Name: investigating stress-relaxation and failure responses in the trachea
Uploaded: 2022-10-18T12:30:00
Description: Watch this Scientific Journal Video about Investigating Stress-relaxation and Failure Responses in the Trachea at JoVE.com

Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health under Award Number R15HD093024 and the National Science Foundation CAREER Award Number 1752513.

All methods described were approved by the Institutional Animal Care and Use Committee (IACUC) at Drexel University. All cadaveric animals were acquired from a United States Department of Agriculture (USDA)-approved farm located in Pennsylvania, USA. A cadaver of a male Yorkshire pig (3 weeks old) was used for the present study.
1. Tissue harvest
<ol>
	<li>Acquire a cadaver of a pig from an approved farm and perform the experiments within 2 h from euthanasia. Keep the cadave...

<ol>
	<li>Brand-Saberi, B. E. M., Sch&#228;fer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trachea:+Anatomy+and+physiology.">Trachea: Anatomy and physiology.</a> Thoracic Surgery Clinics. 24, 1-5 (2014).</li><li>Barnett, S. B., Nurmagambetov, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Costs+of+asthma+in+the+United+States:+2002-2007.">Costs of asthma in the United States: 2002-2007.</a> The Journal of Allergy and Clinical Immunology. 127 (1), 145-152 (2011).</li><li>Chronic Obstructive Pulmonary Disease (COPD). Centers for Disease Control and Prevention Available from: <a target="_blank" href="https://www.cdc.gov/copd/index.html">https://www.cdc.gov/copd/index.html</a> (2022)</li><li>Wilson, L., Devine, E. B., So, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+medical+costs+of+chronic+obstructive+pulmonary+disease:+chronic+bronchitis+and+emphysema).">Direct medical costs of chronic obstructive pulmonary disease: chronic bronchitis and emphysema).</a> Respiratory Medicine. 94 (3), 204-213 (2000).</li><li>Codd, S. L., Lambert, R. K., Alley, M. R., Pack, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+stiffness+of+ovine+tracheal+wall.">Tensile stiffness of ovine tracheal wall.</a> Journal of Applied Physiology. 76 (6), 2627-2635 (1994).</li><li>Noble, P. B., Sharma, A., McFawn, P. K., Mitchell, H. W. <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+the+bronchial+mucosa:+epithelial+unfolding+and+stretch+in+response+to+airway+inflation.">Elastic properties of the bronchial mucosa: epithelial unfolding and stretch in response to airway inflation.</a> Journal of Applied Physiology. 99 (6), 2061-2066 (2005).</li><li>Teng, 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=Anisotropic+material+behaviours+of+soft+tissues+in+human+trachea:+An+experimental+study.">Anisotropic material behaviours of soft tissues in human trachea: An experimental study.</a> Journal of Biomechanics. 45 (9), 1717-1723 (2012).</li><li>Wang, L., 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=Mechanical+properties+of+the+tracheal+mucosal+membrane+in+the+rabbit.+I.+steady-state+stiffness+as+a+function+of+age.">Mechanical properties of the tracheal mucosal membrane in the rabbit. I. steady-state stiffness as a function of age.</a> Journal of Applied Physiology. 88 (3), 1014-1021 (2000).</li><li>Ambrosi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+biological+growth+and+remodeling.">Perspectives on biological growth and remodeling.</a> Journal of the Mechanics and Physics of Solids. 59 (4), 863-883 (2011).</li><li>Bai, T. R., Knight, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+the+airways+in+asthma:+Observations+and+consequences.">Structural changes in the airways in asthma: Observations and consequences.</a> Clinical Science. 108 (6), 463-477 (2005).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extent+of+impaired+axoplasmic+transport+and+neurofilament+compaction+in+traumatically+injured+axon+at+various+strains+and+strain+rates.">Extent of impaired axoplasmic transport and neurofilament compaction in traumatically injured axon at various strains and strain rates.</a> Brain Injury. 31 (10), 1387-1395 (2017).</li><li>Singh, A., Kallakuri, S., Chen, C., Cavanaugh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+changes+in+nerve+roots+due+to+tension+at+various+strains+and+strain+rates:+An+in-vivo+study.">Structural and functional changes in nerve roots due to tension at various strains and strain rates: An in-vivo study.</a> Journal of Neurotrauma. 26 (4), 627-640 (2009).</li><li>Singh, A., Lu, Y., Chen, C., Cavanaugh, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+spinal+nerve+roots+subjected+to+tension+at+different+strain+rates.">Mechanical properties of spinal nerve roots subjected to tension at different strain rates.</a> Journal of Biomechanics. 39 (9), 1669-1676 (2006).</li><li>Singh, A., Lu, Y., Chen, C., Kallakuri, S., Cavanaugh, J. M. <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+model+of+traumatic+axonal+injury+to+determine+the+effects+of+strain+and+displacement+rates.">A new model of traumatic axonal injury to determine the effects of strain and displacement rates.</a> Stapp Car Crash Journal. 50, 601-623 (2006).</li><li>Singh, A., Magee, R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+in+vivo+biomechanical+testing+on+brachial+plexus+in+neonatal+piglets.">Methods for in vivo biomechanical testing on brachial plexus in neonatal piglets.</a> Journal of Visualized Experiments. (154), e59860 (2019).</li><li>Singh, A., Magee, R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+cervical+spinal+cord+in+neonatal+piglet:+in+vitro.">Mechanical properties of cervical spinal cord in neonatal piglet: in vitro.