Name: fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion
Uploaded: 2020-05-10T14:45:00
Description: Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Dette arbejde er økonomisk støttet af NIH (Grants 1R01AR072027-01, 1R03AR069383-01), NSF Career Award (1653702) og University of Connecticut.

1. Sammenfatning af JBNT'er
BEMÆRK: JBNT monomer blev udarbejdet som offentliggjort tidligere11.
<ol><li>Syntese af sammensatte A1<ol>
<li>Der fremstilles en opløsning indeholdende 8,50 g 2 cyanoeddikesyre og 9,80 g ethylcarbamat i 25 ml toluen og 2,5 ml N, N-dimethylformamid. Der tilsættes 4,90 mL phosphorylchlorid på dråbe. Derefter opvarmes blandingen til 70 °C, og der omrørs under omrøring i 1,5 timer.</li>
		<li>Afkøl reaktionsblandingen til stuetemperat...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+convergence+of+fracture+repair+and+stem+cells:+interplay+of+genes,+aging,+environmental+factors+and+disease.">The convergence of fracture repair and stem cells: interplay of genes, aging, environmental factors and disease.</a> Journal of Bone and Mineral Research. 29 (11), 2307-2322 (2014).</li><li>De Becker, A., Riet, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homing+and+migration+of+mesenchymal+stromal+cells:+How+to+improve+the+efficacy+of+cell+therapy.">Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy.</a> World Journal of Stem Cells. 8 (3), 73-87 (2016).</li><li>Chen, Y., Song, S., Yan, Z., Fenniri, H., Webster, T. 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V., Khorsand, B., Geary, S. M., Salem, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printing+of+scaffolds+for+tissue+regeneration+applications.">3D Printing of scaffolds for tissue regeneration applications.</a> Advanced Healthcare Materials. 4 (12), 1742-1762 (2015).</li><li>Shi, 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=Structurally+and+functionally+optimized+silk-fibroin-gelatin+scaffold+using+3D+printing+to+repair+cartilage+injury+in+vitro+and+in+vivo.">Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo.</a> Advanced Material. 29, 1701089 (2017).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. 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I denne undersøgelse udviklede vi en selvsamlet biomimetisk NM, som blev dannet med DNA-inspirerede JBNT'er og FN. Ved tilberedning af JBNT-opløsningen skal det JBNT-lyofile pulver opløses i vandet i stedet for PBS, fordi PBS vil forårsage agglomeration af JBNT'er, hvilket hæmmer deres samling. Desuden bør NM også samles i vand, hvis vi ønsker at observere nano-fibril strukturer NM, fordi saltet i PBS vil bundt med NM fibre, hvilket i høj grad kan reducere opløsningen af billederne.
...

Humane mesenchymale stamceller (hMSCs) har vist potentialet for selvfornyelse og selvdifferentiering langs forskellige mesenchymale afstamninger, som hjælper med regenerering og vedligeholdelse af væv1. Baseret på differentieringspotentialet betragtes hMSC'er som kandidater til mesenchymale vævsskader og hæmatopoietisk lidelsesterapi2. hMSCs har vist evnen til at fremme sårheling ved at øge væv reparation, angiogenese, og reducereinflammation 3. Men uden biokemiske eller biomaterialer bistand, effektiviteten for hMSCs at nå et mål væv og funktion på det ønskede sted er lav4. Selvom forskellige manipulerede stilladser er blevet brugt til at tiltrække hMSC'er til at klæbe til læsionerne, er nogle steder som vækstpladebrud midt i en lang knogle ikke let tilgængelige af de konventionelle præfabrikerede stilladser, som måske ikke passer perfekt ind i et uregelmæssigt formet skadet sted.
Her har vi udviklet et biomimetisk nanomateriale, der kan samle sig på stedet og blive injiceret til et svært tilgængeligt målområde. Det injicerbare biostillads NM består af Janus base nanorør (JBNTs) og fibronectin (FN). JBNTs, også kendt som Rosette Nanotubes (RNTs), er afledt af DNA base par, specielt thymine og adenin, her5,6,7. Som det ses i figur 1, dannes nanorørene , når seks molekyler af den afledte DNA-base parrer sig selv via brintbindinger for at danne et plan6. Seks molekyler stables derefter på hinanden i et plan via en stærk pi-stabling interaktion7, som kan være op til 200-300 μm i længden. JBNT'erne er designet til morfologisk at efterligne kollagenfibre, så FN vil reagere med dem.
FN er et klæbemiddelglycoprotein med høj molekylvægt, som findes i den ekstracellulære matrix (ECM)9. Disse kan mægle i fastgørelse af stamceller til andre komponenter i ECM, især kollagen10. Vi designede JBNT'er til morfologisk at efterligne kollagenfibre, så FN kan reagere med dem for at danne NM på få sekunder via ikke-ækvivalent binding. Derfor er NM et lovende biostillads, der skal injiceres i et knoglebrudssted, der ikke kunne være tilgængeligt for de konventionelt fremstillede stilladser. Her præsenterer den injicerbare NM en fremragende evne til at forbedre hMSC-forankring in vitro, der udviser deres potentiale til at tjene som stillads til vævsregenerering.

