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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

  1. See Figure 1 for all t.......

Representative Results

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 .......

Discussion

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.......

Acknowledgements

None

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Materials

NameCompanyCatalog NumberComments
0.2 µm pore Acrodisk FilterVWRCA28143-310
0.9 % Sodium Chloride Solution (Normal Saline)BaxterJF7123
20 L Polypropylene CarboyCole-ParmerRK-62507-20
3-0 Sofsilk Nonabsorbable Surgical TieCovidien LS639
3-way StopcockCole-ParmerUZ-30600-04
Adson ForcepsFine Science Tools11027-12
Antibiotic-Antimycotic Solution, 100XWisent450-115-EL
Atropine Sulphate 15 mg/30mlRafter 8 Products238481
BD Angiocath 20-GaugeVWRBD381134
BD Angiocath 22-GaugeVWRBD381123
BD Angiocath 24-GaugeVWRBD381112
Calcium ChlorideSigma-AldrichC4901DNAse Co-factor
DNase I from bovine pancreasSigma-AldrichDN25
DNA assay (Quant-iT PicoGreen dsDNA Assay Kit)InvitrogenP7589
DPBS, 10XWisent311-415-CL without Ca++/Mg++
Halsted-Mosquito HemostatFine Science Tools13008-12
Heparin, 1000 I.U./mLLeo Pharma A/S453811
Ketamine Hydrochloride  5000 mg/50 mlBimeda-MTC Animal Health Inc.612316
Ismatec Pump Tygon 3-Stop TubingCole-ParmerRK-96450-40Internal Diameter:  1.85 mm
Ismatec REGLO 4-Channel PumpCole-Parmer78001-78
Ismatec Tubing CassettesCole-ParmerRK-78016-98
Isoflurane 99.9%, 250 mlPharmaceutical Partners of Canada Inc.2231929
LB Agar LennoxBioshop CanadaLBL406.500Sterility testing agar plates
Magnesium SulfateSigma-AldrichM7506DNAse Co-factor
Masterflex L/S 16 TubingCole-ParmerRK-96410-16
Midazolam 50 mg/10 mlPharmaceutical Partners of Canada Inc.2242905
Monopolar Cautery PencilValleylabE2100
Normal Buffered Formalin, 10%Sigma-AldrichHT501128
N°11 scalpel bladeSwann Morton303
Papain from papaya latexSigma-AldrichP3125
Peracetic AcidSigma-Aldrich269336
Plastic Barbed Connector for 1/4" to 1/8" Tube IDMcMaster-Carr5117K61
Plastic Barbed Tube 90° Elbow ConnectorsMcMaster-Carr5117K76
Plastic Quick-Turn Tube PlugsMcMaster-Carr51525K143Male Luer
Plastic Quick-Turn Tube SocketsMcMaster-Carr51525K293Female Luer
Punch Biopsy ToolIntegra Miltex3332
Potassium Chloride 40 mEq/20 mlHospira Healthcare Corporation37869
Povidone-Iodine, 10%Rougier833133
Serological Pipet, 2mLFisher Science13-678-27D
Snap Lid Airtight ContainersSnapLock142-3941-4
Sodium Dodecyl Sulfate PowderSigma-AldrichL4509
Surgical Metal Ligation Clips, SmallTeleflex001200
Stevens Tenotomy Scissors, 115 mm, straightB. BraunBC004R
TruWave Pressure Monitoring SetEdwards LifesciencesPX260

References

  1. Richardson, D., Fisher, S. E., Vaughan, D. E., Brown, J. S. Radial Forearm Flap Donor-Site Complications and Morbidity: A Prospective Study. Plastic and Reconstructive Surgery. 99 (1), 109-115 (1997).
  2. Edsander-Nord, &. #. 1. 9. 7. ;., Jurell, G., Wickman, M.

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PorcineVascularized FlapsPerfusion decellularizationBioreactorTissue EngineeringReconstructive SurgeryOmental FlapTensor Fascia Lata FlapProcurementCannulationPerfusionDecellularizationRecellularization

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