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 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 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—the atrialis (A), the spongiosa (S), the fibrosa (F), and the ventricularis (V)—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 (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.
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
2. Polarized spatial frequency domain imaging
3. Microdissection of tricuspid valve leaflet composite layers
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'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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm. Abbreviations: atrialis/spongiosa; F/V = fibrosa/ventricularis; VIC = valvular interstitial cell. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Composite Layer | λcirc | λrad |
A/S | 1.26 ± 0.05 | 1.37 ± 0.05 |
F/V | 1.17 ± 0.03 | 1.32 ± 0.08 |
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. Please click here to download this File.
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. Please click here to download this File.
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. Please click here to download this File.
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 (>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 and extensive studies from the literature4,5,16,17 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 authors have nothing to disclose.
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's Heart Foundation Predoctoral Fellowship (Award #821298). All of this support is gratefully acknowledged.
10% Formalin Solution, Neutral Buffered | Sigma-Aldrich | HT501128-4L | |
Alconox Detergent | Alconox | cleaning compound | |
BioTester – Biaxial Tester | CellScale Biomaterials Testing | 1.5 N Load Cell Capacity | |
Cutting Mat | Dahle | B0027RS8DU | |
Deionized Water | N/A | ||
Fine-Tipped Tool | HTI INSTRUMENTS | NSPLS-12 | |
Forceps – Curved | Scientific Labwares | 16122 | |
Forceps – Thick | Scientific Labwares | 161001078 | |
Forceps – Thin | Scientific Labwares | 16127 | |
LabJoy | CellScale Biomaterials Testing | Version 10.66 | |
Laser Displacement Sensor | Keyence | IL-030 | |
Liquid Cyanoacrylate Glue | Loctite | 2436365 | |
MATLAB | MathWorks | Version 2020a | |
Micro Scissors | HTI Instruments | CAS55C | |
Pipette | Belmaks | 360758081051Y4 | |
Polarized Spatial Frequency Domain Imaging Device | N/A | 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. |
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Scalpel | THINKPRICE | TP-SCALPEL-3010 | |
Single Edge Industrial Razor Blades (Surgical Carbon Steel) | VWR International | H3515541105024 | |
Surgical Pen | LabAider | LAB-Skin-6 | |
T-Pins | Business Source | BSN32351 | |
Wax Board | N/A | Made in-house using modeling wax and baking tray | |
Weigh Boat | Pure Ponta | mdo-azoc-1030 |