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
<ol>
	<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 laboratory space.</li>
	<li>Upon arrival at the lab, submerge the heart in a bucket of phosphate-buffered saline (PBS) solution to rinse off any excess blood. Retrieve forceps, a placemat, a surgical blade, a bucket of PBS solution, bleach, and a plastic bag. Prepare the placemat by laying it on the dissection counter, allowing for an easier cleanup of blood-related mess. After the heart has been sufficiently rinsed, place the heart on the placemat (Figure 1a).</li>
	<li>Using the forceps, locate the parting line between the atria and the ventricle on each side of the heart. Using a razor blade, carefully make an incision along this parting line and reveal the heart valves and the ventricles (Figure 1b). Make the incision along the entire outer circumference of the heart, such that the atria and all heart material superior to the ventricles may be removed.</li>
	<li>With the forceps, carefully pull out any observed blood clots in the ventricles (Figure 1c). If an attempt is made to remove a blood clot but it does not move, ensure the chordae tendineae or leaflets have not been grabbed. Place blood clots in the biohazard bag for waste disposal.</li>
	<li>When all blood clots have been removed from the ventricles, rinse the heart one final time in a bucket with PBS solution. Place the clean heart in the plastic bag and store it in a freezer.</li>
	<li>Using a solution of 10% bleach and 90% water, mix the blood with the bleach solution and stir continuously for approximately 10 min. Look for a successful bleach treatment, indicated by the solution transitioning from red to yellow. Dispose of the bleach-treated blood through drainage. 
	CAUTION: Bleach is a toxic substance and can be harmful if ingested. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Heart dissection and examination of anatomy
<ol>
	<li>Retrieve the previously cleansed heart and allow it to thaw in a warm water bath. The required materials for dissection include forceps, surgical blades, placemats, PBS, and small storage containers. After the heart is completely thawed, put it on a placemat to absorb any remaining blood.</li>
	<li>Hold the heart for a top-down (superior) view to optimally observe the valve structures. Beginning with the MV on the left side of the heart, use forceps to carefully manipulate the leaflets and identify a commissure, or parting line, between the leaflets.</li>
	<li>Make an incision along the commissure and carefully cut through the ventricular wall, making sure not to damage the leaflets. It may be necessary to cut the chordal attachments during this process to fully open the ventricle. Once the full incision is made, open the ventricle (Figure 2a).</li>
	<li>Identify the MV anterior and posterior leaflets and use a surgical blade to sever the chordal attachments to the papillary muscles. Using forceps, stretch the leaflets taut and make cuts to separate the leaflets from the annulus. Place the excised leaflets in an appropriately labeled container filled with PBS solution and store it in a refrigerator at approximately 4 &#176;C.</li>
	<li>Hold the heart for a top-down view and identify the TV on the right side of the heart. Locate the commissures and make an incision through one of the commissures and the ventricular wall (Figure 2b).</li>
	<li>Identify the TV septal, posterior, and anterior leaflets, and perform the leaflet extraction as done in step 2.4. Place all obtained leaflets in a labeled container filled with PBS solution and store the container in a refrigerator at approximately 4 &#176;C. 
	NOTE: The protocol can be paused here. However, tissue biomechanical testing and the subsequent histology analyses should occur within 2 days of the heart dissection.</li>
</ol>
3. Tissue dissection
<ol>
	<li>Retrieve a leaflet from the fridge, the tissue cutter for the specified sectioning size, a surgical pen, forceps, razor blades, and a cutting mat.</li>
	<li>Using forceps, remove the specimen from the PBS solution and lay it flat on the cutting mat with the radial direction (R) aligned to the Y-direction, and the circumferential direction (C) aligned to the X-direction (Figure 3a). Identify the leaflet&#8217;s central region as the testing section.</li>
	<li>Align the tissue cutter so that the desired tissue testing region is within the boundaries of the razor blades. Make one cut horizontally and another vertically to form a square region of the desired dimensions (Figure 3b). Using the surgical pen, label the tissue&#8217;s radial direction (Figure 3b).</li>
	<li>Using the razor blade, trim any chordal attachments by stretching the chordae from the leaflet with the forceps and making a careful cut without causing any damage to the leaflet. 
	NOTE: The protocol can be paused here. If the protocol is paused, store the sectioned tissue in a labeled container filled with PBS solution, and store the container in a refrigerator at approximately 4 &#176;C (as explained in step 2.6). However, tissue testing should occur within 2 days of the dissection.</li>
</ol>
4. Thickness measurement and biaxial tester setup
<ol>
	<li>Retrieve the sectioned tissue specimen, digital calipers, and a small metal spatula. Using the digital calipers, measure and record the thickness of the metal spatula.</li>
	<li>Using forceps, lay the tissue specimen flat on the metal spatula. Using the digital calipers, measure the thickness of the spatula-tissue pair (Figure 3c) at three different leaflet locations. Subtract the spatula&#8217;s thickness from each measurement and record the average thickness.</li>
	<li>Prepare a PBS bath at 37 &#176;C, which corresponds to the tissue&#8217;s physiological conditions.</li>
</ol>
5. Tissue mounting and fiducial marker placement
<ol>
	<li>Retrieve forceps, the tissue specimen, mounting hardware, a fine-tipped tool, glass beads (with diameters of 300&#173;&#8211;500&#8201;&#181;m), and super glue.</li>
