allows a demonstration of how the tissue&#39;s displacement creeps to maintain the peak membrane tension. However, it has been shown that the creep is insignificant for heart valve leaflets under physiologically functioning3,20. Third, in stress-relaxation testing, the tissue is loaded to the peak membrane tension and the associated displacement is held constant for an extended period of time3,21,22. In this type of testing, the tissue stress has a notable reduction from the peak membrane tension. Lastly, in force-controlled testing, tissues are cyclically loaded at various ratios of the peak membrane tension in each direction17,23. These tests reveal the material&#39;s anisotropy and nonlinear stress-strain response, and by loading the tissue under various ratios, potential physiologic deformations may be better understood. These recent investigations made it apparent that stress-relaxation and force-controlled protocols prove most beneficial to perform a mechanical characterization of heart valve leaflets. Despite these advances in heart valve biomechanical characterization, the testing has not been performed under one unified testing scheme, and there are limited methods to investigate the coupling between directions.
The purpose of this method is to facilitate a full material characterization of the heart valve leaflets by a unified biaxial mechanical testing scheme. A unified testing scheme is considered as one where each leaflet is tested under all testing protocols in one session. This is advantageous, as tissue properties are inherently variable between leaflets, so a full characterization for each leaflet proves more accurate as a descriptor than performing each protocol independently on various leaflets. The testing scheme consists of three main components, namely (i) a force-controlled biaxial testing protocol, (ii) a displacement-controlled biaxial testing protocol, and (iii) a biaxial stress-relaxation testing protocol. All testing schemes utilize a loading rate of 4.42 N/min, and 10 loading-unloading cycles to ensure stress-strain curve replicability by the 10th cycle (as found in previous work)23. All protocols are also constructed based on the membrane tension assumption, which requires that the thickness be less than 10% of the effective specimen lengths.
The force-controlled protocol used in this presented method consists of 10 loading and unloading cycles with peak membrane tensions of 100 N/m and 75 N/m for the mitral valve (MV) and tricuspid valve (TV), respectively15,17. Five loading ratios are considered in this force-controlled testing protocol, namely 1:1, 0.75:1, 1:0.75, 0.5:1, and 1:0.5. These five loading ratios prove useful in describing the stresses and strains correspondent to all potential physiologic deformations of the leaflet in vivo.
The displacement-controlled protocol presented in this method consists of two deformation scenarios, namely (i) constrained uniaxial stretching and (ii) pure shear. In the constrained uniaxial stretching, one direction of the tissue is displaced to the peak membrane tension while fixing the other direction. In the pure shear setup, the tissue is stretched in one direction and judiciously shortened in the other direction, so the area of the tissue remains constant under deformation. Each of these displacement-controlled testing procedures is performed for each of the two tissue directions (circumferential and radial directions).
The stress-relaxation protocol used in the presented method is achieved by loading the tissue to the peak membrane tension in both directions and holding the tissue at the correspondent displacements for 15 min to monitor the tissue&#39;s stress relaxation behavior. The detailed experimental procedures are discussed next.

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			<td height="21" style="height:21px;width:360px;">10% Formalin Solution, Neutral Bufffered</td>
			<td style="width:291px;">Sigma-Aldrich</td>
			<td style="width:147px;">HT501128-4L&#160;</td>
			<td style="width:528px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">40X-2500X LED Lab Trinocular Compound Microscope</td>
			<td>AmScope</td>
			<td>SKU: T120C</td>
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			<td height="22" style="height:23px;">BioTester - Biaxial Tester</td>
			<td>CellScale Biomaterials Testing</td>
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			<td>1.5N Load Cell Capacity</td>
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			<td height="21" style="height:21px;">ImageJ</td>
			<td>National Institute of Health, Bethesda, MD</td>
			<td>Version 1.8.0_112</td>
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			<td height="21" style="height:21px;">LabJoy</td>
			<td>CellScale Biomaterials Testing</td>
			<td>Version 10.66</td>
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			<td height="21" style="height:21px;">MATLAB</td>
			<td>MathWorks</td>
			<td>Version 2018b</td>
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			<td height="25" style="height:25px;">Phosphate-Buffered Saline</td>
			<td>n/a</td>
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			<td>Recipe for 1L 1X PBS Solution: 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4</td>
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			<td height="42" style="height:42px;width:360px;">Single Edge Industrial Razor Blades (Surgical Carbon Steel)</td>
			<td style="width:291px;">VWR International</td>
			<td style="width:147px;">H3515541105024</td>
			<td>Razord blades for tissue retrieval and preparation procedures</td>
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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.

