Name: dendrimer-based uneven nanopatterns to locally control surface adhesiveness: a method to direct chondrogenic differentiation
Uploaded: 2018-01-20T14:00:00
Description: Watch this Scientific Journal Video about Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation at JoVE.com

Författarna erkänner Oriol Font-Bach och Albert G. Castaño för deras hjälp i dmin kvantifiering. De erkänner också avancerad Digital mikroskopi enheten vid Institutet för forskning inom biomedicin (IRB Barcelona) att låta författarna spela in video i sina lokaler. Detta arbete stöds av de nätverk biomedicinsk forskning Center (CIBER), Spanien. CIBER är ett initiativ som finansieras av den VI nationella R & D & i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER åtgärder och Instituto de Salud Carlos III, med stöd från Europeiska regionalutvecklingsfonden. Detta arbete har stötts av kommissionen för universitet och forskning av Institutionen för Innovation, universitet, och företaget av Generalitat de Catalunya (2014 SGR 1442). Det har också finansierats av projekt OLIGOCODES (nr. MAT2012-38573-C02) och CTQ2013-41339-P, delas ut av det spanska ministeriet för ekonomi och konkurrenskraft, i tillägg till INTERREG V-A Spanien-Portugal 2014-2020 POCTEP (0245_IBEROS_1_E). C.R.P. erkänner ekonomiskt stöd från det spanska ministeriet för ekonomi och konkurrenskraft grant (nr. IFI15/00151).

1. substrat förberedelse
<ol><li>
Glödgning 1,4 x 1,1 cm Au(111) på Mica substrat.
	<ol>
<li>Placera Au(111) substratet på glaskeramik spis och glödga det med butan låga för 3 min. Tillåt substratet svalna under en argon-atmosfär. Upprepa detta steg för varje Au(111) substrat. Obs: Au(111) substrat bör användas omedelbart efter glödgning.</li>
	</ol>
</li>
	<li>
Beredning av Poly (L-mjölksyra) (PLLA)-belagda glas substrat.
	<ol>
<li>Skär och tvätta glasskivor.<ol>
<l...

