Surgical preparation and experimental protocol

Eight female Merino sheep (31.5 ± 4.5 kg, approximately 3 years old; Talley Ranch, Bastrop, TX) were used in this study. The animals were chronically instrumented for hemodynamic monitoring with a right femoral artery catheter and a 7-French Swan-Ganz thermodilution catheter under aseptic conditions. Two full-thickness flame burn sites of 25 cm² were made on both sides of dorsum with 2 cm distance between sites and 3 cm apart from the spine under anesthesia and analgesia. For the induction of burn sites, we have used a metal sheet with 25 cm² open space in the middle. Underneath the metal sheet, a burn resistant cloth was instilled to prevent the heat to the adjacent tissues. The burn was induced by fire flame, which was applied approximately 3 s until the skin shrinkage stops. The method was previously established in our laboratory that the full-thickness skin burn was confirmed by the histological analysis⁴⁹.

Twenty-four hours after the burn, the eschar was excised down to the fascia. Split-thickness skin grafts 0.03 inches thick were harvested from sites remote from the burned sites to be meshed and grafted to the wounds. 0.1 ml/cm² of 4% Omega-7 in 0.9% saline was topically applied to one of two randomly selected autografted sites (Omega-7 group). The remaining site received the same amount of 0.9% saline as a control (control group). The donor sites were also randomly allocated to 0.1 ml/cm² of 4% Omega-7 or 0.9% saline. After the initial treatment, the grafted sites were covered with tie-over dressings removed at postoperative day (POD) 7. At the 7 days after the grafting, the wounds were gently washed daily with a sterile saline without anesthesia. Thereafter, the wounds were treated with Omega-7 or 0.9% saline, and covered with polyurethane foam for 7 days (total duration of the study is 14 days) (Fig. 8). No wound debridement was done.

Experimental design of clinically relevant ovine grafted burn wound healing model. The third-degree burn was induced by fire flame (above left). 24 h after the burn, the eschar was excised down to the fascia. Split-thickness skin grafts 0.03 inches thick were harvested from sites remote from the burned sites to be meshed and grafted to the wounds (above center). 0.1 ml/cm² of 4% Omega-7 in 0.9% saline was topically applied to one of two randomly selected autografted sites (Omega-7 group). The remaining site received the same amount of 0.9% saline as a control (control group). The donor sites were also randomly allocated to 0.1 ml/cm² of 4% Omega-7 or 0.9% saline (above right). After the initial treatment, the grafted sites were covered with tie-over dressings removed at postoperative day (POD) 7 (below left). At the 7 days after the grafting, the wounds were gently washed daily with a sterile saline without anesthesia (below center). Thereafter, the wounds were treated with Omega-7 or 0.9% saline, and covered with polyurethane foam for 7 days (14 days total duration of experiments). The donor site wounds were covered with polyurethane film dressing and treated daily with 4.0% Omega-7 or 0.9% saline starting at POD1 for 14 days (below right). The study design was drawn using Adobe illustrator by the first author (https://www.adobe.com/jp/products/illustrator.html).

The donor site wounds were covered with polyurethane film dressing (Tegaderm, 3 M, MN) and treated daily with 4.0% Omega-7 or 0.9% saline starting at POD1. Fourteen days after surgery, animals were deeply anesthetized and euthanized by intravenous administration of xylazine (3.0 mg/kg), ketamine (40 mg/kg), and buprenorphine (0.01 mg/kg) following IACUC approved protocols and American Veterinary Medical Association Guidelines for Euthanasia.