This research was supported in part by NIH RO3-EB007299-02 and NSF EPSCoR First Award.

1. Tissue Construct Preparation
 The tissue construct preparation process consists of three main stages: expansion of cell population, seeding of cells onto a biomaterial scaffold, and differentiation through the use of chemical signaling molecules. The procedure for construct preparation is based on methods conducted by Dennis et al., Hong et al., and Marion and Mao8,9,10. 
 <ol>
 <li>After culture and expansion of the cell line, seed the human mesenchymal stem cells (hMSCs) onto a gelatin sponge (4 mm diameter, 3.5 mm thickness) at a density of 1x106 cells/ml for bone and 3x106 cells/ml for adipose formation.</li>
 <li>For differentiation of hMSCs into adipose, apply the adipose induction media consisting of 1 &mu;M dexamethasone, 0.5 &mu;M isobutyl-methylxanthine, 10 &mu;g/ml human recombinant-insulin, and 200 &mu;M indomethacin in cell expansion medium once cells appear confluent on the scaffold. After three days, replace the media with 10 &mu;g/ml of human recombinant-insulin in expansion media for 24 hours then return to induction media. Repeat the cycle three times and then exchange only in maintenance media every two days. </li>
 <li>To induce osteogenesis, prepare osteogenic induction media by making a final concentration of 0.1 &mu;M dexamethasone, 50 &mu;M of L-ascorbic acid-2phosphate, and 10 mM &beta;-glycerophosphate in cell expansion medium. Replace with fresh osteogenic media every two days.</li>
 </ol>
 2. Actuator Characterization 
 Characterization of the actuator is a vital step for the MRE experiment. MRE relies on the propagation of mechanical shear waves to assess local values of mechanical properties; therefore, these mechanical vibrations need to be generated and characterized within the tissue of interest using a piezoelectric actuator. An illustrated example of the characterization process is shown in Figure 3. The goal of this procedure is to optimize the motion of the actuator in order to generate harmless shear waves with significant amplitudes (~250 micron).
 <ol>
 <li>Prior to the experiment, apply 0.5% agarose gel to enclose the construct in a 10 mm test tube. The temperature of the gel should be approximately 37 &deg;C to minimize damage to the construct. </li>
 <li>After allowing the agarose gel five minutes to set at room temperature, insert the tip of the piezoelectric bending motor into the surface of the gel. </li>
 <li>Attach the tube containing the sample and the actuator to a rigid support, and orient the beam of the Laser Doppler Vibrometer toward the tip of the mechanical actuator. Adjust the positioning of the system to optimize the reflected signal, use reflective tape if necessary. </li>
 <li>Based on the expected resonance frequency of the mechanical actuator, set the function generator to sweep the desired frequency range (i.e. 20 to 2000 Hz in this experiment) using an operating voltage of 20 Vpp with a white noise signal. </li>
 <li>View the characterized spectrum on the Polytec Vibrosoft program to identify the resonance frequency of the system and set the program to FFT and velocity as the y-axis. </li>
 <li>For displacement measurement, set the actuator to deliver a continuous sinusoid at the indicated resonance frequency using an operating voltage of 200 Vpp, and note the generated displacement being delivered to the surface of the gel. Set Vibrosoft to display the FFT with displacement as the y-axis. </li>
 </ol>
 3. Image Acquisition
 <ol>
 <li>After completing actuator characterization, place the sample and actuator in the center of the MRI scanner. For tissue construct experiments, use a small and more sensitive RF coil (i.e. 10 mm in this experiment) for the transmission and reception of the RF signal. (The procedure shown uses a 9.4 T vertical bore magnet equipped with triple axis gradients, 100 G/cm). </li>
 <li>Acquire a scout image for identification of the construct location.</li>
 <li>Set the parameters for the acquisition. A typical in vitro sagittal scan will have a repetition time of 1000 ms, echo time of 20-40 ms, slice thickness of 0.5-1.0 mm, and field of view of 12x10 mm2 with a matrix size of 128x128 pixels. </li>
 <li>For the elastography parameters, set the actuator frequency to the value determined by the Laser Doppler Vibrometer characterization. In the current study, one bipolar pair was needed with a gradient amplitude of 50 G/cm. Other parameters to adjust include the delay which should be set to zero milliseconds for the initial acquisition.</li>
 <li>Change the function generator to burst mode and adjust the parameters of the function generator to match those in the elastography acquisition parameters including the frequency and number of cycles. Also, set the function generator to be externally triggered. </li>
 <li>For a sagittal image, set the motion sensitization to be in the positive slice direction and start the scan. Following acquisition, check the image and change the sensitization to the negative slice direction.</li>
 <li>Execute the MATLAB program that will perform the complex division for generation of the shear wave image.</li>