</a> Neurology and Neurobiology. 3 (2), (2020).</li><li>Singh, A., Magee, R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+study+to+investigate+biomechanical+responses+of+peripheral+nerves+in+hypoxic+neonatal+piglets.">An in vitro study to investigate biomechanical responses of peripheral nerves in hypoxic neonatal piglets.</a> Journal of Biomechanical Engineering. 143 (11), 114501 (2021).</li><li>Singh, A., Shaji, S., Delivoria-Papadopoulos, M., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+responses+of+neonatal+brachial+plexus+to+mechanical+stretch.">Biomechanical responses of neonatal brachial plexus to mechanical stretch.</a> Journal of Brachial Plexus and Peripheral Nerve Injury. 13, 8-14 (2018).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The effects of tensile loading on mechanical, neurophysiological and morphological changes in spinal nerve roots. , (2006).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Eskandari, M., Arvayo, A. L., Levenston, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+the+airway+tree:+Heterogeneous+and+anisotropic+pseudoelastic+and+viscoelastic+tissue+responses.">Mechanical properties of the airway tree: Heterogeneous and anisotropic pseudoelastic and viscoelastic tissue responses.</a> Journal of Applied Physiology. 125 (3), 878-888 (2018).</li><li>Toby, E. B., Rotramel, J., Jayaraman, G., Struthers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+stress+relaxation+properties+of+peripheral+nerves+after+transection.">Changes in the stress relaxation properties of peripheral nerves after transection.</a> Journal of Hand Surgery. 24 (4), 694-699 (1999).</li><li>Safshekan, F., Tafazzoli-Shadpour, M., Abdouss, M., Shadmehr, M. B. <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+human+tracheal+tissues.">Viscoelastic properties of human tracheal tissues.</a> Journal of Biomechanical Engineering. 139 (1), (2017).</li><li>Singh, A. <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+approach+to+teaching+biomechanics+through+active,+adaptive,+and+experiential+learning.">A new approach to teaching biomechanics through active, adaptive, and experiential learning.</a> Journal of Biomechanical Engineering. 139 (7), 0710011-0710017 (2017).</li><li>Singh, A., Ferry, D., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+clinical+simulation+based+training+in+biomedical+engineering+education.">Efficacy of clinical simulation based training in biomedical engineering education.</a> Journal of Biomechanical Engineering. 141 (12), 121011-121017 (2019).</li><li>Singh, A., Ferry, D., Mills, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+biomedical+engineering+education+through+continuity+in+adaptive,+experiential,+and+interdisciplinary+learning+environments.">Improving biomedical engineering education through continuity in adaptive, experiential, and interdisciplinary learning environments.</a> Journal of Biomechanical Engineering. 140 (8), 0810091-0810098 (2018).</li><li>Singh, A., Ferry, D., Ramakrishnan, A., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+virtual+reality+in+biomedical+engineering+education.">Using virtual reality in biomedical engineering education.</a> Journal of Biomechanical Engineering. 142 (11), 111013 (2020).</li><li>Majmudar, T., Balasubramanian, S., Magee, R., Gonik, B., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+stress+relaxation+response+of+neonatal+peripheral+nerves.">In-vitro stress relaxation response of neonatal peripheral nerves.</a> Journal of Biomechanical Engineering. 128, 110702 (2021).</li><li>Orozco, V., Magee, R., Balasubramanian, S., Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+the+tensile+biomechanical+properties+of+the+neonatal+brachial+plexus.">A systematic review of the tensile biomechanical properties of the neonatal brachial plexus.</a> Journal of Biomechanical Engineering. 143 (11), 110802 (2021).</li><li>Singh, A. M. T., Magee, R., Gonik, B., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+pre-stretch+on+neonatal+peripheral+nerve:+An+in-vitro+study.">Effects of pre-stretch on neonatal peripheral nerve: An in-vitro study.</a> Journal of Brachial Plexus and Peripheral Nerve Injury. 17 (1), 1-9 (2022).</li><li>Balasubramanian, S., D'Andrea, C., Viraraghavan, G., Cahill, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+finite+element+model+of+the+pediatric+thoracic+and+lumbar+spine,+ribcage,+and+pelvis+with+orthotropic+region-specific+vertebral+growth.">Development of a finite element model of the pediatric thoracic and lumbar spine, ribcage, and pelvis with orthotropic region-specific vertebral growth.</a> Journal of Biomechanical Engineering. 144 (10), 101007 (2022).</li><li>Hadagali, P., Peters, J. R., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphing+the+feature-based+multi-blocks+of+normative/healthy+vertebral+geometries+to+scoliosis+vertebral+geometries:+Development+of+personalized+finite+element+models.">Morphing the feature-based multi-blocks of normative/healthy vertebral geometries to scoliosis vertebral geometries: Development of personalized finite element models.</a> Computer Methods in Biomechanics and Biomedical Engineering. 21 (4), 297-324 (2018).</li><li>Singh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Available+computational+and+physical+models+to+understand+the+mechanisms+of+neonatal+brachial+plexus+injury+during+shoulder+dystocia.">Available computational and physical models to understand the mechanisms of neonatal brachial plexus injury during shoulder dystocia.</a> Open Access Journal of Neurology and Neurosurgery. 9 (4), 555768 (2019).</li></ol>