<ol>
	<li>Jiang, F., H&#246;rber, H., Howard, J., M&#252;ller, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+collagen+into+microribbons:+Effects+of+pH+and+electrolytes.">Assembly of collagen into microribbons: Effects of pH and electrolytes.</a> J. Struct. Biol. 148 (3), 268-278 (2004).</li><li>Smith, M. L., 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=Force-induced+unfolding+of+fibronectin+in+the+extracellular+matrix+of+living+cells.">Force-induced unfolding of fibronectin in the extracellular matrix of living cells.</a> PLoS Biol. 5 (10), 2243-2254 (2007).</li><li>Little, W. C., Smith, M. L., Ebneter, U., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+to+mechanically+tune+and+optically+probe+fibrillar+fibronectin+conformations+from+fully+relaxed+to+breakage.">Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage.</a> Matrix Biol. 27 (5), 451-461 (2008).</li><li>Christman, K. L., Enriquez-Rios, V. D., Maynard, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopatterning+proteins+and+peptides.">Nanopatterning proteins and peptides.</a> Soft Matter. 2, 928-939 (2006).</li><li>Falconnet, D., Csucs, G., Grandin, H. M., Textor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+engineering+approaches+to+micropattern+surfaces+for+cell-based+assays.">Surface engineering approaches to micropattern surfaces for cell-based assays.</a> Biomaterials. 27 (16), 3044-3063 (2006).</li><li>Arnold, 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=Activation+of+integrin+function+by+nanopatterned+adhesive+interfaces.">Activation of integrin function by nanopatterned adhesive interfaces.</a> ChemPhysChem. 5 (3), 383-388 (2004).</li><li>Cavalcanti-Adam, E. 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=Lateral+spacing+of+integrin+ligands+influences+cell+spreading+and+focal+adhesion+assembly.">Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly.</a> Eur. J. Cell. Biol. 85 (3-4), 219-224 (2006).</li><li>Cavalcanti-Adam, E. 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=Cell+spreading+and+focal+adhesion+dynamics+are+regulated+by+spacing+of+integrin+ligands.">Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.</a> Biophys. J. 92 (8), 2964-2974 (2007).</li><li>Arnold, 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=Cell+interactions+with+hierarchically+structured+nano-patterned+adhesive+surfaces.">Cell interactions with hierarchically structured nano-patterned adhesive surfaces.</a> Soft Matter. 5 (1), 72-77 (2009).</li><li>Malmstr&#246;m, 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=Large+area+protein+patterning+reveals+nanoscale+control+of+focal+adhesion+development.">Large area protein patterning reveals nanoscale control of focal adhesion development.</a> Nano Lett. 10 (2), 686-694 (2010).</li><li>Deeg, J. 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=Impact+of+local+versus+global+ligand+density+on+cellular+adhesion.">Impact of local versus global ligand density on cellular adhesion.</a> Nano Lett. 11 (4), 1469-1476 (2011).</li><li>Medda, R., 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=Investigation+of+early+cell&#8211;surface+interactions+of+human+mesenchymal+stem+cells+on+nanopatterned+&#946;-type+titanium-niobium+alloy+surfaces.">Investigation of early cell&#8211;surface interactions of human mesenchymal stem cells on nanopatterned &#946;-type titanium-niobium alloy surfaces.</a> Interface Focus. 4, 20130046 (2014).</li><li>Wang, X., 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=Effect+of+RGD+nanospacing+on+differentiation+of+stem+cells.">Effect of RGD nanospacing on differentiation of stem cells.</a> Biomaterials. 34 (12), 2865-2874 (2013).</li><li>Wang, X., Ye, K., Li, Z. H., Yan, C., Ding, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion,+proliferation,+and+differentiation+of+mesenchymal+stem+cells+on+RGD+nanopatterns+of+varied+nanospacings.">Adhesion, proliferation, and differentiation of mesenchymal stem cells on RGD nanopatterns of varied nanospacings.