The authors would like to acknowledge the help of Walter Scholdei (Max-Planck-Institute for Polymer Research, Mainz, Germany.

Sodyum naftalinid Aktive Çözüm ve Yüzey Aktivasyon 1. hazırlanması Not: iyi havalandırılan bir davlumbaz tepkilere yürütmek. yüksek derecede yanıcı çözücüler ve metalik sodyum gibi aşındırıcı metallerin işlemek için genel kurallara uyun. Naftalin Çok az miktarlarda dahi çok hoş olmayan bir koku (mothball), var! aksi belirtilmedikçe reaksiyonlar, oda sıcaklığında gerçekleştirilir. Sodyum azid son derece zehirlidir! THF moleküler elek (hacim olarak) üzerinde yaklaşık% 20 muhafaza edildi (% 99.9, Malzeme listesine bakınız). sodyum üzerinde gözle görülür bir su içeriği ile THF damıtmak. su eser miktarlarda mevcut olması durumunda, sodyum naftalenid oluşumu oluşmaz. <ol><li> (3 A moleküler elek üzerinde kurutuldu, THF,), 20 ml tetrahidrofuran içinde naftalen 1.4 g (10.9 mmol) içindeki bir çözeltisine, bir PTFE ile donatılmış bir vidalı kapaklı 100 ml&#39;lik bir cam şişede, 0.25 g (10.9 mmol) sodyum metal ilave kaplanmış manyetik karıştırma çubuğu. Not: Dissolu(- 40 ° C ila 35) yon ölçüde küçük parçalar ve mütevazı ısıtma halinde sodyum kesilerek arttırılabilir. Nihai çözüm karanlık, biraz yeşilimsi rengi vardır ve kesinlikle kuru koşullarda saklanabilir. </li><li> 0.5 mm kalınlığında folyo malzemeden 12 mm çapında PTFE diskleri yumruk. Mark bir tarafı (örneğin, bir mektup damga yumruk kullanarak) izopropanol ile ve temiz. </li><li> 2 dk kullanarak forseps - 1 için aktive etme çözeltisi (adım 1.1) ayrı ayrı PTFE örnekleri inkübe. Not: koyu kahverengi, beyaz renk değişimi başarılı bir şekilde tedavi gösterir. </li><li> Daha sonra, izo-propanol ile iki kez THF ile ve daha sonra durulanır. Not: naftalenid çözüm tükendiğinde bir sulu açık kahverengi rengi değişir. (Muhtemelen metal sodyum ihtiva eden) Kullanılmayan naftalenid çözeltisi yavaş yavaş tasfiye etmeden önce, izo-propanol ilave edilerek dekompoze edilmesi gerekir. </li><li> Hidrojen peroksit (% 30) 3 saat süre ile trikloroasetik asit (ağ / hac)% 20 ihtiva eden tedavi örnekleri okside eder. Yıkamasu ve kuru ile. Yüzey şimdi hafif kahverengimsi bir görünüme sahiptir. Not: Aktivasyon ve oksidasyon sonucu yüzeyinin büyük ölçüde artmıştır ıslatılabilme içinde. Bu bulgu, daha önce 14 ayrıntılı olarak incelenmiştir. </li><li> 2 saat boyunca kuru THF içinde% 50 (h / h) heksametilen diizosiyanat (HMDI) ile oksitlenir diskleri tedavi THF ile durulanır ve kurumaya bırakın. </li><li> 3 saat ve kuru - 2 suda izosiyanat rulman örnekleri hidrolize. Not: Nihai amino işlevselleştirilmiş PTFE yüzeyi çok daha stabil izosiyanat taşıyan numuneler daha ayrıntılı bağlanma için birçok standart çapraz bağlayıcı ile uyumludur. </li></ol> 2. Peptit İmmobilizasyonu <ol><li> karbonat tamponu, pH 9, 50 mM% 20 (h / h) dietilenglikol diglisidil eter (diepoksit) içindeki bir çözeltisi hazırlandı. </li><li> ayrı ayrı 24 plaka içine belirgin tarafı yukarı ile amine diskleri yerleştirin ve epoksit çözeltisi 1.5 ml ekleyin. Örneklerin tam kapsama sağlamak. 