이 작품은 SNF [31003A-138528], Octav 및 Marcella의 Botnar 재단, 노바티스 재단과 엠마 Muschamp 재단의 보조금에 의해 지원되었다. 우리는 우수한 기술 지원을 마틴 Lambelet하고, 장 - 크리스토프 Stehle 감사합니다.

로잔 대학의 윤리위원회는 인체 조직의 사용을 1983 년 개정, 1975 년 헬싱키 선언에 명시된 원칙에 따라 아르 실험을 승인했다. 1 인간 대 복재 정맥 수확 <ol><li> 허혈 하체 바이 패스 수술을받은 환자에서 비 정맥류 인간 복재 정맥의 잉여 세그먼트를 얻습니다. 수술실에서, 요오드 용액과 다리 전체를 소독하고 발에 사타구니에서 다리를 노출하는 환자를 내리면. </li><li> 무릎에 사타구니에서 중간 절개를 (중단 피부 부분을 떠나). </li><li> 조직 (노 터치 기술을) 주변의 척추 경에 큰 복재 정맥을 수확. 4-0 실크 넥타이와 혈관의 보안 측면 분기합니다. 즉시 RPMI-1640 G에서 4 ° C에서 9cm 2.5-4 mm의 외부 직경이 큰 복재 정맥의 긴 잉여 세그먼트의 최소 저장lutamax 매체는, 12.5 % 태아 혈청으로 보충하고 실험실로 가져옵니다. </li></ol> 2 EVPS 디자인 <ol><li> . 그림 1에 나와있는 일반적인 장비를 조립 모든 장비를 오토 클레이브 멸균 조건에서 모든 구성 요소를 유지합니다. 또한, 시스템은 방수 및 배지에 화학 물질이 누출되지 않도록. 표지 폴리 메타 크릴 메틸 (PMMA-GS)를 사용합니다. 스틸 (X5 크롬 니켈 18 10) 및 정맥 지지체로서 폴리 옥시 메틸렌 수지 (POM). </li><li> 정맥과 연결의 배치를 허용하도록 원하는 형상으로 관류 챔버를 설계한다. 이 배양 배지 (그림 1)에 의해 일정한 범위와 함께 선박의 최소 굴곡과 팽창을 할 수 있도록 (원통형 구조를 사용하는 경우 또는 반경) 최소 2.5 cm 있는지 깊이를 확인합니다. 밀봉은 중요한 문제이며, 직사각형 PMMA-GS 건설이 사용되는 이유입니다. </li><li> 원하는 geometr에 정맥 지원 디자인y를 입력합니다. 정맥 또는 꼬임 위에 팽만을 피하기 위해, (정맥이 나사와 함께 꼬아 것처럼 스크류, 그 목적으로 사용될 수 없다) 밀거나 당기는 길이 조정을 허용한다. 참고 : L 모양 (선박에 맞게 5mm 직경)이 정맥 실린더를 지원하는 조각 및 정맥 (그림 1B와 그림 2) 슬라이드 2에 연결하는 강재의로드 여기에 사용됩니다. </li><li> P = 0-10 = HX ρ XG, 여기서 P = 압력 (N / m 2, PA) 유체 열 (m)의 H = 높이 ρ : 압력 열, 같은 시스템에 적용 &quot;휴식 압력&quot;이라고 디자인 = 액체 (kg / m 3), g = 중력 상수 (9.81 m / 초 2)의 밀도. 위에서 아래로 디자인 넷 연결 덕트 : (정맥) 유출에 대한 (정맥) 유입 압력을 적용하고 매체 변경을 허용 할 수 있습니다. </li><li> 매체를 준비합니다. 