Name: biological preparation and mechanical technique for determining viscoelastic properties of zonular fibers
Uploaded: 2021-12-16T13:30:00.000+00:00
Duration: 6 min 39 s
Description: Watch this Scientific Journal Video about Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers at JoVE.com

This work was supported by NIH R01 EY029130 (S.B.) and P30 EY002687 (S.B.), R01 HL53325 and the Ines Mandl Research Foundation (R.P.M.), the Marfan Foundation, and an unrestricted grant to the Department of Ophthalmology and Visual Sciences at Washington University from Research to Prevent Blindness. J.R. also received a grant from the University of Health Sciences and Pharmacy in support of this project.

All animal experiments were approved by the Washington University Animal Studies Committee and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Fabrication of specialized parts and construction of apparatus
<ol>
	<li>Fabrication of specialized parts
	<ol>
		<li>Probe fabrication. Hold a glass capillary at an angle as shown in the left panel of Figure 2A. Place a flame from a cigarette lighter about 2&#160;cm from one end and keep it there until the end bends by 90&#176;, as shown in the right panel of Figure 2A.</li>
		<li>Sample platform fabrication. Using 3D drawing software, design a platform measuring 30 x 30 x 5 mm and containing hemispherical indentations of 2.0, 2.5, and 3.0 mm in diameter, as shown in Figure 2B.</li>
		<li>Probe holder fabrication. Using the 3D drawing software, design a mount that holds the capillary probe and attach it to a micromanipulator (see Figure 2C). 
		NOTE: A sample 3D file for platform fabrication and probe holder fabrication in STL format is available on request from the corresponding author.</li>
		<li>Negative lens assembly. Place a negative cylindrical lens (-75 mm in focal length and approximately 50 mm in height and length) as shown in Figure 1C and Figure 1D to correct the distortion caused by the addition of fluid to the Petri dish (addition of fluid distorts the view of the dissected eye when imaged from the side).</li>
		<li>Glue the negative lens to one of the 2-slotted bases (see Figure 2D for positioning of the lens on the base).</li>
		<li>Assemble the remaining parts as shown in Figure 2D.</li>
		<li>Adjust the height of the post so that the lens barely hovers over the scale and tighten the screw in the post-holder.</li>
	</ol>
	</li>
	<li>Construction of apparatus
	<ol>
		<li>Install on a computer the logging program supplied with the scale, the microscope camera software, and the motorized micrometer controller application.</li>
		<li>Connect the motorized micrometer to the servo motor controller and the latter to the computer. Start the&#160;motor controller application and edit the motor settings. 
		NOTE: The motor settings, which are listed below, were chosen following preliminary experiments that revealed that stresses relaxed on a time scale of 10-20 s. Based on this determination, we selected a speed and acceleration that allowed the motor to complete a 50 &#181;m displacement in a time smaller than the relaxation time, but not too short to avoid jolting&#160;the sample. Here we chose a displacement time of about 5-10 s.</li>
		<li>Set the maximum velocity to 0.01 mm/s and the acceleration to 0.005 mm/s2.</li>
		<li>Install the camera in the inspection microscope and test the camera imaging software.</li>
		<li>Place the scale on the bench space devoted to the apparatus.</li>
		<li>Glue a 3D-printed platform (from step 1.1.2) to a Petri dish and add a 2-3 mm glass bead to one of the wells. Place the Petri dish on the scale so that the bead is located near the center of the pan.</li>
		<li>Replace the manual micrometer from the micromanipulator with the motorized one.</li>
		<li>Screw the two 4-40 screws into the probe holder. Attach the probe holder to the manipulator as shown in Figure 1C.</li>
		<li>Prepare a probe as illustrated in Figure 2A, place it inside the probe holder with the bent portion facing down, and tighten the screws.</li>
		<li>Position the micromanipulator on the table such that the tip of the probe is over the bead on the platform. Affix the micromanipulator to the table to prevent accidental movement during the experiment.</li>
		<li>Position the side microscope on the table so that the bead is at the center of its field of view and in focus.</li>
	</ol>
	</li>
</ol>
2. Sample preparation and data acquisition
<ol>
	<li>Eye fixation and dissection
	<ol>
		<li>Maintain wild-type mice and Magp1-null animals on an identical C57/BL6J background. Euthanize 1-month-old or 1-year-old mice by CO2 inhalation.</li>
		<li>Remove the eyes with fine forceps and fix the enucleated globes at 4 &#176;C overnight in 4% paraformaldehyde/phosphate-buffered saline (PBS, pH 7.4). Maintain a positive pressure of 15-20 mmHg in the eye during the fixation process, as described6. 
		NOTE: Experiments are conducted on male mice, to control for possible sex-related differences in the size of the ocular globe. The positive pressure ensures that the globe remains inflated, preserving the gap between the lens and the wall of the eye spanned by the zonular fibers.</li>
		<li>Wash the eyes for 10 min in PBS. Using ophthalmic surgical scissors and working under a stereomicroscope, make a full-thickness incision in the wall of the eye near the optic nerve head.</li>
