Name: electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells
Uploaded: 2014-06-18T15:00:00
Description: Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

This work was conducted using the facilities of the Biomedical Engineering Department at Florida International University.

1. Electrospinning Collector Setup
<ol>
	<li>Cut the aluminum foil into a rectangular piece.</li>
	<li>Fold the rectangular piece into a rectangular strip, and attach it perpendicular to a flat metal plate with tape (Figure 1). Note: The size of the fiber mat depends on the length and width of the strip. Thus, the strip dimensions can be adjusted as needed.</li>
</ol>
2. Polymeric Solution Preparation
<ol>
	<li>Mix glycerol and dodecanedioic acid (DDA) in 1:1 molar ratio in a ...

<ol>
	<li>Migneco, F., Huang, Y. -. C., Birla, R. K., Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+(glycerol-dodecanoate),+a+biodegradable+polyester+for+medical+devices+and+tissue+engineering+scaffolds.">Poly (glycerol-dodecanoate), a biodegradable polyester for medical devices and tissue engineering scaffolds.</a> Biomaterials. 30, 6479-6484 (2009).</li><li>Reneker, D. H., Yarin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+jets+and+polymer+nanofibers.">Electrospinning jets and polymer nanofibers.</a> Polymer. 49, 2387-2425 (2008).</li><li>Wang, Y., Ameer, G. A., Sheppard, B. J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tough+biodegradable+elastomer.">A tough biodegradable elastomer.</a> Nature biotechnology. 20, 602-606 (2002).</li><li>Panunzi, S., De Gaetano, A., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approximate+linear+confidence+and+curvature+of+a+kinetic+model+of+dodecanedioic+acid+in+humans.">Approximate linear confidence and curvature of a kinetic model of dodecanedioic acid in humans.</a> American Journal of Physiology-Endocrinology And Metabolism. 289, (2005).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+preparing+electrospun+nanofibers:+designing+an+electrospinning+process+for+nanofiber+fabrication.">Apparatus for preparing electrospun nanofibers: designing an electrospinning process for nanofiber fabrication.</a> Polymer Internationa l. 56, 1361-1366 (2007).</li><li>Barnes, C. P., Sell, S. A., Boland, E. D., Simpson, D. G., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+technology:+designing+the+next+generation+of+tissue+engineering+scaffolds.">Nanofiber technology: designing the next generation of tissue engineering scaffolds.</a> Advanced drug delivery reviews. 59, 1413-1433 (2007).</li><li>Li, W. -. J., Mauck, R. L., Tuan, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+scaffolds:+production,+characterization,+and+applications+for+tissue+engineering+and+drug+delivery.">Electrospun nanofibrous scaffolds: production, characterization, and applications for tissue engineering and drug delivery.</a> Journal of Biomedical Nanotechnology. 1, 259-275 (2005).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+polymeric+nanofibers+for+tissue+engineering+applications:+a+review.">Electrospinning of polymeric nanofibers for tissue engineering applications: a review.</a> Tissue engineering. 12, 1197-1211 (2006).</li><li>Lim, S. H., Mao, H. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+scaffolds+for+stem+cell+engineering.">Electrospun scaffolds for stem cell engineering.</a> Advanced drug delivery reviews. 61, 1084-1096 (2009).</li><li>Lowery, J. L., Datta, N., Rutledge, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fiber+diameter,+pore+size+and+seeding+method+on+growth+of+human+dermal+fibroblasts+in+electrospun+poly+(epsilon-caprolactone)+fibrous+mats.">Effect of fiber diameter, pore size and seeding method on growth of human dermal fibroblasts in electrospun poly (epsilon-caprolactone) fibrous mats.</a> Biomaterials. 31, 491-504 (2010).</li><li>Tillman, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+stability+of+electrospun+polycaprolactone-collagen+scaffolds+in+vascular+reconstruction.">The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction.</a> Biomaterials. 30, 583-588 (2009).</li><li>Ju, Y. M., Choi, J. S., Atala, A., Yoo, J. J., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayered+scaffold+for+engineering+cellularized+blood+vessels.">Bilayered scaffold for engineering cellularized blood vessels.</a> Biomaterials. 31, 4313-4321 (2010).</li><li>McCullen, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+collagen+polymerization+of+layered+cell-seeded+electrospun+scaffolds+for+bone+tissue+engineering+applications.">In situ collagen polymerization of layered cell-seeded electrospun scaffolds for bone tissue engineering applications.</a> Tissue Engineering Part C: Methods. 16, 1095-1105 (2010).</li></ol>

The limitations of simple collectors or the complexities of rotating collectors that are currently used for electrospinning increase the restriction of obtaining the desired length and size of fiber mat for some applications. Additionally, transferring fibers from the ground collector to the culture dish or other substrates is a challenge5. In this report, a newly designed collector, made simply by attaching an aluminum foil strip to the grounded collector, was able to obtain large size fiber mats up to 10 cm ...

