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 immunosuppression, which leads to drug toxicity, opportunistic infections, and malignancies4,5,6.
Tissue-engineered acellular scaffolds are a potential solution to these limitations7. Acellular tissue scaffolds can be obtained using decellularization methods, which remove cellular material from native tissues while preserving the underlying extracellular matrix (ECM) microarchitecture. In contrast to the use of synthetic materials in tissue engineering, the use of biologically derived scaffolds offers a biomimetic ECM substrate that allows biocompatibility and the potential for clinical translation8. Following decellularization, the subsequent recellularization of scaffolds with recipient-specific cells can then generate functional, vascularized tissues with little to no immunogenicity9,10,11. By developing an effective protocol to obtain acellular tissues using perfusion decellularization techniques, a broad range of tissue types can be engineered. In turn, building on this technique allows the application to more complex tissues. To date, perfusion decellularization of vascularized soft tissues has been investigated using simple vascularized tissues such as a full thickness fasciocutaneous flap in rodent12, porcine13, and human models14, as well as porcine rectus abdominis skeletal muscle15. Additionally, complex vascularized tissues have also been perfusion decellularized as demonstrated in porcine and human ear16,17 models and human full-face graft models18.
Here, the protocol describes the decellularization of vascularized free flaps using biologically derived ECM scaffolds. We present the decellularization of three clinically relevant flaps: 1) the omentum, 2) the tensor fascia lata, and 3) the radial forearm, all of which are representative of workhorse flaps used routinely in reconstructive surgery and have not been previously examined in animal studies within the context of tissue decellularization. These bioengineered flaps offer a versatile and readily available platform that has the potential for clinical applications for use in the field of large soft tissue defect repair and reconstruction.
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
Figure 1: Fabrication of the perfusion bioreactor. The perfusion bioreactor consists of (A) a plastic polypropylene tissue chamber (B) with side holes drilled to accommodate perfusion tubing with air-and water-tight lid. (C) Stopcocks are attached to tubing to allow for the attachment of the perfusion tubing that carries decellularization agents from the detergent reservoir to waste in a single-pass fashion. (D) Compatible pump cassettes are used to connect the three-stop tubing to the peristaltic pump. Please click here to view a larger version of this figure.
2. Preparation of decellularization solutions
3. Procurement of porcine flaps
NOTE: This is a terminal procedure. One pig was used to procure all three flaps. Humanely euthanize the animal following the procurement of all flaps.
Figure 2: Procurement of three porcine vascularized flaps. (A) Omentum. The right (i) and left (ii) gastroepiploic arteries are cannulated in the omental flap (iii). (B) Tensor fascia lata. The pedicle of the flap (iv) is the ascending branch of the lateral femoral circumflex artery (v). (C) Radial forearm flap. Procurement of the radial forearm flap (vi) is based on the radial artery and the vena comitantes (vii) as the vascular pedicle (NOTE: Drapes were omitted for demonstration purposes). Scale bars: 3 cm. Please click here to view a larger version of this figure.
4. Setup of the decellularization system
Figure 3: Assembled perfusion decellularization system. (A) Schematic of the perfusion decellularization system. The inflow tubing carries perfusate from the detergent reservoir into the tissue chamber in a single-pass fashion with pressure sensor monitoring. The outflow tubing removes perfusate actively from the tissue chamber into the waste container. Black arrows denote the direction of perfusion flow. A peristaltic pump is used with the left pump to control inflow. Outflow is actively removed using a second peristaltic pump through the respective tubing. Figure created with BioRender.com. (B) Photograph of the perfusion decellularization system assembled on the benchtop with the inflow peristaltic pump (i) connected to the tissue chambers (ii) and then the outflow peristaltic pump (iii). The inflow perfusate pressure is monitored with an in-line pressure sensor (iv) prior to entering the tissue chamber. Here, three flaps are decellularized in parallel. Both the detergent and waste reservoirs are below the benchtop and not photographed. Please click here to view a larger version of this figure.
5. Decellularization of porcine flaps
Table 1: Summary of perfusion-decellularization protocol parameters. Please click here to download this Table.
6. Evaluation of decellularization
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 bioreactor was designed to allow for flap perfusion within an enclosed environment. Perfusion of flaps within the bioreactor was achieved in a single-pass fashion using two peristaltic pumps connected to the tissue chamber. The perfusion pressure was monitored with an in-line pressure sensor.
During decellularization, the duration of SDS exposure was dependent on the type of tissue being processed. With the described perfusion decellularization technique, the omentum, tensor fascia, and radial forearm flaps were decellularized with 0.05% SDS for 2 days, 3 days, and 5 days, respectively. Successful sterilization following decellularization was demonstrated by the absence of microbial colony growth after swabbing the flaps and culturing the swabs on agar plates for 14 days. Perfusion pressures were monitored at a 2 mL/min flow rate and ranged between 20-60 mmHg during all stages of decellularization for all three flaps. Upon the conclusion of decellularization, the flaps were flushed under manual control and demonstrated evidence of outflow from a venous cannula left to free drainage (Supplementary Video 1, Supplementary Video 2, Supplementary Video 3).
