Figure 3A was created in BioRender.com.

Cadaveric male Lewis rats (300-430 g) obtained from the Toronto General Hospital Research Institute were used for all experiments. For all surgical procedures, sterile instruments and supplies were used to maintain aseptic technique (see the Table of Materials). All procedures were performed in compliance with guidelines from the Animal Care Committee at Toronto General Hospital Research Institute, University Health Network (Toronto, ON, Canada). A total of four hindlimbs were decellularized.
1. Presurgical preparation
<ol>
	<li>Prepare 50 mL of 5% heparinized saline. From a 50 mL saline bag, take out 2.5 mL of saline solution using a 5 mL syringe and discard. Using a 5 mL syringe, add 2.5 mL of heparin to the saline bag. Invert the saline bag to mix its contents.</li>
	<li>Place a cadaveric rat in a supine position under a blue pad. Shave the hindlimb and groin area circumferentially using an electric shaver and remove hair.</li>
	<li>Bring the rat to the surgical station and apply Povidone iodine scrub solution using a gauze to the hindlimb and groin area. Subsequently, apply 70% isopropyl alcohol to wipe off the scrub solution using gauze.</li>
	<li>Discard the blue pad from step 1.2 and change into new, sterile gloves.</li>
</ol>
2. Procurement of rat hindlimb
<ol>
	<li>Make a skin incision using a #10 surgical blade and a #3 blade runner along the inguinal ligament level, moving from a lateral to a medial direction (Figure 1A). Use Adson forceps to hold the surrounding skin to ensure a smooth incision.</li>
	<li>When the underlying fat is exposed, use blunt dissection to carefully dissect through the fat. Locate the superior epigastric vessels.</li>
	<li>Use microscissors to dissect proximally and expose the underlying femoral nerve, artery, and vein at the inguinal ligament level.</li>
	<li>Under a dissection microscope, identify the femoral vessels and dissect the artery and vein proximally using fine forceps to obtain sufficient length from the bifurcation points of the arterial network (Figure 1B).</li>
	<li>Ligate the femoral artery and vein separately using 6-0 sutures.</li>
	<li>Conduct circumferential dissection around the remainder of the hindlimb without disrupting the ligated femoral vessels.</li>
	<li>Transect the femoral bone mid-length using a bone cutter.</li>
	<li>To fully isolate the hindlimb, transect the ligated femoral vessels below the ligatures using microscissors.</li>
	<li>Cannulate the femoral artery using a 24 G angiocatheter under the dissection microscope. Use fine forceps to insert the cannula carefully. Flush with heparinized saline until clear outflow is observed from the femoral vein.</li>
	<li>Secure the cannula by tying one suture around the cannulated vessel and another suture distally around the cannula itself. Ensure the cannula is placed proximally to prevent blocking the bifurcation points.</li>
	<li>Submerge the procured hindlimbs in phosphate-buffered saline (PBS) until decellularization.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64069/64069fig01.jpg" /> 
Figure 1: Procurement of rat hindlimb. (A) Marking of skin incision at the inguinal ligament level from lateral to medial. (B) View of the femoral vein and the femoral artery, which have been dissected proximally toward the inguinal ligament, indicated by the dotted line. Abbreviations: L = lateral; M = medial; FV = femoral vein; FA = femoral artery. <a href="https://www.jove.com/files/ftp_upload/64069/64069fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Preparation of solutions
<ol>
	<li>In a 6 L glass flask, prepare a 5 L detergent reservoir of 0.25% sodium dodecyl sulfate (SDS) by dissolving 12.5 g of SDS powder in 5 L of ultrapure distilled water. Cover the opening of the flask with parafilm to seal it.</li>
	<li>In a 1 L glass jar, prepare 1 L of 0.25% SDS solution separately by dissolving 2.5 g of SDS in 1 L of ultrapure distilled water. Add a stir bar to mix the solution on a magnetic stirrer until all the SDS is dissolved.</li>
	<li>Prepare 1 L of 1x PBS wash solution with 1% Antibiotic-Antimycotic (AA) solution. In a 1 L flask, add 990 mL of 1x PBS solution and 10 mL of AA.</li>
	<li>Separately, in a 500 mL glass jar, prepare 1x PBS + 1% AA solution again using 495 mL of 1x PBS solution and 5 mL of AA.</li>
	<li>Prepare 200 mL of 1% peracetic acid (PAA)/4% ethanol (EtOH) solution. Prepare this solution under a fume hood.</li>
</ol>
4. Bioreactor and perfusion circuit construction
NOTE: Refer to Figure 2 for the configuration of the bioreactor and perfusion circuit throughout the listed steps.
<ol>
	<li>In a 500 mL chamber (plastic container), drill three holes (1/4 in) at the labeled locations in Figure 2: Port A is the inlet line, Port B is the replenishing line, and Port C is the outlet line. Remove and discard excess plastic. Spray and wipe the chamber with 70% ethanol.</li>
	<li>Cut three 5 cm long silicone tubes and insert one halfway into each port of the chamber. Connect a male Luer connector on the opening of Port A facing the inside of the chamber and a female Luer connector on the other end of the tube outside of the chamber.</li>
	<li>Connect a female Luer connector at Port B and Port C on the ends of the tubes facing out of the chamber.</li>
	<li>In an air-tight lid for the plastic container, drill one hole on the surface of the lid.</li>
	<li>Cut a 3 cm silicone tube and insert it into the hole of the lid. Ensure approximately 2 cm of the tube is located out of the lid, as shown in Figure 2.</li>
	<li>Sterilize both the chamber and the lid with the tubing.</li>
	<li>Cut two 30 cm silicone tubes using scissors. Connect a 1/16 in to a 1/8 in connector on one end of each tube.</li>
	<li>Separately, cut two 12 cm silicone tubes using scissors. Connect a 1/16 in to a 1/8 in connector on one end of both tubes. Connect a male Luer connector on the other end of one tube and a female Luer connector to the other tube.</li>
	<li>Sterilize all the tubing material from step 4.7 and step 4.8. Include two 3-stop pump tubings (1.85 mm) in the sterilization.</li>