이전의 연구 5,11-14을 바탕으로, 12.5 Glutamax 보충, RPMI-1640을 선택% 소 태아 혈청, 1 %의 항생제 항진균제 용액 (10,000 U / ㎖ 페니실린 G, 플러스 10 ㎎ / ㎖ 스트렙토 마이신 황산염, 플러스 25 ㎎ / ㎖ 암포 테리 신 B, 플러스 0.5 ㎍ / ㎖의 : 겐타 마이신). 전단 응력 (SS)가 SS = 4 μQ / π의 * r을 3 Q 주어진다하면 유속 (㎖ / 초), 정맥 세그먼트의 반경 (cm)을 r에, 그리고 μ는 관류 매질의 점도이다. <ol><li> 70 kDa의 덱스 트란을 첨가하여 점도를 조정 SS를 변조. 점도계와 점도를 측정한다. 여기에, DYN / cm 2 9-15에 SS를 설정 8퍼센트 70 kDa의 덱스 트란을 추가합니다. </li></ol></li><li> 60 펄스 / 분 정 진폭이 컴퓨터 시스템에인가되고 제어 압력 독립적 150 ± 15 ㎖ / 분의 단방향 흐름을 생성 박동성 카디오이드 신호를 유도하는 기어 펌프를 설정한다. 구동 소프트웨어가 지속적인 수집 및 압력 모니터링, 유속, 펄스 속도, 신호를 통합 있는지 확인합니다. 원하는 경우, 제 2 펌프를 사용하여 (비 동기화hronized) 비 층류, 난류를 생성한다. </li></ol> 3 EVPS 조립 (그림 1) <ol><li> 시작하기 전에 모든 장비는 멸균 있는지 확인합니다. 층류 후드에서 무균 아래의 모든 다음 단계를 수행합니다. </li><li> 배지로 가득 배양 접시에있는 정맥을 놓습니다. 수술 블레이드를 사용하여 3 동일한 세그먼트로 정맥을 나눕니다. </li><li> 즉시 PBS에서 하나의 세그먼트를 씻어. , 세 부분으로 세그먼트를 분할 형태 계측을 위해 포르말린을 수정합니다. 양적 성적 증명서 (RT-PCR)와 단백질 (서양 얼룩) 분석을위한 다른 두 동결. 제어되지 않은 혈관 관류 이들 세그먼트를 고려한다. </li><li> 관류이 나머지 부분을 사용합니다. <ol><li> 아주 부드럽게 정맥 ​​매체를 주입하고 정상 유동 방향을 결정하고; 밸브의 존재 정맥이 반전됩니다. </li><li> 혈관 씰링 실험 성공에 가장 중요합니다. 담보를 통해 누출을 점검한다. 6-0 실크 봉합과 누출을 고정합니다. </li> &lt;/ OL&gt; </li><li> 시간 (2.3, 그림 1)에서 정맥 두 개의 금속 실린더 사이의 세그먼트, 한 끝을 연결합니다. 들여 쓰기 (그림 1A 및 B)의 주위에 Ethibon 3-0로 실린더를 고정합니다. <ol><li> 이전에 매체 가득 관류 챔버로 전체 정맥 세그먼트를 배치합니다. 제 2 세그먼트에 대한 동일한 절차를 반복한다. 제대로 실린더에 정맥을 밀봉하지 않으면이 누수의 원인이 될 것입니다 재 시술이 필요하며, 크게 감염과 실험 실패의 위험을 증가 : 주. </li></ol></li><li> 두 번째 세그먼트를 강화 (메쉬)하기 위해, L 모양의 조각 (2.3 및 그림 1)에서 (첨부 된 정맥 포함)이 실린더를 놓습니다. <ol><li> 온화하고 악기 정맥을 만지지 마십시오. 정맥에 다음 실린더에 먼저 메쉬를 밀어 넣습니다. 