Name: experimental investigation of secondary flow structures downstream of a model type iv stent failure in a 180&#176; curved artery test section
Uploaded: 2016-07-19T13:00:00
Description: Watch this Scientific Journal Video about Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180Â° Curved Artery Test Section at JoVE.com

The authors acknowledge support from NSF grant CBET-0909678 and funding from the GW Center for Biomimetics and Bioinspired Engineering (COBRE). We thank the students, Mr. Christopher Popma, Ms. Leanne Penna, Ms. Shannon Callahan, Mr. Shadman Hussain, Mr. Mohammed R. Najjari, and Ms. Jessica Hinke for help in the laboratory and Mr. Mathieu Barraja for assisting in CAD drawings.

1. Design and Fabrication of Stent Models
Note: The following steps have been followed to create laboratory-scale models of straight and curved stents. The installation of the two stent models will embody a &#34;Type IV&#34; fracture (fragmentation and linear displacement of fractured stent parts).
Note: The authors used Pro/Engineer software at the time of the research for creating CAD models of the stent geometry. The procedure below is generalized and may not inclu...

<ol>
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The protocol presented in this paper describes the acquisition of high fidelity experimental data using particle image velocimetry technique (PIV) and coherent structure detection methods, viz., continuous wavelet transforms, <img alt="Equation 1" src="/files/ftp_upload/51288/51288eq1.jpg" />, suited for identification of vortex and shear-dominated flows. Analysis of experimental data from physiological inflows in the presence of an idealized &#34;Type IV&#34; fracture reveals that secondary flow structures with complica...