2 saat boyunca inkübe ve iki yıkamakatı, suyla ve bir kez karbonat tamponu ile yıkandı. </li><li> Taze 24 plaka, tek tek oyuklara altına, NaN3% 0.01 ihtiva eden 50 mM bir 0.5 mg / ml peptid (ör REDV) karbonat tamponu, pH 9, 50 ul ekle ve dikkatli bir şekilde, epoksi-fonksiyonlu diskler ters yer aşağıya doğru peptid solüsyonu damlasına üzerine (yani aşağı yan işaretlenmiştir). Kuyunun dibine PTFE disk arasındaki boşluk tamamen nedeniyle kılcal eylem ıslanan olduğundan emin olun. </li><li> Islak odasında inkübe (örneğin, sıkı bir kapama kapağı ile bir sızdırmaz plastik kutu) nemli bir atmosferde gece boyunca en az 3 saat ya da için (alt ıslak kağıt mendil yerleştirerek). Not: REDV motifi gibi iyi karakterize kabul edilebilir olsa bir karıştırılmış veya ters bir amino asit sekansı, ek bir negatif kontrol olarak dahil edilebilir. </li><li> su ile üç kez yıkanır ve en az 30 dakika boyunca,% 50 izo-propanol / su içinde sterilize edilir. Tohumlama, rin hücre Öncekisteril fosfat tamponlu tuzlu su (PBS) içinde örnekleri e. </li></ol> 3. Hücre Yayımlama <ol><li> Standart hücre kültürü prosedürleri 17,18 kullanarak HUVECler büyütün. </li><li> 24 yuvalı plakalar içinde tadil edilmiş tarafı yukarı peptit-konjuge numuneler. Bir kontrol olarak işlenmemiş PTFE diskleri kullanın. </li><li> Tohum 5 x 10 4 oyuk başına 2 ml&#39;lik ortam içinde HUVECler ve bir kuluçka makinesi içinde 37 ° C de 4 saat ve% 5 CO2 inkübe edilir. </li><li> diskleri çıkarın, unattached hücreleri çıkarmak ve yeni bir 24 plaka aktarmak dikkatlice yıkayın. İstenen süre boyunca taze ortam 2 ml ilave edilir ve kuluçkaya (örneğin, 24 saat, w, 1, vb.) her iki günde orta değiştirin. </li><li> dimetil sülfoksit (DMSO) içinde 1 mg / ml Calcein-AM bir stok çözelti hazırlayın. </li><li> Büyümeden sonra hücreler, orta kaldırmak, PBS ile yıkayın ve PBS ihtiva eden 1 ul / ml Calcein-AM leke ekleme (örneğin, ml PBS her bir stok çözeltisi 1 ul). Hafifçe ajitasyon ve incubatKaranlıkta 45 dakika boyunca 37 ° C &#39;de, e. </li><li> Standart FITC filtreleri ile donatılmış bir floresan mikroskop mikrograflar almak (Ör 488 nm, Em 515 nm) hemen PBS ile yıkayın ve. 100-kat büyütme kullanın. değiştirilmemiş gelen triplicates gibi muamele örnekleri gerçekleştirin. </li><li> ImageJ yazılımı kullanarak hücreler tarafından kolonize alanı belirlemek. Alternatif elle hücreleri saymak veya ImageJ kullanın. Her iki yöntem de ilgili yüzeylerde eki ve hücrelerin büyümesi ile ilgili benzer bilgiler verecektir. </li></ol>

