Bu eser mali NSFC projeler (81522023, 81530059, 91639113, 81772000, 81371699 ve 81401534) tarafından desteklenmiştir.

Deneysel hayvan kullanımı hayvan deneyleri Etik Komitesi Nankai Üniversitesi tarafından onaylanmış ve bakım ve Laboratuvar hayvanlarının kullanmak için Kılavuzu uygun olarak yürütülmektedir.
1. Electrospun PCL Greftler imalatı
Not: Burada, vasküler Greftler imal etmek bir electrospinning tekniği kullanılmıştır.
<ol><li>PCL çözümleri 25 wt % ve 10 wt %, sırasıyla, metanol ve kloroform, karışımı PCL çözülerek hazırlamak (1:5 hacim oranı), oda sıcaklığında (RT) 12 h için.</li>
	<li>PCL çözüm 10 mL Cam şırınga yükleyin.</li>
	<li>Şırınga ile 21-G iğne yerleştirin.</li>
	<li>Paslanmaz çelik mandrel (2 mm çapında, uzunluğu 25 cm) koleksiyon araç üzerinde yerleştirin.</li>
	<li>Lif kalın Greftler için kullanın PCL çözüm 25 wt %, çalışma mesafesi toplayıcı için iğne ucu 17 cm, 8 mL/h debi ve gerilim 11 kV electrospinning parametre olarak. İnce lifli greft için 10 wt %, iğne ucu 20 cm toplayıcı için çalışma mesafesi, 2 mL/h debi ve gerilim 18 PCL çözüm kullanın kV electrospinning parametre olarak.</li>
	<li>Elde edilen Greftler gecede kalan solvent kaldırmak için vakum yerleştirilir emin olun. Önce yordamı tüm aletleri sterilize ve aseptik teknik boyunca korumak.</li>
	<li>İmplantasyon öncesinde Greftler dezenfekte onları 10 ml % 75 etanol için 30 dk çeker ve sonra onları ifşa UV ışığı gecede.</li>
	<li>Lif ve gözenek boyutu ölçüleri: ImageJ yazılım elektron mikroskobu (SEM) görüntüleri taramak tabanlı kullanarak ortalama lif çapı hesaplamak.</li>
	<li>Mekanik iskele test:<ol>
<li>Bir tıraş bıçağı kullanarak uzunluğu 3 mm bölümlere borulu iskele kesti. Bir mikrometre kullanarak iskele kalınlığını ölçmek.</li>
		<li>100 N. yük kapasiteli bir gerilme test makinede borulu iskele yerleştirin</li>
		<li>1 mm arası kelepçe mesafe ve 10 mm/dk hızında boyuna çekme ile iskele rüptürü kadar kelepçe. Çekme dayanımı ve kopma nihai uzama ölçmek. Young katsayısı ilk doğrusal bölgesinden hesaplamak (en fazla % 5 zorlanma) stres gerginlik eğrisi.</li>
	</ol>
</li>
</ol>2. fare Abdominal aort implantasyon modeli
Not: Tüm malzeme ve kullanılan aletler steril. Ameliyat sırasında operatör bir gazlı bez maske ve enfeksiyonları önlemek için steril eldiven giyer emin olun. Oda sıcaklığında hayvan vücut ısısını korumak için 27-30 ° C'de tutulur emin olun. Analjezi ile ilgili yerel IACUC yönergeleri izleyin.
<ol><li>Vasküler greft alıcıları 240-270 g ağırlığında erkek Sprague Dawley rat kullanın. Fareyi ameliyattan önce 24 saat oruç emin olun. 24 h açlık sıçanlara amacı boş dışkı bağırsak yeterince, böylece operatörün ufku genişletmek.</li>
	<li>Sıçan arka boyun kavramak ve kafasını aşağıya doğru tutmak, alt karın karın boşluğuna springe iğne yerleştirin. Kloral hidrat (330 mg/kg) ile anestezi için fare mayi iğne tarafından ikna etmek.</li>