		<li>Extend the cut forward to the equator, and then around the equatorial circumference of the eye. Take care to spare the delicate ciliary processes and associated zonular fibers.</li>
		<li>Remove the back of the globe, exposing the posterior surface of the lens.</li>
		<li>Use the forceps to remove a dissected eye from the buffer solution and place it on a dry task wipe with the cornea facing down. Gently drag the cornea over the surface of the wipe to dry it.</li>
		<li>Add 3 &#181;L of instant glue to the platform wells that will accommodate the eye in the Petri dish.</li>
		<li>Place the dish on the stage plate of the stereomicroscope so that the well with the glue is in view.</li>
		<li>Transfer the eye from the wipe to the edge of the well that contains glue. Then, carefully drag the eye into the well and quickly adjust its orientation so that the back of the lens is uppermost.</li>
		<li>Dry the exposed side of the lens by gently blotting it with the corner of a dry wipe.</li>
		<li>Apply a dab of instant glue to the bottom of a 50 mm Petri dish and cement the platform to it.</li>
	</ol>
	</li>
	<li>Measurement of zonular viscoelastic response
	<ol>
		<li>Turn on the scale, start the scale logging program and the camera software. Ensure that the logging program can acquire data for 30 min, as some trials can last that long.</li>
		<li>Switch on the servo motor controller and start the controller application on the computer. Make sure the controller is set to move in 50 &#181;m increments using motion parameters similar to those outlined in the NOTE in step 1.2.2.</li>
		<li>Create a 90&#176; bend in a capillary rod as described in step 1.1.1.</li>
		<li>Slip the bent capillary into the capillary probe holder and tighten the securing screws. 
		NOTE: To minimize sample dehydration, we recommend that steps 1-4 be completed prior to, or during, eye dissection.</li>
		<li>Add a small (~1 mm) bead of UV-curing glue to the tip of the capillary.</li>
		<li>Using the manual adjustments on the manipulator, move the tip of the capillary probe so that it is directly over the center of the lens. Check whether the bottom portion of the UV glue appears centered over the top of the lens when viewed from the front (by visual inspection) and the side (through the microscope camera).</li>
		<li>While looking through the camera, lower the probe tip until the UV glue makes contact with the lens and covers one-third to one-half of its upper surface.</li>
		<li>Use a low-intensity (~ 1 mW), directional, near-visible UV (380-400 nm) light source to cure the glue. 
		NOTE: These specifications suffice to cure the glue in a few seconds while minimizing the potential for inducing protein crosslinking. The UV light sources supplied with commercial UV glue pens meet these specifications.</li>
		<li>Add PBS solution to the dish until the eye is covered by fluid to a depth of at least 2 mm.</li>
		<li>Place the cylindrical lens in front of the inspection microscope and as close as possible to the Petri dish without touching it.</li>
		<li>Simultaneously start the logging program and a timer program. Take a picture of the eye/probe using the camera.</li>
		<li>After 60 s, initiate another 50 &#181;m displacement, and thereafter every 60 s until the experiment is complete, i.e., until all the fibers have been broken. Note that the signal will not return to baseline levels due to buffer evaporation during the experiment. Correct the ensuing drift in the readings during the data analysis, as exemplified in step 2.2.14.</li>
		<li>Upon completion of a run, save the scale logging data and export it into a format compatible with the spreadsheet, e.g., a .csv format. Save the lens pictures that were collected during the run.</li>
		<li>Import data into a spreadsheet. Use the first and the last scale reading to interpolate the drift in the background reading over time due to evaporation (see Figure 3). Subtract the interpolated reading from the reading at&#160;each time point. 
		NOTE: If using a spreadsheet, the interpolation can be performed automatically by entering the formula = B2 - $B$2 + ($B$2 - @INDIRECT(&#34;B&#34;&#38;COUNTA(B:B)))/(COUNTA(B:B)-2) * A2 in the cell to the right of the first scale reading, then moving&#160;the cursor to the right-lower corner of the cell and dragging it down to the last data value. The formula assumes that the data is organized in a column with the first data point appearing in cell B2. If desired, the data processed in step 2.2.14 may be analyzed with the quasi-elastic viscoelastic model developed by one of the co-authors, Dr. Matthew Riley4.</li>
	</ol>
	</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ciliary+body+in+accommodation.">The ciliary body in accommodation.</a> Transactions of the Ophthalmological Societies of the United Kingdom. 105, 208-219 (1986).</li><li>Michael, R., 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=Elastic+properties+of+human+lens+zonules+as+a+function+of+age+in+presbyopes.">Elastic properties of human lens zonules as a function of age in presbyopes.</a> Investigative Ophthalmology &amp; Visual Science. 53 (10), 6109-6114 (2012).</li><li>van Alphen, G. W., Graebel, W. 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The authors have nothing to disclose.

