이 작품은 NHMRC와 스탠 폭스 의료 재단 보조금에 의해 지원되었다. 저자는 고소 맥케이, 릴리 아나 페페, 안나 Deftereos 및 실험 의학의 아만다 Rixon 외과 단위, 성 빈센트 병원, 멜버른의 수술 지원을 인정합니다. 지원은 혁신, 산업의 빅​​토리아 주 정부의 부서 및 지역 개발의 운영 인프라 지원 프로그램에 의해 제공됩니다.

여기에 설명 된 프로토콜은 세인트 빈센트 병원 멜버른, 호주의 동물 윤리위원회에 의해 승인되었으며, 호주 국립 보건 의료 연구위원회 가이드 라인을 엄격하게 준수에 따라 실시 하였다. 참고 : 두 개의 챔버 프로토콜이 아래에 설명되어 있습니다. 두 가지 모델과 특정 실 디자인은도 1에 도시된다. 챔버 (1) (래트 동정맥 루프 챔버 모델) 폴리 카보네이트로 제조된다. 그것은 내부 직경 13mm, 높이 4mm 원통형이다. 벽에 한 지점에서의 창은 척추 경에 대한 방해받지 않고 액세스 할 수 있습니다. (쥐 관류 척추 경 챔버 모델) 제 모델에서, 챔버는 폴리 이루어지고, (10 × 8 × 4mm 3 내부 치수) 사각형이다. 그들이 챔버 범으로 대퇴 동맥 및 정맥을 수용하기 위해 양측에 두 개의 1.5 mm의 개구부를 갖는다. 1. 쥐 동정맥 루프 회의소 모델 (한 참BER 당 동물) 참고 : 수술을 시작하기 전에, 모든 악기가 제대로 살균되어 있는지 확인합니다. 마찬가지로, 멸균 수건 악기 받침대를 확인하고 절차를 수행하는 동안 오염을 방지하기 위해 수술 분야에서 적당한 거리에있다. <ol><li> 수술에 대한 동물의 준비 <ol><li> 동정맥 루프의 창조를위한 선박의 그들의 큰 크기에 최소 250g의 무게 쥐를 사용합니다. </li><li> 4 % 이소 플루 란 흡입과 동물을 마취. 발가락 핀치에 응답하지 않는 문제를 평가하여 마취의 적절한 깊이를 확증. 마취 후, 충분히 2 % 이소 플루 란과 절차 전반에 걸쳐 마취 동물을 유지합니다. </li><li> 온난화 패드에 부정사 위치에 동물을 배치하고 수술 중 탈수를 방지하기 위해 눈에 멸균 윤활제를 적용합니다. </li><li> 전기 면도기를 사용하여, 두 사타구니 면도 촉촉한 거즈 조각으로 머리를 제거합니다. </li><li> 클로르헥시딘과 수술 사이트를 준비/ 70 % 에탄올 용액 및 멸균 수건 동물 드레이프. 진통제로 카프로 펜의 단일 용량 (5 ㎎ / ㎏, 피하)를 관리 할 수​​ 있습니다. </li></ol></li><li> 대퇴 정맥 이식의 수확 <ol><li> A # (15) 블레이드를 사용하여, 사타구니 인대에 왼쪽 사타구니 평행에 4cm 길이의 피부 절개를합니다. 이 사타구니 지방 패드를 노출합니다. </li><li> 가위는 상복부의 선박에 따라 혈관 척추 경에 부착 떠나와 원주 지방 패드를 통해 절단. </li><li> 마이크로 가위를 사용하여 복벽 및 하부 대퇴 혈관의 필름 상 결합 조직 유착 해방. </li><li> 복부 벽에 트랙터를 놓고 내측 당깁니다. 이 사타구니 인대와 대퇴 혈관의 전체 길이를 노출합니다. </li><li> 마이크로 집게를 사용하여 곡선 가위 부드럽게 당겨 및 절단하여 상복부 정맥을 해부와 그 주변 지방에서 격리. 루프를 구성 할 때이 정맥은 밧줄 역할을합니다. </li><li> 날기g 마이크로 집게 곡선 마이크로 가위는 대퇴 혈관과 신경 상복부 분기에 그 분기의 말단에 사타구니 인대에서 모든 방법을 포함하는 혈관 주위 덮개를 엽니 다. </li><li> 마이크로 집게를 사용하여 외막에 의해 대퇴 정맥을 선택하고 조심스럽게 주위 조직에서 분리하고 동맥을 동반. 마이크로 집게와 떨어져 조직을 당겨 그것을 통해 절단에 의해 곡선 라운드 지적 마이크로 가위로이 작업을 수행합니다. 