	<li>Yeterli anesthetization sıçan kasları ve düzenli nefes rahat sağlayarak onaylayın. Çalışma mikroskop altında fare sırtüstü pozisyonda yerleştirin.</li>
	<li>Petrolatum oftalmik veteriner merhem Gözlerde kuruluk anestezi iken altında önlemek için geçerlidir. AntiCoagulation (100 UI/kg) ameliyattan önce kuyruk ven enjeksiyonla heparinized fizyolojik serum çözüm (50 UI/mL) ile yönetebilirsiniz.</li>
	<li>Bir tıraş bıçağı kullanarak ön karın duvarındaki kürk kapalı tıraş ve deri iyot çözüm ve Tıbbi alkol çözümü kullanarak temiz.</li>
	<li>Laparotomi ensizyon cerrahi makas ile gerçekleştirmek ve belgili tanımlık kesme yaklaşık 4-5 cm uzunluğunda olduğundan emin olun ve karın boşluğu karşı karşıya.</li>
	<li>Geri çekmek ve bağırsak ile tuzlu çözüm tercihen ıslatılmış gazlı bez ile sarın.</li>
	<li>Abdominal aort dikkatle incelemek.</li>
	<li>Tanımlamak ve tüm küçük dalları 9-0 monofilament naylon dikiş kullanarak ligate.</li>
	<li>İzole bölüm kelepçe (ilâ 1 cm uzunluğunda) iki damar kelepçeler kullanılarak aort. Aorta için 20-30 dk klempe kalabilir.</li>
	<li>İki kelepçeler anastomoz siteleri oluşturmak için mikro-makas kullanarak arasında karın aortu transect.</li>
	<li>Artık kan kaldırmak için heparinized serum fizyolojik (50 UI/mL) çözüm kullanarak aort iki ucunun floş.</li>
	<li>Mikro-makas kullanarak adventitia soyma.</li>
	<li>Greft ile 2 mm iç çap ve 1 cm uzunluğunda sıçan abdominal aort için 9-0 monofilament naylon dikiş kullanarak bir sekiz şekli dikiş desen ile anastomose.</li>
	<li>İlk olarak, dört anastomoses, 9, 3, 12 ve 06: 00 proksimal yan konumlarda sırasına göre oluşturmak, sonra iki dikiş arasında 4 dikiş kesilmiş kenarları anastomose. Proksimal dikiş bittikten sonra aynı yöntemle distal yan dikiş. Not: Her dikiş yerli yan biraz greft gömülü olduğu için gereklidir.</li>
	<li>Kan nakli akışını sağlamak için distal klempi kaldırın, sonra proksimal klemp kaldırın.</li>
	<li>Dikiş biter steril bir pamuk veya bir küçük gazlı bez sünger kullanarak kanamayı durdurmak için tuşuna basın. Yaklaşık 3 dakika için hemostazın kadar basın.</li>
	<li>Bağırsak karın boşluğuna dönün.</li>
	<li>Karın boşluğu sıcak fizyolojik serum çözüm gentamisin ile (320 U/mL) kullanarak floş.</li>
	<li>3-0 naylon dikiş kas ve deri katmanının içinde sırasıyla kullanarak karın duvarına kadar dikmek.</li>
	<li>Fareyi bir temiz ve kuru kafesin içine yerleştirin ve bir ısıtma yastığı hayvan vücut ısısını korumak için kafes altında koymak; daha sonra fareyi anesteziden kurtarmak bekleyin. Sternal recumbency korumak için yeterli kendine geldi kadar hayvan için katılmak.</li>
	<li>Bilinci yerine sonra yiyecek ve su ile tek bir kafes içinde fare koydu. İyot ameliyattan sonra enfeksiyonu önlemek için yaranın üzerine uygulayın. Tamamen iyileşene kadar fareyi diğer hayvanlar şirkete geri.</li>
	<li>Fareler önceden belirlenmiş zaman noktalarda kurumsal esaslarına göre ötenazi.</li>
</ol>

<ol>
	<li>Coombs, K. E., Leonard, A. T., Rush, M. N., Santistevan, D. A., Hedberg-Dirk, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolated+effect+of+material+stiffness+on+valvular+interstitial+cell+differentiation.">Isolated effect of material stiffness on valvular interstitial cell differentiation.</a> J Biomed Mater Res A. 105 (1), 51-61 (2017).</li><li>Zhang, 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=A+sandwich+tubular+scaffold+derived+from+chitosan+for+blood+vessel+tissue+engineering.">A sandwich tubular scaffold derived from chitosan for blood vessel tissue engineering.</a> J Biomed Mater Res A. 77 (2), 277-284 (2006).</li><li>Thottappillil, N., Nair, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+in+vascular+regeneration:+current+status.">Scaffolds in vascular regeneration: current status.</a> Vasc Health Risk Manag. 11, 79-91 (2015).</li><li>Pektok, 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=Degradation+and+healing+characteristics+of+small-diameter+poly+(e-caprolactone)+vascular+grafts+in+the+rat+systemic+arterial+circulation.">Degradation and healing characteristics of small-diameter poly (e-caprolactone) vascular grafts in the rat systemic arterial circulation.</a> Circulation. 118 (24), 2563-2570 (2008).</li><li>Innocente, F., 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=Paclitaxel-eluting+biodegradable+synthetic+vascular+prostheses:+a+step+towards+reduction+of+neointima+formation?.">Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?.</a> Circulation. 120 (11 Suppl), S37-S45 (2009).</li><li>de Valence, 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=Advantages+of+bilayered+vascular+grafts+for+surgical+applicability+and+tissue+regeneration.">Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration.</a> Acta Biomater. 8 (11), 3914-3920 (2012).</li><li>Assmann, 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=Acceleration+of+autologous+in+vivo+recellularization+of+decellularized+aortic+conduits+by+fibronectin+surface+coating.">Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating.</a> Biomaterials. 34 (25), 6015-6026 (2013).</li><li>Hasan, 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=Electrospun+scaffolds+for+tissue+engineering+of+vascular+grafts.">Electrospun scaffolds for tissue engineering of vascular grafts.</a> Acta Biomater. 10 (1), 11-25 (2014).</li><li>Baker, B. 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=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29 (15), 2348-2358 (2008).</li><li>Wang, K., 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=Creation+of+macropores+in+electrospun+silk+fibroin+scaffolds+using+sacrificial+PEO-microparticles+to+enhance+cellular+infiltration.">Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO-microparticles to enhance cellular infiltration.</a> Journal of Biomedical Materials Research Part A. 101 (12), 3474-3481 (2013).</li><li>Lee, B. L. P., 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=Femtosecond+laser+ablation+enhances+cell+infiltration+into+three-dimensional+electrospun+scaffolds.">Femtosecond laser ablation enhances cell infiltration into three-dimensional electrospun scaffolds.</a> Acta Biomaterialia. 8 (7), 2648-2658 (2012).</li><li>Rnjak-Kovacina, J., 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=Increasing+the+pore+size+of+electrospun+scaffolds.">Increasing the pore size of electrospun scaffolds.</a> Tissue Eng Part B Rev. 17 (5), 365-372 (2011).</li><li>Zhong, S., Zhang, Y., Lim, C. T. <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+large+pores+in+electrospun+nanofibrous+scaffolds+for+cellular+infiltration:+a+review.">Fabrication of large pores in electrospun nanofibrous scaffolds for cellular infiltration: a review.</a> Tissue Eng Part B Rev. 18 (2), 77-87 (2012).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+poly(epsilon-caprolactone)+microfiber+and+multilayer+nanofiber/microfiber+scaffolds:+characterization+of+scaffolds+and+measurement+of+cellular+infiltration.">Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.</a> Biomacromolecules. 7 (10), 2796-2805 (2006).</li><li>Rnjak-Kovacina, 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=Tailoring+the+porosity+and+pore+size+of+electrospun+synthetic+human+elastin+scaffolds+for+dermal+tissue+engineering.">Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering.</a> Biomaterials. 32 (28), 6729-6736 (2011).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+thick+fibers+and+large+pores+of+electrospun+poly(epsilon-caprolactone)+vascular+grafts+on+macrophage+polarization+and+arterial+regeneration.">The effect of thick fibers and large pores of electrospun poly(epsilon-caprolactone) vascular grafts on macrophage polarization and arterial regeneration.</a> Biomaterials. 35 (22), 5700-5710 (2014).</li><li>Hutcheson, J. D., 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=Genesis+and+growth+of+extracellular-vesicle-derived+microcalcification+in+atherosclerotic+plaques.">Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques.</a> Nat Mater. 15 (3), 335-343 (2016).</li><li>Tara, 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=Well-organized+neointima+of+large-pore+poly(L-lactic+acid)+vascular+graft+coated+with+poly(L-lactic-co-epsilon-caprolactone)+prevents+calcific+deposition+compared+to+small-pore+electrospun+poly(L-lactic+acid)+graft+in+a+mouse+aortic+implantation+model.">Well-organized neointima of large-pore poly(L-lactic acid) vascular graft coated with poly(L-lactic-co-epsilon-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(L-lactic acid) graft in a mouse aortic implantation model.</a> Atherosclerosis. 237 (2), 684-691 (2014).</li></ol>

Yazarlar hiçbir çakışan mali ilgi alanlarına sahip.

Hücre infiltrasyonu rejenerasyon için kritik ve remodeling vasküler greft vivo içinde16. Sınırlı hücre infiltrasyonu kez hücreleri geçiş greft duvara engel greft nispeten küçük gözeneklerin ilgilidir. Bu zorluk adrese, electrospun PCL vasküler greft büyük gözenek yapısı ile hazırlamak için değiştirilmiş bir yöntem geliştirdi. Ayrıntılı olarak, kolayca işleme parametreleri tarafından kontrol edilebilir fiber kalınlığı artış ile gözenek boyutu artmı?...

Vasküler Greftler sentetik polimerler yapılan yaygın olarak klinik kardiyovasküler hastalıklar (CVDs) tedavisi için kullanılmaktadır. Ne yazık ki, söz konusu olduğunda küçük çaplı vasküler Greftler (D < 6 mm) mevcuttur herhangi başarılı bir ürün nedeniyle sık sık kan pıhtılaşması, intimal hiperplazi ve diğer yol açar azaltılmış kan akım hızı tarafından tetiklenen düşük açıklık komplikasyonlar1.
Doku mühendisliği uzun vadeli açıklık ve bir iskele güdümlü vasküler yenilenme ve yeniden yapılanma dayalı homeostazı gerçekleştirmek için alternatif bir strateji sağlar. Ayrıntılı olarak, vasküler greft, üç boyutlu bir şablon olarak mekanik destek ve dahil hücre adezyon, göç, yayılması, damar dokusu ve etkisi hücresel fonksiyonları yeniden oluşturma sırasında yapısal rehberlik sağlayabilir ve hücre dışı matriks2salgılanmasını. Şu ana kadar çeşitli sentetik polimerler damar doku mühendisliği uygulamalarında değerlendirilir. Bu polimerler arasında poly(ε-caprolactone) (PCL) yoğun bir şekilde iyi hücre uyumluluk ve birkaç ay sonra iki yıl3' e kadar yavaş bozulması nedeniyle soruşturma. Mükemmel yapısal bütünlüğü ve açıklık ve neovaskülarizasyon içinde de kadar sürekli artan hücre işgal PCL vasküler greft electrospinning tarafından işlenen bir sıçan aort modeli4,5,6' da sergilenen greft duvar 6 aya kadar için. Ancak, olumsuz doku, hücre ve kılcal damarlar ve kalsifikasyon, regresyon dahil olmak üzere remodeling Ayrıca gözlendi daha uzun timepoints yukarı 18 ay.
Cellularization vasküler greft doku rejenerasyonu belirlenmesi ve7remolding önemli bir faktördür. Electrospinning, çok yönlü bir teknik olarak yaygın nano-lifli yapısı8ile vasküler greft hazırlanması için istihdam edilmiştir. Ne yazık ki, nispeten küçük gözenek yapısı genellikle yetersiz hücre infiltrasyonu sonraki doku rejenerasyonu sınırlar electrospun vasküler greft içinde yol açar. Bu sorunu gidermek için çeşitli teknikler gözenek boyutu ve9,10, toplayıcı cihazları, lazer radyasyon11 tarafından tedavi sonrası modifikasyonu leaching tuz/polimer dahil olmak üzere toplam gözeneklilik artırmak için kurulması denenen , vs. Aslında, electrospun Greftler (lif çapı, gözenek boyutu ve porozite dahil) yapısını işleme koşulları12,13ile yakından ilgilidir. Electrospinning sırasında lif çapı kolayca Polimer çözüm, akış hızı, voltaj, vbkonsantrasyonu gibi parametreleri değiştirerek kontrol edilebilir. 14 , 15, ve bu nedenle, gözenek boyutu ve porozite buna göre geliştirilmiştir.
Biz son zamanlarda değiştirilmiş bir PCL electrospun greft macroporous yapısı (5-7 µm çapını ve 30-40 µm gözeneklerin lifleri) ile bildirdi. Karın sıçan Aortası değiştirerek in vivo implantasyon 3 ay ameliyat sonrası16, açıklık, hem de iyi endothelialization ve düz kas yenilenme oranı yüksek gösterdi. Daha da önemlisi, hiçbir olumsuz doku kalsifikasyon ve hücre regresyon dahil olmak üzere remodeling implantasyon bile bir yıl sonra gözlenen.

<table>
	<tbody>
		<tr>
			<td>Poly(&#949;-caprolactone) (PCL) pellets (Mn=80,000)</td>
			<td>Sigma</td>
			<td>704067</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1060</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Tianjin Chemical Reagent Company</td>
			<td>A1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Tianjin Fengchuan Company</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Alfa Aesar</td>
			<td>A16046</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rats</td>
			<td>Laboratory Animal Center of the Academy of Military Medical Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Hebei Tiancheng Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloral hydrate</td>
			<td>Tianjin Ruijinte chemical company</td>
			<td>2223</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium Injection</td>
			<td>Tianjin Biochem Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin Sulfate Injection</td>
			<td>Jiangsu Lianshui Pharmaceutical company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-&#945;-SMA primary antibody</td>
			<td>Abcam</td>
			<td>ab7817</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-smooth MYH primary antibody</td>
			<td>Abcam</td>
			<td>ab683</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-rat elastin antibody</td>
			<td>Abcam</td>
			<td>ab23748</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-von Willebrand factor primary antibody</td>
			<td>Abcam</td>
			<td>ab6994</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150117</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (Alexa Fluor 488)</td>
			<td>Invitrogen</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>5% normal goat serum</td>
			<td>Zhongshan Golden bridge</td>
			<td>ZLI9022</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin and eosin (H&#38;E)</td>
			<td>Beijing leagene biotech</td>
			<td>DH0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Masson&#39;s trichrome</td>
			<td>Beijing leagene biotech</td>
			<td>DC0032</td>
			<td></td>
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		<tr>
			<td>Verhoeff-van Gieson (VVG)</td>
			<td>Beijing leagene biotech</td>
			<td>DC0059</td>
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		<tr>
			<td>Von Kossa</td>
			<td>Beijing leagene biotech</td>
			<td>DS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,3-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>G3002b</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical sutures needles with thread,9-0 silk</td>
			<td>Shanghai Jinhuan medical supplies company</td>
			<td>H901</td>
			<td></td>
		</tr>
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implantation of electrospun vascular grafts with optimized structure in a rat model

Burada, macroporous PCL vasküler greft imal ve abdominal aort değiştirme bir fare modeli kullanarak bir değerlendirme protokolü tanımlamak için bir iletişim kuralı mevcut. Electrospun vasküler Greftler kez hücre infiltrasyonu Greftler içine sınırlamak ve yenilenme ve neo-damarların remodeling engel nispeten küçük gözenekleri, sahip. Bu çalışmada, PCL vasküler Greftler kalın elyaf (5-6 µm) ve daha büyük gözenekler (~ 30 µm) ile modifiye işleme tekniği kullanarak fabrikasyon. Uzun vadeli performansı greft implantasyonu bir sıçan abdominal aort modeli tarafından incelendi. Ultrason analiz Greftler patent anevrizması veya stent implantasyonu bile 12 ay sonra meydana gelen olmadan kaldı gösterdi. Macroporous yapısı hücre ingrowth geliştirilmiş ve böylece 3 ayda yeniden doku terfi. Daha da önemlisi, olumsuz, 12 ay sonra greft duvar içinde kireçlenme gibi şeklinin değiştiğine dair işaret yok oldu. Bu nedenle, electrospun PCL vasküler Greftler değiştirilmiş macroporous işleme ile bir arter yerine uzun vadeli implantasyon için olmak potansiyel tutun.

PCL Greftler 3 ay ve 12 ay post-operatively explanted ve hematoksilen eozin (H & E) için standart histolojik teknikler Masson trichrome, Verhoeff-van Gieson (VVG), Von Kossa ve α-SMA için boyama ayirt analiz, MYH, vWF ve elastin. Histolojik görüntüleri dik bir mikroskop kullanarak alındı ve ﬂuorescence mikroskop kullanarak ayirt görüntüleri alındı.
Tüm verileri ± SD demek gibi ifade edildi Eşleştirilmiş iki k...

Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Bir sıçan modelinde en iyi duruma getirilmiş yapısı ile Electrospun vasküler greft implantasyonu

Burada, PCL vasküler Greftler kalın elyaf ve iri gözenekli ile imal ve abdominal aort değiştirme bir sıçan modelinde vivo içinde performansını değerlendirmek için bir iletişim kuralı tanımlamak için modifiye electrospinning yöntemi mevcut.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

implantation-electrospun-vascular-grafts-with-optimized-structure-rat

Research

JoVE Journal

Biology

7.9K Görüntüleme.  Nankai University. Burada, PCL vasküler Greftler kalın elyaf ve iri gözenekli ile imal ve abdominal aort değiştirme bir sıçan modelinde vivo içinde performansını değerlendirmek için bir iletişim kuralı tanımlamak için modifiye electrospinning yöntemi mevcut.

Video: Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

The Structure of Skilled Forelimb Reaching in the Rat: A Movement Rating Scale

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and primates. The video describes a test situation that allows filming of repeated acts of reaching for food by the rat that has been mildly food deprived. A rat is trained to reach through a slot in a holding box for food pellet that it grasps and then places in its mouth for eating. Reaching is accomplished in the main by proximally driven movements of the limb but distal limb movements are used for pronating the paw, grasping the food, and releasing the food into the mouth. Each reach is divided into at least 10 movements of the forelimb and the reaching act is facilitated by postural adjustments. Each of the movements is described and examples of the movements are given from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation. Because the reaching act for the rat is very similar to that displayed by humans and nonhuman primates, the scale can be used for comparative purposes. from a number of viewing perspectives. By rating each movement element on a 3-point scale, the reach can be quantified. A number of studies have demonstrated that the movement elements are altered by motor system damage, including damage to the motor cortex, basal ganglia, brainstem, and spinal cord. The movements are also altered in neurological conditions that can be modeled in the rat, including Parkinson's disease and Huntington's disease. Thus, the rating scale is useful for quantifying motor impairments and the effectiveness of neural restoration and rehabilitation.

Experiments on animals were performed in accordance with the guidelines and regulations set forth by the University of Lethbridge Animal Care Committee in accordance with the regulations of the Canadian Council on Animal Care.

The skilled reaching scale divides the movement by a forelimb in a reach for food act into composite elements each of which are evaluated with a three-point scale. The rating scale is described for a normal rat and can be applied toward evaluating neurological motor disorders.

Sıçan ulaşmak Nitelikli ön ayakları Yapısı: Hareket Ölçeği

Skilled reaching for food is an evolutionary ancient act and is displayed by many animal species, including those in the sister clades of rodents and ...

Biyoloji

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Sıçan Kalp Engineered Doku implantasyonu

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Embriyonik Ekstremite Cilt damarları Tüm montaj İmmünohistokimyasal Analizi: Embriyo vasküler dallanma Morphogenesis Eğitim Model Sistemi

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Mycoplasma optimize edilmiş PCR tabanlı Algılama

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

Vasküler Hyperpermeability indükleyici ajanlar cilt mil tahlil ile değerlendirilmesi

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Kazı-kazan tahlil Vitro hücre göç bir otomatik optik kamera ile test etmek için en iyi duruma getirilmiş

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Peri-implantasyon Aşamasında Trophoblast Hücrelerinin Tek Hücre Li Hücre Koleksiyonu İnsan Embriyoları

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Primer Sıçan Hepatositlerinin Çok Parametreli Perfüzyon Kontrolü ile İzolasyonu

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...