The zonule is an unusual ECM system where fibers are arranged symmetrically and can be manipulated identically by displacing the eye lens along the optical axis. The space can also be readily accessed without cellular disruption, allowing the fibers to be studied in an environment close to their native state. The pull-up technique takes advantage of this ECM presentation to manipulate the delicate fibers from mice, a genetically tractable system, and accurately quantify their mechanical properties. This has allowed us to examine the contribution of key ECM proteins (fibrillin-118, LTBP-24, and MAGP-1 reported here) to the biomechanical properties of the zonular fibers. Our analysis of fibrillin-1-deficient mice revealed that zonular fibers lacking fibrillin-1 weaken with age and eventually rupture, leading to the displacement of the lens within the eye (in humans, a condition known as ectopia lentis). Significantly, lens dislocation is also a common occurrence in patients with Marfan syndrome, a disease caused by mutations in the FBN1 gene20. Thus, the pull-up assay offers an opportunity to model aspects of human connective tissue disease in mice. In mice lacking LTPB-2 (a protein thought to be involved in the genesis of microfibrils), we were able to demonstrate that zonular fibers were produced in the absence of that protein, but ruptured at significantly lower stresses and eventually disintegrated with age4. These results suggest that LTBP-2 contributes to the longevity of the fibers rather than their synthesis. In the current study, we determined that MAGP-1-deficient fibers had similar viscoelastic properties to wild-type fibers but ruptured at lower stresses, with no sign of further age-related degradation. This would be consistent with a model in which fibers lacking MAGP-1 are intrinsically weaker as soon as they develop.
We note that the ultimate tensile strengths listed in Table 1 are estimated under the assumption that the fibers break somewhere mid-span. However, we cannot rule out the possibility that fiber failure is due to detachment from anchorage points on the lens surface or the ciliary body. If this were the case, the fracture tensile strength of the fiber could be higher than the values listed in Table 1. Microscopic analysis will be required to differentiate between these possibilities. Such analysis is far from trivial since the fibers involved are very thin (~0.5-0.6 &#181;m in width) and nearly index-matched to water, making them essentially invisible. In the absence of this additional information, we can only state that the ultimate tensile strengths listed in Table 1 represent their lower limits. It would also be interesting, in principle, to check whether the force measurements differ depending on which direction the lens is pulled. In practice, however, pulling the lens from the anterior side would require removing the iris without damaging the zonular fibers lying immediately beneath. Such precise dissection is beyond what we can currently achieve with the mouse eye.
The relative simplicity of the method and the high reproducibility of its results are desirable qualities for comparative studies of ECM mechanical properties. Additionally, as demonstrated here, it is also possible to use the pull-up assay to obtain absolute values of viscoelastic parameters by assuming a viscoelastic model and fitting the time curves to it. For instance, using a standard quasi-linear viscoelastic (QLV) model, we were able to extract values for instantaneous (G0) and equilibrium (G&#8734;) stiffnesses, the relaxation time constant (&#964;), and the ultimate tensile strength (&#963;f) of zonular fibers from wild-type mice, as well as those lacking LTBP-24 or MAGP-1. The G0 and G&#8734;&#160;values obtained for wild-type animals in both studies vary from 6.7 x 104 Pa to 2.3 x 105 Pa, a range broadly comparable to those found in much larger fibers derived from human, bovine, and porcine zonules (1.8 x 105-1.5 x 106 Pa)12,13,14,15,16. This agreement among species suggests that these are universal features of these fibers, and gives us confidence that meaningful viscoelastic parameters can be extracted with our method.
A critical step for obtaining quality viscoelastic responses is the orientation of the dissected eye glued to the platform (step 2.1.9). Minor tilting (less than 10&#176;) does not appear to affect the results significantly. Experiments performed outside this limit can generate curves with shapes that deviate from the ones shown in Figure 4. For example, some of those curves may possess two broad peaks instead of one.
Ideally, the procedure outlined in this paper would have been performed without fixation of the eyes, which limits our ability to assess the true viscoelastic parameters of fresh zonular fibers. However, after our preliminary experiments showed no significant difference between the paraformaldehyde-fixed and fresh samples, we decided to adopt fixation as it afforded several advantages. As alluded to in the Protocol, the use of fixed tissues helps to preserve the native stretch of the fibers for the pull-up experiments. Additionally, we found that fixation promoted greater adhesion of the UV glue to the eye capsule, thus reducing the chances of the probe detaching from the lens during the pull-up action, as commonly experienced with fresh samples (probe detachment can be readily recognized as a sudden return of the force to a baseline level). The fixation also prevented buckling of the eyewall in the direction of the pull. Despite this limitation, our method provides a robust approach for determining the relative contribution of protein components to the viscoelastic properties of the zonular fibers.
Although our work to date has focused on the contribution of specific proteins, the method could readily be adapted to study the effect of factors external to the fibers on their mechanical properties. Such factors include temperature, pH, calcium concentration, and the presence or absence of cross-linking enzymes. High-precision measurements could be achieved using our method in differential mode, i.e., by pre-tensioning the zonular fibers with an initial stress/strain, and then reading the difference in tension that ensues when external conditions are altered. Some of these interventions may conceivably affect the elasticity of the tissues that surround the zonule and hence produce changes in tension that compete with those generated in the zonule. Control measurements with isolated tissues would be needed to assess their relevance to the proposed experiments. We expect that such effects may be negligible, based on observations with the side camera showing that contiguous tissues behave as highly stiff materials that undergo essentially no deformation even when the zonular fibers are fully stretched.