푸시 / 풀 흔들림과는 정맥에 메쉬를 얻을 것이다. </li><li> 메쉬 커버 일단 정맥의 전체 표면은 J 확보Ethibon 3-0으로 실린더에 acketed 정맥. </li><li> L 모양의 지원에 정맥 / 실린더 화합물을 재 조립 이전에 매체 가득 관류 챔버로 전송합니다. </li></ol></li><li> 3.2 mm의 내부 직경을 가진 과산화물 처리 된 실리콘 튜브를 이용하여 Y-스플리터 각 금속 실린더 (인 및 유출)을 연결한다. </li><li> 튜브의 동일한 유형을 사용하여 초 Y-스플리터 유출 분배기를 연결한다. 이 Y-스플리터에서 모두 혈관을 통해 관류 압력을 측정하는 하나의 튜브를 사용한다. 폐쇄 루프 시스템 (그림 2)을 형성하기 위해 다시 열로 다른 하나를 연결합니다. </li><li> 인큐베이터 내부 펌프 헤드에 압력 열을 연결하는 긴 (한-m 길이) 튜브를 사용합니다. </li><li> 긴 다른 길이의 튜브 유입 Y-스플리터 펌프 헤드를 연결하여 설정 (도 1)를 완료한다. </li></ol> (4) 혈관 관류 <ol><li> EVPS 조립체 공동 후에mpleted이 매체와 열을 채우기 (재충전 할 수 있도록 정맥 유출 덕트 미만을 유지). 시스템이 가득 찰 때까지 열에 더 많은 매체를 추가합니다. pH가 37 ± 0.1 ° C로 유지 인큐베이터에 모든 시스템을 이동은 (헨더슨 - Hasselbach 방정식을 기반으로 CO 2 / 산도 알고리즘을 사용하여) 7.40 ± 0.01로 일정하게 유지. </li><li> 인큐베이터 외부 기어 펌프 헤드를 가져오고 기어 펌프 드라이브에 연결합니다. 어셈블리를 고정하는 봉 나사. </li><li> 펌프 전원 스위치,이 구동 소프트웨어에서 활성화되어 있는지 확인하고, 매체가 동등하게 모든 구획에 분산 될 때까지 5 분을 할 수 있습니다. </li><li> 압력을 모니터하기 위해 동맥 라인 모니터링을 사용합니다. 컴퓨터에 연결된 압력 변환기에 EVPS 압 출력 (이 동맥 카테터에 대응)에 연결. <ol><li> 튜브가 완전히 매체로 가득하고 거품이 포함되어 있는지 확인합니다. &quot;동맥 리를 통해 드 거품 배양 시스템을네브라스카 &quot;튜브 (그림 2). 디스플레이에 주목하고 60 펄스 / 일정한 진폭의 분의 박동 지향성 신호를 찾습니다. 이 시점에서, 평균 압력은 0~10mm 수은 사이이다. 압력이 &lt;0이고, 열이 점진적으로 (정맥 튜브 사이의 정맥 담보하거나 불충분 한 인) 누수를 찾아 비워합니다. </li></ol></li><li> 정맥 테스트 또는 동맥 테스트를위한 90mm 수은에 6mm 수은에 최소한의 압력을 설정합니다. 이러한 조건에서, 공기 분사기는 열 및 시스템에 필요한 압력을 적용한다. </li><li> 압력 컬럼에 연결된 튜브를 사용하여 모든 이일 매체를 변경합니다. , 압력 변화의 손상을 방지 첫번째 열 플러그를 엽니 다. </li></ol> 관류의 5 완료 <ol><li> 관류의 3 ~ 7 일 후 : 인큐베이터 밖으로 EVPS을 가지고 정맥을 분리. 5mm의 근위부를 버리고 말초 정맥은 장비에 부착 종료합니다. 중앙, 5mm 두께 린을 잘라나머지 부문에서 GS와 포르말린 (형태 학적)에 고정한다. 나머지 조각을 동결하고 더 분자 분석을위한 분말로 줄일 수 있습니다. </li></ol>