<ol>
	<li>Doctor, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+various+prosthetic+materials+and+newer+meshes+for+hernia+repairs.">Evaluation of various prosthetic materials and newer meshes for hernia repairs.</a> J. Minim. Access Surg. 2, 110-116 (2006).</li><li>Zaghal, 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=Update+on+totally+implantable+venous+access+devices.">Update on totally implantable venous access devices.</a> Surg. Oncol. 21, 207-215 (2012).</li><li>Niu, G., Sapoznik, E., Soker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+blood+vessels.">Bioengineered blood vessels.</a> Exp. Opin. Biol. Th. 14, 403-410 (2014).</li><li>Wang, M. -. J., Tsai, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomaterials in Blood-Contacting Devices: Complications and Solutions. , (2010).</li><li>de Mel, A., Jell, G., Stevens, M. M., Seifalian, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofunctionalization+of+biomaterials+for+accelerated+in+situ+endothelialization:+a+review.">Biofunctionalization of biomaterials for accelerated in situ endothelialization: a review.</a> Biomacromolecules. 9, 2969-2979 (2008).</li><li>Zdrahala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+caliber+vascular+grafts.+Part+I:+state+of+the+art.">Small caliber vascular grafts. Part I: state of the art.</a> J. Biomat. Appl. 10, 309-329 (1996).</li><li>Cleary, M. 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=Vascular+tissue+engineering:+the+next+generation.">Vascular tissue engineering: the next generation.</a> Trends Mol. Med. 18, 394-404 (2012).</li><li>Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+and+other+recognition+sequences+for+integrins.">RGD and other recognition sequences for integrins.</a> Annu. Rev. Dev. Bi. 12, 697-715 (1996).</li><li>Lei, Y., Remy, M., Labrugere, C., Durrieu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+immobilization+on+polyethylene+terephthalate+surfaces+to+study+specific+endothelial+cell+adhesion,+spreading+and+migration.">Peptide immobilization on polyethylene terephthalate surfaces to study specific endothelial cell adhesion, spreading and migration.</a> J. Mat. Sci. Mater. M. 23, 2761-2772 (2012).</li><li>Gabriel, 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=Covalent+RGD+Modification+of+the+Inner+Pore+Surface+of+Polycaprolactone+Scaffolds.">Covalent RGD Modification of the Inner Pore Surface of Polycaprolactone Scaffolds.</a> J. Biomat. Sci.. Polym. E. 23, 941-953 (2012).</li><li>Ceylan, H., Tekinay, A. B., Guler, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+adhesion+and+growth+of+vascular+endothelial+cells+on+bioactive+peptide+nanofiber+functionalized+stainless+steel+surface.">Selective adhesion and growth of vascular endothelial cells on bioactive peptide nanofiber functionalized stainless steel surface.</a> Biomaterials. 32, 8797-8805 (2011).</li><li>Chlupac, J., Filova, E., Bacakova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+replacement:+50+years+of+development+and+tissue+engineering+paradigms+in+vascular+surgery.">Blood vessel replacement: 50 years of development and tissue engineering paradigms in vascular surgery.</a> Physiol. Res. / Academia Scientiarum Bohemoslovaca. 58, 119-139 (2009).</li><li>Wise, S. G., Waterhouse, A., Kondyurin, A., Bilek, M. M., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-based+biofunctionalization+of+vascular+implants.">Plasma-based biofunctionalization of vascular implants.</a> Nanomedicine UK. 7, 1907-1916 (2012).</li><li>Mikulikova, 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=Cell+microarrays+on+photochemically+modified+polytetrafluoroethylene.">Cell microarrays on photochemically modified polytetrafluoroethylene.</a> Biomaterials. 26, 5572-5580 (2005).</li><li>Gabriel, M., Dahm, M., Vahl, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wet-chemical+approach+for+the+cell-adhesive+modification+of+polytetrafluoroethylene.">Wet-chemical approach for the cell-adhesive modification of polytetrafluoroethylene.</a> Biomed. Mater. 6, 035007 (2011).</li><li>Gabriel, M., van Nieuw Amerongen, G. P., Van Hinsbergh, V. W., Amerongen, A. V., Zentner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+grafting+of+RGD-motif-containing+peptide+on+the+surface+of+polycaprolactone+films.">Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films.</a> J. Biomat. Sci.. Polym. E. 17, 567-577 (2006).</li><li>Larsen, C. C., Kligman, F., Kottke-Marchant, K., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+RGD+fluorosurfactant+polymer+modification+of+ePTFE+on+endothelial+cell+adhesion,+growth,+and+function.">The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.</a> Biomaterials. 27, 4846-4855 (2006).</li><li>Gabriel, M., Nazmi, K., Veerman, E. C., Nieuw Amerongen, A. V., Zentner, A. <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+LL-37-grafted+titanium+surfaces+with+bactericidal+activity.">Preparation of LL-37-grafted titanium surfaces with bactericidal activity.</a> Bioconjugate Chem. 17, 548-550 (2006).</li><li>Lotz, A., Heller, M., Brieger, J., Gabriel, M., F&#246;rch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+Plasma+Polymerized+Thin+Films+and+Attachment+of+Biomolecules+to+Influence+HUVEC-Cell+Adhesion.">Derivatization of Plasma Polymerized Thin Films and Attachment of Biomolecules to Influence HUVEC-Cell Adhesion.</a> Plasma Process Polym. 9, 10-16 (2012).</li></ol>

