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

Este trabalho foi apoiado por subvenções do NHMRC e Stafford Fox Fundação Médica. Os autores reconhecem a assistência cirúrgica de Sue McKay, Liliana Pepe, Anna Deftereos e Amanda Rixon do Experimental Médica e Unidade Cirúrgica do Hospital de St. Vincent, Melbourne. O suporte também é fornecido pelo Departamento de Inovação, Indústria do Governo do Estado de Victoria e Programa de Apoio à infra-estrutura operacional do Desenvolvimento Regional.

Os protocolos aqui descritos foram aprovados pelo Comitê de Ética Animal de St. Vincent Hospital Melbourne, Austrália, e foram conduzidos em estrita observância às orientações australianos Conselho Nacional de Saúde e Pesquisa Médica. NOTA: Dois protocolos de câmara são descritos abaixo. Os dois modelos diferentes e os seus modelos específicos de câmara são ilustradas na Figura 1. Câmara (1) é feita de policarbonato (para o modelo de rato câmara anelar arteriovenosa). Ele é cilíndrico com um diâm...

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Engenharia da microcirculação está sendo investigado, essencialmente através de duas abordagens. A primeira envolve o desenvolvimento de uma rede vascular altamente interligada dentro da construção in vitro de modo a que, quando implantados, os capilares do leito vascular hospedeiro ligar com aqueles do transplantado construir através de um processo chamado inosculation, garantindo assim a entrega de nutrientes, não só para a periferia mas também para o núcleo. 21,32,33 Isto é chamado de ...

Fabricação de tecido vascularizado funcional, utilizando uma abordagem de engenharia de tecidos é um paradigma emergente na medicina regenerativa. 1,2 Muitas abordagens para projetar o tecido novo e saudável para a substituição de tecido lesionado ou órgãos defeituosos têm sido desenvolvidos, 3-6 experimentalmente em modelos animais pequenos com promissor potencial clínico 7,8. No entanto, a vascularização continua a ser um dos maiores desafios para a engenharia de tecidos que limitam o seu potencial de crescimento de tecidos tamanho clinicamente relevante. 9 As abordagens actuais para vascularizar tecidos seguir qualquer uma via extrínseca, onde novos vasos crescem a partir do leito vascular destinatário e invadir durante todo o tecido implantado constrói 10 ou uma via de vascularização intrínseca, onde a vasculatura cresce e se expande em uníssono com o tecido acabado de desenvolvimento. 11 A abordagem extrínseca tradicionalmente envolve as células de semeadura em um andaimein vitro e implantando a construção completa para o animal vivo com a expectativa de que os nutrientes, previamente fornecido pelo meio de cultura, será obtida a partir da circulação. 12,13 O conceito é simplista como ingressos vascular é muito lento e apenas implantes muito finas (&lt; 1-2 mm de espessura) permanecerá viável. Fornecendo nutrientes e oxigênio através de uma vascularização suficiente e rápida é o cerne de todas as tentativas de sucesso para crescer substitutos mais complexos e maiores da engenharia de tecidos, como ossos, músculos, gordura e órgãos sólidos. 14,15 vascularização Intrinsic oferece o potencial para construções maiores para desenvolver pelo crescimento tecidual progressiva à altura do seu fornecimento de sangue em expansão. Um projeto é a implantação in vivo em uma câmara de um pedículo vascular, com ou sem um andaime semeado celular. 5,6 Isso abriu o caminho para novos procedimentos para a geração de mais grossos tecidos intrinsecamente vascularizados 16,17. &lt;/ P&gt; Mais recentemente, as estratégias têm sido desenvolvidos para enxertos de tecidos, antes da implantação-vascularizar previamente. Estas redes de vasos sanguíneos incorporados são destinados a unir com os vasos do hospedeiro ao implante permitindo o rápido fornecimento de um suprimento de sangue completo para melhorar a sobrevivência de todas as partes de um enxerto de tecido grosso transplantado 18. Nós pioneira um modelo de engenharia de tecidos in vivo vascularizado em pequenos animais que envolve uma câmara fechada semi-rígida implantadas subcutaneamente contendo um pedículo vascular perfundido e biomateriais contendo as células. A câmara cria um ambiente isquémico que estimula a germinação angiogénica dos vasos implantados. 3 O pedículo vascular pode ser um circuito arteriovenoso reconstruída ou uma artéria de fluxo intacto e veia. 3-6,19 A vasculares couves pediculares um funcionamento e um grande artério rede -capillary-venosa que liga tanto a arteeriole e venosa termina com o pedículo vascular. 3,20 Além disso, a câmara de suporte oco envolvente protege o tecido em desenvolvimento de potencialmente deformando forças mecânicas e prolonga a unidade isquêmico para melhorar a vascularização. 3,21,22 Se o pedículo embarcação é simplesmente implantados em tecido normal e não dentro do espaço protegido da câmara, surgimento angiogênico deixa ao longo da mesma linha do tempo como uma ferida normal e nenhum novo tecido irá acumular em torno do pedículo. Os investigadores usaram esta configuração in vivo para produzir tridimensionais construções de tecido vascularizado funcionais com vasculatura de suporte e de tamanho clinicamente relevante. 4,23 Além disso, as construções de tecido vascularizado artificiais, com a sua pedículo vascular intacta pode ser colhida para transplante subsequente no local da lesão 24,25. Um cenário mais clinicamente viável seria a criação da câmara no local definitivo para a reconstrução sdominam vários como o da mama. Assim, este novo abordagem de engenharia de tecidos pode ter um potencial clínico para proporcionar uma nova fonte de tecido alvo funcional para cirurgia reconstrutiva. 26-28 O protocolo que se segue irá fornecer um guia geral para a construção de uma câmara de engenharia de tecidos vascularizados in vivo no rato, que pode ser adaptada em diferentes modelos animais e utilizados para examinar o intrincado processo de angiogénese, a produção da matriz, e a migração e diferenciação celular.

