Animal use
Yorkshire pigs (3040kg; age approximately 12weeks old) were used for all decellularization and recellularization experiments. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University Health Network and Toronto General Hospital Research Institute. Humane care was provided to all animals in accordance to the Principles of Laboratory Animal Care defined by the National Society for Medical Research and the Guide for the Care of Laboratory Animals issued by the National Institutes of Health. Reporting of use of experimental animals in this study followed recommendations specified by the ARRIVE guidelines.
Pigs were fasted for 12h prior to surgery. Sedation was achieved with ketamine (20mg/kg IM), atropine (0.04mg/kg IM) and midazolam (0.3mg/kg IM). Anesthesia was induced by inhalation of 5% isoflurane through a mask at a flow rate of 22 to 44mL/kg/min to facilitate peripheral line insertion and intubation. Anesthesia was maintained with isoflurane (0.5 to 2%). Pigs were intubated with an appropriate endotracheal tube (78mm) and ventilated to a tidal volume of 8mL/kg, positive end-expiratory pressure of 5cm H2O, FiO2 of 0.5 and respiratory rate of 14 breaths per minute. Pigs were prepped and draped in the usual sterile fashion prior to flap procurement. Surgical procedure for porcine omentum and TFL flaps procurement were as previously described30. Briefly, the omental flap was procured by midline laparotomy and the left gastroepiploic artery and vein was used as the dominant vascular conduit. The right gastroepiploic vessels were ligated to prevent perfusion flow-through.
The TFL flap was procured with pigs in the lateral decubitus position. The main vascular pedicle was defined by the ascending branch of the lateral circumflex femoral artery and veins. The overlying skin island was removed to produce a pure fascial flap. Following flap detachment, the vascular pedicle was cannulated with 2022 G Angiocath (Becton Dickenson) under direct vision and flushed with 20 U/mL heparin sodium (LEO Pharma, Denmark) in 0.9% normal saline and transported under sterile conditions to the lab.
Porcine flaps were perfusion-decellularized using low-concentration SDS followed by DNase (Sigma Aldrich) reconstituted to a concentration of 10mg/mL, as previously described30. Cannulated flaps were each connected to a perfusion system to allow antegrade perfusion via the arterial inlet at 2ml/min, in which solutions: 0.05% SDS followed by 0.1mg/mL deoxyribonuclease (DNase) were perfused through the flap vasculature with 1phosphate buffered saline (PBS) perfusion in between to remove residual detergent. Flaps were sterilized in 0.1% paracetic acid (PAA) / 4% ethanol (EtOH) (Sigma Aldrich) and then washed in 1PBS prior to recellularization. As described previously30, omental and TFL flaps were perfused with SDS for 2 and 3days, respectively. Following SDS perfusion, flaps were washed with PBS for 24h and then perfused with DNase for 2h, PBS for again for 24h, and finally PAA/EtOH for 3h. With the exception of DNase, each step included an exchange of the submersion fluid to match the given perfusate. For the DNase step, flaps were submerged in fresh PBS.
Commercially available HUVECs (American Type Culture Collection/ATCC, USA) were cultured in EGM-2 (Lonza, Switzerland) supplemented with SingleQuots (Lonza) of Growth Supplements including: FBS 2%, hEGF, hydrocortisone, Gentamicin/Amphotericin-B, VEGF, hFGF-B, R3-IGF-1, ascorbic acid, and heparin (concentrations proprietary). Commercially obtained human bone-marrow derived MSCs (Promocell, Germany) were cultured in MSCGM (Promocell) containing proprietary media supplement and 5% FBS. HMSCs and HUVECs between passage 4 and 6 were used for recellularization. Both cell types were verified for correct functional and phenotype expression. HUVECs expressed CD31/VE-Cadherin using flow cytometry and were functionally capable to undergo angiogenesis. MSCs were CD90/73/44 positive and CD34/45/11b negative using flow cytometry and capable of undergoing trilineage differentiation (Supplementary Fig.1). These findings were consistent with the minimal criteria to define MSCs according to the International Society for Cellular Therapy Criteria47.
All cells were maintained in 150 cm2 dishes until reaching 90% confluency (resulting in approximately 50,000 cells/cm2). Cells were detached from culture vessels with 0.25% trypsinEDTA solution (Gibco) prior to recellularization. Cell media was replaced every other day, and the cultures were maintained in a humidified 95% air/5% CO2 incubator at 37C.
A closed-system bioreactor was set up in an incubator for recellularization within the flap scaffold matrix. We used a modified airtight snap-lid container, previously used for decellularization with a closed-circuit L/S-16 (Masterflex, Fisher Scientific) silicone tubing. The end of the tubing external to the tissue chamber was fitted with a female Luer thread-style panel (Cole-Parmer), which connected to a 3-stop tubing compatible with peristaltic pump (Ismatec, Cole-Parmer) tubing cassette as previously used for perfusion-decellularization. The opposite end of tubing was reconnected to the second port from the tissue chamber to allow closed-loop circulation of medium from tissue chamber into the flap via the arterial cannula at a flow rate of 2mL/min. Just proximal to the tissue chamber, silicone tubing was connected to a three-way stopcock (Baxter, USA). The chamber was filled with 200mL of EGM-2 media, which was primed through the tubing to remove air bubbles. Decellularized flaps were perfused with EGM-2 at 2mL/min in conventional cell culture incubator at standard conditions (95% air/5% CO2) overnight before cell seeding to equilibrate flaps with culture medium.
