This research was supported by the FP7 European Research Council Grant 281501, ENGVASC.

<ol>
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The authors declare that they have no competing financial interests.

Los avances en la ingeniería de tejidos se han encontrado con una creciente demanda de tejidos sustitutos para la reconstrucción de los diversos tipos de tejidos. Una variedad de materiales sintéticos y biológicos 1,17,18 14-16, así como métodos de fabricación han sido evaluados por su capacidad para hacer frente a estas demandas. Sin embargo, a pesar de los avances en la atención clínica y en la ingeniería de tejidos, la restauración de defectos de la pared abdominal de espesor completo...

Defectos de la pared abdominal a menudo surgen después de un trauma severo, el tratamiento del cáncer, quemaduras y la eliminación de la malla infectada. Estos defectos implican a menudo la pérdida de tejido significativa, requiriendo procedimientos quirúrgicos complicados y presentar un gran reto para los cirujanos de reconstrucción de plástico 1-4. Investigadores de ingeniería de tejidos que buscan nuevas fuentes de tejidos artificiales han explorado diferentes materiales, fuentes de células y factores de crecimiento. Se informó anteriormente restauraciones exitosas de diversos tejidos, tales como 5,6 tráquea, vejiga 7, 8 córnea, hueso 9 y la piel 10, mediante la implantación de tejidos de ingeniería. Sin embargo, la fabricación de un tejido vascularizado ingeniería de espesor, en particular para la reconstrucción de defectos grandes, sigue siendo un desafío significativo en la ingeniería de tejidos. Uno de los principales factores que limitan el espesor de una construcción de tejido viable es el control de suministro de oxígeno a sus contrascélulas tituent. Cuando se depende de la difusión, la construcción de espesor se limita a la de una capa muy delgada. La distancia máxima entre oxígeno y nutrientes capilares de suministro in vivo es de aproximadamente 200 micras, que se correlaciona con el límite de difusión de oxígeno 11,12. Vascularización insuficiente puede resultar en isquemia tisular y escalar a la resorción de tejido o necrosis 13. Además, el material ideal utilizado para la reconstrucción de tejidos debe ser biocompatible y no inmunogénico. También debe ser capaz de promover una mayor integración de las células huésped con el biomaterial, y el mantenimiento de la integridad estructural. Varios biológicos 14-16 y sintéticos 1,17,18 matrices se han explorado previamente para la reconstrucción de tejidos, sin embargo su uso quedará limitado debido a la falta de suministro de sangre eficaz, las infecciones o la fuerza del tejido insuficiente. En este estudio, un biocompatible, célula-embandamio edded compuesto de Food and Drug Administration (FDA) de ácido L-láctico con aprobación para poli (PLLA) / poli ácido láctico-co-glicólico (PLGA), se implantó alrededor de los vasos de la arteria femoral y la vena (AV) de un ratón desnudo y separado del tejido circundante, lo que garantiza la vascularización de sólo los vasos AV. Una semana después de la implantación, el injerto era viable, grueso y bien vascularizado. Este tejido vascularizado grueso con los vasos de AV, fue trasladado como un colgajo pediculado a un defecto de espesor total abdominal en el mismo ratón. Una semana después de la transferencia, la aleta era viable, vascularizado y bien integrado con el tejido circundante, teniendo resistencia suficiente para soportar las vísceras abdominales. Por lo tanto, el espesor, colgajo de tejido vascularizado ingeniería, teniendo un pedículo autólogo, se presenta un método novedoso para la reparación de defectos de la pared abdominal de espesor completo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>small fine straight scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>spring scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr>
			<td>straight forceps with fine tip</td>
			<td>Fine Science Tools (FST)</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>serrated forceps&#160;</td>
			<td>Fine Science Tools (FST)</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>needle holder</td>
			<td>Fine Science Tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Small vessel cauterizer&#160;</td>
			<td>Fine Science Tools (FST)</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Duratears</td>
			<td>Alcon</td>
			<td align="right">5686</td>
			<td></td>
		</tr>
		<tr>
			<td>Sedaxylan</td>
			<td>Euravet</td>
			<td>DJ03</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorketam 1000</td>
			<td>Vetoquinol</td>
			<td>4A0726B</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>vetmarket</td>
			<td>B15100</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 silk sutures</td>
			<td>Assut sutures</td>
			<td align="right">647</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 polypropylene sutures</td>
			<td>Assut sutures</td>
			<td>9351F</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 silk sutures</td>
			<td>Assut sutures</td>
			<td align="right">684568</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (6mm needle)</td>
			<td>BD</td>
			<td align="right">324911</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 2100 high-resolution ultrasound system</td>
			<td>VisualSonics inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MS250 non-linear transducer</td>
			<td>VisualSonics inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromarker non-targeted contrast agent</td>
			<td>VisualSonics inc.</td>
			<td>VS-11694</td>
			<td></td>
		</tr>
		<tr>
			<td>tail vein catheter</td>
			<td>VisualSonics inc.</td>
			<td>VS-11912</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 2100 software</td>
			<td>VisualSonics inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein isothiocyanate-conjugated dextran</td>
			<td>Sigma</td>
			<td>FD500S</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech</td>
			<td align="right">34120</td>
			<td></td>
		</tr>
		<tr>
			<td>antigen unmasking solution</td>
			<td>Vector laboratories</td>
			<td>H-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CD31 antibody</td>
			<td>Abcam&#160;</td>
			<td>ab28364</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated goat anti-rabbit (secondary) antibody</td>
			<td>Vector laboratories</td>
			<td>BA-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin-peroxidase</td>
			<td>Jackson&#160;</td>
			<td>016-030-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayer&#39;s hamatoxylin solution</td>
			<td>Sigma-Aldrich</td>
			<td>MHS-16</td>
			<td></td>
		</tr>
		<tr>
			<td>aminoethylcarbazole (AEC) substrate kit</td>
			<td>Life technologies, Invitrogen&#160;</td>
			<td>00-2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectamount</td>
			<td>Vector laboratories</td>
			<td>H-5501</td>
			<td></td>
		</tr>
	</tbody>
</table>

