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 arbejde blev delvist støttet af startup midler fra University of Nebraska Medical Center og National Institute of Health (tilskud nummer 1R15 AR063901-01).

1. Fremstilling af opløsningen <ol><li> Der fremstilles en opløsning af poly (ε-caprolacton) (PCL) (Mw = 80.000 g / mol) ved en omtrentlig koncentration på 100 mg / ml. Opløs PCL i en blanding af dichlormethan (DCM) og N, N-dimethlyformamide (DMF) i et forhold på 4: 1 (v / v) med en koncentration på 10% (w / v). </li><li> Placer opløsning i et 20 ml glasrør for at blande. Placer glasrør i ultralydsrenser i 30 minutter, eller indtil opløsningen er gennemskinnelig. </li></ol> ...

<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...

Nanofibre er et populært værktøj til vævsmanipulering grund af deres evne til at efterligne den ekstracellulære matrix i sin struktur og relative størrelse 1. Men nogle native vævsgrænseflader, såsom sene-til-knogle insertionssted, indeholder collagenfibre, som udviser en variabel organisationsstruktur, der øger i tilpasning til senen og falder ved knoglen stedet 2-5. Så for effektiv vævsregeneration der er behov for at fremstille et stillads, der effektivt kunne efterligne denne strukturelle gradient. Tidligere har der været forskning udført på gradvise ændringer i fibersammensætning, specifikt indhold af mineraler 6. Men genskabe den strukturelle komponent af bindevæv stort set uudforsket. En tidligere undersøgelse undersøgte morfologiske gradienter ved at studere effekten af ​​overfladen silica partikeldensitet om spredning af rotte calvariale osteoblaster og fandt en InverSE forholdet mellem silica partikeldensitet og celleproliferation 7. Men de morfologiske ændringer, medieret celleproliferation i tidligere arbejde var for det meste relateret til overfladen ruhed mangler kapacitet i efterligne fiber organisationsændringer 7,8. En nylig undersøgelse har forsøgt at fremstille et stillads, som efterlignede de unikke collagen fiberorienteringer ved hjælp af en hidtil ukendt kollektor til elektrospinding 9. Selv om denne undersøgelse lykkedes at fremstille et stillads med både linie og tilfældige fibre, undlod at efterligne gradvise ændringer udstillet i de indfødte væv. Også i produktion separate komponenter, med en øjeblikkelig ændring fra linie til tilfældig orientering, de biomekaniske egenskaber af denne stillads faldt betydeligt. Ingen tidligere arbejde har været i stand til at producere gældende nanofiber stilladser med kontinuerlige overgange i fiberorienteringer fra aligned og tilfældig. Vores seneste undersøgelse har vist vellykket genskabelse af nanofiber stilladsermed inddeling i fiber organisation, der kan potentielt efterligner native collagen organisation sene-til-bone indsættelse 10. Dette arbejde har til formål at præsentere de protokoller, der anvendes til produktion af nanofiber stilladser med en struktur, der ligner den, fiber organisation i native sene-til-knoglevæv interface. Gradient nanofiber strukturer potentielt vidtrækkende applikationer på tværs af en række forskellige områder. Vi fokuserede på de ansøgninger til tissue engineering af senen-til-bone insertionssted ved at kombinere vores stilladser med adipøst afledte stamceller (ADSCs), der allerede anvendes til vævsregeneration på forskellige substrater 11-14. Desuden ADSCs er meget ens i naturen til knoglemarv stamceller i form af multipotency og deres ressourcer er rigeligt der kan høstes ved hjælp af en simpel fedtsugning 15,16. Seeding disse celler til nuancerede nanofiber stilladser yderligere øger deres tissagsøge ingeniørmæssige anvendelser ved at tillade kontrolleret distribution af de celler, der potentielt kan differentiere til forskellige væv. Ud over at podning stamceller kan nanofibre være indkapslet med signalmolekyler for regulering af cellulær reaktion. Kobling nanoencapsulation med den organisatoriske gradient af disse scaffolds muliggør studiet af cellulære adfærd eller mulige implantat design og belægninger. Indkapsling af funktionelle molekyler som knoglemorfogenetisk protein 2 (BMP-2), som er blevet vist at inducere osteoblastdifferentiering 15,16, yderligere forbedre vævsdyrkningsapplikationer disse scaffolds 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 anvendelse af denne protokol blev en fibermåtte med en organisatorisk gradient dannet. Figur 3 viser SEM billeder taget på forskellige steder på nanofiber stilladset. Kvalitativt kan det bestemmes, at der er en progression fra uniaksialt aligned fibre ved 0 mm (figur 3A) til en tilfældig fiber sortiment på 6 mm (figur 3D). FFT giver en kvantitativ værdi for fiber justering detaljerne om kvantitative fremgangsmåder er detaljeret her 19. Fibre ved 0 ...

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 præsenterer vi en protokol til at fabrikere elektrospundet nanofiber stilladser med fordelte toner organisering af fibre og udforske deres ansøgninger i regulering cellemorfologi / orientering. Forløb med hensyn til fysiske og kemiske egenskaber af nanofiber stilladser tilbyder en bred vifte af applikationer inden for det biomedicinske område.

Elektrospundne Nanofiber Stilladser med inddeling 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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