This study was conducted in compliance with the Animal Welfare Act, the implementing Animal Welfare Regulations and in accordance with the principles of the Guide for the Care and Use of Laboratory Animals. The study was conducted according to a research protocol reviewed and approved by Absorption Systems Institutional Animal Care and Use Committee (IACUC) for the designated Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) accredited research facility. Twelve female pure RD pigs (40–60 kg) were purchased from Fresno State Swine Unit (Fresno, CA, USA). Animals were identified by ear tags, cage cards, and/or color identifiers in the animal’s back. The study animals were observed at least twice daily for signs of illness or distress, and any such observations were promptly reported to the veterinarian staff.
Anesthesia and analgesia
Animals were fasted overnight prior to the use of anesthesia. Animals were anesthetized via an intramuscular injection of a cocktail containing ketamine (20 mg/kg), xylazine (2 mg/kg), and atropine (0.04 mg/kg). Upon loss of responsiveness and spontaneous movement, the animal was intubated and maintained on isoflurane (1 to 5%) in oxygen (1 to 3 L/min). Animals were ventilated as necessary. Heart rate, respiratory rate, oxygen saturation (SPO2), inspired/end tidal CO2, body temperature, and anesthetic depth were continuously monitored.
Porcine model of deep-partial thickness excisional wounds
Following antiseptic preparation, two pairs of bilateral excisional cutaneous wounds (approximately 2 mm depth; 7.6 cm × 7.6 cm) were created in the flanks of each animal using a Zimmer dermatome set at a depth of 0.02 inch (0.5 mm) for four consecutive passes. Excisional biopsies (approximately 2 × 2 cm) were collected from each harvested skin layer in order to verify the thickness of each excision. In order to be acceptable for inclusion in the study each wound had to have a cumulative depth of 1.8–2.7 mm with no evidence of injury extending into subcutaneous adipose tissue (characteristic of full-thickness injury). In order to ensure that each pair of wounds within the same animal was matched at baseline, any wound pair for which the difference in cumulative wound depth was greater than 0.4 mm was excluded from the study. Only one (1) wound pair out of 12 did not meet these criteria. A multilayer dressing was applied to protect the wounds from infection and mechanical damage; Layer 1 (contact with the wound) Mepilex® Foam Dressing (Mölnlycke Health Care, Gothenburg, Sweden); layer 2 Ioban2™ (3 M Corporation, Maplewood, MN, USA), layer 3 stockinet (cotton wrap); layer 4 protective jacket (Sullivan Supplies, Houston, TX, USA). Bandaging was changed once every 5–7 days up to approximately week 4–5 when wounds were fully epithelialized. Three animals were euthanized at 2 weeks post-injury and a further three at 2 months post-injury for assessment of histology and expression of inflammatory growth factors. The remaining six animals were maintained up to 6 months post-injury.
Adipose-derived regenerative cell (ADRC) isolation
Following wound injury (within 2 hours), adipose tissue (30–50 g) was excised from the inguinal fat region of the animals (while under general anesthesia) and isolated as previously described using Celase® enzyme [20,21,22]. ADRC yield and viability was determined using the NucleoCounter® NC‐100™ (ChemoMetec, Lillerød, Denmark), as previously described [22, 25].
Flow cytometric characterization of ADRCs
Freshly isolated ADRCs were resuspended in staining buffer [0.2% bovine serum albumen (BSA) in phosphate-buffered saline (PBS); BD Biosciences, San Diego, CA, USA] and then stained with the following antibodies CD45-FITC (clone K252.1E4; Bio-Rad, Hercules, CA, USA), CD31-PE (clone LCI-4; Bio-Rad), CD90-PerCP-Cy™5.5 (clone 5E10; BD Biosciences, San Jose, CA, USA) and CD146-PE (P1H12; BD Biosciences). After 20 minutes incubation at 4 °C, cells were washed twice in staining buffer and fixed using a BDFACS Lysis Solution (BD Biosciences). Acquisition and analysis of the cells were performed on FACSAria using FACSDiva software.