	<li>Prepare three 3-way stopcocks, one syringe filter, two 1 mL serological pipettes, and one 10 mL syringe for the decellularization setup. Remove the filter from the 1 mL serological pipettes.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64069/64069fig02.jpg" /> 
Figure 2: Preparation of bioreactor and perfusion circuit construction. Apparatus shown of the perfusion circuit including (A) peristaltic pump and (B) corresponding cassettes for both inlet and outlet lines. (C, D) Silicone tubings of 12 cm and 30 cm are also shown with respective connectors. (E) Tubing for peristaltic pump (1.85 mm). Bioreactor chamber with labeled ports for (F) inflow, (G) replenishing port, and (H) outflow. (I) Bioreactor lid shown with ventilation port. <a href="https://www.jove.com/files/ftp_upload/64069/64069fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Decellularization of rat hindlimbs
<ol>
	<li>Place the sterilized chamber and screw on three single-use 3-way stopcocks at the inlet, outlet, and replenishing lines. Ensure the stopcock for the replenishing port is capped at its remaining two ports to prevent leakage.</li>
	<li>Attach the tubes made in step 4.8 to the stopcocks at the inlet and outlet lines.</li>
	<li>Connect peristaltic tubing to the tubes in the step above. Secure the cassette on the peristaltic tubing and place it on the peristaltic pump. Do not secure the cassettes with tubing in place yet.</li>
	<li>Connect one tube from step 4.7 to the end of the peristaltic tubing of the outlet line from the step above. Connect a 1 mL serological pipette on the other end. Suspend the end attached with a serological pipette in the waste reservoir flask.</li>
	<li>Repeat step 4.7 for the inlet line. Suspend the end of the tube attached to the serological pipette into the detergent reservoir. Seal the opening of the detergent reservoir flask with parafilm immediately. See Figure 3A,B for an overview of the decellularization circuit.</li>
	<li>Add 0.25% SDS from the 1 L glass jar (step 3.2) into the bioreactor chamber at the halfway level.</li>
	<li>Take the procured hindlimb using Adson forceps and suspend it into the bioreactor chamber carefully.</li>
	<li>Use two pairs of Adson forceps to guide the cannulated portion of the hindlimb to the inlet line. While holding the cannula with one pair of forceps, use the other pair of forceps to twist and secure the inlet line to the cannula. Ensure the cannula is not pulled or twisted to prevent decannulation.</li>
	<li>Once secured, add more 0.25% SDS from the 1 L glass jar to fully submerge the limb, as needed. Ensure the outlet port is also submerged in the bioreactor reservoir to ensure that consistent outflow is maintained (Figure 3C).</li>
	<li>Attach a single-use syringe filter to the ventilation port on the lid of the bioreactor chamber, referring to step 4.4 and step 4.5.</li>
	<li>Secure the lid on the bioreactor and ensure the chamber is sealed from all sides (Figure 3B).</li>
	<li>To remove air from tubing and prime the perfusion circuit, use a new, single-use 10 mL syringe to draw detergent from the detergent reservoir using the 3-way stopcock at the inlet line.</li>
	<li>Once drawn, use the same fluid to insert into the 3-way stopcock at the outlet line. Ensure that there is detergent present throughout the tubing at both the inlet and outlet lines.</li>
	<li>Press down and secure the cassettes with the tubing into the peristaltic pump. Turn the peristaltic pump on using the power button.
	<ol>
		<li>On the peristaltic pump screen, proceed to the second tab using the arrow key to set the perfusion rate for the first channel. Input flow rate as the mode of delivery and set the perfusion rate at 1 mL/min. Ensure the direction of flow is correct according to apparatus set-up. Repeat for the second channel.</li>
		<li>Calibrate the peristaltic pump to ensure the amount of fluid delivered through the inlet line and/or taken from the outlet line is flowing at a consistent rate between the two. Ensure that the tubing ID is set to 1.85 mm.</li>
	</ol>
	</li>
	<li>Begin decellularization via machine perfusion at 1 mL/min for both the inlet and outlet lines by pressing the power button on the keypad. Monitor and ensure that the flow is consistent and ongoing at both the inlet and outlet lines.</li>
	<li>Continue decellularization and monitor daily. Use the 1 L of 0.25% SDS (step 3.2) to replenish the bioreactor reservoir through the replenishing port, as needed. Look for a white, translucent appearance of the tissue, which will appear by day 5, indicating decellularization of the rat hindlimbs.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64069/64069fig03.jpg" /> 
Figure 3: Overview of perfusion decellularization bioreactor circuit of rat hindlimb. (A) Schematic representation of bioreactor perfusion circuit. Blue arrows indicate the direction of detergent and waste flow. (B) Overview of the decellularization circuit with bioreactor containing rat hindlimb. The SDS reservoir (left flask) leads into the peristaltic pump and into the inlet tubing of the bioreactor. The outflow is connected to the waste reservoir (right flask) through the peristaltic pump. (C) (I) Bioreactor containing rat hindlimb with inlet tubing connected to the cannulated femoral artery. (II) Replenishing port located in the corner for perfusing detergent. (III) Outflow tubing suspended in suspension reservoir. Abbreviation: SDS = sodium dodecyl sulfate. <a href="https://www.jove.com/files/ftp_upload/64069/64069fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Post-decellularization washing and sterilization
<ol>
	<li>Following the confirmation of decellularization, replace the detergent reservoir with the 1x PBS + 1% AA reservoir. Seal the opening of the flask with parafilm. Begin 1x PBS + 1% AA perfusion at 1 mL/min and continue for 2 days.</li>
	<li>Replace the 1x PBS + 1% AA reservoir with 200 mL of 0.1% PAA/4% EtOH reservoir and begin perfusion at 1 mL/min for 2 h.</li>
	<li>Disconnect the hindlimb from the inlet line using two pairs of sterile Adson forceps, with one pair of forceps to twist the inlet line and the other holding the cannula. Ensure that the cannula is not pulled to prevent decannulation.</li>
	<li>Store the limb in a 500 mL glass jar containing 1x PBS + 1% AA at 4 &#176;C until further use.</li>
</ol>