Stress-stretch data from the force-controlled biaxial mechanical testing reveals a nonlinear curve with some resemblance to an exponential curve (Figure 12). Regarding the response in each principal direction, the material behavior is transversely isotropic, with the radial stretch greater than the circumferential deformation. In some cases, the anisotropy&#39;s directions may flip, with the circumferential direction exhibiting greater compliance than the rad...

Watch this Scientific Journal Video about Biaxial Mechanical Characterizations of Atrioventricular Heart Valves at JoVE.com

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

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 ...

This method can provide a better understanding of heart valve biomechanics which can be extremely useful in computational modeling and in the refinement of treatment methods for heart valve disease. This protocol is advantageous compared to other established testing protocols because it can be used to perform mechanical characterizations of heart valve tissues using a unified testing scheme. This biaxial testing protocol with the unified testing scheme will be beneficial to the soft tissue biomechanics research of mechanical quantifications such as full characterization of arterial vessels and skin tissue.Begin by using forceps to remove the leaflet specimen of interest from PBS storage. Lay the leaflet flat on a cutting mat with the radial direction aligned to the Y direction and the circumferential direction aligned to the X direction. Identify the central region of the leaflet as the testing section and align a tissue cutter so that the desired tissue testing region is within the boundaries of the razor blades.Make one horizontal and one vertical cut to form a square region of the desired dimensions and use a surgical pen to label the radial orientation of the tissue. Then, use the forceps to stretch the chordae from the leaflet and use a razor blade to trim any chordal attachments, taking care not to damage the leaflet. Using forceps, lay the tissue specimen flat on a spatula and use a digital caliper to measure the thickness of the spatula-tissue pair at three different leaflet locations.Next, mount the tissue to the biaxial testing system, ensuring that the circumferential and radial directions of the specimen are aligned with the machine's X and Y directions. For the fiducial marker placement, place glass beads into one small open-faced container and add a small pool of superglue to another container. Coat the tip of a fine-tipped tool with a small amount of superglue and stick an individual bead to the tip of the tool.Then, carefully use the tool to transfer the bead to one corner of the middle third of the tissue testing region, repeating this placement until a square array of four beads is formed. On the computer connected to the biaxial testing system, create a preconditioning protocol so that the tissue will undergo 10 loading/unloading cycles at the forces associated with the peak membrane tension and a loading rate of 4.42 newtons per minute including a preload of 2.5%of the maximum force. Create a new arbitrary testing directory to temporarily store the preconditioning data, and establish a loading rate of 4.42 newtons per minute for subsequent testing.Create a new set of testing parameters and set the name of the protocol as Preconditioning0. For the X and Y axes, set the Control Mode to Force, and the Control Function to Step. Set the Load Magnitude as the force associated with the targeted peak membrane tension, and the Preload Magnitude as 2.5%of the maximum force for the first repetition only.Set both the Stretch Duration and Recovery Duration to 25 seconds and set the number of Repetitions to 10. When the preconditioning step finishes, make a note of the deformation of the tissue in the X and Y directions and prepare a protocol to move the specimen to the maximum force, beginning from the recorded size. Next, begin the maximum force loading protocol, starting from the post-preconditioning deformation while simultaneously starting a stopwatch when the machine begins actuation.Stop the stopwatch when the actuation stops as indicated by auditory cues. Then, record the post-preconditioning peak tissue deformation, along with the time from the stopwatch representing the optimal tissue stretch time. For biaxial mechanical testing, prepare a force-controlled protocol at a loading rate of 4.42 newtons per minute as demonstrated and open a new testing directory.Name the test and set the data to save to a known location for use in later stress and strain calculations. Move the specimen back to the original mounting configuration and create a protocol set titled First Image. Set the X and Y axis Control Mode to Force and the Control Function to Step.Set the Load Magnitude to zero millinewton and set both the Stretch Duration and Recovery Duration to one second. Set the number of Repetitions to one and both the Data Output Frequency and Image Output Frequency to one hertz. Instruct a new testing set and name it Preconditioning A, establishing the testing parameters such that the tissue will undergo 10 repetitions of cyclic loading/unloading to the targeted force for the desired membrane tension as demonstrated.Instruct another testing set named Preconditioning B with testing parameters identical to the Preconditioning A testing set, but with the Image Output Frequency set to 15 hertz and with no applied preload. After the preconditioning protocols, create the testing protocols so that the tissue is loaded to the peak membrane tension in the indicated circumferential to radial loading ratios at a loading rate of 4.42 newtons per minute. Retrieve the data from the last two cycles of each loading ratio for subsequent data processing and analyses described.Prepare a displacement-controlled biaxial stretching testing protocol at a loading rate of 4.42 newtons per minute in the X and Y directions for the displacements associated with the peak circumferential and radial stretches respectively. Prepare a pure shear testing protocol along the X direction, stretching in the X direction associated with the peak circumferential stretch and shortening in the Y direction while keeping the dashed area constant under deformation. Prepare a constrained uniaxial stretching testing protocol along the X direction.Then, prepare a pure shear testing protocol along the Y direction and a constrained uniaxial stretching testing protocol along the Y direction. Between each of these protocols, construct a rest cycle of one minute that holds the tissue at the original mounted configuration and retrieve the data from the last two cycles of each loading ratio for data processing and analyses. Then, prepare a stress relaxation protocol so that the tissue is loaded in each direction at a loading rate of 4.42 newtons per minute to the displacements associated with the peak membrane tensions and held at that displacement for 15 minutes.After 15 minutes, the protocol should be set to recover the tissue to its original mounting configuration. Stress stretch data from a representative force-controlled biaxial mechanical testing reveals a non-linear curve with some resemblance to an exponential curve, with the material behavior curves transversely isotropic and the radial stretch greater than the circumferential deformation. In some cases, the direction of the anisotropy may flip, with the circumferential direction exhibiting greater compliance than the radial direction.From displacement-controlled testing, stress stretch data follows a non-linear response for the principal direction undergoing tension. In the constrained uniaxial tension protocol, an increasing stress stretch response is exhibited in the constrained direction, demonstrating the coupling of applied stretching in the other principal direction. From the stress relaxation testing, normalized membrane tension time data follows a non-linear decaying curve.Both the mitral and tricuspid valve leaflet tissues exhibit a greater stress reduction in the radial direction compared to that in the circumferential direction. Representative histologic analysis of mitral valve and tricuspid valve anterior leaflet tissue sections using Masson's trichrome staining demonstrates typical constituents found in atrioventricular heart valves such as collagen fibers and valvular interstitial cells. It's important that the glass beads are not accidentally glued together to avoid significant errors in the tissue deformation calculation during the post-processing step.The acquired data can be later used in heart valve computational modeling that can better inform how the valve functions and for improvements in the surgical procedures for treating valvular heart diseases. This protocol opens doors in the field of soft tissue biomechanics for comparing the mechanical behaviors between healthy and diseased tissues as well as for designing biomimetic materials.