 <li>Assess the image for the presence of shear waves and possible artifacts such as phase wrapping.</li>
 <li>If no adjustments to the image are necessary, adjust parameter array size to eight equally spaced values ranging from zero seconds to a full period of the characterized resonance frequency. </li>
 <li>Acquire a scan in both the positive and negative slice orientations.</li>
 <li>Once the images are acquired, use a MATLAB program designed for generating the shear wave data from an array of images.</li>
 </ol>
 4. MRE Experiment Image Processing
 <ol>
 <li>The final step of MRE is to calculate the shear stiffness from the shear wave images. Placing the data into the MATLAB program that will assess the three dimensional dataset (2 spatial, 1 temporal).</li>
 </ol>
 Note: By assuming a planar shear wave, the equations of motion decouple allowing the estimation of the complex-valued shear modulus as a function of the displacement and its Laplacian. The algorithm approximates spatial second derivatives with finite difference and computes the shear modulus on a pixel-by-pixel basis. From this complex number, many mechanical parameters can be deduced such as the shear wave speed, wave attenuation, shear stiffness, shear elasticity, shear viscosity, etc. The algorithm also allows the selection of regions of interest for which the mean and standard deviation of each parameter is calculated. 
 <ol start="2">
 <li>The imaging parameters need to be specified at the beginning of the program. Additionally, the upper limit of the elastogram can be adjusted to optimize contrast in the sample. </li>
 </ol>
 Note: The program provides intermediate results (wave after low-pass filters, wave after directional filtering, temporal FFT, line profiles, etc.) that help the user estimate the faithfulness of the recovery. 
 <ol start="3">
 <li>Some parameters can be adjusted based on this information, such as the levels of low-pass filters, the temporal frequency of the motion, the direction of propagation of the wave, etc. The standard deviation of a parameter in a specific region of interest is also an indicator of the quality of the calculation. </li>
 </ol>
 5. Representative Results
 Figure 4 notes the change in mechanical properties throughout four weeks of osteogenic and adipogenic construct development. MRE was conducted at 730-820 Hz. While both seeded sponges started at approximately 3 kPa, osteogenic directed tissues resulted in a stiffness of 22 kPa; whereas, adipose directed tissues decreased in stiffness to 1 kPa. Furthermore, the osteogenic constructs showed a notable decrease in size in comparison from beginning to end of the study. Additional properties derived from elastography study are shown in Table 1.
 <img src="/files/ftp_upload/3618/3618fig1.jpg" alt="Figure 1"/> Figure 1. The image acquisition process for magnetic resonance elastography. During image acquisition, a pulse sequence (a) controls the synchronization (b) of the function generator with the bipolar gradients pulses of the MRI scanner. Following acquisition of bipolar gradients toggled in positive and negative orientations, (c) a shear wave image is produced using complex division.
 <img src="/files/ftp_upload/3618/3618fig2.jpg" alt="Figure 2"/> Figure 2. Flow diagram of the MRE process for tissue engineered constructs. First, cells (a) are first grown and expanded to the population size essential for the designed project. Then the cells are seeded (b) onto a biomaterial scaffold and chemical reagents are applied to signal differentiation. Scaffolds are characterized with MRE, whose first step (c) is the determination of the resonance frequency of the actuator coupled to the construct. Next, MRI images (d) are acquired to generate a shear wave image (e). Finally, an algorithm is applied to yield an elastogram (f) that maps the stiffness of the construct. Concurrently, constructs are sectioned for histological assessment (g) in order to validate differentiation. 
 <img src="/files/ftp_upload/3618/3618fig3.jpg" alt="Figure 3"/> Figure 3. Actuator characterization procedure. The gelatin scaffold is enclosed by a 0.5% agarose gel. To characterize the motion being transferred into the sample a white noise is first sent into the system (1a) and the resulting motion is detected using a Laser Doppler Vibrometer (1b). Once the resonance frequency is determined, a continuous sinusoid signal at resonance (2a) is sent to determine the displacement (2b) transferred to the gelatin environment. 
 <img src="/files/ftp_upload/3618/3618fig4.jpg" alt="Figure 4"/> Figure 4. Construct development map over four week period. Adipogenic (A) and osteogenic (O) constructs are shown from left to right with corresponding magnitude and shear wave images, elastogram, and average shear stiffness. The colormap for the elastogram corresponds with the color scheme of the bar chart and error bars represent the standard deviation within each construct's region of interest. 
<img src="/files/ftp_upload/3618/3618Table1.jpg" alt="Table 1"/> Table 1. Mechanical properties of adipose and osteo constructs over a four week period of growth.

