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 by the National Science Foundation CAREER Award Number 1752513.

Institutional Animal Care and Use Committee at Drexel University approved all procedures (#20704).
1. Animal Arrival and Acclimation
<ol>
	<li>Quarantine 1&#8211;2 day-old piglets for at least 24 h after arrival.</li>
	<li>House piglets in clean and sanitized stainless-steel cages (36 in x 48 in x 36 in) on aspen chip bedding and feed ad libitum with pig milk replacer.</li>
	<li>Maintain the room temperature at 85 &#176;F to ensure a thermo-neutral environment.</li>
</ol>
2. Day of Experiment
<ol>
	<li>Remove the feed 2 h prior to the experiment.</li>
	<li>Inject piglets with an intramuscular injection of ketamine (10&#8211;40 mg/kg)/xylazine (1.5&#8211;3.0 mg/kg IM) and transport via a transport cage to the surgical space.</li>
</ol>
3. Induction and Maintenance of Anesthesia
<ol>
	<li>Administer 4% isoflurane inhalation anesthetic mixed in oxygen by nose cone and confirm that the animal is deeply anesthetized by assessing the absence of palpebral and withdrawal reflexes.</li>
	<li>Intubate the animal by placing it in the supine position and use a laryngoscope (straight blade) to help guide the intubation tube (diameter 2.5&#8211;2 mm) into the trachea.</li>
	<li>Place the animal on the ventilator once intubation tube is secured.</li>
	<li>Ensure that piglets receive a mix of isoflurane (0.25%&#8211;3% maintenance), oxygen, and nitrous oxide.</li>
	<li>Provide a dose of fentanyl (10 &#956;g/kg) and continue giving a dose every 1&#8211;2 h to ensure continued adequate depth of analgesia and sedation and to avoid motion artifacts that could risk dislodgment of the endotracheal tube.</li>
	<li>Establish intravenous (IV) access in the subcutaneous abdominal vein or any other peripheral vein.</li>
	<li>Establish the arterial line through the femoral artery. This can be done non-invasively or by performing a cut-down.</li>
</ol>
4. Monitoring and Care
<ol>
	<li>Monitor the depth of anesthesia by confirming the absence of canthal reflex and absence of withdrawal response to toe pinch.</li>
	<li>Perform continuous monitoring of physiological parameters during anesthesia and throughout the experiment, which includes arterial blood pressure, electrocardiography (ECG), end-tidal CO2, pulse oximetry, and body temperature.</li>
	<li>Monitor blood gases and blood sugars every 0.5&#8211;1 h and give intravenous fluids (50% dextrose and 50% normal saline) to animals anesthetized longer than 1 h at ~100 cc/kg/day, as needed, to ensure euglycemia.</li>
	<li>Monitor the animal&#39;s anesthetic plane closely and frequently. Provide analgesia and/or increase inhalant anesthesia.</li>
	<li>Maintain the animal at normal oxygen tension by controlling the ventilator parameters and drug dosages as needed to ensure normoxia, then place the animal on a temperature-regulated circulating water blanket such that normal body temperature is maintained at 39 &#176;C for the duration of the experiment.</li>
</ol>
5. Brachial Plexus Surgery
<ol>
	<li>Place the animal in a supine position on the operating table after proper anesthesia as described in section 3, with the upper limb in abduction, exposing the axillary region.</li>
	<li>Use any surgical drape to cover the animal. Use clean but non-sterile techniques.</li>
	<li>Expose the brachial plexus complex on both sides of the spine by making a midline incision (using a #10 blade) over the skin and fascia overlying the trachea, down to the upper third of the sternum, corresponding to spine levels between C3&#8211;T3.</li>
	<li>Extrapolate the incision using the forceps and hemostat horizontally on each side from the suprasternal notch along the edge of the clavicle to the upper arm, while sparing the cephalic and basilic veins.</li>
	<li>Release the superior and inferior flaps by blunt dissection using scissors and forceps, allowing access to the cervical and thoracic regions of the brachial plexus, respectively.</li>
	<li>Identify the axis (C2) and first rib at the T1. Using these landmarks, identify the lower three cervical (C6&#8211;C8) and first thoracic (T1) spinal vertebral foramen, then examine the plexus carefully to locate bifurcations of the divisions (M shape) to achieve exposure.</li>
	<li>Label (using nerve loops) the brachial plexus regions above these bifurcations closer to the spine as root/trunk and label those below these bifurcations as chord followed by the nerve, which are located closer to the arm.</li>
</ol>
6. Biomechanical Testing
<ol>
	<li>Set-up of the biomechanical testing device 
	NOTE: A custom-built mechanical testing device was designed and fabricated to perform in vivo stretch of the BP (Figure 1).
	<ol>
		<li>Attach the base of the set-up to a cart.</li>
		<li>Attach the electromechanical actuator onto the base using large C-clamps. The actuator is capable of providing 150 lb of force, 10&#34; stroke, and speed of 15 mm/s. The speed can be reduced to 0.2 mm/s and still function as desired.</li>
		<li>Attach the 200 N load cell to actuator.</li>