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D., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+vein+graft+adaptation+to+the+arterial+circulation:+insights+into+the+neointimal+algorithm+and+management+strategies.">Mechanisms of vein graft adaptation to the arterial circulation: insights into the neointimal algorithm and management strategies.</a> Circulation Journal : Official Journal of the Japanese Circulation Society. 74, 1501-1512 (2010).</li><li>Tao, 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=A+simplified+murine+intimal+hyperplasia+model+founded+on+a+focal+carotid+stenosis.">A simplified murine intimal hyperplasia model founded on a focal carotid stenosis.</a> The American Journal of Pathology. 182, 277-287 (2013).</li><li>Berard, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salvage+treatment+for+venous+aneurysm+complicating+vascular+access+arteriovenous+fistula:+use+of+an+exoprosthesis+to+reinforce+the+vein+after+aneurysmorrhaphy.">Salvage treatment for venous aneurysm complicating vascular access arteriovenous fistula: use of an exoprosthesis to reinforce the vein after aneurysmorrhaphy.</a> European Journal of Vascular and Endovascular Surgery : the Official Journal of the European Society for Vascular Surgery. 40, 100-106 (2010).</li></ol>

저자가 공개하는 게 없다.

이 연구는 인간의 혈관에 광범위한 혈역학 적 연구를 수행 할 수있는 생체 정맥 관류 시스템 (EVPS)을 알게되어. 이 시스템은 생체 내에서 세포를 순환시켜 출시 악화 염증과 성장 요인의 부재에서 정의 혈역학에서 복재 정맥 관류 할 수 있습니다. 따라서, 인간의 혈관 이식 5,11,12,15 IH에서의 제어에 관여하는 기본 경로의 더 나은 이해를 제공한다. 재현성?...

심장 혈관 질환은 서구 국가 하나의 이환율과 사망률의 주요 원인입니다. 혈관 내 치료에 만든 발전에도 불구하고, 바이 패스 수술을 따라서 만 절반 이상 정맥 이식은 미국에서 매년 수행, 현대 치​​료의 의지가 남아있다. 그러나 수십 년간의 연구에도 불구하고, 하체의 정맥 이식의 30~60% 인해 내막 증식증 (IH) 2에 첫번째 년 안에 실패한다. 기계의 힘, 특히 낮은 전단 응력 (SS) 및 높은 벽 장력이 증식 응답 3,4의 시작과 발전에 중추적입니다. 이 문제를 해결하기 위해, 생체 혈관의 관류 시스템 (EVPS)을 엄격하게 통제 혈역학 적 상태 (압력, 전단 응력), 인간 복재 정맥의 동작에 따라, 공부 생성되었습니다. 이 연구에서, 동맥 순환 형으로 삽입 다음, 고압 prolif을 자극하기에 충분 하였다 (= 100 mmHg로 의미)eration과 내막 층 (IH) (5) 내로 평활근 세포의 이주. 포유 동물의 연구는 &quot;동맥 화 정맥&quot;지원 급성 혈역학이 한번 정맥 동맥에 주입을 향하는 적 환경 변화에 대처하는 효율적인 방법으로서 외부 보강의 사용을 제안했다. 메시는, 과잉 팽창이 방지 증가 전단 응력 및 벽 장력 결과적 IH 6-10 줄였다. 그러나 기본 메커니즘과 바이 패스 개통을 개선 인간의 혈관에 그것의 적용이 완전히 특징되지 않았다. 우리 EVPS는 정맥 번 동맥 섭생에 삽입 대향 변화 (고 전단 응력 및 압력), 외부 거대 다공성 폴리 관형 메쉬의 부재 및 존재하에 인간 복재 정맥의 행동을 모방하는 조건에서 비교하기 위해 사용되었다. 병적 리모델링 및 IH 방지함으로써, 메시는 잠재적 임상 효율 (11)의 증거를 제공 </sup&gt;. 본 연구 1)는 외부 매크로 다공성 폴리 에스테르 메쉬 IH을 줄이고 잠재적 인 임상 응용 프로그램에 대한 중요한 정보를 제공한다는 것을 보여줍니다 제어 압력과 전단 응력 2)에서 생체 인간 복재 정맥 혈류의 모델을 소개합니다.