참고 :이 혈전증에이 경향이 만드는 내막에 외상이 발생할 수 있습니다 정맥 벽의 전체 두께를 잡아하지 마십시오. </li><li> 결찰 측 분기 10/0 나일론 봉합사와 절개 중에 발견 또는 바이폴라 coagulator로 응고. </li><li> 대퇴 정맥 완전 무료로, 4/0 실크 봉합사와의 근위 및 원위 끝을 결찰. 적어도 10mm 길이의 정맥 이식을 얻을 수 있는지 확인하고 약 상복부 분기의 0.5 cm 길이가 남자 로프 밧줄로 사용할 수 포함챔버의 개방 루프를 개최합니다. </li><li> 마이크로 집게 똑바로 마이크로 가위를 사용하여 부드럽게 당겨 절단하여 이식의 끝에서 외막 트림. 이것은 또한 미세 수술 문합 전에 나중에 수행 될 수있다. </li><li> 헤파린 생리 식염수 (헤파린의 10 U / ㎖)와 정맥 이식을 세척하고, 용액에서 휴식을 둡니다. 지속적인 실행을 4/0 실크 봉합사 플러스 두 개 또는 세 개의 추가 간단한 중단 바늘을 사용하여 상처를 닫습니다. </li></ol></li><li> 상공 회의소의 동정맥 루프 및 주입의 창조 <ol><li> 반복 반대쪽 다리에 동일한 방식으로 1.2.4에 1.2.1 단계를 반복합니다. </li><li> 마이크로 집게를 사용하여, 해부 및 상복부 동맥을 모두 분리하고 주변의 지방 패드를 형성 정 맥. 부드럽게 혈관에서 떨어져 조직을 당겨이 작업을 수행 </li><li> 마이크로 집게를 사용하여 주변 조직에서 대퇴의 외막에 의해 동맥과 무료 픽업. 마이크로 집게 및 곡선 라운드 지적 마이크와 함께이 작업을 수행떨어져 조직을 당겨 그것을 통해 절단에 의해 RO 위입니다. 결찰 또는 옆으로 가지를 응고. </li><li> 대퇴부 동맥을 결찰 및 4/0 실크 봉합사를 이용하여 상복부 혈관의 출현 말초 정맥. </li><li> 대퇴 동맥과 정맥의 각 근위 하나의 클램프를 놓습니다. 날카로운 직선 마이크로 가위를 사용하여, 상복부 가지의 출현에 각 혈관 원위부 절단 깨끗한 가로을합니다. 선박에서 멸균 플라스틱 대비 배경을 놓습니다. </li><li> 모든 혈액이 루멘에서 제거 될 때까지 헤파린 생리 식염수의 관대 한 양 적극적으로 혈관을 플래시합니다. </li><li> 수술 필드에 정맥 이식을 가져와 필요한 경우, 단계 1.2.10에 따라 선박 &#39;끝에서 어떤 여분의 외막을 제거합니다. </li><li> 10/0 나일론 봉합사와 두 미세 수술 문합을 수행합니다. 대퇴 정맥과 대퇴 동맥에 선단 정맥 이식편의 기단부를 문합. 이 F 피가 흐를 수 있습니다정맥 이식 내 밸브에서 저항없이 정맥 측에 동맥을 ROM. 참고 : 어떤 왜곡없이 대퇴 혈관과 자연 위치에있는 정맥 이식의 나머지 부분을 확인합니다. </li><li> 문합 부위 모두에서 누수가 있는지 확인합니다. , 상단에 지방의 작은 조각을 배치하고 부드럽게 5 ~ 10 분 동안 압축하여 혈액이 문합 부위에서 나오는 비 맥동​​처럼 작은 누수를 해결합니다. 빠르게 전체 필드 홍수 큰 맥동 누수 추가 바늘이 필요합니다. </li><li> 동정맥 루프의 개통을 확인합니다. 대퇴 동맥의 부드러운 폐색은 대퇴 정맥에서 같은 그것을 충혈해야하면서 축소해야한다. </li><li> 왜곡 또는 꼬임이없는 자연 위치에 후자의 휴식과 동정맥 루프 아래의 조직 공학 챔버의 기초를 놓으십시오. </li><li> 6/0 나일론 봉합사와 사타구니 인대 및 기본 근육의 근막에 챔버의베이스를 고정합니다. </li><li> 오버 덮개를 놓습니다기본 대퇴 혈관 (챔버의 측면에 창) 노치 (notch)를 통해 챔버를 입력 있도록. 