The eye of a vertebrate contains a living optical lens that helps focus images on the retina1. The lens is suspended on the optical axis by a system of delicate, radially-oriented fibers, as illustrated in Figure 1A. At one end, the fibers attach to the lens equator and, at the other, to the surface of the ciliary body. Their lengths span distances ranging from 150 &#181;m in mice to 1 mm in humans. Collectively, these fibers are known as the zonule of Zinn2, the ciliary zonule, or simply the zonule. Ocular trauma, disease, and certain genetic disorders can affect the integrity of the zonular fibers3, resulting in their eventual failure and accompanying loss of vision. In mice, the fibers have a core comprised mostly of the protein fibrillin-2, surrounded by a mantle rich in fibrillin-14. Although zonular fibers are unique to the eye, they bear many similarities to elastin-based ECM fibers found elsewhere in the body. The latter are covered by a fibrillin-1 mantle5&#160;and have similar dimensions to&#160;zonular fibers6. Other proteins, such as latent-transforming growth factor &#946;-binding proteins (LTBPs) and microfibril-associated glycoprotein-1 (MAGP-1), are found in association with both types of fibers7,8,9,10,11. The elastic modulus of zonular fibers is in the range of 0.18-1.50 MPa12,13,14,15,16, comparable to that of elastin-based fibers (0.3-1.2 MPa)17. These architectural and mechanical similarities lead us to believe that any insight into the roles of zonule-associated proteins may help elucidate their roles in other ECM elastic fibers.
The main purpose of developing the method described here is to gain insights into the role of specific zonular proteins in the progression of inherited eye disease. The general approach is to compare the viscoelastic properties of zonular fibers in wild-type mice with those of mice carrying targeted mutations in genes encoding zonular proteins. While several methods have been used previously to measure the elasto-mechanical properties of zonular fibers, all were designed for the eyes of much larger animals12,13,14,15,16. As such models are not genetically tractable; we sought to develop an experimental method that was better suited to the small and delicate eyes of mice.
The method we developed for assessing the viscoelasticity of mouse zonular fibers is a technique we refer to as the pull-up assay4,18, which is summarized visually in Figure 1. A detailed description of the pull-up method and the analysis of the results is provided below. We begin by describing the construction of the apparatus, including the three-dimensional (3D)-printed parts used in the project. Next, we detail the protocol used for obtaining and preparing the eyes for the experiment. Lastly, we provide step-by-step instructions on how to obtain data for the determination of the viscoelastic properties of zonular fibers. In the Representative Results section, we share previously unpublished data obtained with our method on the viscoelastic properties of zonular fibers from mice lacking MAGP-119 as well as a control set obtained from age-matched wild-type animals. Finally, we conclude with general remarks on the advantages and limitations of the method, and suggestions for potential experiments that may elucidate how environmental and biochemical factors affect the viscoelastic properties of ECM fibers.

<table><tbody><tr><td>1/4-20 hex screws 3/4 inch long</td><td>Thorlabs</td><td>SH25S075</td><td></td></tr><tr><td>1/4-20 nut</td><td>Hardware store</td><td></td><td></td></tr><tr><td>3D SLA printer</td><td>Anycubic</td><td>Photon</td><td></td></tr><tr><td>4-40 screws 3/8 inch long, 2</td><td>Hardware store</td><td></td><td></td></tr><tr><td>Capillaries, OD 1.2 mm and 3 inches long, no filament</td><td>WPI</td><td>1B120-3</td><td></td></tr><tr><td>Cyanoacrylate (super) glue</td><td>Loctite</td><td></td><td></td></tr><tr><td>Digital Scale accurate to 0.01 g</td><td>Vernier</td><td>OHAUS Scout 220</td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td>Spreadsheet</td></tr><tr><td>Gas cigarette lighter</td><td></td><td></td><td></td></tr><tr><td>Inspection/dissection microscope</td><td>Amscope</td><td>SKU: SM-4NTP</td><td>Working distance ~ 15 cm</td></tr><tr><td>Micromanipulator, Economy 4-axis</td><td>WPI</td><td>Kite-L</td><td></td></tr><tr><td>Motorized micrometer</td><td>Thorlabs</td><td>Z812B</td><td></td></tr><tr><td>Negative cylindrical lens</td><td>Thorlabs</td><td>LK1431L1</td><td>-75 mm focal length</td></tr><tr><td>Petri dishes, 50 mm</td><td></td><td></td><td></td></tr><tr><td>Post holder, 3 inches</td><td>Thorlabs</td><td>PH3</td><td></td></tr><tr><td>Post, 4 inches</td><td>Thorlabs</td><td>TR4</td><td></td></tr><tr><td>Scale logging software</td><td>Vernier</td><td>LoggePro</td><td></td></tr><tr><td>Servo motor controller</td><td>Thorlabs</td><td>KDC101</td><td></td></tr><tr><td>Servo motor controller software</td><td>Thorlabs</td><td>APT</td><td></td></tr><tr><td>Slotted base, 1</td><td>Thorlabs</td><td>BA1S</td><td></td></tr><tr><td>Slotted bases, 2</td><td>Thorlabs</td><td>BA2</td><td></td></tr><tr><td>Stand for micromanipular</td><td>WPI</td><td>M-10</td><td></td></tr><tr><td>USB-camera for microscope</td><td>Amscope</td><td>SKU: MD500</td><td></td></tr><tr><td>UV activated glue with UV source</td><td>Amazon</td><td></td><td></td></tr></tbody></table>

