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.

All methods described herein were approved by the Institutional Animal Care and Use Committee at the University of Oklahoma. Animal tissues were acquired from a USDA-approved slaughterhouse.
1. Biaxial mechanical characterization
<ol>
	<li>Tissue preparation
	<ol>
		<li>Retrieve a TV leaflet from the freezer, razor blades, a surgical pen, forceps, a pipette with deionized (DI) water, and a cutting mat. Thaw the TV leaflet using 2-3 drops of room-temperature DI water. 
	...

<ol>
	<li>Vesely, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+elastin+in+aortic+valve+mechanics.">The role of elastin in aortic valve mechanics.</a> Journal of Biomechanics. 31 (2), 115-123 (1998).</li><li>Zhang, W., Ayoub, S., 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=A+meso-scale+layer-specific+structural+constitutive+model+of+the+mitral+heart+valve+leaflets.">A meso-scale layer-specific structural constitutive model of the mitral heart valve leaflets.</a> Acta Biomaterialia. 32, 238-255 (2016).</li><li>Stella, J. A., 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=On+the+biaxial+mechanical+properties+of+the+layers+of+the+aortic+valve+leaflet.">On the biaxial mechanical properties of the layers of the aortic valve leaflet.</a> Journal of Biomechanical Engineering. 129 (5), 757-766 (2007).</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>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=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>Meador, W. 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=A+detailed+mechanical+and+microstructural+analysis+of+ovine+tricuspid+valve+leaflets.">A detailed mechanical and microstructural analysis of ovine tricuspid valve leaflets.</a> Acta Biomaterialia. 102, 100-113 (2020).</li><li>Hudson, L. 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 (&#...

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 disse...

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 ...

This method can isolate the contributions of tissue layers to the overall tissue behavior, providing new insight into the tissue microstructure. Our microdissection and testing framework allows us to directly compare the mechanical and microstructural properties of heart valve leaflets to their own composite layers. The microdissection can be difficult to master, hence, it is highly recommended to practice as much as possible before launching your study.First, gather the required materials, wax board, DI water, pipette, scalpel, micro scissors, thin forceps, curved forceps, thick forceps, and pins. Unmount the tissue from the biaxial tester and measure its thickness using the laser displacement sensor. Place the tissue on the wax board.Examine the ventricularis side of the tissue for large chorda insertions. Take a photograph for reference. Spread the tissue flat on the wax board with the atrialis facing up.To affix the tissue to the board, place one pin angled away from the tissue in each corner so that it slightly pulls the tissue taut. Ensure that the pins are outside the holes created by the tines when mounting the tissue and the tissue is square and does not shift during the layer microdissection. If necessary, place pins along the side of the tissue during the dissection to stretch the tissue more.Remove the glass bead fiducial markers. Place DI water on the surface of the tissue with a pipette so that it completely covers the tissue in a bubble-like manner. To make the initial corner, select a corner of the pinned specimen, avoiding large chorda insertions in fragile areas.Then, cut the A/S layer by lightly dragging the scalpel over the tissue surface along the mounting holes for mechanical testing. The edges of the cut start to pull apart, revealing the F/V layer underneath. With blunt, thin forceps, firmly rub along the cut to further separate its edges.If the cut in the A/S layer does not start to pull apart, lightly trace over the same cut again with a scalpel. Make a second cut perpendicular to the first cut. If the two cuts are not connected, run the thin tweezers under the small area of tissue separating the two cuts.Then, carefully use the scissors to cut the tissue. Rub along the cuts of the corner with a thin forceps until the tissue separates from the F/V layer. As soon as a small piece of tissue is separated, grasp it with the large tweezers and gently pull it to separate the composite layers further.Continue to peel the tissue while grasping it with larger tweezers to prevent undesired ripping and tearing of the A/S composite layer, and rub the seam until it reaches the end of the two cuts made for the corner. If the first corner has major issues with separation, try a different corner as a starting point. Then, to make a second corner, extend the two cuts made for the first corner by placing the scalpel tip at the bottom of each cut and lightly dragging it along the tissue surface so that the cut extensions connect to the original cuts and continue to follow the tine or suture holes.Continue extending the cuts and peeling the top composite A/S layer back while rubbing the seam until one side is finished. Observe that the tissue will be entirely separated along one cut. Next, create a second corner perpendicular to the end of the fully peeled side.Extend the remaining cuts while avoiding large chorda insertions. Exclude the chorda insertions from the A/S separation area only when this exclusion will allow for an A/S specimen large enough for experimental characterizations. Continue separating the A/S and F/V layers by using the rubbing and pulling techniques employed for the first corner.Stop separating the tissue immediately if a tear or hole forms. To prevent tweezers from being caught, place the scissors in any hole that forms and cut the tissue away from the center. If the defect forms along the seam of separation, then begin separating the tissue along another edge.Look for interlayer connections that may appear when separating the tissue and prevent further tissue separation. Observe that these are thin but strong strands that must be carefully cut using scissors. Avoid creating a hole in the A/S layer or cutting downwards into the F/V layer.Continue this process until the largest possible sample of the A/S layer has been separated. Mark the orientation of the sample using the surgical pen. Finally, cut along the seam of separation for the remaining tissue edge with the scissors.Place the separated A/S composite layer flat on the cutting mat. If necessary, use the scalpel to straighten the edges of the tissue and create a square tissue shape suitable for biaxial mechanical testing. Place the A/S layer in DI water until it is ready to be tested.Mark the orientation of the F/V layer that remains on the wax board. Cut the largest square possible out of the area where the A/S layer was removed. Then, place it in DI water.Histological analysis of the intact leaflet and the two dissected layers verified if the tissue was correctly separated along the border between the spongiosa and fibrosa. The histology micrographs were used to determine the tissue layer thickness and constituent mass fractions using ImageJ software. The displacement controlled mechanical testing and post-processing produced membrane tension versus stretch data of the tricuspid valve anterior leaflet, tricuspid valve posterior leaflet, and tricuspid valve septal leaflet, describing the nonlinear mechanical behavior of the tissue.The polarized spatial frequency domain imaging data yielded color maps of the collagen fiber orientation and degree of optical anisotropy. Specifically, these color maps provided a comprehensive understanding of the collagen fiber architecture across the entire tissue specimen. It is important to note that the composite layer shrinks once they are separated from the pinned tissue.Collect the largest possible sample to ensure there is enough tissue for the subsequent testing.

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1.5K Views.  The University of Oklahoma. This method can isolate the contributions of tissue layers to the overall tissue behavior, providing new insight into the tissue microstructure. Our microdissection and testing framework allows us to directly compare the mechanical and microstructural properties of heart valve leaflets to their own composite layers. The microdissection can be difficult to master, hence, it is highly recommended to practice as much as possible before launching your study.First, gather the required mater...