The authors have nothing to disclose.

PTFE yüzey modifikasyonu protokolünün ayrıntılı açıklaması Şekil 6&#39;da gösterildiği gibi, polimer omurgasından flor ortadan kaldırılması ile başlayarak ardışık adımlardan oluşur. Bunun bir sonucu olarak, bir katman konjuge karbon-karbon çift bağlarının bol miktarda içerir, meydana gelen naftalenid tedavisi üzerine geliştirilen koyu kahverengimsi renk ile uyum. Asidik hidrojen peroksit ile standart oksidasyon soluk kahverengi aydınlatıcı eşliğinde hidroksile yüzey, b...

Yani gelişmiş biyouyumluluk, hücre yapışma eksikliği, fibrotik kapsülleme ve trombojenitesine uyarılmasını göstermezler süredir onaylanan tıpta kullanılan çeşitli polimerler, birkaç söz. biyo ve biyolojik sistemi arasındaki etkileşimler özellikle implantın yüzeyinde yer alır. Bunun bir sonucu olarak, araştırma etkilenmez malzemenin yığın özelliklerini bırakarak istenilen uygulama için uygun özellikleri oluşturmak için yüzey değişimi üzerine odaklanmıştır. Fizyolojik olarak inert bir polimer olarak politetrafloroetilen (PTFE) gibi fıtığı cerrahi mesh 1, ve en önemlisi, tıbbi bağlantı noktaları 2 ve, vasküler greftler 3 gibi birçok tıbbi alanlarda kullanılır. Özellikle kan temas durumlarda PTFE hidrofobik doğası çoğu zaman thromboti sonuçlanan bir sonucu platelet yapışması, plazma bileşenlerinin gibi spesifik olmayan adsorpsiyon neden olurc olayları ve greft 4 tıkanması. Bundan başka PTFE, en polimerleri gibi, arzu edilen bir özellik, vasküler greft 5, iç (lümen) yüzey üzerinde endotelyal hücreleri (EC) faydalı bir tabakanın oluşumuna yol olacaktır hücre yapışması ve kapsamı desteklemez. Bir biomimetic endotel özellikle onun antitrombojenik özelliklerini 6, doğal eşdeğeri işlevlerin çoğunu yerine getirmesi beklenir. Genel biomimetic değişiklik stratejisi etkilenmemiş malzemeler dökme özelliklerini bırakarak sadece hücre yapışma malzemeyi endowing kavramına dayanmaktadır. Buna ek olarak, platelet yapışma önleyici bir yapışkan (anti-kirlenme) 7 özellikleri dahil edilmesiyle azaltılabilir. Çeşitli peptiplere - çoğunlukla hücre-dışı matrisin proteinlerden türetilen - güçlü integrin 8 sınıfına ait, hücresel reseptörlere bağlanarak hücre yapışmasını geliştirmek olduğu tarif edilmiştir. olmakBu konuda bir örnek st bilinen çoğu hücre tipi ile etkileşim peptid Arg-Gly-Asp (RGD) &#39;dir. Diğer amino asit dizileri özel olarak spesifik hücreler üzerinde eksprese edilen integrinler tarafından tanınır. Örneğin, Arg-Glu-Asp-Val (REDV) ve Tyr-Ile-Gly-Ser-Arg (YIGSR) belirli bir şekilde 9 EC bağlamak için tespit edilmiştir. Peptidler kovalent immobilizasyon metaller ve polimerler 10,11 içeren doğal olmayan yapışkan malzemelerin bir bolluk üzerinde gerçekleştirilmiştir. Gözenekli PTFE, daha doğrusu PTFE (ePTFE) genişletilmiş - polietilen tereftalat (PET) ile birlikte - vasküler üretimi için en önemli malzeme 12 greftleri olduğunu. Plazma modifikasyonu 13 veya fotokimyasal yöntemlerle 14 tarafından uygun tedaviler için kurulan fiziksel teknikler, gözenekli ve / veya boru