A total of 15 flaps were decellularized, with five replicates for each of the three tissue types. On examination, the gross morphology of native tissues appeared pink-colored (Figure 4A,E,I), whereas decellularized tissues were characteristically white/opaque in appearance (Figure 4C,G, K). Histological examination of native tissues with H&E shows the presence of blue nuclei (Figure 4B,F,J). In decellularized flaps, H&E staining showed a loss of cellular material with the absence of blue nuclear staining (Figure 4D,H,L), indicating an acellular tissue scaffold. Additional quantification of the DNA content in the five replicates showed a statistically significant decrease in DNA in the acellular scaffolds compared to native tissues by a Student's t-test for each flap (Figure 5). In the omentum, DNA decreased from 460 ng/mg ± 124 ng/mg dry tissue in the native flap to 25.8 ng/mg ± 5.90 ng/mg dry tissue in the decellularized flap (n = 5, p < 0.05). In the tensor fascia lata, DNA decreased from 297 ng/mg ± 68.2 ng/mg to 58.3 ng/mg ± 13.5 ng/mg between native and decellularized flaps, respectively (n = 5, p < 0.05). The radial forearm flap showed a DNA decrease from 1180 ng/mg ± 241 ng/mg in the native flap to 162 ng/mg ± 34.9 ng/mg in the decellularized flap (n = 5, p < 0.05).
Figure 4: Decellularization of three porcine flaps. Gross examination of the (A) native omentum, (E) tensor fascia lata, and (I) radial forearm flaps demonstrate the pink appearance of the flaps immediately after procurement. The histological staining of native tissues demonstrates clear hematoxylin staining of cellular nuclei with H&E (B,F,J). After decellularization, the (C) omentum, (G) tensor fascia lata, and (K) radial forearm appear grossly white and opaque. Histologically, the three decellularized flaps show an absence of nuclear staining with H&E (D,H,L). Scale bars: 200 µm. Please click here to view a larger version of this figure.
Figure 5: Quantification of DNA content in acellular scaffolds. Values are normalized to mg of dry scaffold mass. Fresh tissue samples from native, non-decellularized tissues (n = 5) and decellularized tissues (n = 5) were dried and weighed before overnight digestion in papain prior to quantification. Statistical testing used Student's t-tests with a significance (**) level defined as a p-value < 0.05. Please click here to view a larger version of this figure.
Supplementary Figure 1. Time-course examination of the progression of perfusion-decellularization of omentum using 0.05% sodium dodecyl sulfate over 5 days by gross examination (scale bars = 3 cm) and H&E histology (scale bars = 200 µm). Please click here to download this File.
Supplementary Figure 2. Time-course examination of the progression of perfusion-decellularization of tensor fascia lata using 0.05% sodium dodecyl sulfate over 5 days by gross examination (scale bars = 3 cm) and H&E histology (scale bars = 200 µm). Please click here to download this File.
Supplementary Figure 3. Time-course examination of the progression of perfusion-decellularization of radial forearm flap using 0.05% sodium dodecyl sulfate over 5 days by gross examination (scale bars = 3 cm) and H&E histology (scale bars = 200 µm). Please click here to download this File.
Supplementary Video 1. Representative manual perfusion of the decellularized omentum flap via the arterial cannula (pink) demonstrating outflow from the freely draining venous cannula (yellow). Please click here to download this Video.
Supplementary Video 2. Representative manual perfusion of the decellularized tensor fascia lata flap via the arterial cannula (pink) demonstrating outflow from the freely draining venous cannula (blue). Please click here to download this Video.
Supplementary Video 3. Representative manual perfusion of the decellularized radial forearm flap via the arterial cannula (blue) demonstrating outflow from the freely draining venous cannula (yellow). Please click here to download this Video.
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 with histological techniques (Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3). This method offers a straightforward approach that achieves decellularization within a short time frame and at a reasonable cost. In our experience, the decellularization and sterilization of porcine flaps take between 2-5 days, with longer duration decellularization needed for tissues with greater density (e.g., forearm skin at 5 days vs. omentum at 2 days). Furthermore, the low concentration of SDS used in this protocol was selected based on the rationale that a low concentration of SDS at 0.05% falls below the critical micellar concentration of SDS (approximately at 0.2%21) and, thus, allows the surfactant to effectively solubilize cell membranes while leaving important bioactive ECM components intact. The use of low concentration SDS in this protocol contrasts with the use of relatively higher concentrations of SDS (e.g., 1%) for perfusion-decellularization of vascularized fascio-cutaneous flaps as reported by previous authors13,22. High concentrations of SDS, while effective at decellularization, come at the cost of incurring detrimental effects on the ECM scaffold, such as GAG and growth factor depletion, as well as the denaturation of proteins such as collagen and vimentin23,24. Indeed, future work in the perfusion-decellularization of porcine vascularized flaps will benefit from additional characterization studies to examine the effects of the decellularization agent on the ECM at the structural and molecular levels and their implications for downstream recellularization25,26,27. Assays for ECM components such as collagen, elastin, or GAG content can be used to quantitatively assess changes to the ECM following decellularization. Furthermore, mechanical strength testing is a useful modality to study changes to the biomechanical properties after decellularization. Finally, assessment of the vascularity using techniques such as angiography or microCT scanning will also help to characterize the vascular architecture at a systemic level as a requisite to generating perfusable vascularized flaps28.
