This work was supported by the American Heart Association Scientist Development Grant (16SDG27760143) and the Presbyterian Health Foundation. KMC was supported in part by the University of Oklahoma (OU) Undergraduate Research Opportunity Program and Honors Research Apprenticeship Program. DWL was supported in part by the National Science Foundation Graduate Research Fellowship (GRF 2019254233) and the American Heart Association/Children&#39;s Heart Foundation Predoctoral Fellowship (Award #821298). All of this support is gratefully acknowledged.

<ol>
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T., 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+pilot+study+on+linking+tissue+mechanics+with+load-dependent+collagen+microstructures+in+porcine+tricuspid+valve+leaflets.">A pilot study on linking tissue mechanics with load-dependent collagen microstructures in porcine tricuspid valve leaflets.</a> Bioengineering. 7 (2), 60 (2020).</li><li>Pant, A. D., 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=Pressure-induced+microstructural+changes+in+porcine+tricuspid+valve+leaflets.">Pressure-induced microstructural changes in porcine tricuspid valve leaflets.</a> Acta Biomaterialia. 67, 248-258 (2018).</li><li>Kramer, K. E., 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+layer-specific+tissue+biomechanics+of+porcine+atrioventricular+heart+valve+leaflets.">An investigation of layer-specific tissue biomechanics of porcine atrioventricular heart valve leaflets.</a> Acta Biomaterialia. 96, 368-384 (2019).</li><li>Ross, C. J., Laurence, D. W., Wu, Y., Lee, C. -. H. <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+characterizations+of+atrioventricular+heart+valves.">Biaxial mechanical characterizations of atrioventricular heart valves.</a> Journal of Visualized Experiments: JoVE. (146), e59170 (2019).</li><li>Goth, W., Lesicko, J., Sacks, M. S., Tunnell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-based+analysis+of+soft+tissue+structures.">Optical-based analysis of soft tissue structures.</a> Annual Review of Biomedical Engineering. 18, 357-385 (2016).</li><li>Jett, S. V., 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=Integration+of+polarized+spatial+frequency+domain+imaging+(pSFDI)+with+a+biaxial+mechanical+testing+system+for+quantification+of+load-dependent+collagen+architecture+in+soft+collagenous+tissues.">Integration of polarized spatial frequency domain imaging (pSFDI) with a biaxial mechanical testing system for quantification of load-dependent collagen architecture in soft collagenous tissues.</a> Acta Biomaterialia. 102, 149-168 (2020).</li><li>Reddy, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An Introduction to Continuum Mechanics. , (2013).</li><li>Duginski, G. A., Ross, C. J., Laurence, D. W., Johns, C. H., Lee, C. -. H. <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+effect+of+freezing+storage+on+the+biaxial+mechanical+properties+of+excised+porcine+tricuspid+valve+anterior+leaflets.">An investigation of the effect of freezing storage on the biaxial mechanical properties of excised porcine tricuspid valve anterior leaflets.</a> Journal of the Mechanical Behavior of Biomedical Materials. 101, 103438 (2020).</li><li>Salinas, S. D., Clark, M. M., 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=Mechanical+response+changes+in+porcine+tricuspid+valve+anterior+leaflet+under+osmotic-induced+swelling.">Mechanical response changes in porcine tricuspid valve anterior leaflet under osmotic-induced swelling.</a> Bioengineering. 6 (3), 70 (2019).</li><li>Pokutta-Paskaleva, A., Sulejmani, F., DelRocini, M., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+mechanical,+morphological,+and+microstructural+characterization+of+porcine+mitral+and+tricuspid+leaflets+and+chordae+tendineae.">Comparative mechanical, morphological, and microstructural characterization of porcine mitral and tricuspid leaflets and chordae tendineae.</a> Acta Biomaterialia. 85, 241-252 (2019).</li><li>Ross, C. J., 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+glycosaminoglycan+contribution+to+biaxial+mechanical+behaviors+of+porcine+atrioventricular+heart+valve+leaflets.">An investigation of the glycosaminoglycan contribution to biaxial mechanical behaviors of porcine atrioventricular heart valve leaflets.</a> Journal of the Royal Society Interface. 16 (156), 0069 (2019).</li><li>Sommer, G., Regitnig, P., Ko&#776;ltringer, L., Holzapfel, G. A. <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+intact+and+layer-dissected+human+carotid+arteries+at+physiological+and+supraphysiological+loadings.">Biaxial mechanical properties of intact and layer-dissected human carotid arteries at physiological and supraphysiological loadings.</a> American Journal of Physiology-Heart and Circulatory Physiology. 298 (3), 898-912 (2009).</li><li>Holzapfel, G. A., Sommer, G., Gasser, C., Regitnig, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+layer-specific+mechanical+properties+ofhuman+coronary+arteries+with+intimal+thickening,+and+related+constitutive+modelling.">Determination of the layer-specific mechanical properties ofhuman coronary arteries with intimal thickening, and related constitutive modelling.</a> American Journal of Physiology-Heart and Circulatory Physiology. 289 (5), 2048-2058 (2005).</li><li>Sommer, G., 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=Multiaxial+mechanical+response+and+constitutive+modeling+of+esophageal+tissues:+Impact+on+esophageal+tissue+engineering.">Multiaxial mechanical response and constitutive modeling of esophageal tissues: Impact on esophageal tissue engineering.</a> Acta Biomaterialia. 9 (12), 9379-9391 (2013).</li></ol>

