이 작품은 NSFC 프로젝트 (81522023, 81530059, 91639113, 81772000, 81371699, 및 81401534)에 의해 재정적으로 지원 되었다.

실험 동물의 사용은 동물 실험 윤리 위원회의 난카이 대학에 의해 승인 하 고 관리 및 실험 동물의 사용에 대 한 가이드에 따라 실시.
1입니다. Electrospun PCL 이식의 제조
참고: 여기, 전기 기술 혈관 이식 조작에 이용 되었다.
<ol><li>각각, 메탄올과 클로 프롬의 혼합물에서 PCL을 용 해 하 여 25 wt %와 10 wt %의 PCL 솔루션을 준비 (1:5 볼륨 비율), 실 온 (RT) 12 시간에서.</li>
	<li>10 mL 유리 주사기를 PCL 솔루션을 로드 합니다.</li>
	<li>21-G 바늘 주사기를 놓습니다.</li>
	<li>컬렉션 악기에 스테인리스 스틸 맨 드릴 (2 mm 직경, 길이 25 c m)를 놓습니다.</li>
	<li>두꺼운 섬유 이식에 대 한 사용 하 여 25 wt %의 PCL 솔루션, 수집기 바늘 끝에서 17 cm의 거리, 8 mL/h의 유량 및 11의 전압 전기 매개 변수로 kV. 얇은 섬유 이식 10 wt %, 수집기에 바늘 끝에서 20 cm의 거리, 2 mL/h의 유량 및 18의 전압의 PCL 솔루션 사용 전기 매개 변수로 kV.</li>
	<li>얻은 이식 하룻밤 잔여 용 매를 제거 하는 진공 상태에서 배치 됩니다 확인 하십시오. 소독 절차 전에 모든 악기 고 무 균 기술을 통해 유지 합니다.</li>
	<li>주입 전에 이식 30 분 75% 에탄올 10 ml에서 그들을 immersing 및 다음 하룻밤 UV 빛에 노출에 의해 소독.</li>
	<li>섬유 및 기 공 크기 측정: 스캐닝 전자 현미경 (SEM) 이미지에 따라 ImageJ 소프트웨어를 사용 하 여 평균 섬유 직경을 계산.</li>
	<li>기계 장비의 테스트:<ol>
<li>면도날을 사용 하 여 길이 3 m m 섹션으로 관 건설 기계를 절단. 건설 기계는 마이크로미터를 사용 하 여 두께 측정 합니다.</li>
		<li>100 북 아의 부하 용량 인장 테스트 기계에 관 건설 기계를 배치</li>
		<li>파열까지 1mm 간 클램프 거리와 풀 10 m m/min의 속도로 경도 건설 기계 클램프. 인장 강도 및 파단 궁극적인 측정 합니다. 초기 선형 영역에서 탄성 계수를 계산 (최대 5% 변형) 응력-변형 곡선의.</li>
	</ol>
</li>
</ol>2. 쥐 복 부 대동맥 이식 모델
참고: 모든 재료와 수술에 사용 되는 계기는 살 균. 수술 동안 거 즈 마스크와 감염을 피하기 위해 멸 균 장갑을 착용 하는 연산자 확인 하십시오. 실내 온도 동물 체온을 유지 하기 위해 27-30 ° C에서 유지 됩니다 확인 하십시오. 진통에 관한 현지 IACUC 지침을 따르십시오.
<ol><li>남성 Sprague Dawley 쥐 혈관 이식의 수신자로 240-270 g의 무게를 사용 합니다. 쥐 수술 하기 전에 24 시간 금식은 확인 합니다. 24 시간 단식 쥐의 목표는 빈 창 자에서 대변 충분히, 그로 인하여 확장 연산자의 수평선.</li>
	<li>쥐의 백 목 잡고 아래쪽으로 머리를 유지, 낮은 복 부의 복 부 구멍으로 springe 바늘을 삽입. 복 주사에 의해 chloral 하이드 레이트 (330 mg/kg)과 마 취에 대 한 쥐를 유도.</li>
	<li>쥐는 근육과 꾸준히 호흡을 완화 했다 함으로써 적절 한 anesthetization를 확인 합니다. 운영 현미경 쥐 부정사 위치에 놓습니다.</li>