	<li>Mount the tissue to the biaxial testing system (Figure 3d,e). While mounting, ensure that the tissue&#8217;s circumferential and radial directions are aligned with the machine&#8217;s X- and Y-directions.</li>
	<li>For the fiducial marker placement, place glass beads into one small open-faced container and a small pool of super glue in another container. Using the fine-tipped tool, coat the tip with a small amount of super glue and stick an individual bead to the tip of the tool.</li>
	<li>Carefully use the tool to transfer the bead to one corner of the middle third of the tissue&#8217;s testing region (Figure 3f). Repeat this step until a square array of four beads is formed (Figure 3g). 
	NOTE: It is crucial that excess glue is avoided, and that the fiducial markers do not stick together as later digital&#160;image correlation (DIC) techniques will produce useless tracking results. It is important that the square array must be within the middle third of the tissue&#8217;s testing region.</li>
</ol>
6. Preconditioning step and duration timing
<ol>
	<li>To compute the appropriate membrane tension, obtain the tissue&#8217;s effective testing edge length and use the following equation. 
	<img alt="Equation 1" src="/files/ftp_upload/59170/59170eq1.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;(1) 
	NOTE: Here, T is the membrane tension in a unit of force/length, f is the force, and L is the specimen&#8217;s effective testing length.</li>
	<li>Create a preconditioning protocol so that the tissue will undergo 10 loading/unloading cycles at the forces associated with peak membrane tension at a loading rate of 4.42 N/min, including a preload of 2.5% of the maximum force (Figure 4).
	<ol>
		<li>Create a new arbitrary testing directory to temporarily store the preconditioning data, because it is not necessary for future calculations. Establish a loading rate of 4.42 N/min for subsequent testing.</li>
		<li>Create a new set of testing parameters and set the name of the protocol as Preconditioning0 (Figure 4a). For the X- and Y-axes, set the control mode to be force and the control function to be step. Set the load magnitude as the force associated with targeted peak membrane tension (cf. step 6.1) (Figure 4b). Set the preload magnitude as 2.5% of the maximum force for the first repetition only (Figure 4c). Set the stretch duration and recovery duration both to be 25 s. Set the number of repetitions to be 10 (Figure 4e).</li>
	</ol>
	</li>
	<li>When the preconditioning step finishes, make a note of the tissue&#8217;s deformation in the X- and Y-directions. Prepare a protocol to move the specimen to the maximum force, beginning from the recorded size.
	<ol>
		<li>Retrieve a stopwatch for timing purposes. Begin the maximum force loading protocol and start the stopwatch simultaneously when the machine begins actuation (Figure 5a). Stop the stopwatch when the actuation stops. Stopping will be obvious through auditory cues.</li>
		<li>Record the post-preconditioning peak tissue deformation alongside the time from the stopwatch representing the tissue&#8217;s optimal stretch time (Figure 5b).</li>
	</ol>
	</li>
</ol>
7. Biaxial mechanical testing
<ol>
	<li>Prepare a force-controlled protocol at a loading rate of 4.42 N/min.
	<ol>
		<li>Open a new testing directory and name the test. 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.</li>
		<li>Create a protocol set titled FirstImage. Set the X-axis and Y-axis control mode to force and the control function to step. Set the load magnitude to 0 mN. Set the stretch duration and recovery duration each to 1 second. Set the number of repetitions to 1. Set the data output frequency and image output frequency each to 1 Hz.</li>
		<li>Construct a new testing set, named PreconditioningA. Establish the testing parameters such that the tissue will undergo 10 repetitions of cyclic loading/unloading to the targeted force for the desired membrane tension exactly as was prepared in step 6.2. Note that now, the stretch time and recover time should be the time recorded in step 6.3.2. No images are captured in the A testing set, but data is captured at 15 Hz.</li>
		<li>Construct another testing set, named PreconditioningB. All testing parameters should be identical to those as mentioned in the previous step, with the exception that image output frequency is set to 15 Hz, and no preload is applied.</li>
		<li>After the preconditioning protocol, create testing protocols so that the tissue is loaded to the peak membrane tension in the following circumferential-to-radial loading ratios at a loading rate of 4.42 N/min: 1:1, 0.75:1, 1:0.75, 0.5:1, and 1:0.5 (Figure 6). Retrieve data from the last two cycles of each loading ratio for subsequent data processing and analyses described in section 10. Refer to Table 1 for a detailed description of the protocols to be established.</li>
	</ol>
	</li>
	<li>Prepare a displacement-controlled testing protocol at a loading rate of 4.42 N/min as follows. (i) Biaxial stretching in the X-direction and Y-direction to the displacements associated with the peak circumferential and radial stretches, respectively (Figure 7a). (ii) Pure shear along the X-direction&#8212;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 (Figure 7b). (iii) Constrained uniaxial stretching along the X-direction (Figure 7c). (iv) Pure shear along the Y-direction (Figure 7d). (v) Constrained uniaxial stretching along the Y-direction (Figure 7e).
	<ol>
		<li>Between each of these steps, construct a rest &#8220;cycle&#8221; of 1 min that holds the tissue at the original mounted configuration. Retrieve data from the last two cycles of each loading ratio for data processing and analyses (section 9). Refer to Table 2 for a detailed description of the protocols to be established.</li>
	</ol>
	</li>
	<li>Prepare a stress-relaxation protocol so that the tissue is loaded in each direction, at a loading rate of 4.42 N/min, to the displacements associated with the peak membrane tensions (step 7.2) and held at that displacement for 15 min (Figure 8 and Figure 9). After 15 min, the protocol should be set to recover the tissue to its original mounting configuration. 
