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 ...

<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>

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

12.3K visualizações.  University of Nebraska-Lincoln. O procedimento demonstra a metodologia de elastografia de ressonância magnética para monitorar o resultado de engenharia de tecidos adiposo e construções osteogénicas engenharia através da avaliação não invasiva locais das propriedades mecânicas, utilizando elastografia ressonância magnética microscópica (μMRE).