Name: electrospun nanofiber scaffolds with gradations in fiber organization
Uploaded: 2015-04-19T15:00:00
Description: Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

Ce travail a été soutenu en partie par des fonds de démarrage de l&#39;Université du Nebraska Medical Center et l&#39;Institut national de la santé (numéro de subvention 1R15 AR063901-01).

1. Préparation de la solution <ol><li> Préparer une solution de poly (ε-caprolactone) (PCL) (Mw = 80 000 g / mol) à une concentration approximative de 100 mg / ml. Dissoudre PCL dans un mélange de dichlorométhane (DCM) et de N, N-dimethlyformamide (DMF) dans un rapport de 4: 1 (v / v) à une concentration de 10% (p / v). </li><li> Placer la solution dans un tube en verre de 20 ml pour le mélange. Placez le tube de verre dans nettoyeur à ultrasons pendant 30 minutes, ou jusqu&#39;à ce que la solutio...

<ol>
	<li>Xie, J., Li, X., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Putting+electrospun+nanofibers+to+work+for+biomedical+research.">Putting electrospun nanofibers to work for biomedical research.</a> Macromol. Rapid Commun. 29 (22), 1775-1792 (2008).</li><li>Genin, G. M., 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=Functional+grading+of+mineral+and+collagen+in+the+attachment+of+tendon+to+bone.">Functional grading of mineral and collagen in the attachment of tendon to bone.</a> Biophys. J. 97 (4), 976-985 (2009).</li><li>Thomopoulos, S., Marquez, J. P., Weinberger, B., Birman, V., Genin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fiber+orientation+at+the+tendon+to+bone+insertion+and+its+influence+on+stress+concentrations.">Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations.</a> J. Biomech. 39 (10), 1842-1851 (2006).</li><li>Thomopoulos, S., Williams, G. R., Gimbel, J. A., Favata, M., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variation+of+biomechanical,+structural,+and+compositional+properties+along+the+tendon+to+bone+insertion+site.">Variation of biomechanical, structural, and compositional properties along the tendon to bone insertion site.</a> J. Orthop. Res. 21 (3), 413-419 (2003).</li><li>Thomopoulos, S., Genin, G. M., Galatz, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+morphogenesis+of+the+tendon-to-bone+insertion+-+What+development+can+teach+us+about+healing.">The development and morphogenesis of the tendon-to-bone insertion - What development can teach us about healing.</a> Musculoskelet Neuronal Interact. 10 (1), 35-45 (2010).</li><li>Li, X., Xie, J., Lipner, J., Yuan, X., Thomopoulos, S., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+scaffolds+with+gradations+in+mineral+content+for+mimicking+the+tendon-to-bone+insertion+site.">Nanofiber scaffolds with gradations in mineral content for mimicking the tendon-to-bone insertion site.</a> Nano Lett. 9 (7), 2763-2768 (2009).</li><li>Kunzler, T. P., Huwiler, C., Drobek, T., V&#246;r&#246;s, J., Spencer, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+study+of+osteoblast+response+to+nanotopography+by+means+of+nanoparticle-density+gradients.">Systematic study of osteoblast response to nanotopography by means of nanoparticle-density gradients.</a> Biomaterials. 28 (33), 5000-5006 (2007).</li><li>Huwiler, C., Kunzler, T. P., Textor, M., V&#246;r&#246;s, J., Spencer, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalizable+nanomorphology+gradients+via+colloidal+self-assembly.">Functionalizable nanomorphology gradients via colloidal self-assembly.</a> Langmuir. 23 (11), 5929-5935 (2007).</li><li>Xie, J., 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='Aligned-to-random'+nanofiber+scaffolds+for+mimicking+the+structure+of+the+tendon-to-bone+insertion+site.">'Aligned-to-random' nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site.