	NOTE: In the case of tissue tearing, abort the test immediately to prevent any potential damage caused to the biaxial testing system.</li>
</ol>
8. Tissue fixation for histology analysis
<ol>
	<li>Unmount the tissue from the biaxial testing system. Place the tissue into a container filled with 10% formalin, and then place the container in a refrigerated environment at approximately 4 &#176;C. Fix the tissue for 24&#8211;48 h, depending on the tissue&#8217;s thickness. 
	CAUTION: Formalin is a known carcinogen and, if breathed in, an excess may cause lungs to become fixed. All work with formalin should be performed in a fume hood with adequate ventilation.</li>
	<li>After the tissue has been fixed in formalin for 24&#8211;48 h, transfer the tissue to an 80% ethanol solution for later histology. The tissue should be stored in solution in a refrigerated environment at 4 &#176;C. 
	NOTE: The protocol can be paused here. Once the tissues are fixed, the specimens can be analyzed at any time. If the protocol is paused, store the tissue in a labeled container filled with 80% ethanol, and store the container in a refrigerator at approximately 4 &#176;C (as explained in step 8.2).</li>
	<li>Prepare the tissue for commercial histology analysis as per the vendor&#8217;s instructions. If a certain leaflet constituent, such as collagen, elastin, glycosaminoglycans, etc., is of the study&#8217;s interest, ensure that the appropriate histology stain is employed. 
	NOTE: Histology slides may be visualized using a microscope to observe desired constituents (Figure 10).</li>
	<li>Using the image processing program ImageJ, perform color deconvolution methods to determine the percentage of each stained constituent in the tissue. For more details on these procedures, please refer to Ruifrok and Johnston24.</li>
</ol>
9. Biaxial testing data post-processing procedures
<ol>
	<li>Perform DIC-based tracking on the four fiducial markers from the images taken during the biaxial mechanical testing (Figure 11) to determine the time-dependent marker positions. 
	<img alt="Equation 2" src="/files/ftp_upload/59170/59170eq2.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;(2)
	<ol>
		<li>If it is desired to perform the analysis with respect to the mounting configuration, let XI be the marker positions in the undeformed state at the beginning of the biaxial test. If it is desired to perform the analysis with respect to the post-preconditioning deformation, let XI be the marker positions at the end of the preconditioning protocol. 
		NOTE: The subsequent steps will be conducted in the same manner, regardless of the reference configuration chosen. 
		NOTE: Here, XI and xI are the undeformed and deformed positions of the markers, respectively, and dI is the displacement vector of each marker.</li>
	</ol>
	</li>
	<li>Compute the deformation gradient (F) of the fiducial markers using a four-node bilinear finite element2,23,25. 
	<img alt="Equation 3" src="/files/ftp_upload/59170/59170eq3.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;(3) 
	NOTE: Here, BxI&#8217;s and ByI&#8217;s are the finite element shape function derivatives in the X- and Y-directions for node I, respectively, and uI(t) and vI(t) are the time-dependent X- and Y-displacements, respectively, as previously determined from step 9.1. Note that the X- and Y- coordinates are aligned to the tissue&#8217;s circumferential and radial directions.</li>
	<li>Compute the right Cauchy&#8211;Green deformation tensor (C) and the Green strain tensor (E). 
	<img alt="Equation 4" src="/files/ftp_upload/59170/59170eq4.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;(4) 
	NOTE: Here, I is the second-order identity tensor. Determine the circumferential and radial stretches by taking the square roots of the principle values of C.</li>
	<li>Determine the first Piola-Kirchhoff (1st-PK) stress tensor (P). 
	<img alt="Equation 5" src="/files/ftp_upload/59170/59170eq5.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;(5) 
	NOTE: Here, t is the specimen&#8217;s thickness, and TC and TR are the applied membrane tensions in the circumferential and radial directions, respectively.</li>
	<li>Also, compute other stress tensors, such as the Cauchy stress tensor (&#963;) and the second Piola-Kirchoff (2nd-PK) stress tensor (S). 
	<img alt="Equation 6" src="/files/ftp_upload/59170/59170eq6.jpg" />&#160;&#160;&#160;&#160;&#160;&#160;(6) 
	NOTE: Here, J is the Jacobian of the deformation gradient tensor F.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+histochemical+staining+by+color+deconvolution.">Quantification of histochemical staining by color deconvolution.</a> Analytical and Quantitative Cytology and Histology. 23 (4), 291-299 (2001).</li><li>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=Biaxial+mechanical+evaluation+of+planar+biological+materials.">Biaxial mechanical evaluation of planar biological materials.</a> Journal of Elasticity. 61 (1), 199 (2000).</li></ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>10% Formalin Solution, Neutral Bufffered</td><td>Sigma-Aldrich</td><td>HT501128-4L&amp;nbsp;</td><td></td></tr><tr><td>40X-2500X LED Lab Trinocular Compound Microscope</td><td>AmScope</td><td>SKU: T120C</td><td></td></tr><tr><td>BioTester - Biaxial Tester</td><td>CellScale Biomaterials Testing</td><td></td><td>1.5N Load Cell Capacity</td></tr><tr><td>ImageJ</td><td>National Institute of Health, Bethesda, MD</td><td>Version 1.8.0_112</td><td></td></tr><tr><td>LabJoy</td><td>CellScale Biomaterials Testing</td><td>Version 10.66</td><td></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td>Version 2018b</td><td></td></tr><tr><td>Phosphate-Buffered Saline</td><td>n/a</td><td></td><td>Recipe for 1L 1X PBS Solution: 8.0g NaCl, 0.2g KCl, 1.44g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.24g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Single Edge Industrial Razor Blades (Surgical Carbon Steel)</td><td>VWR International</td><td>H3515541105024</td><td>Razord blades for tissue retrieval and preparation procedures</td></tr></tbody></table>