	<li>바 셀 린 안과 수 의사 연 고 마 취에서 건조를 방지 하기 위해 눈에 적용 됩니다. 수술 전에 꼬리 정 맥 주입에 의해 heparinized 생리 염 분 해결책 (50 UI/mL)와 anticoagulation (UI/100kg)을 관리 합니다.</li>
	<li>면도칼 블레이드를 사용 하 여 전방 복 벽에 털을 면도 하십시오 그리고 요오드 솔루션 및 의료 알코올 솔루션을 사용 하 여 피부를 청소.</li>
	<li>수술가 위로 중간 개복 술 절 개를 수행 하 고 절 개 길이, 약 4-5 m 인지 확인 다음 복 부 구멍을 노출.</li>
	<li>철회 하 고 우선적으로 생리 식 염 수를 적신 거 즈로 내장을 포장.</li>
	<li>신중 하 게 복 부 대동맥 해 부.</li>
	<li>식별 하 고 9-0 monofilament 나일론 봉합을 사용 하 여 모든 작은 가지를 선.</li>
	<li>격리 된 섹션을 클램프 (길이 최대 1 c m) 2 개의 혈관 클램프를 사용 하 여 대동맥의. 대동맥 20-30 분 채워 남아 있을 수 있습니다.</li>
	<li>Anastomotic 사이트를 만들 마이크로-가 위를 사용 하 여 두 개의 클램프 사이의 복 부 대동맥 transect</li>
	<li>잔여 혈액을 제거 하 heparinized 염 (UI/50ml) 솔루션을 사용 하 여 대동맥의 두 끝을 플러시.</li>
	<li>마이크로-가 위를 사용 하 여 adventitia 벗기다.</li>
	<li>9-0 monofilament 나일론 봉합을 사용 하 여 숫자의 8 봉합 패턴 2 m m 내경 및 쥐의 복 부 대동맥에 길이 1 cm 이식 anastomose.</li>
	<li>첫째, 9, 3, 12, 및 근 위 측에 6 시 위치 순서 대로 4 개의 anastomoses을 구성 다음 두 봉합 사이 4 바늘 컷된 가장자리 anastomose. 근 위 봉합을 마치고 같은 방법으로 원심 측을 봉합. 참고: 모든 스티치는 네이티브 쪽은 약간은 이식에 포함 된 보장 합니다.</li>
	<li>이식, 흘러 혈액을 있도록 원심 클램프 다음 인접 클램프를 제거.</li>
	<li>멸 균 면봉 또는 스폰지 작은 거 즈를 사용 하 여 출혈을 막으려고 봉합 끝을 누릅니다. 약 3 분, hemostasis 때까지 누릅니다.</li>
	<li>복 부 구멍으로 내장을 반환 합니다.</li>
	<li>플러시 gentamicin (320 U/mL)와 따뜻한 생리 식 염 수 솔루션을 사용 하 여 복 부 구멍.</li>
	<li>각각 근육과 피부 층에 3-0 나일론 봉합 사를 사용 하 여 복 벽을 바느질.</li>
	<li>깨끗 하 고 건조 감 금 소에 쥐를 놓고 동물 체온; 유지 케이지 난방 패드를 넣어 마 취에서 회복 하기 위해 쥐를 기다립니다. Sternal recumbency를 유지 하기 위해 충분 한 의식 회복 될 때까지 동물에 참석.</li>
	<li>후 의식 회복, 음식과 물을 함께 단일 케이지에 쥐를 넣어. 수술 후 감염을 방지 하기 위해 상처에 요오드를 적용 합니다. 그것은 완벽 하 게 복구 될 때까지 다른 동물의 회사에는 쥐를 반환 합니다.</li>
	<li>미리 정해진된 시간 지점에서 기관 지침에 따라 쥐 안락사</li>
</ol>

<ol>
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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>

저자 아무 충돌 금융 관심 있다.