<table><tbody><tr><td>RPMI 1640 - Glutamax</td><td>Life Technologies</td><td>61870-010</td><td></td></tr><tr><td>Penicilline/Streptomycine/Fungizone</td><td>Bioconcept</td><td>4-02F00-H</td><td></td></tr><tr><td>Dextran from Leuconostoc spp. 500 g</td><td>Sigma-Aldrich</td><td>31390</td><td></td></tr><tr><td>Tampon PBS CHUV pH 7.1-7.3 1 L</td><td>Laboratorium und Grosse Apotheke Dr. G. Bichsel AG</td><td>100 0 324 00</td><td></td></tr><tr><td>Cryosectionning embedding medium - Tissue-Tek OCT Compound</td><td>Fisher Scientific</td><td>14-373-65</td><td></td></tr><tr><td>Silicon Tubing (Peroxide) L/S 16 (96400-16 ) - 7.5 m</td><td>Idex Health &amp;amp; Science GMBH</td><td>MF0037ST</td><td></td></tr><tr><td>Y-splitter&amp;nbsp;</td><td>Idex Health &amp;amp; Science GMBH</td><td>Y-connector</td><td></td></tr><tr><td>35 mm Culture dish</td><td>Sigma-Aldrich</td><td>CLS430165-100EA</td><td></td></tr><tr><td>15 ml Falcon tube</td><td>BD Bioscence</td><td>352096</td><td></td></tr><tr><td>50 ml Falcon tube</td><td>BD Bioscence</td><td>352098</td><td></td></tr><tr><td>Gearing pump - Reglo-Z</td><td>Idex Health &amp;amp; Science GMBH</td><td>SM 895&amp;nbsp;</td><td>&amp;nbsp;App-Nr 03736-00194</td></tr><tr><td>Pump Head</td><td>Idex Health &amp;amp; Science GMBH</td><td>MI0008&amp;nbsp;</td><td></td></tr><tr><td>Monitoring Kit TRANSPAC IV</td><td>icumedical</td><td>011-0J736-01</td><td></td></tr><tr><td>20 ml Syringes</td><td>B. Braun Medical SA</td><td>4612041-02</td><td></td></tr><tr><td>Etibon 3-0 FS-2</td><td>Ethicon- Johnson&amp;amp;Johnson</td><td>EH7346H</td><td></td></tr><tr><td>Mesh ProVena 6-8mm</td><td>B. Braun Medical SA</td><td>1105012-14</td><td></td></tr><tr><td>NaCl: Sodium chloride solution perfusion 0.9% (100 ml)</td><td>B. Braun Medical SA</td><td>534534</td><td></td></tr><tr><td>Masterflex L/S Standard Drive</td><td>Cole-Parmer Instrument Co</td><td>7521-10</td><td></td></tr><tr><td>Acquisition card</td><td>National Instruments</td><td>PCI-6024 E</td><td></td></tr><tr><td>Flowmeter module</td><td>Transonic Systems Inc.</td><td>TS410 and T402</td><td></td></tr><tr><td>Stopcock with 3-ways</td><td>BD Connexta Luerlock</td><td>394600</td><td></td></tr><tr><td>Millex Filter</td><td>Milian</td><td>SE2M229I04</td><td></td></tr></tbody></table>

procedure for human saphenous veins ex vivo perfusion and external reinforcement

광범위한 폐색 성 동맥 질환에 대한 현대적인 치료의 주류는 정맥 우회술이다. 그러나, 내구성은 결국 혈관 폐색 이식 실패에 이르게 내막 증식증 (IH)에 의해 위협됩니다. 기계의 힘, 특히 낮은 전단 응력과 높은 벽 장력은, 시작하고 이러한 세포 및 분자 변화를 유지하는 것으로 생각되지만, 자신의 정확한 기여를 풀어 일이다. 선택적 IH의 생물학에 대한 압력과 전단 응력의 ​​역할을 평가하기 위해, 생체 외 관류 시스템 (EVPS)는 동맥 요법에서 인간 복재 정맥 (높은 전단 응력 및 고압)의 세그먼트를 관류하기 위해 만들어졌습니다. 또한, 기술 혁신은 같은 맥락, 외부 메쉬 철근이 하나의 세그먼트를 동시에 관류시켰다. 정맥 노 터치 기술을 사용하여 수확하고 즉시 EVPS에 조립 실험실로 옮겼다. 갓 ISOL의 한 세그먼트나타나는데 정맥 (제어, 일 영)을 관류되지 않았습니다. 다른 두 세그먼트가 최대 칠일 관류하고, 하나는 완전히 4mm (직경) 외부 메쉬 숨겨된다. 압력, 유속 및 맥박수를 연속적으로 모니터링하고 대퇴 동맥에 지배적 혈역학 조건을 모방하도록 조정 하였다. 재관류 완료되면, 정맥 분리하고, 조직 학적 및 분자 분석을 사용 하였다. 생체 외 조건, 고압 하에서 관류 (동맥, = 100 mmHg로 평균) IH 인간 혈관의 리모델링을 생성하기에 충분하다. 이러한 변화들은 외부 메쉬 폴리 에스테르의 존재하에 감소된다.

