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

Dette arbeidet ble støttet delvis fra oppstart midler fra University of Nebraska Medical Center og National Institute of Health (stipend nummer 1R15 AR063901-01).

1. Utarbeidelse av løsningen <ol><li> Fremstille en løsning av poly (ε-kaprolakton) (PCL) (Mw = 80000 g / mol) ved en omtrentlig konsentrasjon på 100 mg / ml. Oppløs PCL i en blanding av diklormetan (DCM) og N, N-dimetylformamid (DMF) i et forhold på 4: 1 (v / v) med en konsentrasjon på 10% (w / v). </li><li> Plassere løsningen i et 20 ml glassrør for blanding. Plasser glassrør inn i ultralydrenser i 30 minutter, eller inntil oppløsningen er gjennomskinnelig. </li></ol> 2. I...

<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...

Nanofibers er populære verktøy for regenerering av vev på grunn av deres evne til å etterligne den ekstracellulære matriks i sin struktur og relative størrelse 1. Men noen opprinnelige vev-grensesnitt, for eksempel sene-knokkel-innføringssted, inneholde kollagenfibre, som oppviser en variabel organisasjonsstruktur som øker i flukt mot sene og avtar på benet området 2-5. Så, for effektiv regenerering av vev er det behov for å fremstille et stillas som effektivt kunne etterligne denne strukturelle gradient. Tidligere har det vært foretatt forskning på gradvise endringer i fibersammensetningen, spesielt mineralinnhold 6. Imidlertid gjenskape den strukturelle komponenten av bindevev forblir stort sett uutforsket. En tidligere studie undersøkte morfologiske gradienter ved å studere effekten av overflaten silika partikkeltetthet på spredning av rotte calvarial osteoblaster og fant en inverterse sammenhengen mellom silika partikkeltetthet og celleproliferasjon 7. Men de morfologiske endringer som medierte celleproliferasjon i tidligere arbeid var for det meste relatert til overflaten ruhet mangler evnen i hermet fiberorganisasjonsendringer 7,8. En fersk studie forsøkt å dikte et stillas som etterlignet den unike kollagen fiber orientering ved hjelp av en roman oppsamler for electro 9. Selv om denne studien lykkes i å produsere et stillas med både justert og tilfeldige fiber, klarte det å etterligne de gradvise endringer utstilt i de innfødte vev. Også, i å produsere separate komponenter, med en umiddelbar endring fra justert til tilfeldig orientering, biomekaniske egenskapene til dette stillaset sunket betraktelig. Ingen tidligere arbeid har vært i stand til å produsere gjeldende nanofiber stillasene med kontinuerlige graderinger i fiber orienteringer fra aligned og tilfeldig. Vår fersk studie har vist vellykket rekreasjon av nanofiber stillasermed graderinger i fiber organisasjon som potensielt kan etterligne den opprinnelige kollagen organisasjon på sene-til-ben innsetting 10. Dette arbeidet tar sikte på å presentere de protokollene som brukes til produksjon av nanofiber stillasene med en struktur som minner sterkt om fiber organisasjon i mors sene-til-benvev grensesnitt. Gradient nanofiber strukturer har potensielt vidtrekkende applikasjoner på tvers av en rekke felt. Vi fokuserte på søknadene til tissue engineering av den sene-til-ben innstikkstedet ved å kombinere våre stillaser med adipose-avledet stamceller (ADSCs) som allerede benyttes for vev regenerering på ulike underlag 11-14. I tillegg ADSCs er svært like i naturen til benmarg stamceller i form av multipotency og deres ressurs er rikelig som kan høstes ved hjelp av en enkel fettsuging prosedyre 15,16. Seeding disse cellene til graderte nanofiber stillaser ytterligere forbedrer sin tissaksøke tekniske applikasjoner ved å tillate for kontrollert distribusjon av cellene som potensielt kan differensiere i ulike vev. I tillegg til såing stamceller, kan nanofibers innkapsles med signalmolekyler for regulering av cellulær respons. Kobling nanoencapsulation med organisasjons gradient av disse stillasene åpner for studiet av mobilnettet atferd eller mulige implantat design og belegg. Innkapsling av funksjonelle molekyler som benmorfogenetisk protein 2 (BNIP2), som er blitt vist å indusere osteoblastdifferensierings 15,16, ytterligere kan forbedre vev tekniske anvendelser av disse stillaser 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.

Ved hjelp av denne protokollen, ble en fibermatte med en organisatorisk gradient dannet. Figur 3 viser SEM bilder tatt på forskjellige steder på nanofiber stillaset. Kvalitativt, kan det bestemmes at det er en progresjon fra de uniaksialt orienterte fibre ved 0 mm (figur 3A) til en tilfeldig fiber sortiment på 6 mm (figur 3D). FFT gir en kvantitativ verdi for den fiberinnrettings, detaljene på de kvantitative fremgangsmåter er beskrevet her 19. Fibrene v...

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

Her presenterer vi en protokoll for å dikte elektrospunnede nanofiber stillasene med graderte organisering av fiber og utforske sine applikasjoner i å regulere cellemorfologi / orientering. Gradienter med hensyn til fysiske og kjemiske egenskaper av nanofiber stillasene tilbyr et bredt utvalg av applikasjoner i biomedisinske feltet.

Elektrospunnede nanofiber Stillas med graderinger i 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 ...

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