뚜껑을 닫을 때, 테더가 위치로 동정맥 루프를 유지하는 역할을 챔버 기지와 뚜껑 사이 상복부 지점을 포착해야합니다. </li><li> 지속적인 실행을 4/0 실크 봉합사 플러스 두 개 또는 세 개의 추가 간단한 중단 바늘을 사용하여 상처를 닫습니다. </li><li> 동물이 온난 패드에 마취에서 회복 할 수 있습니다. </li><li> 이 흉골 드러 누움을 유지하기 위해 충분한 의식을 회복 할 때까지 무인 동물을 두지 마십시오. 마찬가지로, 완전히 회복 될 때까지 다른 동물의 회사에 수술을 한 동물을 반환하지 않습니다. 24 시간 후, 진통제 등의 카프로 펜의 또 다른 하나의 용량 (5 ㎎ / ㎏, 피하)를 관리 할 수​​ 있습니다. </li><li> 5 일간 국소 항생제 연고와 상처 치료. 상처가 개방되는 경우, 단계별 1.1.5 1.1.2 에서처럼 동물을 마취 및 1.3과 상처습니다0.14. 매일 동물의 상태를 모니터링합니다. 동물 inacitivity, 식욕 부진, 체중 감소 및 색의 손실 이상의 적당한 흔적이있는 경우 (23 G 바늘 0.25 ㎖에 163 ㎎ / ㎏)을 복강 lethabarb 분사의 치사량을 사용하여 동물을 안락사. </li></ol></li></ol> 2. 플로우를 통해 척추 경 회의소 (동물 당 2 개의 챔버) <ol><li> 수술에 대한 동물의 준비 <ol><li> 반복 1.1.4을 통해 1.1.1 단계를 반복합니다. 두 개의 챔버는 단일 래트 모두 서혜부 영역에 주입 할 수있다. </li></ol></li><li> 대퇴 선박의 분리 및 상공 회의소의 삽입 <ol><li> 반복 1.2.8을 통해 1.2.1 단계를 반복합니다. </li><li> 동맥 완전히 조직을 둘러싼 및 분기 결찰의 해제 정맥 모두, 수술 필드에 실을 가지고. </li><li> 챔버베이스가 더 왜곡 또는 꼬임이 없는지 확인하고의 대응하는 슬릿에 그대로 대퇴 혈관의 각 놓습니다. </li><li> attachi에 의해 챔버를 닫습니다베이스에 뚜껑을 ng를. 지속적인 실행을 4/0 실크 봉합사 플러스 두 개 또는 세 개의 추가 간단한 중단 바늘을 사용하여 상처를 닫고 동물이 앞서 설명한 복구 할 수 있습니다. </li></ol></li></ol> 챔버 및 조직 처리 3. 수확 <ol><li> 실험의 시점 (4~6주 후 주입)에 도달하면, 단계 1.1.2에서와 같이 동물을 마취와 1.1.5 단계를 1.1.3를 반복합니다. </li><li> A # 15 블레이드를 사용하여 상처를 열고 챔버가 완전히 노출 될 때까지 가위로 조직을 잘라. </li><li> 다음 원위 방향으로 피를 우유, 부드럽게 두 microforceps과 용기를 폐색 마지막 근위 집게를 해제 : 혈관 개통의 구조 및 시험에 근위 대퇴 혈관을 노출. 선박이 다시 혈액으로 채워지면,이 개통을 확인합니다. 일 경우에 모두 근위 및 원위 동정맥 루프의 경우 근위 대퇴부 혈관을 결찰전자의 흐름을 통해 구성 및 블록 엉 포함하는 조직으로 챔버를 제거합니다. </li><li> 실험의 끝에서, (23 G 바늘 0.25 ㎖ 중의 163 ㎎ / ㎏)을 복강 내 주사 lethobarb의 치사량을 사용하여 동물을 안락사. </li><li> 24 시간, 실온에서 4 % 파라 포름 알데히드에서 조직을 고정한다. (1-2 mm 두께) 각각 파라핀 왁스 또는 최적의 절단 온도 화합물 파라핀 섹션 (5 μm의) 또는 동결 절편 (10 μm의)에 포함 된 여러 가로 섹션으로 나눈다 조직. 3,4,8,17,22, (24) </li><li> 조직의 일반적인 형태를 조사하는 등의 헤 마톡 실린 및 에오신으로 일상적인 조직 학적 염색을 수행합니다. 심근 예를 들어 심장 트로포 닌 T의 면역 염색에 대한 관심의 세포 유형, 3,4,8,17,22,24,29을 식별하기 위해 특정 항체와 면역 조직 화학 염색을 수행합니다. </li></ol>