Very few studies have reported the stress-relaxation properties of the trachea21,23. Studies are needed to further strengthen our understanding of the time-dependent responses of the tracheal tissue. This study offers detailed steps to perform such investigations; however, the following critical steps within the protocol must be ensured for reliable testing: (1) proper tissue hydration, (2) similar tissue-type (number of cartilaginous rings and muscle) distributi...

Despite its critical role in pulmonary disease, the largest airway structure, the trachea, has limited studies detailing its viscoelastic properties1. An in-depth understanding of the time-dependent, viscoelastic behavior of the trachea is critical to pulmonary mechanics research since understanding the airway-specific material properties can help advance the science of injury prevention, diagnosis, and clinical intervention for pulmonary diseases, which are the third leading cause of death in the United States2,3,4.
Available tissue characterization studies have reported the stiffness properties of the trachea5,6,7,8. The time-dependent mechanical responses have been minimally investigated despite their importance in tissue remodeling, which is also altered by pathology9,10. Moreover, the lack of time-dependent response data also limits the predictive capabilities of the pulmonary mechanics computational models that currently resort to using the generic constitutive laws. There is a need to address this gap by performing stress-relaxation studies that can provide the required material characteristics to inform biophysical studies of the trachea. The current study offers details of testing methods, data acquisition, and data analyses to investigate the stress-relaxation behavior of the porcine trachea.

