This work was funded by Shriners Hospitals for Children grants #85127 (BEU and CLC) and #84702 (AA). The authors would like to thank the &#34;Gueules Cass&#233;es&#34; foundation for the salary support to the fellows involved in that project.

All animals received human care following the National Institute of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee approved the experimental protocol (IACUC- protocol #2020N000015). Seven female Yorkshire pigs (20-25 kg) were used for all experiments.
1. Preoperative care
<ol>
	<li>Fast the animal for solid food 12 h prior to the surgery.</li>
	<li>Sedate the animal with 4.4 mg/kg of Telazol, 2.2 mg/kg of Xylazine, and 0.04 mg/kg (IM) of Atropine sulfate (see Table of Materials).</li>
	<li>Place an 18 G peripheral intravenous catheter in an ear vein.</li>
	<li>Intubate the swine with an appropriate endotracheal tube (6-15 mm can be used for 10-200 kg pigs) and connect the tube to a ventilator. Administer pre-operative analgesia with buprenorphine (0.05 mg/kg, IM) (see Table of Materials).</li>
</ol>
2. Intraoperative monitoring
<ol>
	<li>Maintain anesthesia with an inhalation mixture of 1.5%-3% isoflurane with 1.5 L/min oxygen flow.</li>
	<li>Continuously monitor the heart rate, pulse oximetry, and end-tidal CO2. Assess blood pressure and body temperature every 5 min. 
	NOTE: The target range for the heart rate is between 90-100 beats/min, the oxygen saturation must be higher than 93%, and the end-tidal CO2 range is between 5%-6% of CO2.</li>
	<li>Administer 5-10 mL/kg per hour 0.9% saline throughout the procedure to regulate the mean arterial pressure between 60 mmHg and 90 mmHg.</li>
</ol>
3. Bilateral saphenous FCF procurement
<ol>
	<li>Place the animal in a supine position. Shave and scrub both groins and hindlimbs, include the entire hindlimbs in the surgical site, and drape in a sterile fashion.</li>
	<li>Palpate the pulse of the saphenous artery ~3 finger-widths medial from the patella and tag it.</li>
	<li>Identify and draw the limits of the flap. 
	NOTE: The superior limit is an axis parallel to the inguinal crease 3 cm below it. The lateral limit is an axis from the anterior superior iliac spine to the medial part of the patella.</li>
	<li>Draw a 10 cm diameter oval-like flap centered on the saphenous pedicle and contained in the previously described flap limits (step 3.3).</li>
	<li>Make a 1.5 cm skin incision regarding the distal portion of the pedicle on the flap landmark.</li>
	<li>Open the fascia and blunt dissect to expose the saphenous artery and its two venae comitantes. Perform a double ligature and separate in one bundle.</li>
	<li>Incise the remaining skin of the flap with a blade.</li>
	<li>Use cautery to open the subcutaneous tissue and the surrounding fascia. Perform thorough hemostasis using bipolar forceps (see Table of Materials).</li>
	<li>Attach the skin component of the flap to the underlying fascia with 3-0 non-absorbable sutures to avoid inadvertent traction and disruption of perforating vessels.</li>
	<li>Free the flap from the gracilis by dissecting the fascia away from the muscle. 
	NOTE: The distal part of the saphenous pedicle runs in a plane between the gracilis muscle and the fascia. Appropriate tension and cautious bipolar hemostasis of side branches are crucial elements to ease the pedicle dissection.</li>
	<li>Use a scalpel to make a 12 cm incision in the inguinal crease. Perform a perpendicular incision joining the inguinal crease to the proximal part of the flap. Lift away the connecting skin and open the subcutaneous layer using cautery.</li>
	<li>Continue the pedicle dissection by following the saphenous vessels down towards the femoral vessels. 
	NOTE: The proximal portion of the saphenous pedicle can either run through the intermuscular septum or dive into the gracilis muscle.</li>
	<li>Skeletonize the femoral vessels and ligate them distally to the saphenous branch in two separate bundles. Continue the dissection of the femoral vessels from distal to proximal until reaching the level of the inguinal ligament. Use bipolar forceps to cauterize or vascular clips and 2-0 silk ties to ligate the deep femoral vessels, then cut. 
	NOTE: Vascular clips can also be used before cutting the vessels.</li>
	<li>Repeat steps 3.2-3.13 on the contralateral hindlimb to harvest the second saphenous flap.</li>
	<li>Heparinize the animal with an intravenous (IV) heparin injection (100 IU/kg) 5 min before step 3.16.</li>
	<li>Ligate the femoral pedicle (artery and vein) as proximal to the inguinal ligament as possible and separate the flap from the donor pig.</li>
	<li>Dilate the femoral vessel ends and insert a 20 G angiocatheter in both artery and vein. Use 3-0 silk ties to secure the catheter to the vessels.</li>
	<li>Slowly flush the fascio-cutaneous flap artery with 10 mL of heparin saline (100 IU/mL) until a clear venous outflow is observed (Figure 1).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63557/63557fig01.jpg" /> 
Figure 1: Native and decellularized saphenous fascio-cutaneous flap. (A)&#160;Isolated skin flap with a 20 G angiocatheter inserted in the femoral artery, allowing to wash the flap from the blood and proceed with different experiments (angiography, perfusion decellularization). (B)&#160;Decellularized skin flap. Perfusion decellularization yielding white, acellular scaffolds after 10 days of detergent perfusion. H&#38;E-stained full-thickness cross-sections of (C) native skin flap and (D)&#160;decellularized skin flap. <a href="https://www.jove.com/files/ftp_upload/63557/63557fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="19">
	<li>Euthanize the animal with an IV injection of sodium phenobarbital (100 mg/kg). Confirm death by the absence of heartbeat and respiratory movements.</li>
</ol>