<ol>
	<li>Kueckelhaus, 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=Vascularized+composite+allotransplantation:+Current+standards+and+novel+approaches+to+prevent+acute+rejection+and+chronic+allograft+deterioration.">Vascularized composite allotransplantation: Current standards and novel approaches to prevent acute rejection and chronic allograft deterioration.</a> Transplant International. 29 (6), 655-662 (2016).</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=Bioengineering+a+human+face+graft:+The+matrix+of+identity.">Bioengineering a human face graft: The matrix of identity.</a> Annals of Surgery. 266 (5), 754-764 (2017).</li><li>Zhang, Q., 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=Decellularized+skin/adipose+tissue+flap+matrix+for+engineering+vascularized+composite+soft+tissue+flaps.">Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps.</a> Acta Biomaterialia. 35, 166-184 (2016).</li><li>Londono, R., Gorantla, V. 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The authors have no conflicts of interest to declare.

Rat hindlimbs are useful as experimental models in VCA5. Tissue engineering of acellular scaffolds represents the first step in addressing the shortcomings of long-term immunosuppression regimens associated with VCA. The use of composite grafts poses an added challenge given the presence of multiple tissues, each having unique functional, immunogenic, and structural properties. The present protocol shows a successful method for obtaining acellular composite rat hindlimbs. These scaffolds can be fu...