<ol>
	<li>Xu, H., Othman, S. F., Magin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+tissue+engineering+using+magnetic+resonance+imaging.">Monitoring tissue engineering using magnetic resonance imaging.</a> J. Biosci. Bioeng. 106, 515-527 (2008).</li><li>Xu, H., Othman, S. F., Hong, L., Peptan, I. A., Magin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+microscopy+for+monitoring+osteogenesis+in+tissue-engineered+construct+in+vitro.">Magnetic resonance microscopy for monitoring osteogenesis in tissue-engineered construct in vitro.</a> Phys. Med. Biol. 51, 719-732 (2006).</li><li>Othman, S. F., Xu, H., Royston, T. J., Magin, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+magnetic+resonance+elastography+(microMRE.">Microscopic magnetic resonance elastography (microMRE.</a> Magn. Reson. Med. 54, 605-615 (2005).</li><li>Muthupillai, R., Lomas, D. J., Rossman, P. J., Greenleaf, J. F., Manduca, A., Ehman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+elastography+by+direct+visualization+of+propagating+acoustic+strain+waves.">Magnetic resonance elastography by direct visualization of propagating acoustic strain waves.</a> Science. 269, 1854-1857 (1995).</li><li>Othman, S. F., Curtis, E. T., Plautz, S. A., Pannier, A. P., Xu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+elastography+monitoring+of+tissue+engineered+constructs.">Magnetic resonance elastography monitoring of tissue engineered constructs.</a> NMR Biomed. , (2011).</li><li>Oliphant, T. E., Manduca, A., Ehman, R. L., Greenleaf, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex-valued+stiffness+reconstruction+for+magnetic+resonance+elastography+by+algebraic+inversion+of+the+differential+equation.">Complex-valued stiffness reconstruction for magnetic resonance elastography by algebraic inversion of the differential equation.</a> Magn. Reson. Med. 45, 299-310 (2001).</li><li>Ringleb, S. I., Chen, Q., Lake, D. S., Manduca, A., Ehman, R. L., An, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+shear+wave:+comparison+to+a+dynamic+shear+material+test.">Quantitative shear wave: comparison to a dynamic shear material test.</a> Magn. Reson. Med. 53, 1197-1201 (2005).</li><li>Hong, L., Peptan, I., Clark, P., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+adipose+tissue+engineering+by+human+marrow+stromal+cell+seeded+gelatin+sponge.">Ex vivo adipose tissue engineering by human marrow stromal cell seeded gelatin sponge.</a> Ann. Biomed. Eng. 33, 511-517 (2005).</li><li>Dennis, J. E., Haynesworth, S. E., Young, R. G., Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenesis+in+marrow-derived+mesenchymal+cell+porous+ceramic+composites+transplanted+subcutaneously:+effect+of+fibronectin+and+laminin+on+cell+retention+and+rate+of+osteogenic+expression.">Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutaneously: effect of fibronectin and laminin on cell retention and rate of osteogenic expression.</a> Cell Transplant. 1, 23-32 (1992).</li><li>Marion, N. W., Mao, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+tissue+engineering.">Mesenchymal stem cells and tissue engineering.</a> Methods Enzymol. 420, 339-361 (2006).</li><li>Rydberg, J., Grimm, R., Kruse, S., Felmlee, J., McCracken, P., Ehman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+spin-echo+magnetic+resonance+elastography+of+the+brain.">Fast spin-echo magnetic resonance elastography of the brain.</a> , 1647-1647 (2001).</li><li>Kruse, S. A., Grim, R. C., Lake, D. S., Manduca, A., Ehman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+EPI+based+3D+MR+elastography+of+the+brain.">Fast EPI based 3D MR elastography of the brain.</a> , 3385-3385 (2006).</li></ol>

The authors have no conflicts of interest to disclose.