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저자는 더 경쟁 이익을 선언합니다.

미세 순환 공학은 현재 두 가지 방법을 통해 본질적으로 조사되고있다. 주입 된 때 상기 제 1 호스트 혈관 층에서 모세관의 것과 연결 체외 구조물 내의 높은 상호 혈관 네트워크 개발을 포함 이와 영양분의 전달을 보장하는 처리라고 inosculation 통해 구축 이식뿐만 아니라 주변뿐만 또한 핵심. 21,32,33에이 사전 혈관라고합니다. 그 모세관 발아 전 또는 병용 이식 세포 분화 및 조직 성...

재생 의학의 새로운 패러다임은 조직 공학 방법을 사용하여 기능 혈관 조직입니다 제조. 1,2 많은 방법을 손상된 조직이나 결함이있는 장기의 교체를 위해 새롭고 건강한 조직을 설계하는 것은, 3-6 실험적으로 작은 동물 모델을 개발되었다 유망한 임상 잠재력. 7,8 그러나, 혈관이 임상 적 크기의 조직을 성장 잠재력을 제한하는 조직 공학에 대한 큰 과제 중 하나 남아있다. (9) 현재 접근 방식은 새로운 혈관이받는 혈관 침대에서 성장 곳 외인성 경로를 따라 조직을 vascularize와 이식 된 조직이 10 혈관이 성장하고 새로 개발 조직과 한마음으로 확장 고유 혈관 통로를 구축을 통해 침입합니다. (11)를 외부 접근 전통적으로 지지체 상에 파종 세포를 포함시험관과 영양분은 이전 배지에 의해 공급되는 기대와 살아있는 동물에 완전한 구조를 주입에 혈관 내 증식이 너무 느리고 매우 얇은 임플란트 (AS 12,13 개념은 단순하다. 순환로부터 공급 &lt;될 1-2mm 두께는) 가능한 유지됩니다. 충분한 신속한 혈관을 통해 영양분과 산소를 제공하는 것은 뼈, 근육, 지방, 고체 기관으로 더 복잡하고 더 큰 조직 공학적 대체를 성장하는 성공적인 시도의 핵심입니다. 14, 15 내장 혈관이의 가능성을 제공합니다 더 큰 구조는 확대 혈액 공급에 상응하는 진보적 조직 성장에 의해 개발. 하나의 디자인 또는 셀 시드 발판없이 혈관 유경의 챔버에 생체 주입입니다.이 두꺼운 본질적으로 혈관 조직의 생성을위한 새로운 절차에 도로를 포장하고있다 5,6. 16, 17 &lt;/ P&gt; 최근 전략 이식하기 전에 조직 이식, - vascularize 미리 개발되었다. 이러한 통합 혈관 네트워크는 이식 두꺼운 조직 이식편의 모든 부분의 생존을 향상시키기 위해 전체 혈액 공급의 신속한 공급을 허용 주입시에 호스트 용기에 접합하다보고자한다. 18 우리는 관류 척추 경 혈관 세포 함유 생체 물질을 함유하는 피하 주입 된 반 강성 밀폐 챔버를 포함 작은 동물에서 생체 내 혈관 조직 공학 모델을 개척했다. 챔버는 이식 된 혈관에서 혈관 신생 발아를 자극 허혈성 환경을 만듭니다. 3 혈관 척추 경을 재구성 동정맥 루프 또는 손상 흐름을 통해 동맥과 정맥이 될 수 있습니다. 3-6,19이 혈관 척추 경 콩나물 기능과 광범위한 동맥의를 모두 예술에 연결 -capillary 정맥 네트워크eriole 정맥의 혈관 척추 경으로 끝난다. 3,20를 또한 둘러싸는 중공지지 챔버 혈관 생성을 향상시키기 위해. 3,21,22을 혈관 척추 경 간단히 주입되면 잠재적으로 기계적 힘을 변형되는 현상 조직을 보호하고 허혈성 드라이브 길어 정상 조직이 아닌 상기 챔버의 내부 공간에 보호, 혈관 발아는 정상 상처 척추 경 주위에 축적한다 새로운 조직과 같은 타임 라인을 따라 중단. 연구자들은 임상 관련 크기의 지원 혈관 3 차원 기능 혈관 조직 구조를 생산하는이 생체 구성을 사용하고 있습니다. 4,23는 또한 그것의 본래 혈관 척추 경으로 설계 혈관 조직 구조가 부상 사이트에서 후속 이식 수확 할 수있다 .보다 임상 적으로 가능한 시나리오는 재건의에 대한 확실한 사이트에서 실을 만드는 것 (24, 25)가슴으로 UCH. 따라서,이 새로이 조직 공학 방법은 재건 수술에 대한 기능적 대상 조직의 새로운 소스를 제공하기 위해 임상 적 잠재력을 가질 수있다. 26-28 다음 프로토콜은 상이한 동물 모델에서 적응 혈관 행렬 생성 및 세포 이동과 분화의 복잡한 과정을 조사하기 위해 이용 될 수있는 래트에서 생체 내 혈관 조직 공학 챔버를 구성하는 일반적인 지침을 제공 할 것이다.

<table border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">1 15 Blade Scalpel</td>
			<td style="width:187px;">Braun</td>
			<td style="width:163px;">BB515</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Toothed Adson Forceps</td>
			<td>Braun</td>
			<td>BD527R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Needle Holder</td>