</a> Organogenesis. 9 (4), 280-286 (2013).</li><li>Wang, X., Li, S. Y., Yan, C., Liu, P., Ding, J. 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+RGD+micro/nanopattern+and+corresponding+study+of+stem+cell+differentiation.">Fabrication of RGD micro/nanopattern and corresponding study of stem cell differentiation.</a> Nano Lett. 15 (3), 1457-1467 (2015).</li><li>Li, Z. H., 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=Effects+of+RGD+nanospacing+on+chondrogenic+differentiation+of+mesenchymal+stem+cells.">Effects of RGD nanospacing on chondrogenic differentiation of mesenchymal stem cells.</a> J. Mater. Chem. B. 3 (12), 5197-5209 (2015).</li><li>Stephanopoulos, N., 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=Bioactive+DNA-peptide+nanotubes+enhance+the+differentiation+of+neural+stem+cells+into+neurons.">Bioactive DNA-peptide nanotubes enhance the differentiation of neural stem cells into neurons.</a> Nano Lett. 15 (1), 603-609 (2015).</li><li>Rolland, O., Turrin, C. O., Caminade, A. M., Majoral, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimers+and+nanomedicine:+Multivalency+in+action.">Dendrimers and nanomedicine: Multivalency in action.</a> New J. Chem. 33, 1809-1824 (2009).</li><li>Saovapakhiran, A., D&#8217;Emanuele, A., Attwood, D., Penny, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+PAMAM+dendrimers+modulates+the+mechanism+of+cellular+internalization.">Surface modification of PAMAM dendrimers modulates the mechanism of cellular internalization.</a> Bioconjug. Chem. 20 (4), 693-701 (2009).</li><li>Albertazzi, L., Fernandez-Villamarin, M., Riguera, R., Fernandez-Megia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+functionalization+of+dendrimers+regulates+internalization+and+intracellular+trafficking+in+living+cells.">Peripheral functionalization of dendrimers regulates internalization and intracellular trafficking in living cells.</a> Bioconjug. Chem. 23 (5), 1059-1068 (2012).</li><li>Mikhail, A. S., Jones, K. S., Sheardown, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimer+grafted+cell+adhesion+peptide-modified+PDMS.">Dendrimer grafted cell adhesion peptide-modified PDMS.</a> Biotechnol. Prog. 24 (4), 938-944 (2008).</li><li>Kino-oka, M., Kim, J., Kurisaka, K., Kim, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+growth+of+skeletal+myoblasts+and+fibroblasts+in+co-culture+on+a+dendrimer-immobilized+surface.">Preferential growth of skeletal myoblasts and fibroblasts in co-culture on a dendrimer-immobilized surface.</a> J. Biosci. Bioeng. 115 (1), 96 (2013).</li><li>Kim, M. H., 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=Morphological+regulation+and+aggregate+formation+of+rabbit+chondrocytes+on+dendrimer+immobilized+surfaces+with+D-glucose+display.">Morphological regulation and aggregate formation of rabbit chondrocytes on dendrimer immobilized surfaces with D-glucose display.</a> J. Biosci. Bioeng. 107 (2), 196-205 (2009).</li><li>Lomba, 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=Cell+adhesion+on+surface+patterns+generated+by+the+photocrosslinking+of+hyperbranched+polyesters+with+a+trisdiazonium+salt.">Cell adhesion on surface patterns generated by the photocrosslinking of hyperbranched polyesters with a trisdiazonium salt.</a> React. Funct. Polym. 73 (3), 499-507 (2013).</li><li>Maheshwari, G., Brown, G., Lauffenburger, D. A., Wells, A., Griffith, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+and+motility+depend+on+nanoscale+RGD+clustering.">Cell adhesion and motility depend on nanoscale RGD clustering.</a> J. Cell Sci. 113 (Pt 10), 1677-1686 (2000).</li><li>Kim, M. H., Kino-oka, M., Kawase, M., Yagi, K., Taya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+D-glucose+and+epidermal+growth+factor+display+on+dynamic+behaviors+of+human+epithelial+cells.">Synergistic effect of D-glucose and epidermal growth factor display on dynamic behaviors of human epithelial cells.