셀 침투는 중생에 대 한 중요 하 고16 vivo에서혈관 이식의 개장 이다. 제한 된 셀 침투는 종종 이식 벽으로 세포의 이동 방해는 이식의 상대적으로 작은 구멍 관련이 있습니다. 이 문제를 해결 하려면 우리는 큰 기 공 구조 electrospun PCL 혈관 이식 준비 하 수정된 방법을 개발. 세부 사항에, 기 공 크기는 쉽게 처리 매개 변수에 의해 제어 될 수 있는 섬유 두께의 증가 함께 증가...

합성 중합체에서 만든 혈관 이식 심장 혈관 질병 (CVDs)의 치료를 위한 병원에 널리 활용 됩니다. 불행히도, 작은 직경의 혈관 이식의 경우 (D < 6 m m) 사용할 수 없습니다 종종 이르게 혈전 증, 내 증식, 그리고 다른 감소 된 혈액 흐름 속도 의해 발생 하는 낮은 patency 성공적인 제품 있다 합병증1.
조직 공학 장기 patency 및 항상성 비 계 기반 혈관 재생 및 재건에 따라 실현 하는 대체 전략을 제공 합니다. 자세히, 혈관 이식, 3 차원 서식 파일로 제공할 수 있는 기계적인 지원 및 혈관 조직 및 기능의 영향 세포, 세포 접착, 이동, 확산를 포함 하 여 재생 하는 동안 구조 지도 기질2분 지금까지, 다양 한 합성 폴리머 혈관 조직 공학에서 응용 프로그램에 대 한 평가. 이러한 고분자 중 poly(ε-caprolactone) (PCL)가 집중적으로 조사 좋은 셀 호환성 및 느린 저하 몇 개월에서 2 년3에 이르기까지. 쥐 대동맥 모델4,,56, PCL 혈관 이식 전기 처리 우수한 구조적 무결성 및 patency, 뿐만 아니라 지속적으로 증가 세포 침입과에서 neovascularization 전시는 최대 6 개월까지 이식 벽입니다. 그러나, 불리 한 조직 세포와 모세 혈관의 석 회화, 회귀를 포함 하 여 리 모델링 또한 관찰 되었다 더 긴 timepoints에서 최대 18 개월.
Cellularization 혈관 이식의 조직 재생을 결정 하 고7을 remolding 핵심 요소입니다. 전기, 다양 한 기법으로는 널리 고용 나노 섬유 구조8혈관 이식의 준비에 대 한. 불행히도, 상대적으로 작은 기 공 구조는 종종 후속 조직 재생을 제한 하는 electrospun 혈관 이식에 부족 한 세포 침투를 끈다. 이 문제를 해결 하려면 다양 한 기술은 시도 기 공 크기와 전반적인 다공성,9,10, 수집 장치, 레이저 방사선11에 의해 치료 후의 수정 leaching 소금/폴리머를 포함 하 여 증가 , 등등. 사실, electrospun 이식 (를 포함 하 여 섬유 직경, 기 공 크기 및 다공성)의 구조는 밀접 하 게 관련이 처리 조건12,13. 중 전기, 섬유 직경 폴리머 솔루션, 흐름 속도, 전압 등의 농도 같은 매개 변수를 변경 하 여 쉽게 제어할 수 있습니다. 14 , 15, 그러므로, 숨 구멍 크기와 다공성 향상 되었습니다 따라와.
우리는 최근 macroporous 구조 (5-7 µ m의 직경 및 30-40 µ m의 모 섬유) 수정된 PCL electrospun 이식을 했다. Vivo에서 쥐 복 부 대동맥을 대체 하 여 이식 3 개월 후 수술16patency, 좋은 endothelialization, 부드러운 근육 중생의 높은 비율을 보였다. 더 중요 한 것은, 아니 불리 한 조직의 석 회화 및 셀 회귀를 포함 하 여 개장 이식의 1 년 후에 관찰 될 수 있었다.