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 ...

biaxial-mechanical-characterizations-of-atrioventricular-heart-valves

Research

JoVE Journal

Bioengineering

14.3K Views.  The University of Oklahoma. Deze methode kan zorgen voor een beter begrip van hartklep biomechanica die zeer nuttig kan zijn bij computationele modellering en in de verfijning van de behandeling methoden voor hartklep ziekte. Dit protocol is voordelig in vergelijking met andere vastgestelde testprotocollen omdat het kan worden gebruikt om mechanische karakteriseringen van hartklepweefsels uit te voeren met behulp van een uniform testschema. Dit biaxiale testprotocol met het uniforme testschema zal gunstig zijn voor het ...

Video: Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

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 ...

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 ...

Surgery and Sample Processing for Correlative Imaging of the Murine Pulmonary Valve

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the surgical and instrumental expertise required to accurately quantify the structure and organization across multiple length scales have stunted its advancement. This work provides a detailed description of the murine dissection, en bloc staining, sample processing, and correlative imaging procedures for depicting the heart valve at different length scales. Hydrostatic transvalvular pressure was used to control the temporal heterogeneity by chemically fixing the heart valve conformation. Micro-computed tomography (&#181;CT) was used to confirm the geometry of the heart valve and provide a reference for the downstream sample processing needed for the serial block face scanning electron microscopy (SBF-SEM). High-resolution serial SEM images of the extracellular matrix (ECM) were taken and reconstructed to provide a local 3D representation of its organization. &#181;CT and SBF-SEM imaging methods were then correlated to overcome the spatial variation across the pulmonary valve. Though the work presented is exclusively on the pulmonary valve, this methodology could be adopted for describing the hierarchical organization in biological systems and is pivotal for the structural characterization across multiple length scales.