biological preparation and mechanical technique for determining viscoelastic properties of zonular fibers

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular matrix (ECM), the protein meshwork that binds cells and tissues together. How the elastic properties of an ECM network relate to its composition, and whether the relaxation properties of the ECM play a physiological role, are questions that have yet to be fully addressed. Part of the challenge lies in the complex architecture of most ECM systems and the difficulty in isolating ECM components without compromising their structure. One exception is the zonule, an ECM system found in the eye of vertebrates. The zonule comprises fibers hundreds to thousands of micrometers in length that span the cell-free space between the lens and the eyewall. In this report, we describe a mechanical technique that takes advantage of the highly organized structure of the zonule to quantify its viscoelastic properties and to determine the contribution of individual protein components. The method involves dissection of a fixed eye to expose the lens and the zonule and employs a pull-up technique that stretches the zonular fibers equally while their tension is monitored. The technique is relatively inexpensive yet sensitive enough to detect alterations in viscoelastic properties of zonular fibers in mice lacking specific zonular proteins or with aging. Although the method presented here is designed primarily for studying ocular development and disease, it could also serve as an experimental model for exploring broader questions regarding the viscoelastic properties of elastic ECM&#39;s and the role of external factors such as ionic concentration, temperature, and interactions with signaling molecules.

The pull-up technique described here provides a straightforward approach for determining viscoelastic properties of zonular fibers in mice. In brief, the mouse eye is first preserved by injection of a fixative at physiological intraocular pressure. This approach maintains the natural inflation of the eye and keeps the fibers properly pre-tensioned (fixation was deemed acceptable after preliminary experiments demonstrated it did not alter the elasticity or strength of the fibers significantly). The back of the mouse eye is then removed by dissection to expose the lens and the zonular fibers that suspend it. The front of the eye is affixed to a platform and placed inside a Petri dish that rests on a digital scale. Next, a glass capillary attached to a micromanipulator is cemented to the posterior surface of the lens. The lens is then raised in 50 &#181;m increments while the force on the scale is recorded. A reduction in the apparent weight of the preparation provides information about the forces that stretch the fibers. Each displacement is followed by an equilibration period lasting about 1 min to observe any relaxation of the stress induced by the displacement. Finally, the results are analyzed using a quasi-linear viscoelastic model designed specifically for the geometry of the mouse zonular fibers and the direction of the pull in for the assay4.
Typical viscoelastic data obtained with our method are shown in Figure 3. The curve appears inverted (negative) since the lift force on the lens reduces the weight of the dish/platform/eye assembly on the scale by an equivalent amount. The response includes instantaneous force spikes during each of the 50 &#181;m vertical displacements of the lens, followed by a relaxation phase with a lifetime on the order of 10 s. A similar stress relaxation has been observed for bovine zonular fibers12. The magnitude of the instantaneous and relaxed forces increases with each step up to about 1000 s (~800 &#181;m total displacement) and then begins to drop as fibers start to fail. Zonule failure is complete by the 1,500 s time point (~1.25 mm total displacement). Note that due to the evaporation of buffer in the course of the experiment, the curve does not return to the initial reading after the lens is freed from the eye.
Figure 4 contrasts the responses obtained for a Magp-1 knockout mouse (red curve) and an age-matched wild-type animal (blue curve). These curves have been corrected for evaporation, inverted, and the raw measurements of mass (see Figure 3) are now expressed as force (with units of mN). The initial viscoelastic response of the Magp-1-depleted zonule (time 0-600 s) closely resembles that of the wild-type, suggesting that the viscoelastic properties of the zonule were not significantly altered by the absence of Magp-1. However, the fibers appear to rupture at a much lower tension compared to their wild-type counterparts.
To illustrate the reliability of the method, we gathered data from multiple animals on the maximum instantaneous force applied to the eyes before their fibers ruptured. The results are shown in Figure 5. The data for 1-month-old mice exhibit very small values for the standard error of the mean (SEM) despite the relatively low number of samples used (n = 5 or 6), suggesting high reproducibility. The results indicate that the strength of the fibers differs significantly between the two genotypes (p-value = 2.4 x 10-6). Results not shown in the figures&#160;also suggest that there is a subtle but statistically significant increase in breaking force strength with age for wild-type animals (p-value = 0.024).
The pull-up method also can generate quantitative estimates of the viscoelastic parameters that account for the observed variations in temporal responses. Table 1 summarizes best-fit parameters to our MAGP-1 data, obtained with a quasi-linear viscoelastic model described previously4. The results show that both&#160;MAGP-1 deletion and aging can have&#160;highly significant impacts on some of the mechanical properties of zonular fibers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63171/63171fig01.jpg" /> 