		<li>Attach (screw-in) a clamp to the load cell that consists of padded plexiglass, which prevents the stress concentration at the clamping site.</li>
		<li>Attach a camera to a tripod. Ensure that the camera has the capability of recording up to 120 f/s at a resolution of 658 x 4926 pixels.</li>
		<li>Attach USB cables from the camera, actuator, and load cell to the computer to integrate and synchronize all components of the set-up.</li>
		<li>Plug the computer, actuator, and load cell into a power source.</li>
	</ol>
	</li>
	<li>Calibrate the load cell prior to recording the applied loads. To do so, perform the steps below:
	<ol>
		<li>Set the actuator at a 90&#176; angle using the adjustable handle and checking the angle with a protractor.</li>
		<li>Open the software that works with the load cell (Table of Materials). Press the Start button to show a live readout of voltage.</li>
		<li>Hang weights from the clamp ranging from 0&#8211;1,000 g in increments of 100 g from the setup and record the measured voltages.</li>
		<li>Calculate the linear equation of the voltages and weights by finding the slope (m) and intercept (b). This is done using a spreadsheet program and the included slope function to calculate b from the Equation 1 below. Insert Equation 2 shown below into the mechanical set-up code. 
		Equation 1: b = y - mx 
		Where: y is the weight, x is the voltage, m is the slope, and b is the intercept (constant). 
		Equation 2: y = mx + b 
		Where: y is the weight, x is the voltage, m is the slope, and b is the constant.</li>
	</ol>
	</li>
	<li>Testing: the BP nerve is cut and anchored to the testing set-up by custom-built clamp.
	<ol>
		<li>Cut the BP nerve using fine scissors.</li>
		<li>Clamp the cut side of the BP nerve in the custom-built clamp as shown in Figure 1.</li>
		<li>Manually place black acrylic paint or India ink on the clamped BP segment (Figure 2).</li>
		<li>Place a calibration grid, which is a 1 cm ruler, flat within the animal to set the scale for data analysis.</li>
		<li>Use the camera&#39;s software to view the camera&#39;s placement directly over the tested segments, thereby allowing the monitoring of the motion/displacement of the markers and determining the actual tissue strain at any timepoint.</li>
		<li>Record initial measurements such as the height at which the nerve inserts into the body from the table and the height of the clamp from the table, the angle of the actuator, and the full length of the tissue.</li>
		<li>Open the programming software (table containing the graphical user interface [GUI] as shown in Figure 3).</li>
		<li>Run the GUI by pressing the Run button.</li>
		<li>Initialize the system by pressing the Initialize button.</li>
		<li>Tare the system by pressing the Tare button.</li>
		<li>Stretch the BP segment by pressing the start test button. This pulls the tissue at an assigned rate of 500 mm/min until complete failure occurs in any segment of the BP. This stretch rate is selected based on the available literature4,8,18. The program also saves a video file, the applied tensile load, displacement of the tissue, and duration of the test.</li>
		<li>Record the failure site, which is the point at which the tissue ruptures.</li>
	</ol>
	</li>
	<li>Euthanasia: euthanize piglets at the end of the experiment with a lethal dose of pentobarbital (120 mg/kg i.v.).</li>
	<li>Data analysis: use motion tracking software for the analysis of the videos acquired during testing.
	<ol>
		<li>Open the video file from the experiment within the motion tracking software by selecting File | Open Video File.</li>
		<li>Use the calibration grid to setup the scale in the motion tracking software using the Line tool, right-clicking on the line after it is drawn, selecting Calibrate Measure, and entering a known value in centimeters (Figure 4).</li>
		<li>Track the markers on the tissue within the motion tracking software by right-clicking on the video and selecting Track Path and aligning the center of the marker with the marker on the tissue and tacking it until rupture.</li>
		<li>Export the x- and y-coordinates from the markers by selecting File Export to Spreadsheet so that it can be used to calculate the strains.</li>
		<li>Import the data into a programming software to calculate the distance between the x- and y-coordinates over time to calculate the strains.</li>
		<li>Calculate strain values at each timepoint by dividing the change in distance by the original distance after accounting for changes in inclination during stretch. The actual strain values are determined between each pair of adjacent markers at each timepoint. The average of these strains is also calculated.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+brachial+plexus+stretching+during+shoulder+dystocia+using+a+computer+simulation+model.">Prediction of brachial plexus stretching during shoulder dystocia using a computer simulation model.</a> American Journal of Obstetrics and Gynecology. 189 (4), 1168-1172 (2003).</li><li>Grimm, M. J., Costello, R. 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The authors have nothing to disclose.