EVPS 인간 복재 정맥 리모델링하고 IH를 접목에 독립적으로 혈역학 적 힘을 평가하는 유용한 도구를 제공합니다. 그림 1은 관류 챔버와 정맥 지원을 보여줍니다. 도 1A 및도 B에서, 이전에 정맥 지지체 (도 1a) 및 후 (도 1B) 조립체는 각각 묘사된다. 그것은 쉽게 (왼쪽에서 오른쪽으로) 밀어 수 2 L 형상 조각 용 지지체로서 기능 9cm...

Watch this Scientific Journal Video about Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement at JoVE.com

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

내막 증식증 (IH) 및 정맥 이식 실패의 개발로 이어지는 메커니즘은 아직 제대로 이해하고 있습니다. 이 연구는 제어 흐름과 압력 하에서 인간의 혈관을 관류하는 생체 시스템을 설명합니다. 또한 외부 메쉬 보강 효율 IH의 개발 평가 하였다 제한한다.

인간 복재 정맥 절차<em&gt; 전의 VIVO</em&gt; 관류 및 외부 보강

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

procedure-for-human-saphenous-veins-ex-vivo-perfusion-external

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

11.2K 조회수.  Brigham and Women's Hospital/Harvard Medical School.  내막 증식증 (IH) 및 정맥 이식 실패의 개발로 이어지는 메커니즘은 아직 제대로 이해하고 있습니다. 이 연구는 제어 흐름과 압력 하에서 인간의 혈관을 관류하는 생체 시스템을 설명합니다. 또한 외부 메쉬 보강 효율 IH의 개발 평가 하였다 제한한다. 

비디오: Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

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.

쥐 온목 동맥에 개재 외부 경정맥 정맥 이식의 : 정교한 Microsurgical 기술을 교육

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

연구

의학

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

에 내장 혈관 신생에 의하여 조직 공학<em&gt; 생체</em&gt; 조직 공학 상공 회의소

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

생체공학

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 ...

Decellularization and Recellularization Methodology for Human Saphenous Veins

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipient&#39;s cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 &#176;C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 &#181;L/mL), and acetyl salicylic acid (5 &#181;g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin &#38; eosin (H&#38;E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&#38;E-staining showed the presence of cells on the luminal surface of the vein.

Here, we describe a protocol for the saphenous vein decellularization using detergents and recellularization by perfusion of the peripheral blood and the endothelial medium.

Decellularization 및 Recellularization 방법론을 인간의 Saphenous 정 맥에 대 한

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and ...

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. ...

Synergizing Antegrade Endoscopic with Bridging Vein Harvesting for Improvement of Great Saphenous Vein Graft Quality from the Lower Leg