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			<td>Disposable safety scalpels</td>
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			<td>10000-10</td>
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			<td>eXpert 7600</td>
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			<td>N/A</td>
			<td>Norwood, MA</td>
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			<td>Forceps</td>
			<td>&#160;Fine Science Tools Inc</td>
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			<td>Gauge Safe</td>
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			<td>Image J</td>
			<td>NIH</td>
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			<td>Proramming Software - MATLAB&#160;</td>
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			<td>Sterile phosphate buffer solution&#160;</td>
			<td>Millipore, Thomas Scientific</td>
			<td>MFCD00131855</td>
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investigating stress-relaxation and failure responses in the trachea

The biomechanical properties of the trachea directly affect the airflow and contribute to the biological function of the respiratory system. Understanding these properties is critical to understanding the injury mechanism in this tissue. This protocol describes an experimental approach to study the stress-relaxation behavior of porcine trachea that were pre-stretched to 0% or 10% strain for 300 s, followed by mechanical tensile loading until failure. This study provides details of the experimental design, data acquisition, analyses, and preliminary results from the porcine tracheae biomechanical testing. Using the detailed steps provided in this protocol and the data analysis MATLAB code, future studies can investigate the time-dependent viscoelastic behavior of trachea tissue, which is critical to understanding its biomechanical responses during physiological, pathological, and traumatic conditions. Furthermore, in-depth studies of the biomechanical behavior of the trachea will critically aid in improving the design of related medical devices such as endotracheal implants that are widely used during surgeries.

Figure 1 shows the failed tissue near the clamping site and the presence of tissue within the clamp, confirming no slip during tensile testing. Figure 2 indicates various failure sites, including the top or bottom clamping sites or along the length of the tissue, that were observed during tensile testing among the tested samples. Data analysis results are summarized in Figures 3-4 and Tables...

Watch this Scientific Journal Video about Investigating Stress-relaxation and Failure Responses in the Trachea at JoVE.com

Investigating Stress-relaxation and Failure Responses in the Trachea

The present protocol determines the tensile stress-relaxation and failure properties of porcine tracheae. Results from such methods can help improve the understanding of the viscoelastic and failure thresholds of the trachea and help advance the capabilities of computational models of the pulmonary system.

The biomechanical properties of the trachea directly affect the airflow and contribute to the biological function of the respiratory system. Understanding ...

