Funding support from the National Natural Science Foundation of China (grant 31870941), Natural Science Foundation of Shanghai (grant 22ZR1429600), and the Science and Technology Commission of Shanghai Municipality (grant 19441907700) is acknowledged.

1. Gelatin phantom preparation
<ol>
	<li>Weigh gelatin, glycerol, and water according to Table 1. Mix the gelatin powder with water to obtain the gelatin solution. 
	NOTE: The concentrations of the individual components for preparing the two phantoms are shown in Table 1. The higher the concentration of gelatin, the stiffer the phantom.</li>
	<li>Heat the gelatin solution to 60 &#176;C in a water bath. Add glycerol to the gelatin solution while maintaining the tem...

<ol>
	<li>Qiu, S., 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=Viscoelastic+characterization+of+injured+brain+tissue+after+controlled+cortical+impact+(CCI)+using+a+mouse+model.">Viscoelastic characterization of injured brain tissue after controlled cortical impact (CCI) using a mouse model.</a> Journal of Neuroscience Methods. 330, 108463 (2020).</li><li>Garcia, 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=Dynamic+patterns+of+cortical+expansion+during+folding+of+the+preterm+human+brain.">Dynamic patterns of cortical expansion during folding of the preterm human brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (12), 3156-3161 (2018).</li><li>Qiu, S., 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=Characterizing+viscoelastic+properties+of+breast+cancer+tissue+in+a+mouse+model+using+indentation.">Characterizing viscoelastic properties of breast cancer tissue in a mouse model using indentation.</a> Journal of Biomechanics. 69, 81-89 (2018).</li><li>Yin, Z., 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+new+method+for+quantification+and+3D+visualization+of+brain+tumor+adhesion+using+slip+interface+imaging+in+patients+with+meningiomas.">A new method for quantification and 3D visualization of brain tumor adhesion using slip interface imaging in patients with meningiomas.</a> European Radiology. 31 (8), 5554-5564 (2021).</li><li>Streitberger, K. -. 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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=Standard-space+atlas+of+the+viscoelastic+properties+of+the+human+brain.">Standard-space atlas of the viscoelastic properties of the human brain.</a> Human Brain Mapping. 41 (18), 5282-5300 (2020).</li><li>Seyedpour, S. M., 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=Application+of+magnetic+resonance+imaging+in+liver+biomechanics:+A+systematic+review.">Application of magnetic resonance imaging in liver biomechanics: A systematic review.</a> Frontiers in Physiology. 12, 733393 (2021).</li><li>Okamoto, R. J., Clayton, E. H., Bayly, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscoelastic+properties+of+soft+gels:+comparison+of+magnetic+resonance+elastography+and+dynamic+shear+testing+in+the+shear+wave+regime.">Viscoelastic properties of soft gels: comparison of magnetic resonance elastography and dynamic shear testing in the shear wave regime.</a> Physics in Medicine and Biology. 56 (19), 6379-6400 (2011).</li><li>Feng, Y., 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+multi-purpose+electromagnetic+actuator+for+magnetic+resonance+elastography.">A multi-purpose electromagnetic actuator for magnetic resonance elastography.</a> Magnetic Resonance Imaging. 51, 29-34 (2018).</li><li>Zeng, W., 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=Nonlinear+inversion+MR+elastography+with+low-frequency+actuation.">Nonlinear inversion MR elastography with low-frequency actuation.</a> IEEE Transactions on Medical Imaging. 39 (5), 1775-1784 (2020).</li><li>Wang, 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=Fast+magnetic+resonance+elastography+with+multiphase+radial+encoding+and+harmonic+motion+sparsity+based+reconstruction.">Fast magnetic resonance elastography with multiphase radial encoding and harmonic motion sparsity based reconstruction.</a> Physics in Medicine and Biology. 67 (2), (2022).</li><li>Herraez, M. A., Burton, D. R., Lalor, M. J., Gdeisat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+two-dimensional+phase-unwrapping+algorithm+based+on+sorting+by+reliability+following+a+noncontinuous+path.">Fast two-dimensional phase-unwrapping algorithm based on sorting by reliability following a noncontinuous path.</a> Applied Optics. 41 (35), 7437-7444 (2002).</li><li>Gordon-Wylie, S. W., 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=MR+elastography+at+1+of+gelatin+phantoms+using+3D+or+4D+acquisition.">MR elastography at 1 of gelatin phantoms using 3D or 4D acquisition.</a> Journal of Magnetic Resonance. 296, 112-120 (2018).</li><li>McGarry, M., 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=Uniqueness+of+poroelastic+and+viscoelastic+nonlinear+inversion+MR+elastography+at+low+frequencies.">Uniqueness of poroelastic and viscoelastic nonlinear inversion MR elastography at low frequencies.</a> Physics in Medicine and Biology. 64 (7), 075006 (2019).</li><li>Zampini, M. A., Guidetti, M., Royston, T. J., Klatt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+viscoelastic+parameters+in+Magnetic+Resonance+Elastography:+a+comparison+at+high+and+low+magnetic+field+intensity.">Measuring viscoelastic parameters in Magnetic Resonance Elastography: a comparison at high and low magnetic field intensity.</a> Journal of the Mechanical Behavior of Biomedical Materials. 120, 104587 (2021).</li><li>Ozkaya, 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=Brain-mimicking+phantom+for+biomechanical+validation+of+motion+sensitive+MR+imaging+techniques.">Brain-mimicking phantom for biomechanical validation of motion sensitive MR imaging techniques.</a> Journal of the Mechanical Behavior of Biomedical Materials. 122, 104680 (2021).</li><li>Guidetti, M., 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=Axially-+and+torsionally-polarized+radially+converging+shear+wave+MRE+in+an+anisotropic+phantom+made+via+Embedded+Direct+Ink+Writing.">Axially- and torsionally-polarized radially converging shear wave MRE in an anisotropic phantom made via Embedded Direct Ink Writing.</a> Journal of the Mechanical Behavior of Biomedical Materials. 119, 104483 (2021).</li><li>Badachhape, A. A., 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=The+relationship+of+three-dimensional+human+skull+motion+to+brain+tissue+deformation+in+magnetic+resonance+elastography+studies.">The relationship of three-dimensional human skull motion to brain tissue deformation in magnetic resonance elastography studies.</a> Journal of Biomechanical Engineering. 139 (5), 0510021 (2017).</li></ol>