Available literature on the biomechanical responses of stretch on the BP tissue exhibit a wide range of threshold values as well as methodological discrepancies4,6,8,18,19,20,21,22,23. Variations in published results could...

Despite recent advances in obstetrics, the problem of NBPP caused by stretch injury to the BP complex continues to be a global health burden, with an incidence of 1.5 cases per 1,000 live births1,2. Associated risk factors can be maternal (i.e., excessive weight, maternal diabetes, uterine abnormalities, history of BP paralysis), fetal (i.e., fetal macrosomia), or birth-related (i.e., shoulder dystocia, prolonged labor, assisted delivery with forceps or vacuum extractors, breech presentation3). While these complications are unavoidable in certain circumstances, prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of the neonatal BP when subjected to stretch.
Reported biomechanical studies on the BP have used adult animals and human cadaveric tissue and show significant discrepancies4,5,6,7,8,9,10,11,12,13,14,15. Clinical relevancy of biomechanical properties of the complex BP tissue warrants a neonatal animal model as well as an in vivo biomechanical testing approach. Furthermore, limitations with studying BP stretch injury in complicated real-world delivery scenarios increases the reliance on computer models that provide methods that allows investigation of the effects of various delivery complications and techniques. The key to clinical relevance of these models is their biofidelity (human-like response). Available computational models by Gonik et al.16 and Grimm et al.17 rely on rabbit and rat nerve tissue but not neonatal BP tissue. Performing in vivo biomechanical testing in a clinically relevant neonatal animal model can fill the critical gap of unavailable neonatal BP data.
The current study describes an in vivo mechanical testing device and procedure to conduct biomechanical testing in 3-5 day-old male Yorkshire neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads during failure. The camera system also allows monitoring of the failure location during rupture. Overall, the system allows for detailed biomechanical characterization of the neonatal BP when subjected to stretch, thereby providing the BP's threshold strains and stresses for mechanical failure in vivo. The data obtained can further improve human-like behavior (biofidelity) of the existing computational models that are designed to investigate the effects of exogenous and endogenous forces on BP stretch in delivery scenarios associated with NBPP.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Omega Subminature Tension &#38; Compression Load Cell</td>
			<td>Omega</td>
			<td>LCM201-200N</td>
			<td>200N load cell</td>
		</tr>
		<tr>
			<td>Basler acA640-120uc camera</td>
			<td>Basler</td>
			<td>acA640-120uc</td>
			<td></td>
		</tr>
		<tr>
			<td>Feedback Linear Actuator</td>
			<td>Progressive Automations</td>
			<td>PA-14P</td>
			<td>10&#34; stroke, 150lb force, 15mm/s speed</td>
		</tr>
		<tr>
			<td>Motion Tracking Software</td>
			<td>Kinovea</td>
			<td>N/A</td>
			<td>Open Source</td>
		</tr>
		<tr>
			<td>Proramming Software - MATLAB</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>version 2018A</td>
		</tr>
		<tr>
			<td>Surgical instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools Inc</td>
			<td>11006-12 and 11027-12 or 11506-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostats</td>
			<td>Fine Science Tools Inc</td>
			<td>13009-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>14094-11 or 14060-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for in vivo biomechanical testing on brachial plexus in neonatal piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder regions, collectively referred to as the brachial plexus (BP). Despite recent advances in obstetrical care, the problem of NBPP continues to be a global health burden with an incidence of 1.5 cases per 1,000 live births. More severe types of this injury can cause permanent paralysis of the arm from the shoulder down. Prevention and treatment of NBPP warrants an understanding of the biomechanical and physiological responses of newborn BP nerves when subjected to stretch. Current knowledge of the newborn BP is extrapolated from adult animal or cadaveric BP tissue instead of in vivo neonatal BP tissue. This study describes an in vivo mechanical testing device and procedure to conduct in vivo biomechanical testing in neonatal piglets. The device consists of a clamp, actuator, load cell, and camera system that apply and monitor in vivo strains and loads until failure. The camera system also allows monitoring of the failure location during rupture. Overall, the presented method allows for a detailed biomechanical characterization of neonatal BP when subjected to stretch.