</a> Nanoscale. 2 (6), 923-926 (2010).</li><li>Xie, J., Ma, B., Michael, P. L., Shuler, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+nanofiber+scaffolds+with+gradations+in+fiber+organization+and+their+potential+applications.">Fabrication of nanofiber scaffolds with gradations in fiber organization and their potential applications.</a> Macromol. Biosci. 12 (10), 1336-1341 (2012).</li><li>James, R., Kumbar, S. G., Laurencin, C. T., Balian, G., Chhabra, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+tissue+engineering:+adipose-derived+stem+cell+and+GDF-5+mediated+regeneration+using+electrospun+matrix+systems.">Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems.</a> Biomed. Mater. 6 (2), 025011 (2011).</li><li>Bodle, J. C., Hanson, A. D., Loboa, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells+in+functional+bone+tissue+engineering:+lessons+from+bone+mechanobiology.">Adipose-derived stem cells in functional bone tissue engineering: lessons from bone mechanobiology.</a> Tissue Eng. Part B Rev. 17 (3), 195-211 (2011).</li><li>Lee, J. H., Rhie, J. W., Oh, D. Y., Ahn, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+differentiation+of+human+adipose+tissue-derived+stromal+cells+(hASCs)+in+a+porous+three-dimensional+scaffold.">Osteogenic differentiation of human adipose tissue-derived stromal cells (hASCs) in a porous three-dimensional scaffold.</a> Biochem. Biophys. Res. Commun. 370 (3), 456-460 (2008).</li><li>Tapp, H., Hanley, E. N., Patt, J. C., Gruber, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+characterization+and+current+application+in+orthopaedic+tissue+repair.">Adipose-derived stem cells: characterization and current application in orthopaedic tissue repair.</a> Exp. Biol. Med. 234 (1), 1-9 (2009).</li><li>Gimble, J. M., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+adult+stem+cells:+isolation,+characterization,+and+differentiation+potential.">Adipose-derived adult stem cells: isolation, characterization, and differentiation potential.</a> Cytotherapy. 5 (5), 362-369 (2003).</li><li>Zuk, P. A., 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=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Eng. 7 (2), 211-228 (2001).</li><li>Xie, J., 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+differentiation+of+embryonic+stem+cells+seeded+on+electrospun+nanofibers+into+neural+lineages.">The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages.</a> Biomaterials. 30 (3), 354-362 (2009).</li><li>Xie, J., MacEwan, M. R., Li, X., Sakiyama-Elbert, S. E., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+outgrowth+on+nanofiber+scaffolds+with+different+orders,+structures,+and+surface+properties.">Neurite outgrowth on nanofiber scaffolds with different orders, structures, and surface properties.</a> ACS Nano. 3 (5), 1151-1159 (2009).</li><li>Ayres, C., 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=Modulation+of+anisotropy+in+electrospun+tissue+engineering+scaffolds:+analysis+of+fiber+alignment+by+the+fast+Fourier+transform.">Modulation of anisotropy in electrospun tissue engineering scaffolds: analysis of fiber alignment by the fast Fourier transform.</a> Biomaterials. 27 (32), 5524-5534 (2006).</li><li>Ayres, C., 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=Measuring+fiber+alignment+in+electrospun+scaffolds:+a+user&#8217;s+guide+to+the+2D+fast+Fourier+transform+approach.">Measuring fiber alignment in electrospun scaffolds: a user&#8217;s guide to the 2D fast Fourier transform approach.</a> J. Biomater. Sci. Poly. Ed. 19 (5), 603-621 (2008).</li></ol>