Gelatin phantoms are commonly used as tissue-mimicking materials for testing and validation of algorithms and devices17,19,22,23,24,25,26,27. One of the pioneering studies using the gelatin phantom to compare MRE and dynamic shear testing was presented by O...

Most of the soft biological tissues appear to have viscoelastic properties that are important to understand their injury and development1,2. In addition, viscoelastic properties are important biomarkers in the diagnosis of a variety of diseases such as fibrosis and cancer3,4,5,6. Therefore, the characterization of viscoelastic properties of soft tissues is crucial. Among the many characterization techniques used, ex vivo mechanical testing of tissue samples and in vivo elastography using biomedical imaging are the two widely used methods.
Although various mechanical testing techniques have been used for soft tissue characterization, the requirements for sample size and testing conditions are not easy to be satisfied. For example, shear testing needs to have samples fixed firmly between the shear plates7. Biaxial testing is more suitable for membrane tissue and has specific clamping requirements8,9. A compression test is commonly used for tissue testing, but cannot characterize specific positions within one sample10. The indentation test does not have additional requirements to fix the tissue sample and can be used to measure many biological tissue samples such as the brain and liver. In addition, with a small indenter head, regional properties within a sample could be tested. Therefore, indentation tests have been adopted to test a variety of soft tissues1,3,11.
Characterizing the biomechanical properties of soft tissues in vivo is important for translational studies and clinical applications of biomechanics. Biomedical imaging modalities such as ultrasound (US) and magnetic resonance (MR) imaging are the most used techniques. Although US imaging is relatively cheap and easy to carry out, it suffers from low contrast and is hard to measure organs such as the brain. Capable of imaging deep structures, MR Elastography (MRE) could measure a variety of soft tissues6,12, especially the brain13,14. With applied external vibration, MRE could measure the viscoelastic properties of soft tissues at a specific frequency.
Studies have shown that at 50-60 Hz, the shear modulus of the normal brain is ~1.5-2.5kPa5,6,13,14,15 and ~2-2.5 kPa for normal liver16. Therefore, gelatin phantoms that have similar biomechanical properties have been widely used for mimicking soft tissues for testing and validation17,18,19. In this protocol, gelatin phantoms with two different concentrations were prepared and tested. Viscoelastic properties of the gelatin phantoms were characterized using a custom-built electromagnetic MRE device14 and an indentation device1,3. The testing protocols could be used for testing many soft tissues such as the brain or liver.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-channel head &#38; Neck coil</td>
			<td>United Imaging Healthcare</td>
			<td>100120</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>3T MR Scanner</td>
			<td>United Imaging Healthcare</td>
			<td>uMR 790</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Acquisition board</td>
			<td>Advantech Co</td>
			<td>PCI-1706U</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Computer-Windows</td>
			<td>HP</td>
			<td>790-07</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Electromagnetic actuator</td>
			<td>Shanghai Jiao Tong University</td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>RIGOL</td>
			<td>DG1022Z</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>CARTE D&#8217;OR</td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Vance Bioenergy Sdn.Bhd</td>
			<td></td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Indenter control program</td>
			<td>custom-designed</td>
			<td></td>
			<td>Software; accessed via: https://github.com/aaronfeng369/FengLab_indentation_code.</td>
		</tr>
		<tr>
			<td>Laser sensor</td>
			<td>Panasonic</td>
			<td>HG-C1050</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Load cell</td>
			<td>Transducer Technique</td>
			<td>GSO-10</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td></td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Power amplifier</td>
			<td>Yamaha</td>
			<td>A-S201</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Voice coil electric motor</td>
			<td>SMAC Corporation</td>
			<td>DB2583</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