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			<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

Na cirurgia de reconstrução, há uma necessidade clínica para uma alternativa aos actuais métodos de reconstrução autólogos, que são complexos, dispendiosos e de comércio um defeito para o outro. A engenharia de tecidos mantém a promessa de abordar esta demanda crescente. No entanto, a maioria das estratégias de engenharia de tecidos não conseguem gerar substitutos de tecidos estáveis ​​e funcionais por causa da má vascularização. Este artigo centra-se em um modelo de câmara in vivo engenharia de tecidos da vascularização intrínseca, onde uma artéria e uma veia perfusão ou como um loop arteriovenosa ou uma configuração pedículo flow-through é dirigido dentro de uma câmara oca protegida. Neste sistema baseado na câmara de germinação ocorre a partir angiogénico nos vasos arteriovenosas e este sistema atrai a migração celular endógeno isquémica e inflamatório accionado que preenche o espaço gradualmente câmara com tecido fibro-vascular. células exógena / implantação matriz no momento da construção da câmara aumenta sur celularvência e determina a especificidade dos tecidos de engenharia que se desenvolvem. Os nossos estudos demonstraram que este modelo de câmara pode gerar com êxito diferentes tecidos, tais como gordura, o músculo cardíaco, fígado e outros. No entanto, são necessárias modificações e refinamentos para assegurar a formação de tecido alvo é consistente e reproduzível. Este artigo descreve um protocolo normalizado para a fabricação de dois modelos de câmara engenharia de tecidos vascularizados diferentes in vivo.

A criação de câmaras microcirúrgico em engenharia de tecido foi realizado como descrito no protocolo acima. Os tecidos geradas dentro das câmaras podem ser examinados histologicamente como descrito no passo 3. protocolo Vários tipos de tecido foram projetados com sucesso utilizando a câmara de vascularizado in vivo (Figura 2). Estas incluem tecido cardíaco com cardiomiócitos neonatais de rato (Figura 2a), o tecido do músculo esqueléti...

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.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

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

PGS elastomérico Andaimes em Engenharia de Tecidos Arterial

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

Bioengenharia

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Encapsulamento de Cardiomyocytes em um hidrogel de fibrina para engenharia de tecidos cardíacos

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

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

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

Engenharia de Tecidos do Intestino num modelo murino

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

Capilar Força Litografia de Engenharia tecido cardíaco

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

Engenharia de Tecidos de um 3D Humano<em&gt; In vitro</em&gt; Sistema de Teste de Tumor

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

Geração de espessura completa tridimensional Pele Equivalente e Automated Ferimento

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

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

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

Microambientes sanduíche-como aproveitarar Telemóveis / interações materiais

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

Isolamento de Murino adiposo derivada de tecido microvascular Fragmentos como Vascularização Unidades para engenharia de tecidos

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