Secondary flow structures are vortical flow patterns that occur in internal flow geometries with curvatures such as curved pipes and channels. These vortical structures arise due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. In general, secondary flow structures appear in planar cross-sections of curved pipes as symmetric Dean-type vortices under steady inflow and, symmetric Dean- and Lyne-type vortices under oscillatory inflow conditions 1 - 3. Secondary flow morphologies are greatly affected by pulsatility and multiple harmonics of pulsatile, physiological inflow conditions. These structures acquire markedly different size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 6. Atherosclerotic lesion development in arteries is affected by the existence of high frequency shear oscillations in regions experiencing low mean shear 27, 28. Secondary flow structures may influence the progress of diseases such as atherosclerosis and possibly, mediate the endothelial response due to pulsatile blood flow by altering wall shear stresses and exposure times of blood-borne particles.
A common treatment to atherosclerosis, a complication resulting in narrowing of arteries by obstructive lesions, is the implantation of stents. Stent fractures are structural failures of implanted stents that lead to further medical complications such as in-stent restenosis (ISR), stent thrombosis and aneurysm formation 9 - 13. Stent fractures have been categorized into various failure &#34;Types I-to-IV&#34;, wherein &#34;Type IV&#34; characterizes the highest clinical severity and is defined as the complete transverse fracture of stent struts along with linear displacements of the stent fragments 14. The protocol presented in this study describes an experimental method of visualization of secondary flow structures downstream of an idealized &#34;Type IV&#34; stent fracture in a curved artery model.
The suggested protocol has the following four essential features:
Design and fabrication of laboratory-scale stent models: Geometric description of stents can be associated with a set of self-expandable spirals (springs or helices) intertwined using Nitinol (an alloy of nickel and titanium) wires 29. The length of the stent and its strut diameter depend on the length scale of arterial lesions encountered during clinical implantation 5. Parametric variation of strut diameter and the rising of the winding (or pitch) leads to stents of various geometric configurations. A summary of stent design parameters chosen for 3D printing are presented in Table 1.
Preparation of a blood analog working fluid matched with kinematic viscosity of blood and refractive index of the test section: Optical access to the curved artery test section is required in order to make non-invasive velocity measurements. Accordingly, a Newtonian blood-mimicking working fluid with the refractive index of the vascular model and ideally, a dynamic viscosity, matching human blood is used to obtain accurate blood flow measurements 16 - 18, 30. The working fluid used in this study was reported by Deutsch et al. (2006), that comprised of 79% saturated aqueous sodium iodide (NaI), 20% pure glycerol, and 1% water (by volume)16.
Experimental arrangement for the detection of coherent secondary flow structures using a two-component, two-dimensional particle image velocimetry (2C-2D PIV): Experiments were designed to acquire phase-averaged secondary flow velocity data at various planar cross-sectional locations downstream of a combination of straight and curved stent sections embodying an idealized &#34;Type IV&#34; stent fracture 5, 6, 9, 14. The protocol-steps pertaining to the acquisition of secondary flow velocity fields using particle image velocimetry (PIV) technique involves a PIV system that comprises of a laser (light sheet) source, optics to focus and illuminate the regions of flow, a special cross-correlation charge coupled device (CCD-sensor or camera) and tracer particles to be illuminated by the light sheet within a short time interval (&#916;t; see Table 4) 31, 32.
The steps in the protocol assume the following: First, a calibrated, experimental set-up of a two-component, two-dimensional (2C-2D) PIV system that evaluates images by double-frame, single-exposure recordings. Second, the 2C-2D PIV system calculates the mean displacements of tracer particles by performing cross-correlation between two image frames acquired during each recording. A brief summary of PIV specifications and image acquisition software is presented in the materials and equipment table. Third, all safety precautions needed to operate the laser are followed by trained laboratory personnel according to the guidelines provided by the host institution. The authors suggest Refs. 31 and 32 for a holistic understanding of the implementation, functionality and application of PIV technique in aero-, hydro- and microfluid dynamics, correlation peak detection and displacement estimation, material and density of tracer particles and, measurement noise and accuracy. Also note that the laser and camera can be controlled by the PIV data acquisition computer (Figure 3A) and data processing software.
Data acquisition and post-processing for coherent structure detection: Phase-averaged secondary flow velocity measurements using a 2C-2D PIV were generated using the protocol description that follows. Post-processing of the data involved coherent secondary flow structure detection using the following three methods: continuous wavelet transforms, <img alt="Equation 1" src="/files/ftp_upload/51288/51288eq1.jpg" /> 5, 6, 19 - 24, 26.