Cell seeding was performed as follows: HUVECs and human bone-marrow derived MSCs were lifted from tissue culture plastic with 0.25% trypsin and centrifuged at 500g for 5min. The resultant cell pellet was resuspended in 10mL media, strained with 75m pore mesh, and counted via automated hemocytometer (Vi-Cell XR, Beckman Coulter). A total of 8107 cells, divided equally with 4107 HUVEC co-cultured with 4107 MSCs, were used for recellularization of each scaffold. A combined cell suspension of the two cells were slowly manually injected into the vascular arterial inlet through a three-way stopcock. Following the introduction of cells, flaps were placed in a standard cell culture incubator for 2h of static culture to allow cell attachment. Afterwards, perfusion-culture was initiated with the peristaltic pump (Ismatec, Cole-Parmer) running at 2mL/min for 6days. Media passed through the flap was recovered back into the reservoir using a separate pump channel that drained the bioreactor at an equal rate to the perfusion, allowing for recycling and reuse. Media was exchanged every other day for fresh EGM-2. A total of 750mL of culture medium was used over 6days for each flap.
Native, decellularized, and recellularized tissues were biopsied near the distal margin of the flap, fixed in 10% formalin (Fisher Scientific), embedded in paraffin, and sliced into 5m sections on microtome (Leica Biosystems). Slides of the paraffin-embedded samples were processed for histological and IHC staining. Histologic staining was performed on xylene-deparaffinized slides with the following stains: H&E (Sigma Aldrich), Massons Trichrome (American MasterTech Scientific), and Verhoeff Van Gieson Elastin Stain (Abcam).
For IHC, heat induced antigen retrieval was done with citrate buffer (pH 6.0; Thermo Fisher Scientific) in a 95C autoclave for 10min. Endogenous peroxidases were blocked with a peroxide block (Cardinal Health), and nonspecific binding was blocked with Dako Serum-Free Protein-Block (Agilent). Sections were incubated with the primary antibodies at 4C overnight with dilutions as follows: rabbit polyclonal anti-Collagen IV (Abcam, ab6586, 1:300), rabbit polyclonal anti-Fibronectin (Abcam, ab23751; 1:400); and rabbit polyclonal anti-Laminin (Abcam, ab11575, 1:400) and anti-CD31 (Abcam, ab28364, 1:50) at 4C overnight. Slides were washed three times in PBS with 0.1% Tween and goat anti rabbit IgG HRP-conjugated secondary antibody (ImmPRESS Peroxidase Polymer Reagent, Vector Laboratories) was applied for 30min. Slides were again washed thrice in PBS-Tween and then diaminobenzidine solution (Vector Laboratories) applied for 10min. Slides were counterstained with hematoxylin. After staining, all slides were dehydrated in ethanol to xylene exchange, mounted and imaged on Aperio CS2 Slide Scanner (Leica Biosystems).
Immunofluorescence staining was performed using paraffin embedded sections cut to 5m thickness and deparaffinized using xylene and rehydrated in serial dilutions of ethanol. Tissue sections in were incubated in antigen retrieval buffer (10mM citrate buffer, pH 6.0) at 95C for 10min in an autoclave. Tissue sections were then blocked with 5% blocking serum (goat serum) in 1% bovine serum albumin (BSA) before adding primary antibody. Slides were then incubated with primary antibodies for VE-Cadherin (Abcam, ab33168, 1:100) and vimentin (Abcam, ab92547, dilution 1:200) diluted in 1% BSA at 4C overnight. After washing three times with PBS-Tween, slides were then incubated for 1h at RT in the secondary antibody goat anti-rabbit IgG conjugated with AlexaFluor 647 (Thermo Fisher Scientific, 1:500). Finally, slides were washed three times with PBS-Tween in the dark and counterstained with DAPI (Abcam; 1:5000). Negative controls were used by replacing the primary antibody with the corresponding isotype (IgG) of the primary antibody. Images were taken on a Leica SP8 confocal microscope with LAS X software (Leica Biosystems) installed.
Tissue pieces (~3040mg) were obtained by punch biopsy tool and dried in 60C oven overnight. Dried tissue pieces were digested in papain solution at 65C for 18h. Corresponding native flap tissues were dried and digested in parallel as controls. Papain (Sigma Aldrich, 16 units/mg protein) 1530mg/mL stock was solubilized to working concentration of 0.1mg/ml in 0.1M phosphate buffer (pH 6.0), with 5mM cysteine hydrochloride (Sigma Aldrich), and 5mM EDTA (Sigma Aldrich). The lysates were used for detection of sulfated glycosaminoglycan (sGAG) and DNA content. The Blyscan Sulfated GAG Assay kit (Biocolor) was used to measure sGAG according to manufacturers instruction. Briefly, tissue specimen lysates were mixed with Blyscan Dye Reagent to bind the GAG for 1h at room temperature. The GAG-dye complex was then collected by centrifugation at 10,000g. After the supernatant was removed and the tube drained, Dissociation Reagent was added and 100l of analyte solution was transferred to a 96-well plate. Absorbance against the background control was obtained at a wavelength of 656nm with a SpectraMax spectrophotometer (Molecular Devices). GAG amount was interpolated from a standard curve (05g) using a known GAG standard provided in the kit. Final GAG content was standardized to the total dry tissue mass (mg) used for assay.
For DNA content quantitation, the tissue lysate following papain digestion (above) was used. The Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) was used to measure DNA content according to manufacturers instruction. Fluorescence reading (excitation: 485nm and emission: 528nm) was taken on a plate reader (Cytation 5, Biotek), and the absolute amount of DNA (ng) was quantified against a lambda DNA standard curve (01000ng) provided by the manufacturer; final DNA content was standardized to total dry tissue mass (mg) used for assay.
All statistical analysis was performed using GraphPad Prism, version 9.0 (GraphPad, Inc.). Statistical analyses was conducted with multiple unpaired t test with a significance level of p<0.05. Values are presented as mean, with S.D. unless stated otherwise.
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