şeklinde yapılar sırasıyla gözenekler ya da lümene içinde kolayca tedavi edilebilir olmadığı gerçeği ile engellenmektedir. Islak kimyasıPTFE üzerinde çünkü çoğu kimyasal saldırılar 15 direnen fluorin içeren polimer derece etkisiz doğası zor bir iştir. Bu yazıda bir kovalent modifikasyon stratejisi için nispeten basit bir yöntem açıklanmaktadır. PTFE bondable işlemek için bir prosedür uyarlanan fonksiyonel gruplar biyolojik olarak aktif moleküllerin daha fazla bağlanması için çapa noktaları olarak hizmet malzemeler yüzeyinde oluşturulmuştur.

<table border="0" cellpadding="0" cellspacing="0" width="700">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">PTFE foil 0.5 mm</td>
			<td style="width:119px;">Cadillac Plastic&#160;</td>
			<td style="width:145px;">n/a</td>
			<td style="width:115px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">REDV peptide</td>
			<td>Genecust</td>
			<td>n/a</td>
			<td>custom synthesis &#62;95 % purity</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iso-propanol</td>
			<td>Sigma Aldrich</td>
			<td>34965</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">tetrahydrofurane (THF)</td>
			<td>Sigma Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dimethylsulfoxide</td>
			<td>Sigma Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">molecular sieve 3&#197;</td>
			<td>Sigma Aldrich</td>
			<td>208574</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium metal</td>
			<td>Sigma Aldrich</td>
			<td>483745</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">naphthalene</td>
			<td>Sigma Aldrich</td>
			<td>147141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrogen peroxide 30 %</td>
			<td>Sigma Aldrich</td>
			<td>95321</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trichloroacetic acid</td>
			<td>Sigma Aldrich</td>
			<td>T6399&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">diethylene glycol diglycidyl ether</td>
			<td>Sigma Aldrich</td>
			<td>17741</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hexamethylene diisocyanate (HMDI)</td>
			<td>Sigma Aldrich</td>
			<td>52650</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcein-AM</td>
			<td>Sigma Aldrich</td>
			<td>56496</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6014&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>71290</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 well plates</td>
			<td>Greiner-Bio-One</td>
			<td>662 160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATR-FTIR spectrophotometer Nicolet Magna-IR 850</td>
			<td>&#160;Nicolet</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorescence microscope Olympus X-70</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">humbilical vein endothelial cells (HUVECs)</td>
			<td>Lonza</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ePTFE vascular graft</td>
			<td>Gore</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

wet chemistry and peptide immobilization on polytetrafluoroethylene for improved cell-adhesion