The authors have no conflicts of interest to disclose.

Critical steps for the protocol include: (i) the layer microdissection, (ii) the tissue mounting, (iii) the fiducial marker placement, and (iv) the pSFDI setup. Appropriate layer microdissection is the most important and difficult aspect of the method described herein. Prior to launching an investigation utilizing this technique, the dissector(s) should have long-term practice with the microdissection technique and all three TV leaflets. The dissector should ensure the composite layer specimens are sufficiently large (&#62;3.3 mm) and have a uniform thickness. Histology should be used to confirm that dissections consistently have accurate layer separation.
For tissue mounting, the tissue must be attached to the biaxial tester such that the tissue is flat without any artificial stretch or wrinkling. These errors will result in inaccurate mechanical data. The composite layers are more prone to these errors due to their thinner nature. When affixing the fiducial markers, it is paramount that the markers are placed within the central one-third area of the tissue and do not adhere to each other. Inappropriate marker placement will result in inaccurate quantification of the tissue stretches. Finally, the pSFDI-projected light brightness must be carefully selected and remain unchanged for the intact tissue and the composite layers. If the brightness is changed, the pSFDI results cannot be compared among the intact tissue and its composite layers.
The flexibility of the method described herein lies primarily in the biaxial mechanical characterization, while most of the troubleshooting arises during the pSFDI-based collagen microstructural quantification. The displacement-controlled testing protocols provide two key advantages over alternative force-controlled testing protocols: (i) the stress-stretch curves are smoother with no oscillations, and (ii) the displacement rate (mm/s) and strain rate (%/s) can be directly controlled rather than the loading rate (N/m). However, it is still imperative to perform force-controlled preconditioning prior to the mechanical characterization to acquire repeatable force-displacement curves and determine the tissue configuration that provides equibiaxial tensions. Once the equibiaxial configuration has been determined, the other desired loading ratios (e.g., TXX: TYY = 1: 0.5 and TXX: TYY = 0.5: 1) can be determined by manually jogging the linear actuators. This allows for highly accurate replication of the target biaxial tensions with the added benefits of a displacement-controlled scheme. Furthermore, this versatile mechanical testing protocol can be adjusted to consider more loading ratios or other unique loading conditions, such as pure shear or stress relaxation. Additional pSFDI quantification can be included with these new protocols or at distinct points along the loading paths. Prior to performing these pSFDI characterizations, it is incredibly important to ensure there are no glares, bubbles, or debris on the tissue. Often, one must test different orientations of the polarizer, fluid height of the PBS bath, or methods to prevent/remove debris and bubbles to ensure successful and accurate pSFDI quantification.
There are three main limitations of the layer microdissection. First, the intact tissue can only be separated into two composite layers, meaning that all the four anatomical layers cannot be individually isolated. This is because the tissue is too thin to attempt to separate all four anatomical layers, and the lack of structural components in the spongiosa precludes its microdissection. Secondly, this protocol uses DI water instead of PBS. While PBS is closer to the physiological environment15, the use of PBS during testing resulted in consistent, failed dissections due to frequent tearing of the composite A/S layer. The use of DI water immediately increased the ease and success of dissections by significantly decreasing the likelihood of holes and tears in the composite A/S layer. Third, although the experimental protocol is designed to provide matched data between the intact and composite layers, there are noticeable specimen-to-specimen variabilities in the mechanical and microstructural properties (Table 1). This variability may somewhat confound the data analysis; however, our experience9&#160;and extensive studies from the literature4,5,16,17&#160;show that it falls within the typical tricuspid valve mechanical characterization results.
The presented protocol is significant for three main reasons. First, this is the only protocol to successfully separate the layers of all three TV leaflets. Second, the structure of this protocol allows for the direct comparison of the mechanical and collagen fiber architectural properties of an intact TV leaflet with its composite layers. Third, this unique pSFDI system allows the quantification and visualization of the load-dependent changes in the collagen fiber architecture.
This layer dissection method can be applied to additional tissues with layered morphology, such as the eye or skin. The combined mechanical-structural characterization framework could also be used for tissues with established layer separation procedures, such as the remaining heart valves, arteries, or esophageal tissues18,19,20. While mechanical testing has an established role in understanding the mechanical properties of biological tissues, pSFDI is a much newer development that has yet to be fully realized within the soft tissue biomechanics community. This protocol provides a new method to synthesize these techniques for biological tissues and provide further insight into the tissue-microstructure relationships.