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L., 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=Penis+transplantation:+First+US+experience.">Penis transplantation: First US experience.</a> Annals of Surgery. 267 (5), 983-988 (2018).</li><li>Lantieri, L., 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=Face+transplant:+Long-term+follow-up+and+results+of+a+prospective+open+study.">Face transplant: Long-term follow-up and results of a prospective open study.</a> Lancet. 388 (10052), 1398-1407 (2016).</li><li>Derek, E., Dhanireddy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression.">Immunosuppression.</a> Current Opinion in Organ Transplantation. 17 (6), 616-618 (2012).</li><li>Lantieri, L., 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=First+human+facial+retransplantation:+30-month+follow-up.">First human facial retransplantation: 30-month follow-up.</a> Lancet. 396 (10264), 1758-1765 (2020).</li><li>Kauke, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+facial+retransplantation+in+a+female+patient-Technical,+immunologic,+and+clinical+considerations.">Full facial retransplantation in a female patient-Technical, immunologic, and clinical considerations.</a> American Journal of Transplantation. 21 (10), 3472-3480 (2021).</li><li>Leonard, D. 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J., 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=Engineered+composite+tissue+as+a+bioartificial+limb+graft.">Engineered composite tissue as a bioartificial limb graft.</a> Biomaterials. 61, 246-256 (2015).</li><li>Duisit, J., 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=Decellularization+of+the+porcine+ear+generates+a+biocompatible,+nonimmunogenic+extracellular+matrix+platform+for+face+subunit+bioengineering.">Decellularization of the porcine ear generates a biocompatible, nonimmunogenic extracellular matrix platform for face subunit bioengineering.</a> Annals of Surgery. 267 (6), 1191-1201 (2018).</li><li>Lupon, E., 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=Engineering+Vascularized+composite+allografts+using+natural+scaffolds:+A+systematic+review.">Engineering Vascularized composite allografts using natural scaffolds: A systematic review.</a> Tissue Engineering Part B: Reviews. , (2021).</li><li>Duisit, J., Maistriaux, L., Bertheuil, N., Lellouch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vascularized+composite+tissues+by+perfusion+decellularization/recellularization:+Review.">Engineering vascularized composite tissues by perfusion decellularization/recellularization: Review.</a> Current Transplantation Reports. 8, 44-56 (2021).</li><li>Sullivan, T. P., Eaglstein, W. H., Davis, S. C., Mertz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pig+as+a+model+for+human+wound+healing.">The pig as a model for human wound healing.</a> Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society. 9 (2), 66-76 (2001).</li><li>Haughey, B. H., Panje, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+model+for+multiple+musculocutaneous+flaps.">A porcine model for multiple musculocutaneous flaps.</a> The Laryngoscope. 99 (2), 204-212 (1989).</li><li>Ibrahim, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+heterotopic+swine+hind+limb+transplant+model+for+translational+vascularized+composite+allotransplantation+(VCA)+research.">A modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (VCA) research.</a> Journal of Visualized Experiments. (80), e50475 (2013).</li><li>Rosh, E. H., Vistnes, L. M., Ksander, G. 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The authors have nothing to disclose.