<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

여기, 우리는 macroporous PCL 혈관 이식 조작 및 복 부 대동맥 교체의 쥐 모델을 사용 하 여 평가 프로토콜을 설명 하는 프로토콜을 제시. Electrospun 혈관 이식 자주는 이식으로 세포 침투를 제한 하 고 재생성 하 고 개장 하는 신 동맥의 방해는 상대적으로 작은 구멍을가지고 있다. 이 연구에서는 두꺼운 섬유 (5-6 µ m)와 더 큰 모 (~ 30 µ m) PCL 혈관 이식 수정된 처리 기술을 사용 하 여 조작 했다. 이식의 장기 성능 쥐 복 부 대동맥 모델에 주입에 의해 평가 되었다. 초음파 분석은 이식 남아 동맥 또는 이식의 12 개월 후에 발생 하는 협 착 증 없이 특허를 보여주었다. Macroporous 구조는 셀 ingrowth를 개선 하 고 따라서 3 개월에서 재생성 조직 승진. 더 중요 한 것은, 12 개월 후 이식 벽 내 석 회화 등 불리 한 개장의 아무 표시도 없었다. 따라서, 처리 하는 수정 된 macroporous와 electrospun PCL 혈관 이식 장기 이식에 대 한 동맥 대체 될 잠재적인 개최.

PCL 이식 했다 explanted 3 개월 및 12 개월에서 post-operatively 및 hematoxylin에 오신 (H & E), 표준 조직학 기술에 의해 trichrome Masson, 분석 Verhoeff 반 Gieson (VVG), 폰 Kossa, 면역 형광 α-SMA를 위한 얼룩 MYH, vWF, 그리고 엘라 스 틴입니다. 조직학 이미지 똑바로 현미경을 사용 하 여 찍은 그리고 면역 형광 이미지 ﬂuorescence 현미경을 사용 하 여 찍은.
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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

쥐 모델에 최적화 된 구조를 가진 Electrospun 혈관 이식 술의 이식

여기, 우리는 PCL 두꺼운 섬유와 큰 모 공, 혈관 이식 조작 및 복 부 대동맥 교체의 쥐 모델에서 vivo에서 성능을 평가 하기 위해 프로토콜을 설명 하는 수정 된 전기 방법을 제시.

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 조회수.  Nankai University. 여기, 우리는 PCL 두꺼운 섬유와 큰 모 공, 혈관 이식 조작 및 복 부 대동맥 교체의 쥐 모델에서 vivo에서 성능을 평가 하기 위해 프로토콜을 설명 하는 수정 된 전기 방법을 제시.

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

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Polycaprolactone, 엘라스틴, 콜라겐을 사용하여 네이티브 아티 건축을 모방하기 위해 트라이 - 계층 Electrospinning : 예비 연구

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

연구

생체공학

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.

랫 중심에 엔지니어링된 조직의 주입

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

생물학

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.

티슈 엔지니어링 혈관 이식 및 이후의 이식을위한 수술 기법<em&gt; 생체</em&gt; 모니터링

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 ...

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.

마우스 모델에서 조직 공학 혈관의 생성 및 손톱 확장

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.

엔지니어링 전방 십자 인대 이식의 평가 흉선 쥐 모델

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.

마우스 모델에서 하대 정맥 끼어 이식의 주입

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

의학

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.

소형 동물 모델에서의 동정맥 (AV) 루프는 혈관 신생과 혈관을 조성한 조직 공학을 공부하기

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.

쥐의 원주 식도 재건을 위한 조직 공학 이식편

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.

정맥 삽입 위치 모델 : 바이 패스 이식 개통을 연구하기위한 적절한 모델

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

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

정맥 이식편을 사용하여 동맥 혈압하에서 인티맥 증식을 평가하는 재혈관 외과를 모방한 토끼 정맥 중개 모델

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...