</a> J. Biosci. Bioeng. 104 (5), 428-431 (2007).</li><li>Pericet-Camara, R., Cahill, B. P., Papastavrou, G., Borkovec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-patterning+of+solid+substrates+by+adsorbed+dendrimers.">Nano-patterning of solid substrates by adsorbed dendrimers.</a> Chem. Commun. 3, 266-268 (2007).</li><li>Lagunas, 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=Large-scale+dendrimer-based+uneven+nanopatterns+for+the+study+of+local+arginine&#8211;glycine&#8211;aspartic+acid+(RGD)+density+effects+on+cell+adhesion.">Large-scale dendrimer-based uneven nanopatterns for the study of local arginine&#8211;glycine&#8211;aspartic acid (RGD) density effects on cell adhesion.</a> Nano Res. 7 (3), 399-409 (2014).</li><li>Lagunas, 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=Tailoring+RGD+local+surface+density+at+the+nanoscale+toward+adult+stem+cell+chondrogenic+commitment.">Tailoring RGD local surface density at the nanoscale toward adult stem cell chondrogenic commitment.</a> Nano Res. , (2017).</li><li>Prats-Alfonso, E., 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=Effective+and+Versatile+Strategy+for+the+Total+Solid-Phase+Synthesis+of+Alkanethiols+for+Biological+Applications.">Effective and Versatile Strategy for the Total Solid-Phase Synthesis of Alkanethiols for Biological Applications.</a> Eur. J. Org. Chem. 2013 (7), 1233-1239 (2013).</li><li>Zhu, Y. B., Gao, C. Y., Liu, X. Y., He, T., Shen, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+biomacromolecules+onto+aminolyzed+poly(L-lactic+acid)+toward+acceleration+of+endothelium+regeneration.">Immobilization of biomacromolecules onto aminolyzed poly(L-lactic acid) toward acceleration of endothelium regeneration.</a> Tissue Eng. 10 (1-2), 53-61 (2004).</li><li>G&#252;ell, A., D&#237;ez-P&#233;rez, I., Gorostiza, P., Sanz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+reliable+probes+for+electrochemical+tunneling+spectroscopy.">Preparation of reliable probes for electrochemical tunneling spectroscopy.</a> Anal. Chem. 76 (17), 5218-5222 (2004).</li><li>Bobick, B. E., Chen, F. H., Le, A. M., 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=Regulation+of+the+chondrogenic+phenotype+in+culture.">Regulation of the chondrogenic phenotype in culture.</a> Birth Defects Res. C Embryo Today. 87 (4), 351-371 (2009).</li><li>De Lise, A. M., Fisher, 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=Cellular+interactions+and+signaling+in+cartilage+development.">Cellular interactions and signaling in cartilage development.</a> Osteoarthritis Cartilage. 8 (5), 309-334 (2000).</li><li>Kosher, R. A., Kulyk, W. M., Gay, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+gene+expression+during+limb+cartilage+differentiation.">Collagen gene expression during limb cartilage differentiation.</a> J. Cell Biol. 102 (4), 1151-1156 (1986).</li><li>Biebricher, A., Paul, A., Tinnefeld, P., Golzhauser, A., Sauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+three-dimensional+immobilization+of+biomolecules+on+chemically+patterned+surfaces.">Controlled three-dimensional immobilization of biomolecules on chemically patterned surfaces.</a> J. Biotechnol. 112 (1-2), 97-107 (2004).</li><li>Tinazli, A., Piehler, J., Beuttler, M., Guckenberger, R., Tamp&#233;, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+protein+nanolithography+that+can+write,+read+and+erase.">Native protein nanolithography that can write, read and erase.</a> Nat. Nanotechnol. 2, 220-225 (2007).</li><li>Oberhansl, 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=Facile+Modification+of+Silica+Substrates+Provides+a+Platform+for+Direct-Writing+Surface+Click+Chemistry.">Facile Modification of Silica Substrates Provides a Platform for Direct-Writing Surface Click Chemistry.</a> Small. 8 (4), 541-545 (2012).</li></ol>

