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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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>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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		<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>
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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 ...

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