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 laborato...

<ol>
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M., Moeller, A. D., Lee, J. M., Wells, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biaxial+creep+resistance+and+structural+remodeling+of+the+aortic+and+mitral+valves+in+pregnancy.">Biaxial creep resistance and structural remodeling of the aortic and mitral valves in pregnancy.</a> Annals of Biomedical Engineering. 43 (8), 1772-1785 (2015).</li><li>Potter, 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=A+Novel+Small-Specimen+Planar+Biaxial+Testing+System+With+Full+In-Plane+Deformation+Control.">A Novel Small-Specimen Planar Biaxial Testing System With Full In-Plane Deformation Control.</a> Journal of Biomechanical Engineering. 140 (5), 051001 (2018).</li><li>Khoiy, K. 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. 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.

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		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40X-2500X LED Lab Trinocular Compound Microscope</td>
			<td>AmScope</td>
			<td>SKU: T120C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">BioTester - Biaxial Tester</td>
			<td>CellScale Biomaterials Testing</td>
			<td></td>
			<td>1.5N Load Cell Capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td>National Institute of Health, Bethesda, MD</td>
			<td>Version 1.8.0_112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LabJoy</td>
			<td>CellScale Biomaterials Testing</td>
			<td>Version 10.66</td>
			<td></td>
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			<td height="21" style="height:21px;">MATLAB</td>
			<td>MathWorks</td>
			<td>Version 2018b</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;">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 Na2HPO4, 0.24g KH2PO4</td>
		</tr>
		<tr height="42">
			<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>
		</tr>
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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...

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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

13.2K Views.  The University of Oklahoma. 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 bi...