VCA is an emerging option for patients requiring complex surgical reconstruction. Traumatic injuries or tumor resections result in volumetric tissue loss that can be difficult to reconstruct. VCA offers the transplantation of multiple tissues such as the skin, bone, muscle, nerves, and vessels as a composite graft from a donor to a recipient1. Despite its promising nature, VCA is limited due to long-term immunosuppressive regimens. Lifelong use of such drugs results in increased risk for opportunistic infections, malignancies, and end-organ toxicity1,2,3. To help reduce and/or eliminate the need for immunosuppression, tissue-engineered scaffolds using decellularization approaches for VCA show great promise.
Tissue decellularization entails retaining the extracellular matrix structure while removing the cellular and nuclear contents. This decellularized scaffold can be repopulated with patient-specific cells4. However, preserving the ECM network of composite tissues is an added challenge. This is due to the presence of multiple tissue types with varying tissue densities, architectures, and anatomic locations within a scaffold. The present protocol offers a surgical technique and a decellularization method for a rat hindlimb. This is a proof-of-concept model for applying this tissue engineering technique to composite tissues. This can also prompt subsequent efforts to regenerate composite tissues through recellularization.

<table><tbody><tr><td>0.9% Sodium Chloride Injection USP 50 mL</td><td>Baxter Corporation</td><td>JB1308M</td><td></td></tr><tr><td>1 mL Disposable Serological Pipets</td><td>VWR</td><td>75816-102</td><td></td></tr><tr><td>10 cc Disposable Syringes</td><td></td><td></td><td>Obtained from Research Institution</td></tr><tr><td>3-way Stopcock</td><td></td><td></td><td>Obtained from Research Institution</td></tr><tr><td>5cc Disposable Syringes</td><td></td><td></td><td>Obtained from Research Institution</td></tr><tr><td>70% Isopropyl Alcohol</td><td></td><td></td><td>Obtained from Research Institution</td></tr><tr><td>Acrodisc Syringe Filter 0.2 &amp;micro;m</td><td>VWR</td><td>CA28143-310</td><td></td></tr><tr><td>Adson Forceps, Straight</td><td>Fine Science Tools</td><td>11006-12</td><td></td></tr><tr><td>Angiocatheter 24 G 19 mm (&amp;frac34;&amp;rdquo;)&amp;nbsp;</td><td>VWR</td><td>38112</td><td></td></tr><tr><td>Antibiotic-Antimycotic Solution (100x) 100 mL</td><td>Multicell</td><td>450-115-EL</td><td></td></tr><tr><td>Bone Cutter</td><td>Fine Science Tools</td><td>12029-12</td><td></td></tr><tr><td>Connectors for&amp;nbsp; 1/16&quot; to 1/8&quot; Tubes</td><td>McMasterCarr</td><td>5117K52</td><td></td></tr><tr><td>Female Luer to barbed adapter (PVDF) - 1/8&quot; ID</td><td>McMasterCarr</td><td>51525K328</td><td></td></tr><tr><td>Fine Forceps</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>Fine Forceps with Micro-Blunted Tips</td><td>Fine Science Tools</td><td>11253-20</td><td></td></tr><tr><td>Heparin Sodium Injection 10,000 IU/10 mL</td><td>LEO Pharma Inc.</td><td>006174-09</td><td></td></tr><tr><td>Male Luer to barbed adapter (PVDF) - 1/8&quot; ID</td><td>McMasterCarr</td><td>51525K322</td><td></td></tr><tr><td>Micro Needle Holder</td><td>WLorenz</td><td>04-4125</td><td></td></tr><tr><td>Microscissors</td><td>WLorenz</td><td>SP-4506</td><td></td></tr><tr><td>Peracetic Acid</td><td>Sigma Aldrich</td><td>269336-100ML</td><td></td></tr><tr><td>Peristaltic Pump, 3-Channel</td><td>Cole Parmer</td><td>RK-78001-68</td><td></td></tr><tr><td>Phosphate Buffered Saline 1x 500 mL</td><td>Wisent</td><td>311-425-CL</td><td></td></tr><tr><td>Povidone Surgical Scrub Solution</td><td></td><td></td><td>Obtained from Research Institution</td></tr><tr><td>Pump Tubing, 3-Stop, Tygon E-LFL</td><td>Cole Parmer</td><td>RK-96450-40</td><td></td></tr><tr><td>Pump Tubing, Platinum-Cured Silicone</td><td>Cole Parmer</td><td>RK-96410-16</td><td></td></tr><tr><td>Scalpel Blade - #10</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>Scalpel Handle - #3&amp;nbsp;</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate Reagent Grade: Purity: &gt;99%, 1 kg</td><td>Bioshop</td><td>SDS003.1</td><td></td></tr><tr><td>Surgical Suture #6-0</td><td>Covidien</td><td>VS889</td><td></td></tr></tbody></table>

procurement and decellularization of rat hindlimbs using an ex vivo perfusion-based bioreactor for vascularized composite allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

The procurement protocol was successful in isolating and cannulating the common femoral arteries for subsequent perfusion steps. The representative dissection images in Figure 1A,B show the incision location and exposure of the femoral vessels with sufficient distance from the bifurcation points. Figure 2 shows the apparatus required for preparing the bioreactor and perfusion circuit.&#160;The endpoint of decellularization was determined by obse...