Several technical considerations should be noted with this protocol. Firstly, extreme care should be taken during flap handling and bioreactor assembly to prevent accidental decannulation, as recannulation in an ex vivo setting is comparatively more difficult. Secondly, during flap procurement, obtaining an intact flap with a perfusable and intact vascular pedicle is critical to successful decellularization. Care should be taken during procurement to ensure distal leak points in the scaffold are either clipped or hand-tied. This will help reduce leakage, which can cause incomplete perfusion of solutions through the flap vasculature. An initial period of perfusion with heparinized saline after procurement prevents the retention of blood clots and verifies a perfusable vasculature as evidenced by venous perfusate outflow. Decellularized tissues can also be flushed again upon completion of decellularization to check for any potential intravascular obstructions (e.g., from cell debris/air emboli) that may preclude flap perfusability. In our experience, decellularized flaps showed no intravascular resistance to syringe flushing following this protocol, while venous outflow could again be observed from the decellularized flap after flushing (Supplementary Video 1, Supplementary Video 2, Supplementary Video 3). This was indicative of a patent arterial to venous vascular loop with connecting capillaries that can be perfused.
Another consideration is made regarding the perfusion parameters during decellularization. In this protocol, a constant flow rate perfusion was selected with a target flow rate of 2 mL/min, which was within the range of the operating flow rates used in previously published decellularization studies of porcine flap tissues13,29. Furthermore, our decellularization system incorporates a real-time in-line pressure monitoring capability in order to monitor potential fluctuations in intravascular resistance over the course of perfusion; this method can help avoid high perfusion pressures arising from possible intravascular occlusion that may damage the microvascular ECM.
Finally, sterilization of the resultant scaffold is another important technical consideration. We incorporated a sterilization phase as the last step of the decellularization protocol to address incidental environmental contamination that may occur during decellularization. Flap sterility is especially important so that flaps can be used in the future for recellularization. The perfusion of 0.1% peracetic acid/4% EtOH as a sterilant has been previously reported by groups for sterilizing acellular scaffolds30,31 and was also found to be effective with this protocol. Sterility was verified by examining flap swab cultures on agar plates and noting absent growth after 14 days of incubation.
The customized perfusion bioreactor designed for the experiments is relatively cost-effective as well as simple and quick to assemble. Furthermore, all the components of this perfusion bioreactor are autoclavable and adaptable for recellularization work while maintaining the sterility of the flaps. The customized bioreactor circuit can be easily modified to permit open-circuit perfusion that can circulate cell culture media through a recellularized scaffold following cell seeding experiments. Nonetheless, a few limitations of the bioreactor system are worth mentioning. First, the use of two commercial pumps, while necessary to prevent inadvertent overflow of the system, particularly when large volumes of perfusate are used, detracts from the otherwise low cost of the perfusion set-up. Additionally, the low throughput of the current perfusion bioreactor system can present logistical challenges when needing to run numerous experimental replicates in parallel; comparatively higher throughput systems developed for porcine kidney decellularization32 may one day be adapted for porcine flap decellularization in order to alleviate these logistical challenges. Finally, while the developed bioreactor is simple to set up, human supervision and manual operation are still needed regularly and frequently to ensure no malfunction of the system occurs. Modifications to the bioreactor incorporating automation capabilities that can both monitor and readjust bioreactor performance with minimal or no human intervention can be pursued in order to advance perfusion-decellularization bioreactor technology in the future33,34.
The main application of decellularized flaps is to permit the subsequent recellularization of these acellular scaffolds with cell populations that can regenerate the vasculature. While much future research is required to determine the appropriate cell numbers, populations, seeding strategies, and bioreactor conditions to regenerate functional and viable vascularized tissue, this protocol represents the initial foundational work toward this goal. Future methods of recellularization will help establish strategies to engineer soft tissue flaps with an intact perfusable vascular network and contribute to potentially regenerating more complex composite tissues, such as extremity and facial allografts. These scaffolds can one day circumvent donor site morbidity and immunosuppression for patients undergoing large-scale soft tissue reconstruction, thereby improving their clinical outcomes and quality of life.
The authors have nothing to disclose.
None
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 | 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 | 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: 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 |