The TV is located between the right atrium and right ventricle of the heart. Throughout the cardiac cycle, the TV regulates the unidirectional blood flow via cyclic opening and closing of the TV anterior leaflet (TVAL), the TV posterior leaflet (TVPL), and the TV septal leaflet (TVSL). These leaflets are complex and have four distinct anatomical layers&#8212;the atrialis (A), the spongiosa (S), the fibrosa (F), and the ventricularis (V)&#8212;with unique microstructural constituents. The elastin fibers in the atrialis and ventricularis help restore the tissue to its undeformed geometry after mechanical loading1. In contrast, the fibrosa contains a dense network of undulated collagen fibers that contribute to the load-bearing capacity of the leaflets2. Mainly consisting of glycosaminoglycans, the spongiosa has been hypothesized to enable shearing between leaflet layers during heart valve function3. While all three leaflet types have the same anatomical layers, there are variations in the thicknesses of the layers and constituent ratios that have implications for leaflet-specific mechanical behaviors.
Researchers have explored the properties of the TV leaflets using planar mechanical characterizations, histomorphological assessments, and optical characterizations of the collagen fiber architecture. For example, planar biaxial mechanical characterizations seek to emulate physiological loading by applying perpendicular displacements to the tissue and recording the associated forces. The resulting force-displacement (or stress-stretch) observations have revealed that all three TV leaflets exhibit nonlinear, direction-specific mechanical behaviors with more apparent leaflet-specific responses in the radial tissue direction4,5,6. These leaflet-specific behaviors are believed to stem from differences in the microstructural properties observed using standard histological techniques6,7. Further, second harmonic generation imaging6, small-angle light scattering8, and polarized spatial frequency domain imaging7&#160;(pSFDI) aim to understand these microstructural properties and have shown leaflet-specific differences in the collagen fiber orientation and fiber crimp that have implications for the observed tissue-level mechanical behaviors. These studies have significantly advanced our understanding of the tissue microstructure and its role in tissue-level behaviors. However, much remains to be addressed in experimentally connecting the tissue mechanics and the underlying microstructure.
Recently, this laboratory performed mechanical characterizations of the TV leaflet layers separated into two composite layers (A/S and F/V) using a microdissection technique9. That earlier work highlighted differences in the mechanical properties of the layers and helped provide insight into how the layered microstructure contributes to the tissue mechanical behaviors. Although this investigation improved our understanding of the TV leaflet microstructure, the technique had several limitations. First, the properties of the composite layers were not directly compared to the intact tissue, leading to a lack of complete understanding of the mechanics-microstructure relationship. Second, the collagen fiber architecture of the composite layers was not examined. Third, only the layers of the TVAL were investigated due to difficulties with collecting the composite layers from the other two TV leaflets. The method described herein provides a holistic characterization framework that overcomes these limitations and provides complete characterizations of the TV leaflets and their composite layers.
This paper describes the microdissection technique that separates the three TV leaflets into their composite layers (A/S and F/V) for biaxial mechanical and microstructural characterizations10,11,12. This iterative protocol includes (i) biaxial mechanical testing and pSFDI characterization of the intact leaflet, (ii) a novel and reproducible microdissection technique to reliably obtain the composite TV layers, and (iii) biaxial mechanical testing and pSFDI characterization of the composite TV layers. The tissue was exposed to biaxial tensile loading with various force ratios for mechanical testing. Then, pSFDI was used to determine the collagen fiber orientation and alignment at various loaded configurations. pSFDI preserves the native collagen fiber architecture, allows load-dependent analysis, and circumvents the typical need to fix or clear tissue for collagen fiber architecture analysis, such as in second harmonic generation imaging or small-angle light scattering. Finally, the tissues were prepared using standard histology techniques to visualize the tissue microstructure. This iterative and holistic framework allows for the direct comparison of the mechanical and microstructural properties of the TV leaflet to its composite layers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Formalin Solution, Neutral Buffered</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>Alconox Detergent</td>
			<td>Alconox</td>
			<td></td>
			<td>cleaning compound</td>
		</tr>
		<tr>
			<td>BioTester - Biaxial Tester</td>
			<td>CellScale Biomaterials Testing</td>
			<td></td>
			<td>1.5 N Load Cell Capacity</td>
		</tr>
		<tr>
			<td>Cutting Mat</td>
			<td>Dahle</td>