The authors note that the velocity gradient tensor is essentially, a 3 x 3 matrix, 
<img alt="Equation 2" src="/files/ftp_upload/51288/51288eq2.jpg" />.
The protocol presents a method of acquiring two-dimensional experimental measurements (from 2C-2D PIV technique). Therefore, full experimental access to the velocity gradient tensor will not be attainable using this method. The velocity gradient tensor for each pixel <img alt="Equation 3" src="/files/ftp_upload/51288/51288eq3.jpg" /> of the PIV image <img alt="Equation 4" src="/files/ftp_upload/51288/51288eq4.jpg" /> should be a 2 x 2 matrix, <img alt="Equation 5" src="/files/ftp_upload/51288/51288eq5.jpg" />. The z-component vorticity <img alt="Equation 6" src="/files/ftp_upload/51288/51288eq6.jpg" /> for each pixel <img alt="Equation 7" src="/files/ftp_upload/51288/51288eq7.jpg" /> is computed using the anti-symmetric part of the velocity gradient tensor <img alt="Equation 8" src="/files/ftp_upload/51288/51288eq8.jpg" />. The result will be a 2D array of vorticity <img alt="Equation 9" src="/files/ftp_upload/51288/51288eq9.jpg" /> that can be visualized in a contour plot. The authors strongly suggest Ref. 25 for an eloquent discussion experimental access to the velocity gradient tensor toward enhancing the knowledge of vorticity dissipation, strain rates and coherent structure detection. Furthermore, the authors do not attempt to explore the inter-relationships between the aforementioned coherent structure detection methods and suggest Ref. 23, 24 for a comprehensive discussion on that subject.
The focus of the steps in the protocol is the quantitative identification of secondary flow (vortical) structures (also known as coherent structures). Three methods of coherent structure detection viz., <img alt="Equation 10" src="/files/ftp_upload/51288/51288eq10.jpg" /> and wavelet transformed vorticity <img alt="Equation 11" src="/files/ftp_upload/51288/51288eq11.jpg" /> are applied to velocity field data toward detection of multi-scale, multi-strength occurrences of secondary flow structures downstream of the idealized &#34;Type IV&#34; stent fracture.
The <img alt="Equation 12" src="/files/ftp_upload/51288/51288eq12.jpg" />, defines a vortex as a spatial region where the Euclidean norm of the vorticity tensor dominates that of the rate of strain 19, 23, 24.The velocity gradient matrix is decomposed into symmetric (strain rate) and anti-symmetric (rotation) parts. Eigenvalues of strain rate matrix are computed; <img alt="Equation 13" src="/files/ftp_upload/51288/51288eq13.jpg" />. Norm of the strain rate is then calculated; <img alt="Equation 14" src="/files/ftp_upload/51288/51288eq14.jpg" />. Vorticity is computed from the anti- symmetric part. Enstrophy or square of z-component vorticity, <img alt="Equation 15" src="/files/ftp_upload/51288/51288eq15.jpg" />) is then computed. The <img alt="Equation 16" src="/files/ftp_upload/51288/51288eq16.jpg" /> is finally computed; <img alt="Equation 17" src="/files/ftp_upload/51288/51288eq17.jpg" />. A contour plot of the entire set of <img alt="Equation 18" src="/files/ftp_upload/51288/51288eq18.jpg" /> with iso-regions of <img alt="Equation 19" src="/files/ftp_upload/51288/51288eq19.jpg" />, will indicate secondary flow structures 19. 
The <img alt="Equation 20" src="/files/ftp_upload/51288/51288eq20.jpg" />, also known as &#39;swirling strength&#39; is a vortex identification method performed by critical-point analysis of the local velocity gradient tensor and its corresponding eigenvalues 20 - 24. Eigenvalues of the velocity gradient tensor at each pixel&#160;<img alt="Equation 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> are computed. The eigenvalues should be of the form, <img alt="Equation 22" src="/files/ftp_upload/51288/51288eq22.jpg" />. A contour plot of <img alt="Equation 23" src="/files/ftp_upload/51288/51288eq23.jpg" /> with iso-regions of <img alt="Equation 24" src="/files/ftp_upload/51288/51288eq24.jpg" /> will indicate secondary flow structures 20 - 22.
Wavelet transform method utilizes an analyzing function (or wavelet) that has smoothness in physical and spectral spaces, is admissible (or has zero mean) and has a finite <img alt="Equation 25" src="/files/ftp_upload/51288/51288eq25.jpg" />5, 6, 26. By convolving a dilated or contracted wavelet with a 2D vorticity field, wavelet transformed vorticity&#160;<img alt="Equation 26" src="/files/ftp_upload/51288/51288eq26.jpg" /> field is generated comprising of coherent structures with a wide range of scales and strengths 5, 6, 26. Shannon entropy of the 2D wavelet-transformed vorticity field is computed to estimate the optimal wavelet scale at which all the coherent structures are adequately resolved. This entropy estimation involves a set of probabilities <img alt="Equation 27" src="/files/ftp_upload/51288/51288eq27.jpg" /> for each pixel <img alt="Equation 21" src="/files/ftp_upload/51288/51288eq21.jpg" /> such that&#160; <img alt="Equation 28" src="/files/ftp_upload/51288/51288eq28.jpg" />, the normalized square modulus of the vorticity associated with the pixel at location m, n 5, 6. The procedural steps are presented graphically in Figure 6. The restrictions placed on the choice of the wavelet are presented in detail in Ref. 26. This protocol step describes the procedure for coherent structure detection using a 2D Ricker wavelet. The justification for the use of this wavelet for vortical pattern matching is presented in Ref. 5, 6 and the pertinent references cited therein.