Under utvecklingen av protokollet beskrivs övervägas ett antal kritiska åtgärder. Först avser nanopattern karakterisering med scanning sonden mikroskopi tekniker. För att visualisera nanopatterns, ytan där mallning produceras måste ha ojämnheter värde understiger genomsnittlig diametern på den tåååålamooooooood, vilket är omkring 4 – 5 nm mätt med STM (figur 1B). Också, det bör beaktas att hög upplösning STM imaging är begränsad till ledande substrat, i detta fall Au(111). Några...

Här beskriver vi en enkel och mångsidig dendrimer-baserade nanopatterning förfarande för att erhålla cell kultur ytor som tillåter kontroll av lokala vidhäftning på nanonivå. Nanoskala Detaljer för ECM organisation har rapporterats,1,2,3 och nanopatterning cell adhesion ytor har lämnat djupa insikter i de cellulära krav relaterade till vidhäftning4, 5. Experiment med micellar litografi-baserade nanopatterns visade ett tröskelvärde på runt 70 nm för RGD peptid nanospacing, celladhesion försenas avsevärt över detta värde6,7,8 ,9. Dessa studier visade också större påverkan av lokal än global ligand densitet cell adhesion9,10,11.
Under morfogenes utlösa cell interaktioner med omgivningen första differentiering händelserna, som fortsätta tills sista komplex vävnad strukturer har bildats. Inom denna ram, har nanopatterned ytor använts för att ta itu med påverkan av de inledande cellyta interaktioner på morfogenes. Litografi-baserade RGD nanopatterns med en lateral avstånd 68 nm i β-typ Ti-40Nb legeringar bidra till att upprätthålla den odifferentierade fenotypen av icke-begått stamceller12, medan RGD nanospacings för mellan 95 och 150 nm förbättra differentieringen av mesenkymala stamceller (MSC) mot adipogena/Osteogena13,14,15 och chondrogenic öden16. Också, egen montering makromolekyler modifierad med signalering komponenter har visat att rikta celladhesion och differentiering genom att tillhandahålla nanoskala arkitektoniska förordning av signalering cues17. I detta avseende har nedfall av tåååålamooooooood med cell-interagera beståndsdelarna i deras yttre sfär18,19,20 på ytor använts för att studera cell adhesion21,22, morfologi23,24, och migration händelser25,26. Trots gör bristen på ytan karakterisering i dessa studier det svårt att fastställa något samband mellan dendrimer yta konfiguration och cellssvar.
Dendrimer nanopatterns med vätska-liknande ordning och definierade avstånd kan erhållas när tåååålamooooooood adsorberas till låg laddade ytor från lösningar med låga Joniska styrkor. 27 på grundval av denna egendom, här presenterar vi en metod för att få storskaliga ojämna nanopatterns av RGD-functionalized tåååålamooooooood på låg laddade ytor som tillåter nanoskala kontroll av lokala RGD Ytors. Vatten kontaktvinkel (CA), X-ray fotoelektronen spektroskopi (XPS) och skanning sonden mikroskopi tekniker (STM och AFM nanopatterns) Visa att lokala ligand tätheter kan justeras ändra den inledande dendrimer koncentrationen i lösning. Lokala RGD Ytors är kvantifieras från AFM bilder av sannolikhet konturkartor av interparticle minimiavstånd och sedan korrelerade med cell experiment. Jämfört med andra nanopatterning tekniker4, dendrimer-baserade nanopatterning är okomplicerat och enkelt kan skalas till stora ytor, vilket är fullt kompatibel med cell kultur-program. Nanopatterns används som bioaktiva substrat för att utvärdera effekten av lokala RGD Ytors på cell adhesion28 och chondrogenic induktion av vuxen människa MSCs29. Våra resultat visar att RGD dendrimer-baserade nanopatterns upprätthålla cellernas tillväxt och att celladhesion förstärks av höga lokala RGD ytan tätheter. I experiment som differentiering gynnade mellanliggande vidhäftning av celler till substratesna MSC kondens och tidig chondrogenic differentiering. På grund av den lätthet med vilken dendrimer perifera grupper kan ändras, kan den metod som beskrivs här förlängas ytterligare till andra ECM-ligander som utövar koncentrationsberoende effekter på celler.