			<td>B0027RS8DU</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized Water</td>
			<td>N/A</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-Tipped Tool</td>
			<td>HTI INSTRUMENTS</td>
			<td>NSPLS-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Curved</td>
			<td>Scientific Labwares</td>
			<td>16122</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Thick</td>
			<td>Scientific Labwares</td>
			<td>161001078</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps - Thin</td>
			<td>Scientific Labwares</td>
			<td>16127</td>
			<td></td>
		</tr>
		<tr>
			<td>LabJoy</td>
			<td>CellScale Biomaterials Testing</td>
			<td>Version 10.66</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Displacement Sensor</td>
			<td>Keyence</td>
			<td>IL-030</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Cyanoacrylate Glue</td>
			<td>Loctite</td>
			<td>2436365</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>Version 2020a</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>HTI Instruments</td>
			<td>CAS55C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Belmaks</td>
			<td>360758081051Y4</td>
			<td></td>
		</tr>
		<tr>
			<td>Polarized Spatial Frequency Domain Imaging Device</td>
			<td>N/A</td>
			<td></td>
			<td>Made in-house using a digital light projector, linear polarizer, rotating polarizer mount, and charge-coupled device camera. 
			See doi.org/10.1016/j.actbio.2019.11.028 (PMCID: PMC8101699) for more details.</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>THINKPRICE</td>
			<td>TP-SCALPEL-3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Edge Industrial Razor Blades (Surgical Carbon Steel)</td>
			<td>VWR International</td>
			<td>H3515541105024</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Pen</td>
			<td>LabAider</td>
			<td>LAB-Skin-6</td>
			<td></td>
		</tr>
		<tr>
			<td>T-Pins</td>
			<td>Business Source</td>
			<td>BSN32351</td>
			<td></td>
		</tr>
		<tr>
			<td>Wax Board</td>
			<td>N/A</td>
			<td></td>
			<td>Made in-house using modeling wax and baking tray</td>
		</tr>
		<tr>
			<td>Weigh Boat</td>
			<td>Pure Ponta</td>
			<td>mdo-azoc-1030</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The microdissection will yield A/S and F/V specimens with relatively uniform thicknesses that can be mounted to a (commercial) biaxial testing device. Histology analysis of the intact leaflet and the two dissected layers will verify if the tissue was correctly separated along the border between the spongiosa and fibrosa (Figure 7). Additionally, the histology micrographs can be used to determine the tissue layer thicknesses and constituent mass fractions using ImageJ software. A failed dissection occurs when it produces an A/S sample that is too small for mounting to the biaxial tester. This most often occurs when the A/S tears during peeling, or when a hole arises in the F/V layer due to thick chordae.
The displacement-controlled mechanical testing and postprocessing produce stress-strain data describing the nonlinear mechanical behavior of the tissue (Figure 8). The samples are generally anisotropic, where the circumferential tissue direction has a stiffer mechanical response than the radial tissue direction (Table 1). These low-tensile and high-tensile properties can be quantitatively determined using additional analysis techniques6,14. Collectively assessing the range of biaxial force ratios provides additional insight into the tissue&#39;s directional coupling (i.e., the X-axis force depends on the Y-axis force and vice versa). It is important to note that the mechanical behavior of one tissue direction in these various force ratios may show compressive deformations during nonequibiaxial deformations. This unique behavior typically arises due to highly aligned collagen fibers along the compressive tissue direction.
The pSFDI data yield color maps of the collagen fiber orientation and DOA (Figure 9). Specifically, these color maps provide a comprehensive understanding of the collagen fiber architecture across the entire tissue specimen. One unique advantage of the nondestructive pSFDI technique is the ability to compare the results across various loading configurations and understand how the collagen fibers reorient and uncrimp/align to support the applied loading. These results are suboptimal if the projected light is too bright or dark during imaging, if the projected light is not kept consistent across the intact leaflet and its layers, if there are large bubbles or debris on the sample, if there is too much glue on the tissue from fiducial marker placement, or if the level of the water bath gets too low and creates pinpoints of bright light. All lead to inaccurate representations of the reflected intensity versus polarizer angle data, which interferes with the determined fiber orientation and computed DOA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63522/63522fig01.jpg" /> 
Figure 1: Selection of microdissection area. (A) Identification of problematic areas to avoid and (B) target area for the layer microdissection. Scale bar = 10 mm (A, B). Abbreviations: Rad. = radial; Circ. = circumferential. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63522/63522fig02.jpg" /> 