engineered vascularized muscle flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

Vascularización del injerto y la perfusión in vivo Los injertos fueron implantados uno o dos semanas antes de su transferencia como aletas axiales. A la una y dos semanas después de la implantación, la observación bruto de la zona del injerto reveló injertos de tejidos viables y vascularizados. Estos injertos demostraron ser altamente vascularizado, como se determina por inmunotinción positiva CD31 (Figura 1A), y altamente perfundidos, como se evidencia por l...

Watch this Scientific Journal Video about Engineered Vascularized Muscle Flap at JoVE.com

Engineered Vascularized Muscle Flap

To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps.

Diseñado vascularizado colgajo de músculo

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Research

JoVE Journal

Bioengineering

8.2K vistas.  Kaplan Medical Center. To date, thick tissue defects are typically reconstructed by applying autologous tissue flaps or engineered tissues. In this protocol, we present a new method for engineering vascularized tissue flap bearing an autologous pedicle, to serve as a substitute to autologous flaps. 

Video: Engineered Vascularized Muscle Flap

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Modelo de polarizado Neural tisular basada Silk-colágeno-Engineered 3D

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Investigación

Bioingeniería

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Aplicaciones de la<em&gt; En Vivo</em&gt; Pruebas funcionales de la rata tibial anterior para la evaluación de la ingeniería tisular de reparación del músculo esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularizado de Bioprinting de microfluidos para la ingeniería de tejidos y organitas

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Injerto con ingeniería de tejido para la reconstrucción esofágica circunferencial en ratas

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

Fabricación de guías de onda de modo cero para microscopía de molécula única de alta concentración

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Generación de equivalentes de piel humana vascularizada autoensamblada

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Evaluación de las métricas funcionales de la salud del músculo esquelético en microtejidos del músculo esquelético humano

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

Tejidos pulmonares diseñados preparados a partir de rodajas pulmonares descelularizadas

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients&#39; quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports&#160;a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

Un modelo confiable de colgajo fascio-cutáneo porcino para estudios de bioingeniería de aloinjertos compuestos vascularizados

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...

Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction can be performed using autologous free flap transfer or vascularized composite allotransplantation (VCA), such methods also have disadvantages. Issues such as donor site morbidity and tissue availability limit autologous free flap transfer, while immunosuppression is a significant limitation of VCA. Engineered tissues in reconstructive surgery using decellularization/recellularization methods represent a possible solution. Decellularized tissues are generated using methods that remove native cellular material while preserving the underlying extracellular matrix (ECM) microarchitecture. These acellular scaffolds can then be subsequently recellularized with recipient-specific cells.
This protocol details the procurement and decellularization methods used to achieve acellular scaffolds in a pig model. In addition, it also provides a description of the perfusion bioreactor design and setup. The flaps include the porcine omentum, tensor fascia lata, and the radial forearm. Decellularization is performed via ex vivo perfusion of low concentration sodium dodecyl sulfate (SDS) detergent followed by DNase enzyme treatment and peracetic acid sterilization in a customized perfusion bioreactor.
Successful tissue decellularization is characterized by a white-opaque appearance of flaps macroscopically. Acellular flaps show the absence of nuclei on histological staining and a significant reduction in DNA content. This protocol can be used efficiently to generate decellularized soft tissue scaffolds with preserved ECM and vascular microarchitecture. Such scaffolds can be used in subsequent recellularization studies and have the potential for clinical translation in reconstructive surgery.

The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor.

Obtención y perfusión-descelularización de colgajos vascularizados porcinos en un biorreactor de perfusión personalizado

Large volume soft tissue defects lead to functional deficits and can greatly impact the patient&#39;s quality of life. Although surgical reconstruction ...