Figure 1: A visual summary of the pull-up method. (A) Cross-sectional view of a vertebrate eye showing the lens and the zonular fibers that suspend it. (B) A general approach for determining viscoelastic behavior in zonular fibers by displacing the lens upward (away from the cornea). (C) Actual view of a dissected eye glued onto a platform with its lens being pulled upward by a glass probe attached to a micromanipulator. (D) Schematic of the entire apparatus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63171/63171fig01large.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/63171/63171fig02.jpg" /> 
Figure 2: Fabrication of various parts. (A) Fabrication of the glass probe. A glass capillary is held at an angle and a flame is applied at a spot roughly 2 cm from one end. Within a few seconds, the end of the capillary starts to fall. The flame is removed when the end of the capillary is bent at about 90&#176;. (B) Fabrication of the eye platform. The part is fabricated with a 3D stereolithography (SLA) printer. It measures 30 x 30 x 5 mm and contains three hemispherical indentations with 2.0, 2.5, and 3.0 mm diameters into which dissected eyes of various sizes are glued. (C) Fabrication of probe holder. This part was also fabricated with a 3D SLA printer. It consists of two orthogonal, 7.3 mm diameter rods. The lower rod contains a 1.5 mm bore and two 2.5 mm through holes on the outer surface to accommodate metal screws that secure the capillary probe in place. (D) Negative lens assembly. Images captured by the side microscope contain an astigmatic distortion due to the curvature of the Petri dish and the buffer solution. The lens assembly is designed to compensate for the distortion, allowing the side microscope to capture images in sharp focus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63171/63171fig02large.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/63171/63171fig03.jpg" /> 
Figure 3: Typical raw data obtained with the assay. The graph shown was recorded with logging software that records data from a digital scale with a 0.01 g accuracy. The left edge of the graph (time 0) reflects the weight of the sample without a lifting force. The y-axis depicts mass in g. The lens is then lifted in 50 &#181;m steps until all zonular fibers are broken and the Petri dish again rests fully on the scale. Note that the end reading is offset from the initial reading. The offset is due to gradual evaporation of the buffer solution during the course of the experiment and can be accounted for during data analysis, as outlined in step 2.2.14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63171/63171fig03large.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/63171/63171fig04.jpg" /> 
Figure 4: Representative zonular force-displacement curves for wild-type and MAGP-1-deficient mice. The graph compares the viscoelastic response obtained after discrete displacements of the lens away from its equilibrium position. The response of an eye from a MAGP-1 knockout (KO) mouse tracks that of an age-matched wild-type animal up to the point where the fibers in the knockout mouse break prematurely. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63171/63171fig04large.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/63171/63171fig05.jpg" /> 
Figure 5: Zonular fiber breaking forces obtained with the pull-up method for MAGP-1 KO versus wild-type mice and at two ages. All measurements shown are based on n = 5 or 6 eyes, with error bars representing the standard error of the mean (SEM). Abbreviations: WT = wild-type; KO = MAGP-1 knockout. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63171/63171fig05large.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 colspan="2">Genotype/age</td>
			<td>G0 (Pa)</td>
			<td>G&#8734; (Pa)</td>
			<td>&#430; (sec)</td>
			<td>&#963;f (Pa)</td>
		</tr>
		<tr>
			<td rowspan="3">WT 1-month</td>
			<td>MEAN</td>
			<td>2.34E+05</td>
			<td>9.33E+04</td>
			<td>16.3</td>
			<td>9.61E+05</td>
		</tr>
		<tr>
			<td>SD</td>
			<td>2.83E+04</td>
			<td>2.94E+04</td>
			<td>3.4</td>
			<td>1.25E+05</td>
		</tr>
		<tr>
			<td>95% Cl</td>
			<td>5.55E+04</td>
			<td>5.76E+04</td>
			<td>6.7</td>
			<td>2.45E+05</td>
		</tr>
		<tr>
			<td rowspan="3">KO 1-month</td>
			<td>MEAN</td>
			<td>2.73E+05</td>
			<td>6.74E+04</td>
			<td>17.6</td>
			<td>4.44E+05</td>
		</tr>
		<tr>
			<td>SD</td>
			<td>6.30E+04</td>
			<td>2.06E+04</td>
			<td>3.8</td>
			<td>7.85E+04</td>
		</tr>
		<tr>
			<td>95% Cl</td>
			<td>1.23E+05</td>
			<td>4.03E+04</td>
			<td>7.5</td>
			<td>1.54E+05</td>
		</tr>
		<tr>
			<td></td>
			<td>p values</td>
			<td>0.25</td>
			<td>0.12</td>
			<td>0.58</td>
			<td>0.000022</td>
		</tr>
		<tr>
			<td rowspan="3">WT 1-year</td>
			<td>MEAN</td>
			<td>1.98E+05</td>
			<td>7.42E+04</td>
			<td>17</td>
			<td>1.41E+06</td>
		</tr>
		<tr>
			<td>SD</td>
			<td>1.17E+05</td>
			<td>2.39E+04</td>
			<td>9.1</td>
			<td>2.44E+05</td>
		</tr>
		<tr>
			<td>95% Cl</td>
			<td>2.29E+05</td>
			<td>4.69E+04</td>
			<td>17.9</td>
			<td>4.79E+05</td>
		</tr>
		<tr>
			<td rowspan="3">KO 1-Year</td>
			<td>MEAN</td>
			<td>1.70E+04</td>
			<td>2.46E+04</td>
			<td>12.9</td>
			<td>5.05E+05</td>
		</tr>
		<tr>
			<td>SD</td>
			<td>9.06E+03</td>
			<td>8.04E+03</td>
			<td>7.4</td>
			<td>1.48E+05</td>
		</tr>
		<tr>
			<td>95% Cl</td>
			<td>1.78E+04</td>
			<td>1.58E+04</td>
			<td>14.4</td>
			<td>2.91E+05</td>
		</tr>
		<tr>
			<td></td>
			<td>p values</td>
			<td>0.0063</td>
			<td>0.001</td>
			<td>0.41</td>
			<td>0.000014</td>
		</tr>
		<tr>
			<td rowspan="2">p values, age</td>
			<td>WT</td>
			<td>0.46</td>
			<td>0.23</td>
			<td>0.85</td>
			<td>0.002</td>
		</tr>
		<tr>
			<td>KO</td>
			<td>0.0007</td>
			<td>0.0068</td>
			<td>0.26</td>
			<td>0.44</td>
		</tr>
	</tbody>
</table>
Table 1: Viscoelastic properties obtained with a quasi-linear viscoelastic (QLV) model. Data scans like those shown in Figure 4 were analyzed with a QLV model developed specifically for the pull-up assay and the mouse zonule. Best fit parameters for the instantaneous (G0) and equilibrium (G&#8734;) stiffnesses, the relaxation time constant (&#964;), and the ultimate tensile strength (&#963;f) are shown. Abbreviations: SD = standard deviation; CI = confidence interval.