Figure 2: The pSFDI system integrated with the biaxial testing device. Key components of both devices are labeled. Abbreviations: pSFDI = polarized spatial frequency domain imaging7; DLP = digital light processing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63522/63522fig03.jpg" /> 
Figure 3: Initiation of the leaflet microdissection. (A) Stretching the tissue taut while placing pins, (B) the pinned tissue ready for microdissection, (C) making the first cut into the A/S composite layer, and (D) creating the first corner of cuts into the A/S composite layer. Scale bars = 10 mm. Abbreviations: Rad. = radial; Circ. = circumferential; A/S = atrialis/spongiosa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63522/63522fig04.jpg" /> 
Figure 4: Separation of the A/S composite layer. (A) Extension of the cuts into the A/S composite layer, (B) separation of the A/S composite layer via careful peeling, and (C) creation of the second corner. Scale bar = 10 mm. Abbreviations: Rad. = radial; Circ. = circumferential; A/S = atrialis/spongiosa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63522/63522fig05.jpg" /> 
Figure 5: Potential problems during the leaflet microdissection. (A) Unsuccessful separation of the A/S and F/V composite layers, (B) adjustment of the microdissection area to avoid chordae insertions, (C) creation of a new separation seam due to undesired hole, and (D) interlayer connection connecting the A/S and F/V composite layers. Scale bars = 5 mm (A-C), 10 mm (D). Abbreviations: Rad. = radial; Circ. = circumferential; A/S = atrialis/spongiosa; F/V = fibrosa/ventricularis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63522/63522fig06.jpg" /> 
Figure 6: Completion of the microdissection. (A) Denotation of the top-right corner for orientation, (B) separation of the A/S using scissors, and (C) retrieval of the F/V composite layer with orientation marked. Scale bar = 10 mm Abbreviations: Rad. = radial; Circ. = circumferential; A/S = atrialis/spongiosa; F/V = fibrosa/ventricularis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63522/63522fig07.jpg" /> 
Figure 7: Histological assessment. Micrographs showing circumferential cross-sections of the (A) intact leaflet and (B) properly separated A/S and F/V layers. Scale bars = 50 &#181;m. Abbreviations: atrialis/spongiosa; F/V = fibrosa/ventricularis; VIC = valvular interstitial cell. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63522/63522fig08.jpg" /> 
Figure 8: Representative biaxial mechanical testing results for the equibiaxial loading ratio. Membrane tension versus stretch data of the (A) tricuspid valve anterior leaflet, (B) tricuspid valve posterior leaflet, and (C) tricuspid valve septal leaflet. Abbreviations: atrialis/spongiosa; F/V = fibrosa/ventricularis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63522/63522fig09.jpg" /> 
Figure 9: Representative pSFDI results. (A) Raw image of the leaflet during pSFDI assessment, (B) quantified fiber orientation shown via the color map, and (C) quantified degree of optical anisotropy, shown via the color map, indicating the fiber alignment. The arrows indicate regions with excess glue from the fiducial markers. The upper row demonstrates good images, while the lower row demonstrates poor images. Scale bars = 4 mm. Abbreviations: deg. = degrees; DOA = degree of optical anisotropy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/63522fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Composite Layer</td>
			<td>&#955;circ</td>
			<td>&#955;rad</td>
		</tr>
		<tr>
			<td>A/S</td>
			<td>1.26 &#177; 0.05</td>
			<td>1.37 &#177; 0.05</td>
		</tr>
		<tr>
			<td>F/V</td>
			<td>1.17 &#177; 0.03</td>
			<td>1.32 &#177; 0.08</td>
		</tr>
	</tbody>
</table>
Table 1: Average composite layer stretches. The average peak stretches of the composite layers showing the anticipated variations in the mechanical behaviors. This table is extracted from 9. Abbreviations: atrialis/spongiosa; F/V = fibrosa/ventricularis.
Supplemental Figure S1: Identification of areas to avoid during microdissection. (A) Examining the ventricular side of the tissue sample for chordae insertions, (B) tracking where difficult areas are when tissue is placed with atrialis facing up, and (C) planning for initial cuts to avoid identified areas. Scale bar = 10 mm. Abbreviations: Rad. = radial; Circ. = circumferential. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/FigS1.tif" target="_blank">Please click here to download this File.</a>
Supplemental Figure S2: Demonstration of rubbing along cuts. (A) The cut before rubbing with blunt tweezers and (B) edges of the cut separating more after rubbing. Scale bar = 10 mm. Abbreviations: Rad. = radial; Circ. = circumferential. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/FigS2.tif" target="_blank">Please click here to download this File.</a>
Supplemental Figure S3: Non-connecting cuts. Tweezers are used to identify the thin area of tissue separating the two cuts before carefully cutting the tissue with scissors. Scale bar = 10 mm. Abbreviations: Rad. = radial; Circ. = circumferential. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63522/FigS3.tif" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Layer Microdissection of Tricuspid Valve Leaflets for Biaxial Mechanical Characterization and Microstructural Quantification at JoVE.com