Freshly isolated ADRCs were plated in six-well plate at low density (100 cells/cm2) in DMEM/F12 containing 10% fetal bovine serum (FBS). After 12–14 days, cells were rinsed with PBS, fixed with formalin, and stained with hematoxylin solution (Hemacolor kit; EMD Millipore, Billerica, MA, USA). Colonies containing > 50 fibroblast colony-forming units (CFU-F) were counted. CFU-F frequency was calculated by dividing the number of colonies by the number of seeded cells.
Delivery of ADRCs onto the wound site
Freshly isolated ADRCs (total volume of 0.5 mL) were resuspended in lactated Ringer’s (LR) solution and delivered (within 2 hours of injury) onto the wound surface (approximately 58 cm2) at a dose of 0.25 × 106 per cm2 using a spray system (nasal spray device, LMA-North America, San Diego, CA, USA), as previously reported . Animals were slightly rotated on their side so that the wound surface was flat to ensure even distribution of ADRCs or control. Finally, cells were allowed to adhere onto the wound site for 3–5 minutes prior to wound covering. Each animal served as its own control; for each animal, wounds on the left flank were treated with ADRCs whereas those on the right flank received control LR solution.
Planimetry wound imaging
Digital imaging of wounds was conducted on day 0 (post-injury), day 7, 14, 21, 28, 35, 60, 90, 120, and 180 post-injury. All wounds were subjected to high-quality digital imaging using the SilhouetteStar™ Wound Camera (ARANZ Medical, Christchurch, New Zealand). Wound images were then reviewed and total wound area was measured using the SilhouetteStar™ System.
At day 60 (2 months) and 180 (6 months) post-injury, three (3) full-thickness specimens (approximately 3 cm × 1 cm) were harvested from across the scar. Samples were then fixed in 10% neutral-buffered formalin (NBF), embedded in paraffin, sectioned (5 μm), and stained with hematoxylin and eosin (H&E), Masson Trichrome and Verhoeff–van Gieson (VVG) stain. Entire slides were then digitally scanned using the Aperio ScanScope AT2 slide scanner (Aperio Technologies, Vista, CA, USA). Slides were viewed and analyzed using the ImageScope viewer (Aperio Technologies).
Tissue specimens were fixed in 10% normal buffered formalin and subsequently embedded in paraffin. Tissue sections were subjected to an antigen retrieval step then incubated with primary polyclonal rabbit alpha-smooth muscle actin (α-SMA) (5 μg/ml, Abcam, Cambridge, MA, USA) antibody. alkaline phosphatase (AP)-based detection of the primary antibody was performed using a Vectastain ABC-AP kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions, followed by nuclear staining with Harris hematoxylin. As controls, tissue sections were stained as described above without adding primary antibody. α-SMA staining was quantified using ImageScope analysis software (Microvessel Analysis Algorithm; Aperio Technologies). Vascular smooth muscle cells were distinguished from other α-SMA-positive cells (such as myofibroblasts) on the basis of morphology.
Skin hardness using the skin fibrometer and durometer
The SkinFibrometer (Delfin Technologies, Kuopio, Finland) was briefly pressed against the skin and the contact pressure was registered. The skin and the underlying upper subcutis resist the deformation and the induration value in newtons (N) was determined. The probe was briefly pressed against the skin for five consecutive times at four different sites within the scar area. In addition, a digital Rex Gauge Durometer (model DD-4, type 0, Rex Gauge Company Inc., Glenview, IL, USA), without a foot attachment was used to monitor scar hardness. During measurements, the durometer was rested by gravity against the skin at four different sites within the scar area. Tissue induration measurements were performed and recorded at approximately 2 and 6 months post-injury.
Protein isolation and expression levels
Wound specimens were lysed using total protein extraction buffer (Thermo Fischer Scientific). Total protein content was determined using the BCA assay kit (Thermo Fischer Scientific). Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) protein levels were determined in lysates of whole wounds (100 μg) using Quantikine porcine IL-6 and TNF-α kits (R&D Systems, Minneapolis, MN, USA). Assays were performed in accordance with the manufacturer’s instructions.
Results are expressed as means ± standard error pf the mean (SEM). Comparisons between two groups were performed using a paired t test (two-tailed) (GraphPad Prism version 6.05; GraphPad Software, San Diego, CA, USA). A value of p < 0.05 was considered significant.