The most critical part of the protocol is generation of the gradient scaffold. It is imperative that the mask covering the collector moves at a constant velocity so there is a gradual change within the fiber scaffold. The correct preparation of PCL solution is also important to ensure electrospinning success. Checking the fiber morphology prior to electrospinning is recommendable, especially after the encapsulation of Coumarin-6, which may require a higher voltage to electrospin correctly. 
Fu...

Les nanofibres sont un utilitaire populaire pour l&#39;ingénierie tissulaire en raison de leur capacité à imiter la matrice extracellulaire dans sa structure et une taille relative. Cependant, certaines interfaces tissulaires natives, telles que le site d&#39;insertion du tendon-os, contiennent des fibres de collagène, qui présentent une structure organisationnelle variable qui augmente en direction de l&#39;alignement des tendons et diminue au niveau du site de l&#39;os 2-5. Ainsi, pour la régénération tissulaire effective, il est nécessaire de fabriquer un échafaudage qui pourrait effectivement mimer la structure de ce gradient. Auparavant, il a été effectué des recherches sur des changements graduels dans la composition des fibres, en particulier, la teneur en minéraux 6. Cependant, recréant la composante structurelle des tissus conjonctifs reste largement inexploré. Une étude antérieure a examiné gradients morphologiques en étudiant l&#39;effet de la densité des particules de silice de surface sur la prolifération des ostéoblastes de la voûte crânienne du rat et ont trouvé une inverSE relation entre la densité de particules de silice et la prolifération des cellules 7. Mais les changements morphologiques médiation prolifération cellulaire dans des travaux antérieurs étaient principalement liés à la rugosité de surface manque la capacité en imitant fibres changements organisationnels 7,8. Une étude récente a tenté de fabriquer un échafaudage qui imitait les orientations uniques de fibres de collagène en utilisant un roman collecteur pour électrofilage neuf. Bien que cette étude a réussi à produire un échafaudage avec des fibres à la fois alignés et aléatoires, il n&#39;a pas réussi à imiter les changements graduels exposées dans les tissus natifs. Aussi, dans la production de composants séparés, avec un changement immédiat de alignée sur l&#39;orientation aléatoire, les propriétés biomécaniques de cette échafaudage diminué de manière significative. Aucun travail précédente a été en mesure de produire des échafaudages de nanofibres applicables avec des gradations continus dans des orientations de fibres de alignés et aléatoire. Notre étude récente a montré loisirs succès des échafaudages de nanofibresavec des gradations dans l&#39;organisation de fibres qui peuvent potentiellement imiter l&#39;organisation de collagène natif au tendon-os insertion 10. Ce travail vise à présenter les protocoles utilisés pour la production d&#39;échafaudages de nanofibres avec une structure qui ressemble étroitement à celui de l&#39;organisation de la fibre dans l&#39;interface native tendon-os tissus. Structures de nanofibres gradient ont potentiellement des applications dans une variété de domaines de grande envergure. Nous nous sommes concentrés sur les applications à l&#39;ingénierie tissulaire du site d&#39;insertion du tendon-os en combinant nos échafaudages avec des cellules souches du tissu adipeux (ADSCs) qui sont déjà utilisés pour la régénération tissulaire sur divers substrats 11-14. En outre, ADSCs sont très similaires dans la nature à des cellules souches de moelle osseuse en termes de ressources et de leur multipotence est abondante qui peut être récolté en utilisant un simple 15,16 procédure de liposuccion. Ensemencement ces cellules aux échafaudages de nanofibres dégradés améliore encore leur tispoursuivre applications d&#39;ingénierie en permettant la distribution contrôlée des cellules qui peuvent potentiellement se différencier en divers tissus. En plus de l&#39;ensemencement des cellules souches, des nanofibres peuvent être encapsulés avec des molécules de signalisation pour la régulation de la réponse cellulaire. Couplage nanoencapsulation avec le gradient d&#39;organisation de ces échafaudages permet l&#39;étude du comportement cellulaire ou dessins et revêtements implants possibles. L&#39;encapsulation des molécules fonctionnelles telles que la protéine morphogénétique osseuse 2 (BMP2), qui a été montré pour induire la différenciation ostéoblastique 15,16, permettrait d&#39;améliorer encore les applications d&#39;ingénierie tissulaire de ces échafaudages 10.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polycaprolactone</td>
			<td>Sigma-Aldrich</td>
			<td>440744</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethlyformamide</td>
			<td>Fisher Chemical</td>
			<td>D-119-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Chemical</td>
			<td>AC61093-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coumarin 6</td>
			<td>Sigma-Aldrich</td>
			<td>546283</td>
			<td></td>
		</tr>
		<tr>
			<td>Adipose Derived Stem Cells</td>
			<td>Cellular engineering Technologies</td>
			<td>HMSC.AD-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>26140-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-Modified Eagle&#39;s Medium</td>
			<td>Invitrogen</td>
			<td>a10490-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher Scientific</td>
			<td>s25120a</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Invitrogen</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slides</td>
			<td>VWR international, LLC</td>
			<td>101412-842</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Fisher Scientific</td>
			<td>14-831-200</td>
			<td>Single syringe</td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaner</td>
			<td>Branson</td>
			<td>1510</td>
			<td></td>
		</tr>
		<tr>
			<td>High Voltage DC Power Supply</td>
			<td>Gamma High Voltage Research</td>
			<td>ES30</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>FEI</td>
			<td>Nova 2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager 2</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrospun nanofiber scaffolds with gradations in fiber organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

En utilisant ce protocole, un tapis de fibre à gradient d&#39;organisation a été formé. La figure 3 montre les images MEB prises à divers endroits sur l&#39;échafaud de nanofibres. Sur le plan qualitatif, on peut déterminer qu&#39;il existe une progression des fibres alignées de manière uniaxiale à 0 mm (figure 3A) à un assortiment aléatoire des fibres à 6 mm (figure 3D). La FFT donne une valeur quantitative à l&#39;alignement des fibres, des détails sur ...

Watch this Scientific Journal Video about Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization at JoVE.com

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Ici, nous présentons un protocole pour fabriquer des échafaudages électrofilé de nanofibres avec l&#39;organisation graduée de fibres et d&#39;explorer leurs applications dans la régulation de la morphologie des cellules / orientation. Dégradés en ce qui concerne les propriétés physiques et chimiques des échafaudages de nanofibres offrent une grande variété d&#39;applications dans le domaine biomédical.

Électrofilées nanofibres échafaudages avec des gradations dans l&#39;organisation fibre

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

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