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

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 ...

layer-microdissection-tricuspid-valve-leaflets-for-biaxial-mechanical

Nanyang Technological University

Research

JoVE Journal

Bioengineering

1.5K Views.  The University of Oklahoma. 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.

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

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2 and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo experimentation using human and animal subjects, in vitro experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4, clinical instrument development 5, laryngeal aerodynamics 6-9, vocal fold contact pressure 10, and subglottal acoustics 11 (a more comprehensive list can be found in Kniesburges et al. 12)
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1 that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8 such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.

The methodology for fabricating synthetic vocal fold models is described. The models are life-sized and mimic the multi-layer structure of the human vocal folds. Results show the models to self-oscillate at pressures comparable to lung pressure and demonstrate flow-induced vibratory responses that are similar to those of human vocal folds.

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

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 ...

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  ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and giant congenital nevi. Despite its widespread use, planning and executing an expansion protocol is challenging due to the difficulty in measuring the deformation imposed at each inflation step and over the length of the procedure. Quantifying the deformation fields is crucial, as the distribution of stretch over time determines the rate and amount of skin grown at the end of the treatment. In this manuscript, we present a method to study tissue expansion in order to gain quantitative knowledge of the deformations induced during an expansion process. This experimental protocol incorporates multi-view stereo and isogeometric kinematic analysis in a porcine model of tissue expansion. Multi-view stereo allows three-dimensional geometric reconstruction from uncalibrated sequences of images. The isogeometric kinematic analysis uses splines to describe the regional deformations between smooth surfaces with few mesh points. Our protocol has the potential to bridge the gap between basic scientific inquiry regarding the mechanics of skin expansion and the clinical setting. Eventually, we expect that the knowledge gained with our methodology will enable treatment planning using computational simulations of skin deformation in a personalized manner.

This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization.

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

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

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

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