Here, we describe a correlative workflow for the excision, pressurization, fixation, and imaging of the murine pulmonary valve to determine the gross conformation and local extracellular matrix structures.

The underlying causes of heart valve related-disease (HVD) are elusive. Murine animal models provide an excellent tool for studying HVD, however, the ...

Engineering

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Medicine

Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three leaflets, each with unique mechanical behaviors. These variations among the three TV leaflets can be further understood by examining their four anatomical layers, which are the atrialis (A), spongiosa (S), fibrosa (F), and ventricularis (V). While these layers are present in all three TV leaflets, there are differences in their thicknesses and microstructural constituents that further influence their respective mechanical behaviors. 
This protocol includes four steps to elucidate the layer-specific differences: (i) characterize the mechanical and collagen fiber architectural behaviors of the intact TV leaflet, (ii) separate the composite layers (A/S and F/V) of the TV leaflet, (iii) carry out the same characterizations for the composite layers, and (iv) perform post-hoc histology assessment. This experimental framework uniquely allows the direct comparison of the intact TV tissue to each of its composite layers. As a result, detailed information regarding the microstructure and biomechanical function of the TV leaflets can be collected with this protocol. Such information can potentially be used to develop TV computational models that seek to provide guidance for the clinical treatment of TV disease.

This protocol describes the biaxial mechanical characterization, polarized spatial frequency domain imaging-based collagen quantification, and microdissection of tricuspid valve leaflets. The provided method elucidates how the leaflet layers contribute to the holistic leaflet behaviors.

The tricuspid valve (TV) regulates the unidirectional flow of unoxygenated blood from the right atrium to the right ventricle. The TV consists of three ...

In Silico Clinical Trials for Cardiovascular Disease

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique characteristic of the platform is the integration of patient-specific biological, genetic, and clinical imaging data. The platform allows the testing and optimization of medical treatment to maximize positive therapeutic outcomes. Thus, adverse effects and drug interactions can be avoided, sudden cardiac death can be prevented, and the time between the commencement of drug treatment and the desired result can be shortened. This article presents a parametric model of the left ventricle automatically generated from patient-specific ultrasound images by applying an electromechanical model of the heart. Drug effects were prescribed through specific boundary conditions for inlet and outlet flow, ECG measurements, and calcium function for heart muscle properties. Genetic data from patients were incorporated through the material property of the ventricle wall. Apical view analysis involves segmenting the left ventricle using a previously trained U-net framework and calculating the bordering rectangle based on the length of the left ventricle in the diastolic and systolic cycle. M-mode view analysis includes bordering of the characteristic areas of the left ventricle in the M-mode view. After extracting the dimensions of the left ventricle, a finite elements mesh was generated based on mesh options, and a finite element analysis simulation was run with user-provided inlet and outlet velocities. Users can directly visualize on the platform various simulation results such as pressure-volume, pressure-strain, and myocardial work-time diagrams, as well as animations of different fields such as displacements, pressures, velocity, and shear stresses.

In Silico Clinical Trials for Cardiovascular Disease

This protocol demonstrates the workflow of the SILICOFCM platform for automatically generating a parametric model of the left ventricle from patient-specific ultrasound images by applying a multi-scale electromechanical model of the heart. This platform enables in silico clinical trials intended to reduce real clinical trials and maximize positive therapeutic outcomes.

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique ...

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

Evaluation of Hydrodynamic Properties in Mounted Porcine Aortic Valves

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

Author Spotlight: Advancing Cardiac Procedure Testing Prior to Embarking on Large Animal Studies 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...

A Novel Right Ventricular Volume and Pressure Loaded Piglet Heart Model for the Study of Tricuspid Valve Function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. Mechanistic studies to improve our&#160;understanding of the underlying pathophysiology responsible for the development of premature TV failure are lacking. Due to the inability to conduct these studies in humans, an animal model is required. In this manuscript, we describe the protocols for a novel chronic recovery infant piglet heart model for the study of changes in the TV when placed under combined volume and pressure stress. In this model, volume loading of the right ventricle&#160;and the TV is achieved through the disruption of the pulmonary valve. Then pressure loading is accomplished through the placement of a pulmonary artery band. The success of this model is assessed at four weeks post intervention surgery through echocardiography, intracardiac pressure measurement, and pathologic examination of the heart specimens.

A novel recovery piglet heart model with combined pressure and volume overload on the right ventricle is described for the study of tricuspid valve function.

Heart conditions in which the tricuspid valve (TV) faces either increased volume or pressure stressors are associated with premature valve failure. ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

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