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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'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.
Traumatic injury and tumor removal can lead to large and complex soft tissue defects. These defects can impair patient quality of life, cause loss of function, and result in permanent disability. While techniques such as autologous tissue flap transfer have been commonly practiced, issues with flap availability and donor site morbidity are major limitations1,2,3. Vascularized composite allotransplantation (VCA) is a promising alternative that transfers composite tissues, e.g., muscle, skin, vasculature, as a single unit to recipients. However, VCA requires long-term immunosup....
All procedures involving animal subjects have been approved by the University Health Network Institutional Animal Care and Use Committee (IACUC) and are performed in accordance with University Health Network Animal Resource Centre protocol and procedures and Canadian Council on Animal Care Guidelines. Five Yorkshire pigs (35-50 kg; age approximately 12 weeks old) were used for all experiments.
1. Perfusion bioreactor fabrication
This protocol to decellularize vascularized porcine flaps relies on the perfusion of an ionic-based detergent, SDS, through the flap vasculature in a customized perfusion bioreactor. Prior to decellularization, three vascularized flaps in a porcine model were procured and cannulated according to their main supplying vessels. The flaps were immediately flushed after procurement in order to maintain a patent, perfusable vasculature to allow for successful decellularization. Using airtight snap-lid containers, a customized .......
The proposed protocol uses the perfusion of low concentration SDS to decellularize a range of porcine-derived flaps. With this procedure, acellular omentum, tensor fascia lata, and radial forearm flaps can be successfully decellularized using a protocol that favors low concentration SDS. Preliminary optimization experiments have determined that SDS at a low concentration (0.05%) between 2 days to 5 days is capable of removing cellular material for the omentum, tensor fascia lata, and radial forearm flap when analyzed wit.......
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....Name | Company | Catalog Number | Comments |
0.2 µm pore Acrodisk Filter | VWR | CA28143-310 | |
0.9 % Sodium Chloride Solution (Normal Saline) | Baxter | JF7123 | |
20 L Polypropylene Carboy | Cole-Parmer | RK-62507-20 | |
3-0 Sofsilk Nonabsorbable Surgical Tie | Covidien | Â LS639 | |
3-way Stopcock | Cole-Parmer | UZ-30600-04 | |
Adson Forceps | Fine Science Tools | 11027-12 | |
Antibiotic-Antimycotic Solution, 100X | Wisent | 450-115-EL | |
Atropine Sulphate 15 mg/30ml | Rafter 8 Products | 238481 | |
BD Angiocath 20-Gauge | VWR | BD381134 | |
BD Angiocath 22-Gauge | VWR | BD381123 | |
BD Angiocath 24-Gauge | VWR | BD381112 | |
Calcium Chloride | Sigma-Aldrich | C4901 | DNAse Co-factor |
DNase I from bovine pancreas | Sigma-Aldrich | DN25 | |
DNA assay (Quant-iT PicoGreen dsDNA Assay Kit) | Invitrogen | P7589 | |
DPBS, 10X | Wisent | 311-415-CL | Â without Ca++/Mg++ |
Halsted-Mosquito Hemostat | Fine Science Tools | 13008-12 | |
Heparin, 1000 I.U./mL | Leo Pharma A/S | 453811 | |
Ketamine Hydrochloride 5000 mg/50 ml | Bimeda-MTC Animal Health Inc. | 612316 | |
Ismatec Pump Tygon 3-Stop Tubing | Cole-Parmer | RK-96450-40 | Internal Diameter:Â 1.85 mm |
Ismatec REGLO 4-Channel Pump | Cole-Parmer | 78001-78 | |
Ismatec Tubing Cassettes | Cole-Parmer | RK-78016-98 | |
Isoflurane 99.9%, 250 ml | Pharmaceutical Partners of Canada Inc. | 2231929 | |
LB Agar Lennox | Bioshop Canada | LBL406.500 | Sterility testing agar plates |
Magnesium Sulfate | Sigma-Aldrich | M7506 | DNAse Co-factor |
Masterflex L/S 16 Tubing | Cole-Parmer | RK-96410-16 | |
Midazolam 50 mg/10 ml | Pharmaceutical Partners of Canada Inc. | 2242905 | |
Monopolar Cautery Pencil | Valleylab | E2100 | |
Normal Buffered Formalin, 10% | Sigma-Aldrich | HT501128 | |
N°11 scalpel blade | Swann Morton | 303 | |
Papain from papaya latex | Sigma-Aldrich | P3125 | |
Peracetic Acid | Sigma-Aldrich | 269336 | |
Plastic Barbed Connector for 1/4" to 1/8" Tube ID | McMaster-Carr | 5117K61 | |
Plastic Barbed Tube 90° Elbow Connectors | McMaster-Carr | 5117K76 | |
Plastic Quick-Turn Tube Plugs | McMaster-Carr | 51525K143 | Male Luer |
Plastic Quick-Turn Tube Sockets | McMaster-Carr | 51525K293 | Female Luer |
Punch Biopsy Tool | Integra Miltex | 3332 | |
Potassium Chloride 40 mEq/20 ml | Hospira Healthcare Corporation | 37869 | |
Povidone-Iodine, 10% | Rougier | 833133 | |
Serological Pipet, 2mL | Fisher Science | 13-678-27D | |
Snap Lid Airtight Containers | SnapLock | 142-3941-4 | |
Sodium Dodecyl Sulfate Powder | Sigma-Aldrich | L4509 | |
Surgical Metal Ligation Clips, Small | Teleflex | 001200 | |
Stevens Tenotomy Scissors, 115 mm, straight | B. Braun | BC004R | |
TruWave Pressure Monitoring Set | Edwards Lifesciences | PX260 |
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