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JoVE Journal

Bioengineering

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

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 ...

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

Biaxial Basal Tone and Passive Testing of the Murine Reproductive System Using a Pressure Myograph

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). Smooth muscle cells exhibit a contractile function within the vaginal and cervical walls. Depending on the biochemical environment and the mechanical distension of the organ walls, the smooth muscle cells alter the contractile conditions. The contribution of the smooth muscle cells under baseline physiological conditions is classified as a basal tone. More specifically, a basal tone is the baseline partial constriction of smooth muscle cells in the absence of hormonal and neural stimulation. Furthermore, the ECM provides structural support for the organ walls and functions as a reservoir for biochemical cues. These biochemical cues are vital to various organ functions, such as inciting growth and maintaining homeostasis. The ECM of each organ is composed primarily of collagen fibers (mostly collagen types I, III, and V), elastic fibers, and glycosaminoglycans/proteoglycans. The composition and organization of the ECM dictate the mechanical properties of each organ. A change in ECM composition may lead to the development of reproductive pathologies, such as pelvic organ prolapse or premature cervical remodeling. Furthermore, changes in ECM microstructure and stiffness may alter smooth muscle cell activity and phenotype, thus resulting in the loss of the contractile force.
In this work, the reported protocols are used to assess the basal tone and passive mechanical properties of the nonpregnant murine vagina and cervix at 4-6 months of age in estrus. The organs were mounted in a commercially available pressure myograph and both pressure-diameter and force-length tests were performed. Sample data and data analysis techniques for the mechanical characterization of the reproductive organs are included. Such information may be useful for constructing mathematical models and rationally designing therapeutic interventions for women&#8217;s health pathologies.

This protocol utilized a commercially available pressure myograph system to perform pressure myograph testing on the murine vagina and cervix. Utilizing media with and without calcium, the contributions of the smooth muscle cells (SMC) basal tone and passive extracellular matrix (ECM) were isolated for the organs under estimated physiological conditions.

The female reproductive organs, specifically the vagina and cervix, are composed of various cellular components and a unique extracellular matrix (ECM). ...

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.

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 ...

Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 &#181;M genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.

This protocol aims to fabricate 3D cardiac spheroids (CSs) by co-culturing cells in hanging drops.&#160;Collagen-embedded CSs are treated with doxorubicin (DOX, a cardiotoxic agent) at physiological concentrations to model heart failure. In vitro testing using DOX-treated CSs may be used to identify novel therapies for heart failure patients.

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...