*4.1. Animals*

Three-week-old, male, non-obese diabetic/severe combined immunodeficiency (NOD/Scid-NOD.CB17-Prkdscid/NCrHsd) mice (*n* = 10) (Harlan Laboratories S.r.l. Barcelona, Spain) were used in this study. These mice present immunological multidysfunction, including absence of mature T and B cells, reductions in macrophage function, complement-dependent hemolytic activity, and NK cell activity. [62] All mice were caged under standard light and temperature conditions with free access to food and water throughout the study. All e fforts were made to minimize su ffering and all animals were sacrificed at the end of the study.

All experimental procedures were approved and regulated by the Animal Experimentation Ethics Committee—University of Alcalá and University Hospital of Getafe (IRB: PROEX reference 237/15, 9 October, 2015), in accordance with the Royal Decree 53/2013 and the European Community Council Directive.

#### *4.2. PU model on Human Skin*

Ten NOD/Scid mice were engrafted under general anesthesia (Ohmeda, BOC Health Care) with female human skin. All human skin (*n* = 5) came from abdominoplasty procedures. Written informed consent was obtained from all patients. The skin was immediately stored after haverst in D-MEM (Dulbecco/Vogt modified Eagle's minimal essential medium) at a temperature of 4 ◦C. Skin grafting was performed during the first 24 h after extraction. Human full-thickness skin grafts (FTSGs) were placed onto a 4 × 3 cm wound. Mice skin was incised down to the muscle and removed, exposing muscular layer. FTSGs were sutured in place with 4/0 nylon. A tie-over bolster dressing was placed for the first 5 days. The same skin donor was used to graft two mice (in order to pair two mice with the same human skin donor). After 60 days, a compression device was applied to the human skin graft, as previously described. [59] The clip exerted a pressure of 150 mmHg, measured with a dynamometer. Three cycles of compression–release (8 h of clamping after 16 h of no compression) were applied to simulate a pressure ulcer, based on the method described by Stadler et al. [63].

#### *4.3. Recombinant Human Growth Hormone (rhGH)*

Genotonorm Kabipen 5.3 mg (Pfizer, Madrid, Spain), solution for injection in a pre-filled pen, was provided by the Department of Pharmacy of the University Hospital of Getafe and stored at a temperature between 2 and 8 ◦C. The use of the rhGH is restricted to the hospital setting, and its use was approved and regulated by the Animal Experimentation Ethics Committee University Hospital of Getafe (PROEX reference 237/15) in accordance with the Royal Decree 53/2013 and the European Community Council Directive.

#### *4.4. Local Treatment of the PU with rhGH*

PU formation was confirmed by visual assessment after the last cycle of compression–release. Paired mice were assigned randomly to the rhGH group (*n* = 5) or to the control group (*n* = 5). We designed a protocol for local administration of the hormone. The concentration of GH was determined based on previous studies. [55] In the rhGH group, the five mice were treated with four local rhGH intradermal injections, each of 0.15 mg (0.5 IU), applied to the PU edges on human skin (Figure 8). The injection protocol started 24 h after the last cycle of compression–release, and the administration of the hormone was repeated once per week for four weeks, always the same day of the week at the same hour. In the control group, the five mice received the same care but without the local administration of the rhGH.

**Figure 8.** Local administration of the rhGH. Note the intradermal injection of the rhGH applied to the pressure ulcer edges on human skin.