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		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ubbelhode viscometer</td>
			<td style="width:155px;">Cole Parmer</td>
			<td>YO-98934-12</td>
			<td style="width:208px;">Toward measurement of kinematic viscosity of the blood-analog fluid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">VELP scientifica - ESP stirrer&#160;</td>
			<td style="width:155px;">VELP Scientifica</td>
			<td>F206A0179</td>
			<td style="width:208px;">Magnetic stirrer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ohaus Scout Pro SP 601&#160;</td>
			<td style="width:155px;">The Lab Depot</td>
			<td>SP4001</td>
			<td style="width:208px;">Weigh scale</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Refractometer</td>
			<td>Atago</td>
			<td style="width:119px;">PAL-RI</td>
			<td style="width:208px;">Toward measurement of refractive index of blood-analog fluid</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Beakers, pipettes, syringes and spatula</td>
			<td style="width:155px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">CLS710110,&#160; CLS10031L, CLS71015, CLS71011 Z193216</td>
			<td style="width:208px;">Toward handling materials required for blood-analog solution preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Iodide</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>383112-2.5KG&#160;</td>
			<td style="width:208px;">Crystalline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>G5516-1L</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deionized Water</td>
			<td style="width:155px;">-</td>
			<td style="width:119px;">-</td>
			<td style="width:208px;">Liquid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium thiosulfate anhydrous</td>
			<td style="width:155px;">Sigma-Aldrich</td>
			<td>72049-250G</td>
			<td style="width:208px;">Powder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Recording medium</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">Imager Intense 10Hz</td>
			<td style="width:208px;">PIV Image acquisition CCD camera</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PIV Illumination source</td>
			<td style="width:155px;">New Wave Research</td>
			<td style="width:119px;">Solo III-15</td>
			<td style="width:208px;">PIV Laser source, Nd:YAG laser, 532 nm, dual pulse 70 mJ/pulse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PIV Imaging software</td>
			<td style="width:155px;">&#160;LaVision&#160;</td>
			<td style="width:119px;">DaVis 7.2&#160;</td>
			<td style="width:208px;">PIV data acquisition and instrument control&#160;</td>
		</tr>
		<tr height="211">
			<td height="211" style="height:211px;width:216px;">PIV Seeding material</td>
			<td style="width:155px;">Thermo-scientific&#160;&#160;</td>
			<td style="width:119px;">Flouro-Max</td>
			<td style="width:208px;">Red fluorescent polymer microspheres (&#8776; 7 &#181;m); Dry dyed polystyrene (DVB) fluorescent microspheres emit bright and distinct colors when illuminated by the light of shorter&#160; wavelengths than the emission wavelength.&#160;</td>
		</tr>
	</tbody>
</table>