Electrospinning is one of the effective processing methods to produce micro-to-nanometer size fiber scaffolds. The basic principle of electrospinning involves a Taylor cone of solution that is held at the orifice of a needle by applying high voltage between the tip of the needle and a grounded collector. When the electrostatic repulsion in the solution overcomes the surface tension, a charged fluid jet is ejected out of the needle tip, travels through the air with solvent evaporation, and is finally deposited on the grounded collector. The syringe pump provides a continuous flow of solution emerging from the spinneret and thus multiple copies of the electrospun fibers can be fabricated within a short period of time. During the course of leaving the spinneret to arrive at the collector, the charged jet will undergo stretching and whipping according to a number of parameters that include the viscosity and surface tension of the polymeric solution, the electrostatic force in the solution, and the interaction of the external electric field, etc2.
In the electrospinning process, a collector serves as a conductive substrate where the micro-to-nanometer fibers could be deposited. In this study, a new type of fiber collector was designed to obtain fiber mats with the desired size (length x width). Traditionally, aluminum foil is used as a collector but it is difficult to transfer the fibers from the flat surface to another substrate. The difficulty of harvesting an intact fiber mat from a traditional collector was mainly due to the fact that the electrospun fibers attach strongly to the collector&#39;s surface. Therefore, we modified the collector by folding a piece of aluminum foil into a rectangular strip and attaching it perpendicular to a flat metal plate. The electrospun fibers are stretched across the area between the tip of the strip and the metal plate, which can be easily transferred to another substrate.
Interest in thermally crosslinked elastomeric polymers is rapidly growing because of the pioneering work of Robert Langer&#39;s group, who introduced poly(glycerol sebacate) (PGS), a polyester which is analogous to vulcanized rubber in 2002&#160;3. Similar to PGS, we have successfully developed poly(glycerol-dodecanoate) (PGD) by thermal condensation of glycerol and dodecanedioic acid and demonstrated its unique shape memory property1. Unlike stiffer synthetic materials poly(hydroxyl butyrate) or poly(L-lactide) (Young&#39;s moduli of 250 MPa and 660 MPa, respectively), PGD exhibits elastomeric property like rubber, with a Young&#39;s modulus of 1.08 MPa when the temperature is above 37 &#176;C, which is a close match to the in-situ peripheral nerve (0.45 MPa). In addition, PGD is biodegradable and the degradation time can be fine-tuned by varying the ratio of glycerol and dodecanedioic acid. Dodecanedioic acid is a twelve-carbon substance with two terminal carboxylic groups, HOOC(CH2)10COOH. Even numbered dicarboxylic acids like sebacic acid and dodecanedioic acid can be metabolized to acetyl-CoA and enter the tricarboxylic acid (TCA)/(citric acid) cycle. The metabolic product of dicarboxylic acids, succinyl-CoA, is a gluconeogenetic precursor and intermediate of TCA cycle4. Thus, some studies suggested that they could be utilized as an alternative fuel substrate for enteral and parenteral nutrition, especially in the pathological conditions. In addition, PGD exhibits unique shape memory because its glass transition temperature is 31 &#176;C, thus it shows distinct mechanical properties at room temperature and at body temperature.&#160;In sum, PGD is biodegradable, biocompatible, exhibiting unique elastic properties with mechanical properties similar to nerve tissues; therefore, it is a suitable material for nerve tissue engineering applications. In this protocol, the electrospun long fibers spanning a large deposit area were fabricated via the newly-designed collector from PGD. The fiber scaffolds can support the mouse pluripotent stem cells growth and differentiation.

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Glycerol</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7757</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dodecanedioic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Gelatin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Poly (ehtylene oxide) (PEO)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">182028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Riboflavin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">132350250</td>
			<td style="width:167px;">0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Mouse embryonic stem cells</td>
			<td style="width:175px;">GlobalStem</td>
			<td style="width:119px;">GSC-5002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Matrigel</td>
			<td style="width:175px;">Becton Dickinson</td>
			<td style="width:119px;">356234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">DMEM/F12</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:119px;">SH30272.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">N2 supplement&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">17502048</td>
			<td style="width:167px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">FGF2</td>
			<td style="width:175px;">Stemgent</td>
			<td style="width:119px;">03-0002</td>
			<td style="width:167px;">10ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Accutase</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A11105-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">10010-031&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Resazurin fluorescence dye&#160;</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">62758-13-8&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">SV Total RNA Isolation System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">Z3100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoScript Reverse Transcription System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A5000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoTaq&#129; qPCR Master Mix</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Syringe pump&#160;</td>
			<td style="width:175px;">Fisher scientific</td>
			<td style="width:119px;">14-831-200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:268px;">High voltage power source&#160;</td>
			<td style="width:175px;">Spellman High Voltage Electronics Corporation</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">SL30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">UV light</td>
			<td style="width:175px;">Philips</td>
			<td style="width:119px;">308643</td>
			<td style="width:167px;">15W/G15T8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Synergy HT Multi-Mode Microplate Reader</td>
			<td style="width:175px;">BioTek</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Perkin Elmer GeneAmp PCR System 9600</td>
			<td style="width:175px;">Perkin Elmer</td>
			<td style="width:119px;">8488</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">StepOne Real-time PCR System</td>
			<td style="width:175px;">Applied Biosystems</td>
			<td style="width:119px;">4376357</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

The major components of the electrospinning are shown in Figure 1. A large size fiber mat was typically obtained through the perpendicularly attached aluminum foil strip and a flat metal plate. Figure 2 shows the collector design and the electrospinning fiber mat. The width and length can be adjusted for different applications. The length of the fiber made with PGD polymer and basal solution mixture is up to 10 cm. The morphology of electrospun fibers is shown in Figure 3</strong...

Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

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