malzemeler hücre yapışma özelliklerine sahip yüzey endowing biyomalzeme araştırma ve doku mühendisliğinde yaygın bir stratejidir. Bu durum, bu maddeler de önlenebilir yeni sentezlenmiş polimerlerden tanıtımı ile bağlantılı özelliği ve yasal sorunlar nedeniyle tıpta uzun süreli kullanımı vardır önceden onaylanmış bir polimer için özellikle ilgi çekicidir. Politetrafloroetilen (PTFE), vasküler greft üretimi için en sık kullanılan malzeme biri ama polimerin özelliklerini teşvik hücre yapışmasını yoksundur. Endotelizasyon örneğin, endotel hücrelerinin konfluent tabakası ile greft iç yüzeyi tam kapsamı esas olarak yapay bir arayüz trombojenisitesini azaltmak suretiyle, en iyi performans için anahtar kabul edilir. Bu çalışma, peptit ile modifiye edilmiş PTFE üzerinde endotelyal hücrelerin büyümesini incelenmiş ve modifiye edilmemiş alt tabaka üzerine elde edilen bu sonuçlar karşılaştırır. ile Kavramaendotelyal hücre yapışma peptid Arg-Glu-Asp-Val (REDV) daha sonra konjügasyon adımları, ardından reaktif sodyum naftalenid ile fluorin-içeren polimerin aktivasyonu yoluyla gerçekleştirilir. Hücre kültürü, bir, iki haftalık bir süre boyunca gösterilmiştir peptid hareketsizleştirilmiş malzeme İnsan Umbilikal Ven Endotelyal Hücreleri (HUVECs) ve mükemmel hücresel büyüme kullanılarak gerçekleştirilir.

Önemli kimyasal reaksiyon adımlarının sonuçlar IR spektroskopisi (Şekil 1) ile izlendi. sodyum naftalenid ile ilk aktivasyon çift bağları oluşturur - ve küçük bir ölçüde - OH-işlevleri. Sinyal gösteren C = C bağının hemen hemen tamamen, hidroksil grubu taşıyan bir yüzey verecek şekilde oksidasyon ortadan kalkar. Daha fazla standart konjugasyon adımların analizi burada gösterilmemiştir. Aktivasyonu ve oksidasyona bağlı renk değişiklikleri ...

Watch this Scientific Journal Video about Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion at JoVE.com

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Geliştirilmiş Hücre yapışma Politetrafloroetilen Islak Kimya ve Peptit İmmobilizasyonu

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

wet-chemistry-peptide-immobilization-on-polytetrafluoroethylene-for

Research

JoVE Journal

Bioengineering

7.6K Görüntüleme.  Sidra Cardiovascular Research. Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Video: Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Atomik Kuvvet Mikroskopi Görüntüleme Bakteriyel İmmobilizasyon

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Biyomühendislik

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

Enzim immobilizasyonu ve stabilizasyonu için hidrofobik Tuz-modifiye Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Cilt Tip I Kolajen hazırlanması için geliştirilmiş bir yöntem

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Putatif Hücre yapışma molekülleri Karakterizasyonu için Boncuk Toplama Tahliller

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

MicroRNA Niceleme DNA bağlayıcı Boya Kimya ve Damlacık Dijital PCR kullanarak Sirkülasyon

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

Zebra balığı larva geliştirilmiş deneysel manipülasyon için uzun vadeli canlı görüntüleme aygıtı

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

Geliştirilmiş 3D hidrojel Neuroinflammation Vitro modelleme için birincil gliyal hücreler kültürleri

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Site yönettiği immobilizasyon kemik morfogenetik protein 2 katı yüzeyler için tıklayın kimya tarafından

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

Islak eğirme esaslı kalıplama işlemi doku rejenerasyonu için jelatin

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Bir Ultra-thin Polydimethylsiloxane mikrosıvısal çip su tutma ile kullanarak canlı Caenorhabditis elegans bireylerin immobilizasyon

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...