Name: tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber
Uploaded: 2016-05-30T15:00:00
Description: Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

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, 높...

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Y., 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=In+vivo+tissue+engineering+chamber+supports+human+induced+pluripotent+stem+cell+survival+and+rapid+differentiation.">In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.</a> Biochem Biophys Res Commun. 422 (1), 75-79 (2012).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3 (7), 787-791 (2014).</li></ol>

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 ...

The overall goal of this procedure is to create a space where tissue can grow and expand concomitantly with the development of a robust blood supply, ultimately leading to the generation of a vascularized, large, three-dimensional block of tissue. This method can help answer key questions in the engineering of vascularized tissue for reconstructive purposes as well as for organ replacement and repair. The main advantage of this technique is that the newly generated tissue is intrinsically supplied by its own vascular network without having to rely on diffusion from the environment for survival.Demonstrating the procedure will be Sue McKay and Liliana Pepe. Anesthetize a rat weighing at least 250 grams, and assess the depth of anesthesia using a toe pinch. If the rat is unresponsive, maintain its sedation using 2%isoflurane throughout the procedure.Next, apply epsomic ointment to the eyes. Then, with the rat supine, use electric razors to shave the groin and remove the hair with moistened gauze. Then, clean the surgical sites with a chlorhexidine solution containing 70%ethanol.Finally, drape the animal with sterile towels and proceed with the surgery. Using a number 15 blade, make a four centimeter long skin incision into the left groin parallel to the inguinal ligament. Next, cut through the exposed inguinal fat pad circumferentially with scissors, leaving it attached to its vascular pedicle, based on the epigastric vessels.Now, with micro-scissors, free the filmy connective tissue adhesions between the abdominal wall and underlying femoral vessels. Then, place a retractor on the abdominal wall and pull it medially to expose the inguinal ligament and the whole length of the femoral vessels. Next, using micro-forceps and curved scissors, dissect the epigastric vein and isolate it from the surrounding fat.This vein will function as a tether. Proceed by opening the perivascular sheath containing the femoral vessels and nerve. Open it all the way from the inguinal ligament to its bifurcation distal to the epigastric branch.At this point, and throughout the whole procedure, gentle handling of the femoral vessels is critical. Their damage can result in thrombosis and failure of the whole experiment. Then, using micro-forceps, pick up the femoral vein by its adventitia and gently separate it from the surrounding tissues and the accompanying artery.During this step, do not grasp the whole thickness of the vein wall, which could cause trauma to the intima, thus making the vein prone to thrombosis. Also during this step, ligate the side branches with 10-0 nylon suture, or coagulate them with a bipolar coagulator. With the femoral vein completely free, ligate it proximally and distally with 4-0 silk sutures to obtain a vein graft that is at least 10 millimeters long, which includes about half a centimeter length of the epigastric branch.The branch works as a guy rope tether to hold the loop open in the chamber. Next, trim the adventitia from the graft's ends. If necessary, this step may be postponed to before the micro-surgical anastomoses.Finish preparing the vein graft by flushing it with heparinized saline solution and leaving it to rest loaded with heparinized saline solution. Then, close the wound using continuous running 4-0 silk suture and two or three additional simple interrupted stitches. To begin, expose the inguinal ligament and the whole length of the femoral vessel of the contralateral limb as previously demonstrated.Then, using micro-forceps, dissect and isolate both the epigastric artery and vein from the surrounding fat pad. Be gentle when pulling the tissue away from the vessels. Continue by picking up the femoral artery by its adventitia and freeing it from the surrounding tissues.In this process, ligate or coagulate the artery side branches. Then, place a clamp proximally on both the femoral artery and vein. Now, using 4-0 silk suture, ligate the femoral artery and vein distal to the emergence of the epigastric vessels.Now place a sterile plastic contrast background under the vessels and use a sharp, straight micro-scissor to cut each vessel transversely. Once cut, flush the vessels out vigorously with generous amounts of heparinized saline until all the blood has been removed from the lumen. Now bring the vein graft into the operative field.Position the vein graft so that the proximal end of the vein graft is ready to be anastomosed to the recipient femoral vein, and the distal end to the recipient femoral artery. This will allow the blood to flow from the arterial to the venous side without resistance from the valves inside the vein graft. Make sure the femoral vessels and the vein graft rest in their natural position without any twists.Using 10-0 nylon suture, perform surgical anastomosis at both sides. A good and atraumatic micro-surgical anastomosis is key. Damage to the vessel intima, dessication of the vessels, or incorrectly placed stitches make the anastomotic site prone to thrombus formation.Then, check for leaks at both anastomotic sites. Resolve small leaks, which look like non-pulsating blood coming out of the anastomotic site, by placing a small piece of fat or a sponge on top and gently compressing for five to 10 minutes. Larger pulsating leaks that rapidly flood the entire field will need additional stitches.Next, check the patency of the arteriovenous loop. Gentle occlusion of the femoral artery should make it shrink. Conversely, occlusion of the femoral vein should engorge the loop.Now place the base of the tissue engineering chamber under the arteriovenous loop. Make sure that the loop rests in its natural position without any twists or kinks. Then, with 6-0 nylon sutures, secure the base of the chamber to the inguinal ligament and the underlying muscle fascia.Next, place the lid over the base of the chamber. Pinch the epigastric branches under the lid to hold the loop in position. Then, close the wound using continuous running 4-0 silk suture, plus two or three additional simple interrupted stitches.Finally, allow the animal to recover from anesthesia in an individual cage, and administer a single dose of carprofen subcutaneously as an analgesic. Prepare the animal for surgery as before. Two chambers can be implanted into a single rat, one in each groin.Then, isolate the femoral vessel as previously described. With both the artery and vein completely freed of surrounding tissues and their branches ligated, bring the chamber into the operative field. Then, place each of the intact femoral vessels into the corresponding slits of the chamber base.Make sure that neither is twisted or kinked. Next, close the chamber by attaching the lid to the base. Then close the wound and allow the animal to recover as previously described.The micro-surgical creation of tissue engineering chambers was performed as described. Various tissue types were successfully engineered using the in vivo vascularized chamber, which include cardiac tissue with neonatal rat cardiomyocytes, muscle tissue with rat skeletal myoblasts, and adipose tissue with hydrogel derived from adipose tissue extracellular matrix. The stereology system allows for unbiased quantification of specific tissue volume.For example, lectin-stained endothelial cells in transverse sections were used to estimate vascular volumes. Following in vivo implantation of cells labeled with fluorescent dyes, their fate can be tracked. For example, neonatal rat cardiomyocytes labeled with dye i were detected in the tissue constructs three days post-implantation.Alternatively, species-specific antibodies can be used to identify implanted cells in xenotransplanation studies. For example, human-induced pluripotent stem cells implanted in immuno-compromised rats could be identified in the tissue harvested from the chamber. Once mastered, the flow-through chamber and the arteriovenous loop can be implanted in approximately 20 and 90 minutes, respectively.While attempting this procedure, it's important to maintain sterility at all times and take the necessary measures should contamination occur. Don't forget that gentle handling of the blood vessels and atraumatic micro-surgical anastomosis are key to ensure success. In this sense, we do recommend basic micro-surgical training before attempting this procedure, particularly for the construction of the arteriovenous loop.

Research

JoVE Journal

Bioengineering

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

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

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 ...

연구

생체공학

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

심장 조직 공학을위한 섬유소 히드로겔에 Cardiomyocytes의 캡슐

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

조직 공학을위한 Electrospun의 Fibres의 Postproduction 처리

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of the Intestine in a Murine Model

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

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

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

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

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

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

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. ...

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