Watch this Scientific Journal Video about Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers at JoVE.com

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

The protocol describes a method for the study of extracellular matrix viscoelasticity and its dependence on protein composition or environmental factors. The matrix system targeted is the mouse zonule. The performance of the method is demonstrated by comparing the viscoelastic behavior of wild-type zonular fibers with those lacking microfibril-associated glycoprotein-1.

Elasticity is essential to the function of tissues such as blood vessels, muscles, and lungs. This property is derived mostly from the extracellular ...

biological-preparation-mechanical-technique-for-determining

Nanyang Technological University

Research

JoVE Journal

Medicine

2.0K Views.  University of Health Sciences and Pharmacy. Het belang van onze methode ligt in het vermogen om de vraag te beantwoorden, wat zijn de visco-elastische eigenschappen van zonulaire vezels, de vezels die de lens in het oog ophangen? Het grote voordeel van onze techniek is dat het de visco-elastische eigenschappen van de kleine en delicate zonulaire vezels van muizen met en zonder gerichte genmodificaties kan kwantificeren. Mutaties in genen die coderen voor microfibrileiwitten liggen ten grondslag aan verschillende erfelijke syndromale aa...

Video: Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Bioengineering

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sample Preparation in Quartz Crystal Microbalance Measurements of Protein Adsorption and Polymer Mechanics

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of the data and determines which properties of the film can be quantified. The quartz crystal microbalance offers a uniquely sensitive platform for measuring fine changes in mass and/or mechanical properties of an applied film by observing the changes in mechanical resonance of a quartz crystal oscillating at high frequency. The advantages of this approach include its experimental versatility, ability to study changes in properties over a wide range of experimental time lengths, and the use of small sample sizes. We demonstrate that, based on the thickness and shear modulus of the layer deposited on the sensor, we can acquire different information from the material. Here, this concept is specifically exploited to display experimental parameters resulting in mass and viscoelastic calculations of adsorbed collagen on gold and polyelectrolyte complexes during swelling as a function of salt concentration.

The quartz crystal microbalance can provide accurate mass and viscoelastic properties for films in the micron or submicron range, which is relevant for investigations in biomedical and environmental sensing, coatings, and polymer science. The sample thickness influences which information can be obtained from the material in contact with the sensor.

