Mouse excisional wound splinting model

Eight- to 12-week-old hairless SKH1 mice (Charles River) were used. All techniques, protocols, and animal proceedings employed were performed according to the Animal Ethics Committee of University of Murcia following the Spanish and European governmental regulations on the use of animals for scientific research (Directive 86/609/EC (RD223/1998, RD1201/2005, LAW32/2007) and Directive 2010/63/EU (RD53/2013, LAW 6/2013, Commission Recommendation 2007/526/EC)).

The mouse excisional wound splinting model was carried out as previously described with some modifications [25, 26]. Firstly, mice were individually anesthetized, and then, two donut-shaped silicone wound splints (Grace Bio-Labs) were fixed to the skin on each side of the dorsal surface midline. Next, a 10-mm-diameter full-thickness wound was made aseptically within the splint, and the stem cells were used according to the experimental protocol, i.e., injected into the edge of the wound or applied through cellularized SF patches. In all cases, an occlusive sterile adhesive membrane (Oper film, IHT) was used to completely cover the wounds and splints. In the untreated group of animals, and to avoid possible secondary infections, wounds were covered with Linitul® (Bama-Geve SLU), a dressing frequently employed in the clinical practice which is indicated to treat wounds, bedsores, ulcers, and varicose. It is composed of 18.5 mg of balsam of Peru and 167.8 mg of castor oil per gram.

Two groups of 75 mice were experimentally related to two different umbilical cord donors. Both groups were divided into five different subgroups (N = 15): (i) wounds covered by Linitul, (ii) wounds covered by SF patches, (iii) Wj-MSCs injected at the edge of the wound (1 × 10⁶ cells in 100 μl PBS), (iv) wounds covered by cellularized SF scaffold (5 × 10⁴ cells seeded onto the scaffold for 4 days before surgery), and (v) wounds treated with Wj-MSCs injected at the edge (1 × 10⁶ cells in 100 μl PBS) and also cellularized SF patches (5 × 10⁴ cells seeded onto the scaffold for 4 days before surgery). Every subgroup underwent the sacrifice of three mice at different times, i.e., 48 h and 7, 14, 21, and 28 days.