<table border="0" cellpadding="0" cellspacing="0" width="1384">
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			<td height="21" style="height:21px;width:504px;">Gold (111) on mica. 1.4x1.1 cm&#160;</td>
			<td style="width:169px;">Spi Supplies</td>
			<td style="width:307px;">466PS-AB</td>
			<td style="width:405px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Glass micro slides, plain</td>
			<td style="width:169px;">Corning</td>
			<td style="width:307px;">2947-75x25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Deionized water</td>
			<td style="width:169px;">Millipore</td>
			<td style="width:307px;"></td>
			<td>18M&#937; cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Ethanol 96%</td>
			<td style="width:169px;">PanReac</td>
			<td style="width:307px;">131085.1212</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">L-Lactide/DL-Lactide copolymer</td>
			<td style="width:169px;">Corbion</td>
			<td style="width:307px;"></td>
			<td>95/05 molar ratio</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">1,4 - dioxane</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">296309-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Silicone oil, high temperature</td>
			<td style="width:169px;">Acros Organics</td>
			<td style="width:307px;">174665000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Spinner</td>
			<td style="width:169px;">Laurell</td>
			<td style="width:307px;">WS-650MZ-23NPP/Lite</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:504px;">Tissue culture laminar flow hood</td>
			<td style="width:169px;">Telstar</td>
			<td style="width:307px;">Bio II Advance</td>
			<td>Class II biological safety cabinet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Filter unit</td>
			<td style="width:169px;">Millex-GP</td>
			<td style="width:307px;">SLGP033RB</td>
			<td>0.22 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Syringe 10 mL</td>
			<td style="width:169px;">Discardit</td>
			<td style="width:307px;">309110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Atomic Force microscope</td>
			<td style="width:169px;">Veeco Instruments</td>
			<td style="width:307px;">Dimension 3000 AFM instrument</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:504px;">Silicon AFM probes</td>
			<td style="width:169px;">Budget Sensors</td>
			<td style="width:307px;">Tap300AI-G</td>
			<td>Resonant Freq. 300 kHz, k = 40 N/m</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Scanning tunneling microscope</td>
			<td style="width:169px;">Molecular Imaging</td>
			<td style="width:307px;">PicoSPM microscope</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Pt0.8:Ir0.2 wire</td>
			<td style="width:169px;">Advent</td>
			<td style="width:307px;">PT671012</td>
			<td>Diameter 0.25 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">WSxM 4.0 software</td>
			<td style="width:169px;">Nanotec electronica</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Optical contact angle (CA) system</td>
			<td style="width:169px;">Dataphysics</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">SCA20 software</td>
			<td style="width:169px;">Dataphysics</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">X-ray photoelectron spectrometer</td>
			<td style="width:169px;">Physical Electronics</td>
			<td style="width:307px;">Perkin-Elmer PHI 5500 Multitechnique System</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Fibronectin from bovine plasma</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">F1141-1MG</td>
			<td>1.0 mL solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Dulbecco&#39;s Phosphate Buffer Saline (DPBS)</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:307px;">21600-10</td>
			<td>Powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Mouse embryo fibroblasts</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC CRL-1658</td>
			<td>NIH/3T3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Dulbecco&#39;s modified eagle medium (DMEM) liquid high glucose</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:307px;">11960044</td>
			<td>liquid high glucose, no glutamine, 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:307px;">16000044</td>
			<td>500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">L-Glutamine</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">25030</td>
			<td>200 mM (100X)</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:504px;">Penicillin-streptomycin</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">15140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Sodium pyruvate</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">11360039</td>
			<td>100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">T75 culture flasks</td>
			<td style="width:169px;">Nunclon</td>
			<td style="width:307px;">156499</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:504px;">Trypsin</td>
			<td style="width:169px;">Life Technologies</td>
			<td style="width:307px;">25200072</td>
			<td>0,25% EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Centrifuge</td>
			<td style="width:169px;">Hermle Labortechnik</td>
			<td style="width:307px;">Z 206 A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Non-tissue culture treated plate, 12 well</td>
			<td style="width:169px;">Falcon</td>
			<td style="width:307px;">351143</td>
			<td>Non-adherent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Adipose-derived hMSCs</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-011</td>
			<td>Cell vial 1 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">MSC basal medium</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">MSC growth kit</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-040</td>
			<td>Low serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Chondrocyte differentiation tool</td>
			<td style="width:169px;">ATCC</td>
			<td style="width:307px;">ATCC PCS-500-051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Formalin solution</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">HT5011-15ML</td>
			<td>neutral buffered, 10%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Ammonium chloride</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">A9434-500G</td>
			<td>for molecular biology, suitable for cell culture, &#8805;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Saponin</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">47036-50G-F</td>
			<td>for molecular biology, used as non-ionic surfactant, adjuvant</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:504px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">A3059-50G</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:504px;">Rabbit monoclonal [Y113] anti-paxillin antibody</td>
			<td style="width:169px;">Abcam</td>
			<td style="width:307px;">ab32084</td>
			<td>Diluted 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Mouse monoclonal [1F5] anti-collagen alpha-1 XX chain&#160;</td>
			<td style="width:169px;">Acris Antibodies</td>
			<td style="width:307px;">AM00212PU-N</td>
			<td>Diluted: 1:400</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) secondary antibody</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">A10667</td>
			<td>2 mg/mL. Diluted 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Alexa Fluor 568-conjugated goat anti-rabbit IgG (H+L) secondary antibody</td>
			<td style="width:169px;">Invitrogen</td>
			<td style="width:307px;">A11036</td>
			<td>2 mg/mL. Diluted 1:1000</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Hoechst 33342</td>
			<td style="width:169px;">Thermo Fisher</td>
			<td style="width:307px;">H3570 10ML</td>
			<td>10 mg/mL. Diluted 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">Cover glass 24x24 mm</td>
			<td style="width:169px;">Deltalab</td>
			<td style="width:307px;">D102424</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:504px;">Fluoromount</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:307px;">F4680-25ML</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Epifluorescence Microscope</td>
			<td style="width:169px;">Nikon</td>
			<td style="width:307px;">Eclipse E1000 upright microscope</td>
			<td>with a CCD camera</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:504px;">Confocal Microscope</td>
			<td style="width:169px;">Leica Microsystems</td>
			<td style="width:307px;"></td>
			<td>Leica SPE Upright Confocal Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">ImageJ 1.50g freeware</td>
			<td style="width:169px;">http://imgej.nih.gov/ij</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">MATLAB software</td>
			<td style="width:169px;">The MATHWORKS, Inc.</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:504px;">OriginPro 8.5 software&#160;</td>
			<td style="width:169px;">OriginLab Coorporation</td>
			<td style="width:307px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