This article describes a reliable and reproducible fasciocutaneous flap harvested on swine hindlimbs. Following this step-by-step surgical protocol will allow the procurement of two flaps on only one animal in less than 2 h. The most critical step of the surgery is the skeletonization of the vascular pedicle within the gracilis muscle fibers, which requires a thorough dissection by a skilled surgeon. Securing the skin to the fascia using cutaneous sutures is a crucial tip to avoid a shearing effect disrupting the perfora...

Vascularized composite allografts (VCA) have revolutionized the treatment of hard-to-repair body part losses, such as hands, face, and penis1,2,3. Unfortunately, the first long-term outcomes4 have shown that lifelong administration of high-dose immunosuppressive agents can lead to severe collateral medical conditions, including diabetes, infections, neoplasia, and reno-vascular dysfunction5. Lately, expert VCA teams have had to manage the risk of chronic rejection leading to graft loss and perform the first face retransplantation cases6,7. Different strategies have been described to overcome the limitations of immunosuppression in VCA. The first relies on establishing long-term graft tolerance by inducing an immune mixed chimerism state in the allograft recipient8,9. The second involves de novo creation of a patient-specific graft via tissue engineering.
Recently, perfusion decellularization of biological tissues has generated native extracellular matrix (ECM) scaffolds, allowing the preservation of the vascular network and tissue architecture of whole organs10. Hence, the recellularization of these ECM with recipient-specific cells would create a customized graft free of immune constraints. In research on VCA bioengineering, multiple teams have decellularized and obtained such ECM preserving the entire architecture11,12,13. However, the recellularization process remains challenging and has not been successful in large animal models14,15. Developing these breakthrough technologies creates a need for reliable and reproducible large animal composite tissue models. Swine models represent the utmost choice in the bioengineering developmental pipeline, as porcine skin presents the closest anatomical and physiological characteristics to human skin16. The use of fascio-cutaneous flaps (FCF) is ideal during the first steps towards the creation of &#39;tailored&#39; vascularized composite tissue grafts. Indeed, FCF is an elementary VCA model containing skin, fat, fascia, and endothelial cells. A description of swine myocutaneous flaps17 and osteomyocutaneous flaps18 can be found in the literature. Nonetheless, there is a lack of focus on fascio-cutaneous flaps harvest techniques.
Hence, this study aims to provide researchers with a detailed description of a swine saphenous FCF procurement technique and depict all the flap&#39;s characteristics for its use in many research fields, especially in vascularized composite tissue engineering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381409</td>
			<td></td>
		</tr>
		<tr>
			<td>20 G angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381411</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform</td>
			<td>ASSI</td>
			<td>ASSI.ATK26426</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine Sulfate</td>
			<td>AdvaCare</td>
			<td>212-868</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar cords</td>
			<td>ASSI</td>
			<td>228000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine HCl</td>
			<td>Pharmaceutical, Inc</td>
			<td>42023-179-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilating Forceps</td>
			<td>Fine science tools (FST)</td>
			<td>18131-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotrachel tube</td>
			<td>Jorgensen Labs</td>
			<td>JO615X</td>
			<td>size from 6 to 15mm depending on the pig weight</td>
		</tr>
		<tr>
			<td>Ethilon 3-0 16 mm 3/8</td>
			<td>Ethicon</td>
			<td>MPVCP683H</td>
			<td></td>
		</tr>
		<tr>
			<td>Euthasol</td>
			<td>Virbac AH</td>
			<td>200-071</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution, USP, 100 units/mL</td>
			<td>BD PosiFlush</td>
			<td>306424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-704-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter</td>
			<td>ASSI</td>
			<td>ASSI.BPNS11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Metzenbaum scissors 180 mm</td>
			<td>B Braun</td>
			<td>BC606R</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfil blue</td>
			<td>Flow tech</td>
			<td>LMV-120</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfil dilution</td>
			<td>Flow tech</td>
			<td>LMV-112</td>
			<td>colored filing solution</td>
		</tr>
		<tr>
			<td>Monopolar knife</td>
			<td>ASSI</td>
			<td>221230C</td>
			<td></td>
		</tr>
		<tr>
			<td>N&#176;15 scalpel blade</td>
			<td>Swann Morton</td>
			<td>NS11</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnipaque</td>
			<td>General Electric</td>
			<td>4080358</td>
			<td>contrast product</td>
		</tr>
		<tr>
			<td>Perma-Hand Silk 3-0</td>
			<td>Ethicon</td>
			<td>A184H</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Ligaclip</td>
			<td>Ethicon</td>
			<td>MCM20</td>
			<td></td>
		</tr>
		<tr>
			<td>Stevens scissors 115 mm</td>
			<td>B Braun</td>
			<td>BC008R</td>
			<td></td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td>106-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylamed (xylazine)</td>
			<td>Bimeda</td>
			<td>200-529</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