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.

Results presented in Figure 7A-D were generated after post processing secondary flow velocity data (see Figures 5, 6) acquired from 2C-2D PIV system shown in Figure 3A. The inflow condition supplied to the curved artery test section with an idealized &#34;Type IV&#34; stent fracture was the carotid artery waveform shown in Figure 4B. Our previous studies have demonstrated the sensitivity of secondary flow structures to de...

Watch this Scientific Journal Video about Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180Â° Curved Artery Test Section at JoVE.com

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.

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 ...

This video shows how to use Particle Image Velocimetry, and Coherent Structure Detection, to detect secondary flow or wortical structures in a curved artery model, to better understand the clinical relevance of Type IV stent failures. This method can help answer key questions in cardiovascular hemodynamics. Such as, how stents, which can deteriorate over time and ultimately fracture, can influence blood flow patterns and affect disease progression in vascular remodeling.The main advantage of this technique is that it facilitates investigation of in-vivo and in-vitro measurements in complex physiological arterial flows involving stent and stent fractures. Demonstrating procedure will be Mohammad Najjari, a graduate student, and Jessica Hinke, an undergraduate student. Prepare a blood-analog solution comprised of 79%saturated sodium iodide solution, 20%glycerol, and 1%deionized water.Start with saturated sodium iodide solution, then, add the glycerol in small increments using a syringe until the entire volume of glycerol has been added. Between additions, wait for the solution to become visibly homogenized, and take note of the volume of each glycerol addition. Next, add the required volume of water, continue stirring until the blood-analog solution is visibly homogenized.Finally, add very small quantities of sodium thiosulfate, using a spatula until the solution is optically clear. To begin, create high resolution STL files from the straight and curved stent Cad models. In the software, select Export and Model from the file menu.Choose the STL option, set the Chord Height to zero, and set the Angle Control to one. The angle control value regulates the amount of tessellation along the surface with small radii, and the setting can be adjusted between zero and one. Then, click OK to create the STL file.Next, fabricate the stent models on a Rapid Prototyping machine. To do this, start the 3D printing software and click on Insert to locate the STL file on the 3D printer computer. Then, select the desired file and drive the mouse to place the STL file on a virtual platform on the screen.Next, using the 3D-printed parts, install the stent into the curved artery test section to recreate an Idealized Type IV fracture scenario. Entailing a complete transverse fracture of stents and linear displacement of fragmented parts. Now, assemble the setup on an optical table.Connect the straight acrylic pipes to the inlet and outlet of a 180 degree curved artery test section. To begin, grossly adjust the laser's position by illuminating a small piece of paper. Make the laser sheet approximately 2mm thick by adjusting the focus.Next, target the laser sheet along the 90 degree portion of the measurement region with the sheet perpendicular to the table. Then, place a camera near the zero degree or 180 degree location to view the cross sectional region illuminated by the laser sheet. Incrementally adjust the alignment of the laser and camera so the camera views the circular cross section of the curved artery with minimal particle distortion, which is assessed using the Grab function in the software.Once the camera and laser are in position, acquire images of the secondary flow fields. First, turn on the programmable pump. In the pump instrument control program, set the Amplitude to one volt.Set the DC Offset to zero volts. Set the number of timed steps to 1000, and set the time period to four seconds. Additional settings are discussed in the Text Protocol.Then, load the text file that has the values for the voltage time wave form, and run the program to supply the blood-analog fluid to the experiment. In the PIV Data Acquisition program, after clicking the New Recordings tab, select the device under the Settings section, and confirm that the laser is set to On with the appropriate power settings. Navigate to laser control to confirm the values.Navigate to Timing and set it to External Cyclic Trigger. Click on Acquisit, select Table Scan, Edit Table Scan, Append Scan, and populate scan settings by entering start time representative of systolic deceleration. Time increment and end time of PIV data acquisition.Reference Time, DT1, or the time between laser pulses should be adjusted. Then, click on Close. Under Acquisit, click on image acquisition and enter the number of images as 200.Now, the PIV system is ready to acquire data. Select Start Recording to acquire phase-wise measurements, using the trigger signal from the pump instrument control. The set number of plane or velocity fields at each time instance, and at the prearranged 90 degree test section location will then be acquired.When the recording is done, power down the laser. Turn off the pump, turn off the camera, and replace the lens cover. Using the supplemental code file, compute the Phase Averaged and RMS Secondary Flow Velocities, Vorticity, and Swirling Strength fields.In the software, initialize setting pertaining to defined scale and send to Mask Definition. Review the Supplemental Code file for details. In the software, right click on any PIV data, and initiate Batch Processing.Select the operation Vector Statistics, Vector Field Results from the group Statistics, and click on Parameter in the Dialog box. Toggle the average V and RMSV options under the Vector Field section. Then, select the operation Vorticity from the group Extract Scalar Fields Rotation and Shear.This finds the two dimensional vorticity in the planar cross section. Now, start the processing by making a right click on any PIV data under the Project window, and selecting Hyperloop All Sets. To compute the Swirling Strength fields for the detection of Secondary Flow Structures, right click on any PIV data, select Velocity Vector Field in the tree, and initiate batch processing.Then, select the operation Swirling Strength from the group Extract Scalar Fields Rotation and Shear, and click on Close. Now, right click on the same Velocity Vector Field, select Hyperloop All Sets, and adjust the parameters for Batch Processing as before. Finally, execute the computation of the Swirling Strength fields.Secondary Flow Velocity data at the 90 degree cross-sectional location, was acquired from the 2C 2D PIV system. The inflow condition supplied to the curved artery test section with an Idealized Type IV stent fracture was the carotid artery wave form. The Secondary Flow Velocity field data was generated using the PIV technique, via synchronization of Trigger produced by the pump instrument control computer.Post-processing of the data was applied to the pixelated data, to determine the Q-criterion and Swirling Strength or Landis ci-criterion during the systolic deceleration. The Swirling Strength and Q-criterion-based flow fields were compared. The wortical patterns marked with DLW characters, representative of deformed Dean-Lynn-and Wall-type DLW wortical patterns, and strain-dominated regions, were examined at four distinct cross-sectional locations during systolic deceleration.The continuous Wavelet Transformed-based method was applied to the secondary flow vorticity field, that enabled vortex detection with greater detail in shape, size, and strength. Finally, the Q-criterion, Swirling Strength, and Wavelet Transformed-based flow fields were compared. The Wavelet Transformed-based method revealed the presence of complex wortical patterns with great detail in size, scale, and strengths.Once mastered, this technique can be done in approximately eight hours, including PIV data acquisition and post-processing, after the model stents are printed on the 3D printer. While attempting this procedure, it's important to remember to make sure the blood-analog fluid refractive index is adequately matched using minute quantities of sodium thiosulfate. In future, to improve paratheory result accuracy, and minimize the optical distortion, we will be using a PIV calibration map like this.Following this procedure, flows in patient-specific blood vessels can be evaluated in order to answer additional questions pertaining to pathological conditions, allowing unprecedented access to hemodynamics of prosthetics, stent implants, and fractured stents.