A representative load-time plot and strains from four segments of BP plexus (between four markers) are shown in Figure 5 and Figure 6, respectively. The obtained failure load of 8.3 N at 35% average failure strain reports the biomechanical responses of neonatal BP when subjected to stretch. Some regions of the nerve undergo higher strains than others, indicative of non-uniform injury along the length of the nerve. The camera data...

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Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Neonatal brachial plexus palsy (NBPP) is a stretch injury that occurs during the birthing process in nerve complexes located in the neck and shoulder ...

methods-for-vivo-biomechanical-testing-on-brachial-plexus-neonatal

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6.0K Views.  Widener University. Presented here are methods to perform in vivo biomechanical testing on brachial plexus in a neonatal piglet model.

Video: Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

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 ...

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 ...

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

In this manuscript, we present a protocol to fracture test cadaveric proximal femora in a sideways fall on the hip configuration using instrumented fixtures mounted on a standard servo hydraulic frame. Nine digitized signals comprising forces, moments, and displacement along with two high speed video streams are acquired during testing.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

Biomechanical Testing of Murine Tendons

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, and surgical repair, often fail. In order to define tendon function and demonstrate efficacy of new treatments, the mechanical properties of tendons from animal models must be accurately determined. Murine animal models are now widely used to study tendon disorders and evaluate novel treatments for tendinopathies; however, determining the mechanical properties of mouse tendons has been challenging. In this study, a new system was developed for tendon mechanical testing that includes 3D-printed fixtures that exactly match the anatomies of the humerus and calcaneus to mechanically test supraspinatus tendons and Achilles tendons, respectively. These fixtures were developed using 3D reconstructions of native bone anatomy, solid modeling, and additive manufacturing. The new approach eliminated artifactual gripping failures (e.g., failure at the growth plate failure rather than in the tendon), decreased overall testing time, and increased reproducibility. Furthermore, this new method is readily adaptable for testing other murine tendons and tendons from other animals.

The protocol describes efficient and reproducible tensile biomechanical testing methods for murine tendons through the use of custom-fit 3D printed fixtures.

Tendon disorders are common, affect people of all ages, and are often debilitating. Standard treatments, such as anti-inflammatory drugs, rehabilitation, ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Methods for In Vivo Biomechanical Testing on Brachial Plexus in Neonatal Piglets

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Chapters in this video

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Stress-induced Antibiotic Susceptibility Testing on a Chip

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

Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

Biomechanical Testing of Murine Tendons

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure