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>

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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(&amp;epsilon;-caprolactone) (PCL) pellets (M&lt;sub&gt;n&lt;/sub&gt;=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-&amp;alpha;-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&amp;amp;E)</td><td>Beijing leagene biotech</td><td>DH0006</td><td></td></tr><tr><td>Masson&#039;s trichrome</td><td>Beijing leagene biotech</td><td>DC0032</td><td></td></tr><tr><td>Verhoeff-van Gieson (VVG)</td><td>Beijing leagene biotech</td><td>DC0059</td><td></td></tr><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></tbody></table>

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

Universidad de Cartagena UDEC

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

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

Sıçanlarda Common Karotid Arter interpozisyon Harici Juguler Ven Greft: Gelişmiş Mikrocerrahi Tekniği Eğitimi

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Tıp

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 ...

Biyoloji

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Doku Engineered Vasküler Greftlerinin ve sonraki İmplantasyonunda Cerrahi Tekniği<em&gt; İn Vivo</em&gt; İzleme

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Biyomühendislik

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Bir Fare Modeli Doku mühendisliği Kapların Üretimi ve Tırnak

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Engineered Ön Çapraz Bağ Greftlerinin Değerlendirme Atimik Rat Modeli

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

Fare Modeli Inferior Vena Cava İnterpozisyon Greft İmplantasyonu

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

Bir Küçük Hayvan Modelinde Arteriovenöz (AV) Döngü Anjiogenez ve Vaskülarize Doku Mühendisliği Eğitim için

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Sıçanlarda Çevresel Özofagus Rekonstrüksiyonu için Doku-Mühendislik Greft

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Ven interpozisyonu Model: baypas açıklığının Eğitim için uygun bir model

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

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

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