In this procedure, the process of MRE for tissue engineered constructs is demonstrated from cell preparation to the generation of an elastogram. By applying a nondestructive mechanical assessment method to the tissue engineering pipeline, it is now possible to evaluate changes in engineered constructs throughout multiple stages of development. In addition, MRE complements other MR methods for monitoring tissue engineered constructs such as diffusion, magnetization transfer, and chemical shift analysis1. 
...

<table border="1">
 <tbody>
 <tr>
 <td>Material Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>MSCGM-Bullet Kit</td>
 <td>Reagent</td>
 <td>Lonza</td>
 <td>PT-3001</td>
 <td>Store at 4&deg;C</td>
 </tr>
 <tr>
 <td>1X DPBS</td>
 <td>Reagent</td>
 <td>Invitrogen</td>
 <td>21600-010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.05% Trypsin-EDTA</td>
 <td>Reagent</td>
 <td>Gibco, Invitrogen</td>
 <td>25300-054</td>
 <td>Store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Dexamethasone</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>D2915</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>3-Isobutyl-1-methylxanthine</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>I5879</td>
 <td>Store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Insulin-bovine pancreas</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>I6634</td>
 <td>Store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Indomethacin</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>I7378</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&Beta;-Glycerophosphate</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>G9891</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Ascorbic Acid 2-phosphate</td>
 <td>Reagent</td>
 <td>Sigma-Aldrich</td>
 <td>A8960</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gelfoam</td>
 <td>Scaffold</td>
 <td>Pharmacia &amp; Upjohn Co.</td>
 <td>09-0315-08</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human mesenchymal stem cells</td>
 <td>Cell Line</td>
 <td>Lonza</td>
 <td>PT-2501</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>9.4T MR Scanner</td>
 <td>Equipment</td>
 <td>Agilent</td>
 <td>&nbsp;</td>
 <td>400MHz WB</td>
 </tr>
 <tr>
 <td>10mm Litz Coil</td>
 <td>Equipment</td>
 <td>Doty Scientific</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laser Doppler Vibrometer</td>
 <td>Equipment</td>
 <td>Polytec</td>
 <td>PDV-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vibrosoft (20)</td>
 <td>Software</td>
 <td>Polytec</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Function generator</td>
 <td>Equipment</td>
 <td>Agilent</td>
 <td>AFG 3022B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Equipment</td>
 <td>Piezo inc</td>
 <td>EPA-104-115</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Piezo Bending motor</td>
 <td>Equipment</td>
 <td>Piezo inc.</td>
 <td>T234-A4Cl-203X</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Computer-Linux</td>
 <td>Equipment</td>
 <td>Processor: Intel Core 2 Duo E8400 
 Memory: 2G</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Computer-Windows</td>
 <td>Equipment</td>
 <td>Processor: Intel Core 2 Duo E8400 
 Memory: 2G</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MATLAB</td>
 <td>Software</td>
 <td>Mathworks, inc</td>
 <td>&nbsp;</td>
 <td>2009b</td>
 </tr>
 </tbody>
 </table>

magnetic resonance elastography methodology for the evaluation of tissue engineered construct growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

Watch this Scientific Journal Video about Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth at JoVE.com

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

magnetic-resonance-elastography-methodology-for-evaluation-tissue

Research

JoVE Journal

Bioengineering

12.4K 次观看.  University of Nebraska-Lincoln. 程序演示磁共振弹性的脂肪和骨组织，通过当地的机械性能，使用显微磁共振弹性（μMRE）的无创性评估的工程结构设计的结果监测方法。

视频: Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Custom Engineered Tissue Culture Molds from Laser-etched Masters

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

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

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

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...