Watch this Scientific Journal Video about Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation at JoVE.com

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...

procurement-decellularization-rat-hindlimbs-using-an-ex-vivo

Research

JoVE Journal

Bioengineering

2.1K Views.  University Health Network, Toronto General Hospital. We hebben een klein diermodel opgesteld voor decellularisatie van een ledemaat. Dit is nuttig voor het faciliteren van proof of concept onderzoek naar de en recellularisatie van gevasculariseerde composietweefsels. Immunosuppressieregimes zijn een van de belangrijkste beperkingen bij gevasculariseerde composiet allotransplantatie.Weefselimmunogeniteit kan worden verminderd met behulp van decellularisatie. Maak om te beginnen een huidincisie met behulp van een nummer 10 chirurgisch mes ...

Video: Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Immunology and Infection

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Medicine

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

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 ...

Partial Heterotopic Hindlimb Transplantation Model in Rats

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities after a complex tissue defect. Face and hand transplantations have changed disfigured patients' lives, giving them a new aesthetic and functional social organ. Despite promising outcomes, VCA is still underperformed due to life-long immunosuppression comorbidities and infectious complications. The rat is an ideal animal model for in vivo studies investigating immunological pathways and graft rejection mechanisms. Rats are also widely used in novel composite tissue graft preservation techniques, including perfusion and cryopreservation studies. Models used for VCA in rats must be reproducible, reliable, and efficient with low postoperative morbidity and mortality. Heterotopic limb transplantation procedures fulfill these criteria and are easier to perform than orthotopic limb transplants. Mastering rodent microsurgical models requires solid experience in microsurgery and animal care. Herein is reported a reliable and reproducible model of partial heterotopic osteomyocutaneous flap transplantation in rats, the postoperative outcomes, and the means of prevention of potential complications.

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.

This protocol presents a robust, reproducible model of vascularized composite allotransplant (VCA) geared toward simultaneous study of immunology and functional recovery. The time invested in meticulous technique in a right mid-thigh hind limb orthotopic transplant with hand sewn vascular anastomoses and neural coaptation yields the ability to study functional recovery.

Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current ...

Preparation of Decellularized Kidney Scaffolds in Rats

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs ...

Vascularized Composite Hand Allograft Procurement and Preparation for Distal and Proximal Forearm Allotransplantation: A Stepwise Approach

Upper limb amputations represent a real medical and surgical challenge. The ideal treatment should restore function, sensation, and body image. At present, neither traditional reconstructions nor prostheses meet all these criteria. However, vascularized composite allografts offer a unique option for restoring form and function satisfactorily despite harmful immunosuppression.
Ideally, donor tissue is procured in excess to repair without tension. The donor upper limb is procured through a fish-mouth incision at the mid-arm level. Then, the brachial artery and vein, median, ulnar, and radial nerve are located and dissected. The biceps, brachioradialis, and triceps muscles are sectioned, and then an osteotomy of the humerus is performed above the elbow. For distal forearm transplantation, the donor upper limb can also be procured below the elbow by disarticulating through the joint.
The brachial artery is cannulated, and the graft is irrigated with a preservative solution. Preparation of the graft then begins with two incisions, anterior and posterior, to raise two lateral skin flaps. A medial skin flap exposes the basilic vein, medial antebrachial cutaneous nerve, medial epicondylar muscles, ulnar nerve, median nerve, brachial artery, and vein. A lateral skin flap, including the cephalic vein, the lateral antebrachial cutaneous nerve, the radial nerve up to its division, the brachioradialis, and the lateral epicondylar muscles, completes graft preparation. In the case of transplantation, cutting guides are attached to the posterior surface of the two forearm bones to perform the osteotomies.
This protocol presents a systematized procedure for procuring and preparing a vascularized forearm composite allograft to ensure optimal results and minimize tissue damage during procurement.

Here, we present a protocol for procuring and preparing vascularized composite hand allografts during distal or proximal forearm transplantation.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

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