<table>
	<tbody>
		<tr>
			<td>1,2-dichloroethane</td>
			<td>Alfa Aesar</td>
			<td>39121</td>
			<td></td>
		</tr>
		<tr>
			<td>2-cyanoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>C88505</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Dimethylaminopyridine</td>
			<td>TCI America</td>
			<td>D1450</td>
			<td></td>
		</tr>
		<tr>
			<td>8 wells Chambered Coverglass</td>
			<td>Thermo Fisher</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>absolute ethanol</td>
			<td>Thermo Fisher</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>allylamine</td>
			<td>Sigma-Aldrich</td>
			<td>145831</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC32G</td>
			<td></td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>concentrated hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>Thermo Fisher</td>
			<td>15230147</td>
			<td></td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-tert-butyl dicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>361941</td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylcarbamate</td>
			<td>Sigma-Aldrich</td>
			<td>U2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermo Fisher</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixative Solution (4 % formaldehyde prepared in PBS)</td>
			<td>Thermo Fisher</td>
			<td>R37814</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidinium hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>A13543</td>
			<td></td>
		</tr>
		<tr>
			<td>hexanes</td>
			<td>Sigma-Aldrich</td>
			<td>227064</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl iodide</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>Alfa Aesar</td>
			<td>A17114</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methylmorpholine N-oxide</td>
			<td>Alfa Aesar</td>
			<td>A19802</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetraoxide</td>
			<td>Alfa Aesar</td>
			<td>45385</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Thermo Fisher</td>
			<td>20012050</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphoryl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>201170</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>347825</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase column</td>
			<td>Thermo Fisher</td>
			<td>25305-154630</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>silica gel</td>
			<td>TCI America</td>
			<td>S0821</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium ethoxide</td>
			<td>Alfa Aesar</td>
			<td>L13083</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium periodide</td>
			<td>Sigma-Aldrich</td>
			<td>71859</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>S0505</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium triacetoxyborohydride</td>
			<td>Alfa Aesar</td>
			<td>B22060</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer(NanoDrop One/One&#7580; UV-Vis)</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Growth Medium BulletKit</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrahydrofuran</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>thioanisole</td>
			<td>Sigma-Aldrich</td>
			<td>T28002</td>
			<td></td>
		</tr>
		<tr>
			<td>toluene</td>
			<td>Sigma-Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Alfa Aesar</td>
			<td>A12646</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A12198</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher</td>
			<td>HFH10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion

En biomimetisk NM blev udviklet til at fungere som et vævsteknisk biologisk stillads, som kan forbedre stamcelleforankring. Den biomimetiske NM er dannet af JBNTs og FN gennem selvmontering i en vandig løsning. JBNT'er måler 200-300 μm i længden med indre hydrofobe hule kanaler og ydre hydrofile overflader. JBNT'er er positivt ladet, og FNs er negativt opkrævet. Derfor, når de injiceres i en neutral vandig opløsning, bindes de sammen via ikke-ækvivalent binding for at danne NM-bundterne. Selvmonteringsprocessen afsluttes inden for få sekunder uden kemiske initiatorer, varmekilde eller UV-lys. Når NM-opløsningens pH-opløsning er lavere end det isoelektriske punkt i FNs (pI 5.5-6.0), vil NM-bundterne selvudløse på grund af tilstedeværelsen af positivt ladet FN.
NM er kendt for at efterligne den ekstracellulære matrix (ECM) morfologisk og dermed kan bruges som et injicerbart stillads, som giver en fremragende platform til at forbedre hMSC vedhæftning. Celletæthedsanalyse og fluorescensbilledbilledforsøg viste, at NMs øgede forankringen af hMSC'er betydeligt sammenlignet med den negative kontrol.

Vores undersøgelser opdagede, at dannelsen af NM af JBNTs og FN er hurtig, hvilket skete på 10 sekunder. Som vist i figur 2blev der opnået hvid floccule, da JBNT-opløsningen blev blandet med FN-opløsningen og pipetteret flere gange. Dannelsen af NM er helt biomimetisk. Ingen eksterne stimuli er nødvendige. Fremstillingsprocessen er meget lettere end for nogle nye biomaterialer, som er baseret på ultraviolet lys eller kemisk initiator til krydsning13.
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Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

Målet med denne protokol er at vise samling af en biomimetisk nanomatrix (NM) med Janus base nanorør (JBNTs) og fibronectin (FN). Når co-dyrket med menneskelige mesenchymale stamceller (hMSCs), NMs udviser fremragende bioaktivitet i at tilskynde hMSCs vedhæftning.

Fremstilling af en biomimetisk nanomatrix med Janus Base Nanotubes og Fibronectin til stamcelle vedhæftning

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

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Bioengineering

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