This work on living animals was preceded by determining the saphenous perforasome on three cadaveric specimens (Figure 2). A colored filling solution was injected into the saphenous artery to opacify the specific vascular network coming from the artery. The solution is composed of 10 mL blue-colored glycerin agent mixed with 10 mL of the diluent agent (see Table of Materials). This generated a colored map of the skin vascularized by the saphenous artery and allowed drawing t...

Watch this Scientific Journal Video about A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies at JoVE.com

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

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

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

a-reliable-porcine-fascio-cutaneous-flap-model-for-vascularized

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2.3K Views.  Massachusetts General Hospital, Harvard Medical School. The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

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

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

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.

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.

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 ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and giant congenital nevi. Despite its widespread use, planning and executing an expansion protocol is challenging due to the difficulty in measuring the deformation imposed at each inflation step and over the length of the procedure. Quantifying the deformation fields is crucial, as the distribution of stretch over time determines the rate and amount of skin grown at the end of the treatment. In this manuscript, we present a method to study tissue expansion in order to gain quantitative knowledge of the deformations induced during an expansion process. This experimental protocol incorporates multi-view stereo and isogeometric kinematic analysis in a porcine model of tissue expansion. Multi-view stereo allows three-dimensional geometric reconstruction from uncalibrated sequences of images. The isogeometric kinematic analysis uses splines to describe the regional deformations between smooth surfaces with few mesh points. Our protocol has the potential to bridge the gap between basic scientific inquiry regarding the mechanics of skin expansion and the clinical setting. Eventually, we expect that the knowledge gained with our methodology will enable treatment planning using computational simulations of skin deformation in a personalized manner.

This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization.

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and ...

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.

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

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

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.

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

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.

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