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative wound complications. This standardized protocol for antegrade endoscopic vein harvesting (EVH) from the lower leg has the potential to be introduced to routine coronary artery bypass grafting (CABG). Patients undergoing CABG surgery are positioned on a surgical table with two additional foam rollers below the extended legs, enabling antegrade EVH from the lower leg. Following minimally invasive surgical access through a bridging vein harvest technique, an endoscopic optical dissector is inserted antegrade into the wound. The main vessel and side branches are dissected under continuous optical control of vein quality status and the working channel. After, an endoscopic optical retractor is inserted with an internal bipolar electrocoagulation device for precise, safe, and tissue-protective interruption of side branches. After release of the vein, the vessel is cut off at the proximal and distal ends under optical control, retrieved from the wound, then cannulated and flushed with heparinized saline. Finally, all side branches of the vein graft are double-clipped. Vascular histology is analyzed in a randomized selection of vein samples. After applying this standardized EVH protocol, the learning curve was shown to be steep, and graft quality was sufficient for coronary artery bypass grafting in every case. There was no conversion to surgical harvesting and low risks for tissue damage and bleeding. Leg positioning and synergizing EVH with bridging vein harvesting improved procedural success and vein graft quality. In our hands, antegrade EVH from the lower leg was feasible, demonstrating straightforward graft dissection as well as adequate macroscopic and microscopic graft quality with preserved endothelial integrity. In conclusion, the introduced technique is safe, shows excellent vein autograft quality, and illustrates feasibility for elective and urgent isolated CABG and combined CABG scenarios.

Presented here is a protocol for antegrade endoscopic vein harvesting from the lower leg, which can safely be introduced in routine coronary artery bypass grafting. Vein grafts present excellent graft quality following this standardized protocol with positioning of the legs, minimally invasive access to the vein, and antegrade endoscopic vein harvesting.

하부 다리에서 훌륭한 사페누스 정맥 이식 품질의 개선을 위한 브리징 정맥 수확과 영양 내시경 시너지 효과

Antegrade endoscopic harvesting of autografts for bypass grafting may be an optimal strategy addressing excellent graft quality and reduced post-operative ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

체외에서 혈관 재건을 수행하기 위한 교육 및 테스트 시스템

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...

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 ...

Occlusion of the Great and Small Saphenous Vein Using Copolymeric Glue Based on N-Butyl Cyanoacrylate and Methacryloxy Sulfolane

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and long-term follow-ups of vascular occlusion of the great saphenous vein (GSV) and small saphenous vein (SSV) affected by severe pathological reflux, using an innovative modified cyanoacrylate surgical glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS). Ninety patients, prospectively recruited for 1 year, underwent the study with EcoColor-Doppler (ECD) to evaluate the maximum diameters of the GSV and SSV in the orthostatic position and the reflux time (RT). An RT greater than 0.5 s was considered pathologic. Clinical, etiology, anatomy, and pathophysiology (CEAP) assessment was used for the complete evaluation of each patient in the study. All the patients were treated by NBCA+MS glue to obtain vein occlusion and observed before treatment (baseline; T0), within 6 h after treatment (T1), 1 month after treatment (T2), 3 months after treatment (T3), 6 months after treatment (T4), and 1 year after treatment (T5). Chi-square (&#967;) analysis was performed to evaluate the effectiveness and safety of the treatment. All the patients participated in the entire duration of the study. Complete occlusion was maintained in 100% of patients at T1, 98.9% at T2 and T3, and 97.8% at T4 and T5 (p &lt; 0.001). None of the patients suffered from post-surgical thrombosis. No blue hyperpigmentation, or paresthesia was observed during the entire observation period. Immediately after treatment, 7.7% of patients needed painkillers; 1 week after treatment, 100% of patients returned to normal life. Vascular occlusion of the great or small saphenous vein using NBCA+MS glue is a safe procedure with persistent benefits after a 1 year follow-up. This procedure can be performed with local anesthesia, allowing a quick return to normal life. Thanks to its low invasiveness, the treatment is not painful.

Here, we present a protocol to treat the great saphenous vein (GSV) and the small saphenous vein (SSV) affected by severe pathological reflux, using an original sclerosing and embolizing cyanoacrylate-based glue composed of N-butyl cyanoacrylate and methacryloxy sulfolane (NBCA+MS).

N- 부틸 시아 노 아크릴 레이트와 메타 크릴 옥시 설폴란을 기반으로 한 공중 접착제를 사용한 크고 작은 복재 정맥의 폐색

We present the preliminary results of a longitudinal observational study aimed at evaluating the effectiveness and safety at different short- and ...

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

인간 복재 정맥 내피 세포 분리 및 제어된 수준의 전단 응력 및 스트레칭에 대한 노출

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

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