			<td>Braun</td>
			<td>BM201R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Bipolar Coagulator&#160;</td>
			<td>Braun</td>
			<td>US335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Needle Holder B-15-8.3</td>
			<td>S &#38; T</td>
			<td>00763</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Dilator Forceps D-5a.2</td>
			<td>S &#38; T</td>
			<td>00125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Jeweler&#39;s Forceps JF-5</td>
			<td>S &#38; T</td>
			<td>00108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Straight SAS-11</td>
			<td>S &#38; T</td>
			<td>00098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Curved SDC-11</td>
			<td>S &#38; T</td>
			<td>00090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 Single Clamps B-3</td>
			<td>S &#38; T</td>
			<td>00400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 10/0 nylon suture</td>
			<td>S &#38; T</td>
			<td>03199</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 6/0 nylon suture</td>
			<td>Braun</td>
			<td>G2095469</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 4/0 Silk Sutures</td>
			<td>Braun</td>
			<td>C0760145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xilocaine 1%</td>
			<td>Dealmed</td>
			<td>150733</td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin Sodium</td>
			<td>Dealmed</td>
			<td>272301</td>
			<td>5000 UI / ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ringer Lactate</td>
			<td>Baxter</td>
			<td>JB2323</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 dome-shaped tissue engineering chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 flow-through chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin I, Griffonia Simplicifolia&#160;</td>
			<td>Vector Laboratories</td>
			<td>B-1105</td>
			<td>1.67 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Troponin T antibody</td>
			<td>Abcam</td>
			<td>Ab8295</td>
			<td>4 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human-specific Ku80 antibody</td>
			<td>Abcam</td>
			<td>Ab80592</td>
			<td style="width:176px;">0.06 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Desmin antibody</td>
			<td>Dako</td>
			<td>M0760</td>
			<td style="width:176px;">2.55 &#956;g/mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Tracker CM-DiI dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C-7000</td>
			<td>3 mg/106 cells</td>
		</tr>
	</tbody>
</table>

tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber

재건 수술에서, 서로에 대한 복잡하고 비용이 많이 드는 무역 하나의 결함있는자가 재건의 현재의 방법에 대한 대안에 대한 임상이 필요하다. 조직 공학이 수요 증가를 해결하기 위해 약속을 보유하고있다. 그러나, 대부분의 조직 공학 전략 인해 열악한 혈관 안정 기능적 조직 대체물을 생성하지 못한다. 본 논문은 관류 동맥과 정맥 중 하나 동정맥 루프 또는 흐름을 통해 척추 경 구성으로는 보호 중공 챔버 내부 지향 내장 혈관의 생체 조직 공학 챔버 모델에 초점을 맞추고있다. 이 챔버 기반 시스템에서 혈관 신생 발아는 동정맥 혈관에서 발생하고,이 시스템은 점차적으로 석면 혈관 조직과 챔버 공간을 채우고 구동 허혈성 염증 내생 세포 이동을 끈다. 외인성 세포 / 챔버 구축시에 매트릭스 주입 셀 SUR 향상천공되지 및 개발하는 엔지니어링 조직의 특이성을 결정한다. 우리의 연구는 이러한 챔버 모델이 성공적 지방, 심장 근육, 간 등의 다른 조직을 생성 할 수 있다는 것을 보여 주었다. 그러나, 수정 및 개선이 형성이 일관되고 재현 표적 조직을 보장하기 위해 필요합니다. 이 문서는 생체 내에서 두 개의 서로 다른 혈관 조직 공학 챔버 모델의 제조를위한 표준화 된 프로토콜을 설명합니다.

상기 프로토콜에 기재된 바와 같이 조직 공학 챔버들의 생성은 미세 수술을 실시 하였다. 챔버 내부에서 발생하는 조직 3. 다양한 조직 유형이 성공적으로 생체 내 혈관 챔버 (도 2)를 이용하여 설계 한 프로토콜 단계에서 설명하는 바와 같이 조직 학적으로 관찰 할 수있다. 이러한 신생아 쥐의 심근 (그림 2A), 지방 조직 세포 외 기질 (?...

Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

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 ...

tissue-engineering-intrinsic-vascularization-an-vivo-tissue

Research

JoVE Journal

Bioengineering

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

8.8K 조회수.  St Vincent's Institute of Medical Research. 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. 

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

심근 조직 공학을위한 생물학 유래 주사용 재료의 제조

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

연구

생체공학

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

탄성 PGS는 아티 조직 공학 공사장 공중 발판

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

손쥐 모델에서 창자의 조직 공학

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

심장 조직 공학을위한 모세관 력 리소그래피

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

인간의 3 차원의 조직 공학<em&gt; 시험관</em&gt; 종양 테스트 시스템

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

3 차원 전체 두께 피부의 세대 동등한 및 자동화 부상

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

샌드위치 같은 미세 환경은 셀 / 재료의 상호 작용을 활용하는

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

조직 공학 Thermoresponsive 나노 구조 표면의 제조

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

조직 공학 혈관 신생 단위로 쥐 지방 조직 유래 미세 혈관 조각의 분리

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...