viscoelastic characterization of soft tissue-mimicking gelatin phantoms using indentation and magnetic resonance elastography

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the biomechanics-related mechanisms of disease, injury, and development. The mechanical testing method is the most straightforward way for tissue characterization and is considered as verification for in vivo measurement. Among the many ex vivo mechanical testing techniques, the indentation test provides a reliable way, especially for samples that are small, hard to fix, and viscoelastic such as brain tissue. Magnetic resonance elastography (MRE) is a clinically used method to measure the biomechanical properties of soft tissues. Based on shear wave propagation in soft tissues recorded using MRE, viscoelastic properties of soft tissues can be estimated in vivo based on wave equation. Here, the viscoelastic properties of gelatin phantoms with two different concentrations were measured by MRE and indentation. The protocols of phantom fabrication, testing, and modulus estimation have been presented.

Following the MRE protocol, a clear shear wave propagation in the gelatin phantoms at 40 and 50 Hz were observed (Figure 3). The viscoelastic properties measured from MRE, and indentation tests are shown in Figure 4. The estimated G&#39; and G&#34; values at each testing for each phantom are summarized in Table 2. Following the indentation protocol, the viscoelastic properties of each phantom at each test point are summarized in Table 3<...

Watch this Scientific Journal Video about Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography at JoVE.com

Viscoelastic Characterization of Soft Tissue-Mimicking Gelatin Phantoms using Indentation and Magnetic Resonance Elastography

This article presents a demonstration and summary of protocols of making gelatin phantoms that mimic soft tissues, and the corresponding viscoelastic characterization using indentation and magnetic resonance elastography.

Characterization of biomechanical properties of soft biological tissues is important to understand the tissue mechanics and explore the ...