experimental-investigation-secondary-flow-structures-downstream-model

Research

JoVE Journal

Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

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 ...

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.

Here, we present a protocol to produce an oral vaccine candidate against Type 1 diabetes in an edible plant.

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the ...

Design of a Biocompatible Drug-Eluting Tracheal Stent in Mice with Laryngotracheal Stenosis

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of breath. LTS results from mucosal injury from a foreign body in the trachea, leading to tissue damage and a local inflammatory response that goes awry, leading to the deposition of pathologic scar tissue. Treatment for LTS is surgical due to the lack of effective medical therapies. The purpose of this method is to construct a biocompatible stent that can be miniaturized to place into mice with LTS. We demonstrated that a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) construct had optimal biomechanical strength, was biocompatible, practicable for an in vivo placement stent, and capable of eluting drug. This method provides a drug delivery system for testing various immunomodulatory agents to locally inhibit inflammation and reduce airway fibrosis. Manufacturing the stents takes 28&#8722;30 h and can be reproduced easily, allowing for experiments with large cohorts. Here we incorporated the drug rapamycin within the stent to test its effectiveness in reducing fibrosis and collagen deposition. Results revealed that PLLA-PCL tents showed reliable rapamycin release, were mechanically stable in physiological conditions, and were biocompatible, inducing little inflammatory response in the trachea. Further, the rapamycin-eluting PLLA-PCL stents reduced scar formation in the trachea in vivo.

Laryngotracheal stenosis results from pathologic scar deposition that critically narrows the tracheal airway and lacks effective medical therapies. Using a PLLA-PCL (70% poly-L-lactide and 30% polycaprolactone) stent as a local drug delivery system, potential therapies aimed at decreasing scar proliferation in the trachea can be studied.

Laryngotracheal stenosis (LTS) is a pathologic narrowing of the subglottis and trachea leading to extrathoracic obstruction and significant shortness of ...

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. Considering an implant as a foreign body, a hostile and dysregulated immune response may result in the rejection of the implant, while a regulated response and regaining of homeostasis can lead to its acceptance. Analyzing the microenvironments of implants dissected out under in vivo or ex vivo&#160;settings can help in understanding the pattern of immune response, which can ultimately help in developing new generations of biomaterials. Flow cytometry is a well-known technique for characterizing immune cells and their subsets based on their cell surface markers. This review describes a protocol based on manual dicing, enzymatic digestion, and filtration through a cell strainer for the isolation of uniform cell suspensions from dissected implant tissue. Further, a multicolor flow cytometry staining protocol has been explained, along with steps for initial cytometer settings to characterize and quantify these isolated cells by flow cytometry.

Isolation of cells from dissected implants and their characterization by flow cytometry can significantly contribute to understanding the pattern of immune response against implants. This paper describes a precise method for the isolation of cells from dissected implants and their staining for flow cytometric analysis.