dendrimer-based uneven nanopatterns to locally control surface adhesiveness: a method to direct chondrogenic differentiation

Cellulär adhesion och differentiering villkoras av nanoskala disposition av extracellulär matrix (ECM) komponenter, med lokala koncentrationer med en större verkan. Här presenterar vi en metod för att få storskaliga ojämna nanopatterns av arginin-glycin-asparaginsyra (RGD)-functionalized tåååålamooooooood som tillåter nanoskala kontroll av lokala RGD yta densitet. Nanopatterns bildas genom ytan adsorption av tåååålamooooooood från lösningar vid olika initiala koncentrationer och kännetecknas av vatten kontaktvinkel (CA), X-ray fotoelektronen spektroskopi (XPS), och skanning sond mikroskopi tekniker såsom scanning tunneling microscopy (STM) och atomic force microscopy (AFM). Lokala Ytors av RGD mäts med hjälp av AFM bilder med hjälp av sannolikhet konturkartor av interparticle minimiavstånd och sedan korrelerade med cell adhesion svar och differentiering. Metoden nanopatterning som presenteras här är en enkel procedur som kan skalas på ett okomplicerat sätt att stora ytor. Det är således fullt kompatibel med cell kultur protokoll och kan tillämpas på andra ligander som utövar koncentrationsberoende effekter på celler.

Vi presenterar en nanopatterning metod som möjliggör ytans vidhäftningsförmåga tas upp på nanonivå (figur 1). RGD-Cys-D1 kemiska struktur visas i figur 1A. Tåååålamooooooood var mönstrad på elektriskt ledande Au(111) ytor för högupplösta STM karakterisering. Låg dendrimer koncentration i lösning (upp till 10-5% w/w) återges isolerade tåååålamooooooood av 4-5 nm i diameter (<strong class=...

Watch this Scientific Journal Video about Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation at JoVE.com

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

En metod för att erhålla dendrimer-baserade ojämn nanopatterns som tillåter nanoskala kontroll av lokala arginin-glycin-aspartic acid (RGD) Ytors beskrivs och tillämpas för studien av celldifferentiering vidhäftning och chondrogenic.

Dendrimer-baserade ojämn Nanopatterns till lokalt kontroll yta vidhäftning: en metod för att rikta Chondrogenic differentiering

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Research

JoVE Journal

Bioengineering

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Mätning av Aggregate sammanhållning genom vävnadsytan tensiometri

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design av en cyklisk tryck Bioreaktor för Ex vivo Studie av Aorta hjärtklaffar

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Konstruerar en tvåskiktad Hydrogel till Control ASC differentiering

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

En verktygslåda så att kolväteomvandling i vattenhaltiga miljöer

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

En ny metod för att lokalisera Reporter Fluorescent Pärlor Nära Cell Culture Yta för Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Anpassning av Electro processen med att ge tre unika miljöer för en Tri-lager<em&gt; In Vitro</em&gt; Modell av Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

En automatiserad metod för att utföra Mikrokärnanalys in vitro med hjälp av Multispektral avbildning flödescytometri

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Experimental Methods to Study Human Postural Control

Experimentella metoder för att studera mänsklig Postural kontroll

Many components of the nervous and musculoskeletal systems act in concert to achieve the stable, upright human posture. Controlled experiments accompanied ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

Samtidig kvantifiering av utvalda kynukreniner analyserade av flytande kromatografi-masspektrometri i medium som samlats in från cancercellkulturer

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Encellig optisk åtgärd potentiell mätning hos mänskliga inducerade pluripotenta stamcellsbaserade kardiomyocyter

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...