**3. Discussion**

Wound therapies acting on a molecular and cellular level, such as growth factors, have a main role in wound healing. [27–29] The human GH, as an anabolic agent, stimulates growth and mitosis in a number of cell types by acting both directly and indirectly through the insulin-like growth factor (IGF)-I. Its effects have been proved in a variety of tissues, including skin, nerve, muscle, bone, cornea, etc. [11,14,17,19,22,23,30–34] and animal models reveal that systemic GH promotes granulation tissue and collagen formation, increases extracellular matrix, and enhances keratinocyte migration, shortening the time for healing. [14,17,19,22,23] Our findings show a statistically significant difference in healing rates between the rhGH group and control group, the healing rate being twice as fast in mice subjected to treatment with the rhGH. The faster healing was for the first 30 days in the rhGH group, time in which the hormone was administered once per week.

In our histological analysis, we found a thicker skin, increased cellularity and decreased collagen type I in the dermis of the rhGH group. An increase in collagen type III with a decrease in collagen type I are indicators of a delay in the maturation period of collagen. All these characteristics are typical of an active prolongation of the dermal proliferation and secretion period (facilitating the sliding of the keratinocytes), and they should accelerate and improve the re-epithelialization process. This effect was observed in previous studies on diabetic rats with a polymeric GH delivery system. [18] Epidermis maintained a normal architecture and keratinocyte differentiation, showing no histological signs of malignancy. These data are consistent with the results published by Rudman et al. [35], Jorgensen et al. [36], and Conte et al. [37]. A recent study in mice published by Messias de Lima et al. [38], topical treatment with GH resulted in faster wound closure rates. The GH accelerates the closure of skin wounds by resolving the inflammatory phase faster, accelerating reepithelialization, collagen deposition, and stimulating angiogenesis.

The extracellular matrix (ECM) defines the mechanical properties of the skin. Collagens are a principal component, and type I and III collagens (fibrillar collagens) are the major determinants of the strength and stiffness of the tissue. [39] Both types of collagens have a very similar biochemical composition, and they are secreted as procollagens, containing a non-collagenous C-teminal propetide and a N-teminal propeptide. Propeptide processing may be complete in collagen I and incomplete in collagen III, leaving a C-telopeptide and a partially processed N-propeptide domain. These domains have been implicated in the regulation of fibrillogenesis [40]. In human skin, the ratio of collagen I/III is 1 for adolescent, increasing up to 2.5 in adult skin. This ratio depends on age, increasing in old age and decreasing in fetuses. The presence of a high content of collagen III could provide more flexibility and tensility. During wound healing the content of this type of collagens could modulate the scar formation. There is a direct relationship between a high secretion of collagen type I and hypertrophic scar formation. [41] Other authors have shown the importance of collagen type III in fibrinogenesis compare to collagen type I [42]. The authors mentioned how collagen type III was present in distensible organs, reducing the mechanical stiffness. The alteration of the ratio of both collagens can be indicative of a tissue response process to an adverse event, such as an abdominal hernia. [43] In situations of chronic stress and injury, tissues are able to react with a change in the ratio of collagens, increasing the gene and protein expression of collagen type III [44]. The ECM protein profiles and distributions were examined in our model. The injury caused by the compression device is slightly different from other types of damage with heavy bleeding. Our model presents a progressive ischemia and necrosis formation, with progressive tissue regeneration from the healthy edges, showing an increase in the protein expression of collagen I and III in the human skin [45].

It is well known that GH is one of those molecules with pleiotropic actions on skin cells, and it participates in inflammatory, proliferative, and maturation phases of wound healing. During the inflammatory phase, macrophages deliver growth factors that attract fibroblasts and facilitate the next phase. GH promotes the release of (a) EGF, responsible for stimulating fibroblasts; (b) VEGF, which promotes angiogenesis in the wound; (c) FGF, which stimulates macrophages, mast cells, and T-lymphocytes, and facilitates granulation and epithelization [12]. Angiogenesis plays a key role during the granulation phase and tissue remodeling, as new vessels are required for the progression of wound healing. Endothelial cells express the GH receptor (GHR) [46] and produce the participation of GH in the latter process. In our PU model, the local treatment of the PU with rhGH produced changes in the ratio of the protein's expression (collagen type I/III). A significant increase of collagen type III protein expression was observed after wound closure (62 days, range 60–70). This finding could be interpreted as a delay in the process of consolidation of the scar tissue, keeping the immature tissue longer. However, we hypothesize that these changes increase the tissue effectiveness, favoring the response capacity to cover the damaged area. This fibrillar collagen could potentially be able to mature and generate a skin with greater tone.