In this study, we present various examples of how thin film preparation for quartz crystal microbalance experiments informs the appropriate modeling of ...

Chemistry

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should mimic the human tissues they are aiming to replace; to provide the required anatomical shape, the materials must be able to sustain the mechanical forces they will experience when implanted at the defect site. Although the mechanical properties of tissue-engineered scaffolds are of great importance, many human tissues that undergo restoration with engineered materials have not been fully biomechanically characterized. Several compressive and tensile protocols are reported for evaluating materials, but with large variability it is difficult to compare results between studies. Further complicating the studies is the often destructive nature of mechanical testing. Whilst an understanding of tissue failure is important, it is also important to have knowledge of the elastic and viscoelastic properties under more physiological loading conditions.
This report aims to provide a minimally destructive protocol to evaluate the compressive and tensile properties of human soft tissues. As examples of this technique, the tensile testing of skin and the compressive testing of cartilage are described. These protocols can also be directly applied to synthetic materials to ensure that the mechanical properties are similar to the native tissue. Protocols to assess the mechanical properties of human native tissue will allow a benchmark by which to create suitable tissue-engineered substitutes.

Tissue biomechanics is important for maintaining cell shape and function and for determining phenotype. This report demonstrates non-destructive mechanical protocols for characterizing elastic and viscoelastic properties of human soft tissues, which can be directly applied to tissue-engineered substrates to allow a close matching of engineered materials to native tissue.

Regenerative medicine aims to engineer materials to replace or restore damaged or diseased organs. The mechanical properties of such materials should ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue shape. Manipulating tissue shape on the micrometer to centimeter scale can direct cell alignment, alter effective mechanical properties, and address limitations related to nutrient diffusion. In addition, the vessel in which a tissue is prepared can impart mechanical constraints on the tissue, resulting in stress fields that can further influence both the cell and matrix structure. Shaped tissues with highly reproducible dimensions also have utility for in vitro assays in which sample dimensions are critical, such as whole tissue mechanical analysis.
This manuscript describes an alternative fabrication method utilizing negative master molds prepared from laser etched acrylic: these molds perform well with polydimethylsiloxane (PDMS), permit designs with dimensions on the centimeter scale and feature sizes smaller than 25 &#181;m, and can be rapidly designed and fabricated at a low cost and with minimal expertise. The minimal time and cost requirements allow for laser etched molds to be rapidly iterated upon until an optimal design is determined, and to be easily adapted to suit any assay of interest, including those beyond the field of tissue engineering.

Herein we present a rapid, facile, and low-cost method for fabricating custom polydimethylsiloxane molds that can be used for producing hydrogel-based engineered tissues with complex geometries. We additionally describe results from mechanical and histological assessments conducted on engineered cardiac tissues produced using this technique.

As the field of tissue engineering has continued to mature, there has been increased interest in a wide range of tissue parameters, including tissue ...

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 ...

Experimental and Data Analysis Workflow for Soft Matter Nanoindentation

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft biomaterials and cells. This approach has gained a central role in the fields of mechanobiology, biomaterials design&#160;and tissue engineering, to obtain a proper mechanical characterization of soft materials with a resolution comparable to the size of single cells (&#956;m). The most popular strategy to acquire such experimental data is to employ an atomic force microscope (AFM); while this instrument offers an unprecedented resolution in force (down to pN) and space (sub-nm), its usability is often limited by its complexity that prevents routine measurements of integral indicators of mechanical properties, such as Young&#39;s Modulus (E). A new generation of nanoindenters, such as those based on optical fiber sensing technology, has recently gained popularity for its ease of integration while allowing to apply sub-nN forces with &#181;m spatial resolution, therefore being suitable to probe local mechanical properties of hydrogels and cells.
In this protocol, a step-by-step guide detailing the experimental procedure to acquire nanoindentation data on hydrogels and cells using a commercially available ferrule-top optical fiber sensing nanoindenter is presented. Whereas some steps are specific to the instrument used herein, the proposed protocol can be taken as a guide for other nanoindentation devices, granted some steps are adapted according to the manufacturer&#39;s guidelines. Further, a new open-source Python software equipped with a user-friendly graphical user interface for the analysis of nanoindentation data is presented, which allows for screening of incorrectly acquired curves, data filtering, computation of the contact point through different numerical procedures, the conventional computation of E, as well as a more advanced analysis particularly suited for single-cell nanoindentation data.

The protocol presents a complete workflow for soft material nanoindentation experiments, including hydrogels and cells. First, the experimental steps to acquire force spectroscopy data are detailed; then, the analysis of such data is detailed through a newly developed open-source Python software, which is free to download from GitHub.

Nanoindentation refers to a class of experimental techniques where a micrometric force probe is used to quantify the local mechanical properties of soft ...

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.

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 ...

Biological Preparation and Mechanical Technique for Determining Viscoelastic Properties of Zonular Fibers

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