This protocol offers a detailed approach to investigating the stress-relaxation responses of the trachea. Investigating the stress-relaxation responses of the trachea is critical to the understanding of pulmonary mechanics research. To begin, make a vertical midline incision along the neck of the cadaver and expose the thyroid cartilage, cricoid cartilage, and trachea from the hyoid bone to the suprasternal notch.Harvest the larynx and the full length trachea using a 10 number blade. Separate the trachea sample from the larynx and then cut the tracheal tube longitudinally along the entire length on one side using the blade. Cut the trachea into two circumferential strips approximately five millimeter wide proximally and two longitudinal strips approximately five millimeter wide distally with the minimum length of these strips being 25 millimeters.Navigate to the downloaded zipped folder, then extract its contents and navigate to the Failure Only folder. Store data from tested samples in a Microsoft Excel file using the mm/dd/yy file naming convention. Open the sample file tested on April 30th, 2022 in the Failure Only folder and recognize the multiple worksheet tabs containing the raw data from each sample subjected to mechanical failure on this particular date.Ensure the samples are labeled using the sample type, sample number, and pre-stretch strain level percentage file convention. Select the worksheet tab TA_1_0%Ensure that the raw data header columns are labeled as described in the text manuscript and close the current Microsoft Excel file, then return to the working directory of the data analysis software and navigate to the relaxation folder. Store data from the experimental group samples tested on a particular date in a Microsoft Excel file as demonstrated earlier.Open the relaxation folder and recognize the multiple worksheet tabs containing the raw load relaxation data from each sample in the experimental group tested on that particular date. Pause and note that each of the samples was subjected to mechanical failure under tensile mechanical loading as indicated by the worksheet tabs included in this Microsoft Excel file. Ensure that raw load relaxation data for each sample indicated by any worksheet tab are formatted correctly, then save and close the current Microsoft Excel file.Return to the working directory of the data analysis software and navigate to the folder failure post-relaxation. Confirm that there is a Microsoft Excel file with the same date as in the relaxation folder. Open the Excel file in the failure post-relaxation folder and recognize multiple worksheet tabs each containing raw mechanical failure data from the same samples present in the relaxation folder.Ensure that each sample's header column for the raw mechanical failure data is formatted correctly. Close the current Microsoft Excel file and return to the working directory of the data analysis software. Open the Microsoft Excel file testingDates.xlsx which will direct the code to analyze user-specified testing dates. List testing dates in the first column in the mm/dd/yy format. In the second column, indicate using a Y or N whether the samples on the testing date were from the experimental group which includes stress-relaxation followed by mechanical failure.In the third column, indicate whether any samples were from the control group showing direct mechanical failure. Save and close the current Microsoft Excel file and return to the working directory of the data analysis software, then open the main script file main_relax_failure. m, select the large green arrow on the software interface to run the code.Alternatively, type run main calc relax in the command window. Upon being prompted, input comma-separated fixed elongation levels in percentage for the various experimental groups and press OK, then input comma-separated stress-relaxation testing durations in seconds for the various experimental groups and press OK.Once the code is successfully run, ensure that the computed results are available in the working directory of the data analysis software as a Microsoft Excel file with mm/dd/yy replaced by the date on which the code was run. Stress-relaxation responses for tracheal samples following axial or circumferential pre-stretch to 10%strain demonstrated an initial peak in the stress load, followed by a percentage reduction in stress over the 300-second hold.The stress-strain responses of the tracheal sample subjected to failure testing under axial or circumferential loads following no pre-stretch or 10%pre-stretch indicated failure stress. The preliminary tests successfully characterized the stress-relaxation responses of the tracheal tissue wherein the initial peak stress was higher in axial loading directions. In contrast, the percentage reduction in stress was higher in the circumferential loading direction when compared to the axial loading direction.The relaxation times were also higher in the axial loading direction than the circumferential loading direction for the same 10%pre-stretch group. In the failure data, the failure stress and e-values were higher in circumferential loading directions in the zero and 10%pre-stretch groups, whereas the failure strain reported in the axial loading directions was higher. Ensure that the tested sample data is labeled correctly and the header columns of the data sheets are formatted correctly.These steps are critical to run the data analysis code. In addition to the acute, a chronic stress-relaxation response can also be studied. Also, a quasi-static stress-relaxation response can be investigated.

investigating-stress-relaxation-and-failure-responses-in-the-trachea

Research

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Bioengineering

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&amp;#176; Curved Artery Test Section

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial cell delivery. Rodent model of myocardial infarction (MI) consisting of Left Anterior Descending Artery (LAD) ligation has commonly been performed via a thoracotomy; a second open-heart surgery via a sternotomy has traditionally been performed for epicardial application of the treatment. Since the description of LAD ligation model, post-surgery mortality rate has dropped from 35-13%, however the second surgery has remained critical. In order to improve post-surgery recovery and reduce pain and infection, minimally invasive surgical procedures are presented.&#160;Two thoracotomies were performed, the initial one for LAD ligation and the second one for treatment epicardial administration. Biografts consisting of cells associated with solid or gel type matrices were applied onto the infarcted area.&#160;LAD ligation resulted in loss of heart function as confirmed by echocardiography performed after 2 and 6 weeks. Goldner trichrome staining performed on heart sections confirmed transmural scar formation. First and second surgeries resulted in less that 10%&#160;post-operative mortality.&#160;

Implantation of a biograft to treat myocardial infarction induced by LAD ligation in a rodent model has conventionally required two open-heart surgeries. In order to reduce mortality and provide optimal conditions for fixation of solid and gelatinous biomatrices associated with cells, minimally invasive procedures have been developed.

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...