This protocol provides a way to prepare tissue-mimicking gel functions in the ex vivo and the in vivo biomechanic characterization methods, which are critical for understanding and finding new biomarkers for tissue The indentation can measure small-sized tissue samples and is easy to carry out while the same sample can be tested using MRE, providing a direct estimation of in vivo test scenarios. This method could provide insight into viscoelastic frequency-dependent mechanical properties of soft biological samples, such as the brain, liver, tumor tissues, et cetera. Similar properties of other sample phantoms can also be measured.Begin by mixing the gelatin powder with water to obtain the gelatin solution. Heat the gelatin solution to 60 degrees Celsius in a water bath and add glycerol to the solution while maintaining the temperature. Stir the solution and heat it to 60 degrees Celsius again.Pour the mixed solution into a container that will be used for MRE and indentation tests. Cool the solution to room temperature and wait till the solution is solidified. Place the gelatin phantom into the head coil.Then, put the vibration plate on top of the gelatin phantom. Ensure that the contact between the phantom and the vibration plate is firm. Put sponges and sandbags around the gelatin phantom to make sure the phantom is firmly placed.Mount an electromagnetic actuator on the head coil and connect the transmission bar to the vibration plate. Connect the power lines of the actuator with the amplifier and then connect the control lines with the controller. Set the wave form, vibration frequency, and amplitude in the function generator.Set the desired vibration amplitude by adjusting the power amplifier. Then, set the function generator to work in the trigger mode. Connect the trigger line to the external trigger port of the MRI machine.Set the MRE scanning frequency the same as that from the function generator, so that the motion encoding gradient is synchronized with the motion of the vibration plate. Next, set the flip angle to 30 degrees, TR and TE to 50 and 31 milliseconds, field-of-view to 300 millimeters, slice thickness to five millimeters, and voxel size to 2.34 by 2.34 square millimeter. Measure the phase images at four temporal points in one sinusoidal cycle.Apply both positive and negative motion and coding gradients at each time point. Based on the phase image acquired, remove the background phase by subtracting the positive and negative encoded phase images. Unwrap the phase with a reliability sorting-based algorithm.Extract the principle components of the motion by applying fast Fourier transform to the unwrapped phase images. Filter the phase image with a digital band pass filter and estimate the shear modulus with a 2D direct inversion algorithm to obtain storage modulus G-prime and loss modulus G-double prime. Use a circular punch to trim the gelatin phantom into a cylindrical sample and use a surgical blade to trim it into a cuboid sample.Trim the sample's surface with a sharp blade to make it as smooth as possible for indentation. Turn on the power of the indentation tester and click on the Back Off button in the GUI to initialize the calibration process. Read the value from the laser sensor and type the value in the BaseLine box.Place a glass slide on the baffle plate and record the value shown by the laser sensor. Next, put the sample on the glass slide and place them together on the baffle plate. Read the value from the laser sensor and type this value in the Sample Slide box.The difference between these two values is the sample thickness at the region of interest. Carefully place the sample along with the underlying glass slide right below the indentor and then click on the Contact button to initiate automatic contact between the indentor and the sample surface. Based on the measured sample thickness, estimate the indentation displacement by multiplying the thickness with the indented testing strain.Type the displacement values in the Displacement box. Set the relaxation time to 180 seconds in the Dwell time box and click on the Indentation button. The displacement and the reactive force during the ramp/hold procedure will be automatically recorded and saved in a file at the specified file path.Wave propagation images for the two gelatin phantoms at 40 and 50 hertz are shown here. The four phases correspond to the four temporal points at one sinusoidal cycle. Viscoelastic properties measured from MRE and indentation experiments are shown here.The representative images depict typical estimated G-prime and G-double prime maps at 40 and 50 hertz for the two gelatin phantoms from MRE. Mean and standard deviation of the G-zero and G-infinity values for the two phantoms from the six repeated indentation tests are presented here. The graphical images shown on the screen represent the mean and standard deviation of the G-prime and G-double prime values at 40 and 50 hertz for the two phantoms from the six repeated MRE tests.The asterisk symbol indicates a significant difference. When attempting this procedure, make sure the vibrating plate is firmly pressed on top of the phantom and not to over-press the plate. When treating the sample, make sure the surface is as flat as possible.This technique paves the way to explore biomechanical properties related to pathological studies, and develop biomechanics-based biomarkers for disease diagnosis and prognosis.

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Research

JoVE Journal

Bioengineering

1.9K visualizações.  Shanghai Jiao Tong University. Este protocolo fornece uma maneira de preparar funções de gel que imitam tecidos nos métodos de caracterização biomecânica ex vivo e in vivo, que são críticos para a compreensão e descoberta de novos biomarcadores para o tecido O recuo pode medir amostras de tecido de pequeno porte e é fácil de realizar, enquanto a mesma amostra pode ser testada usando MRE, fornecendo uma estimativa direta de cenários de teste in vivo. Este método poderia fornecer informações sobre as proprieda...