Studies in patients who present delayed wound healing and/or catabolic states, such as diabetes or burns, showed that systemic GH treatment improves skin healing and reduces time required for wound healing. These patients present catabolic responses, with negative nitrogen balance and reduction in serum levels of GH and IGF. [47–50] A review of randomized controlled trials showed that people with large burns could benefit from using systemic rhGH because of faster healing of the wound and donor site and reduced length of hospital stay, without increased mortality or scarring. However, it seems to be related to an increased risk of hyperglycemia. [25] Patients with PU are patients with multiple comorbidities. In the case of the elderly, especially in hospitals, they are usually malnourished or have catabolic states that worsen the tissue repair processes, resembling the situation of burn or diabetic patients. Therefore, the rhGH could potentially have an important role in accelerating the PU healing in this population. We think that our immunosuppressed model resembles the general condition that many patients with PU present in clinical practice. Since pressure ulcers are not just a problem of pressure, many of the patients are characterized by advanced age, malnutrition, systemic diseases such as diabetes, etc. with delayed wound healing, altered levels of cytokines and inflammatory cells, decreased reepithelialization, and of course immunosuppression. Furthermore, chronic wounds, such as PU, usually present dysregulation of cytokines and growth factors. A critical step for treatments may be to target these factors. [51] Several studies [52,53] have reported that GH, PRL, and IGF-I have a direct influence on cells involved with immunity (high-affinity PRL and GH receptors have been observed on a number of these cells) and modulate humoral and cellular immune functions.

Other anabolic agents, such as anabolic steroids (derivates of testosterone, i.e., oxandrolone), might be useful in promoting healing of PU. A comprehensive review of the literature found one trial in which oxandrolone administered orally was compared with a dose of placebo on pressure ulcer healing in people with spinal cord injuries [54]. The authors were uncertain whether oxandrolone is better than placebo in promoting complete healing of pressure ulcers at 24 weeks of treatment, and it could not draw conclusions about the potential benefits or harms of this treatment on treating PU. Well-designed studies are necessary to provide evidence as to whether anabolic steroids are beneficial or not in treating pressure ulcers.

We are concerned about the adverse effects of systemic GH treatment. Most side effects of GH treatment are local reactions at the injection site, such as pain, erythema, nodules, bruising, lipoatrophy, or swelling. However, different side-effects (i.e., hypoglycemia, changes in mental status, edema, fatigue, and headache) have been reported after systemic GH treatment and they are dependent on dose and time of administration. [26] Therefore, we developed our study for local administration of rhGH into ulcer edges. Several animal studies support the use of local GH in order to accelerate the wound healing process. Rasmussen et al. [55] injected different doses of GH into the back of 36 rats and compared it to a control group. They concluded that the optimal doses to increase the granulation tissue were between 0.2 to 0.7 UI. Kim et al. [56] demonstrated that the wound healing was faster after applying local rhGH in the back of five micro-pigs. A study by Lee et al. [22] described how the GH enhances the local formation of IGF-1, which activates fibroblast proliferation and keratinocyte migration—which highlight the potential of the topical application of GH. Andreassen et al. [32] reported bone formation without an increase in muscle mass, weight, or contralateral bone dimensions after the local injection of GH at the surface of tibial diaphyses in rats. Similar results were found in our study: mice did not present significant weight gain or changes in the usual behavior. Based on these data, we think that local application of GH should be the selected route of administration in future studies.

There is a lack of controlled clinical trials on humans to prove that GH can accelerate the PU healing process. There are a few case reports in the literature using the GH to treat PU. In 1955 Ravina et al. [57] reported the use of systemic GH to treat ulcers of diverse etiology. Six patients received intramuscular administration of the hormone. He reported complete healing after 1 to 3 months in a mixed cohort of patients with no control group and with different doses of GH. In 1987, Waago [58] administered GH topically in a diabetic patient with two recalcitrant ulcers. Four IU were administered topically twice per day, and 8 IU afterward. The author observed faster wound healing and noticeable decreased size of the ulcers. Due to logical ethical considerations, there was not histological analysis in all these cases to prove an improvement of the quality of the skin. The main advantages of our model

are the possibility of having a matched control group and performing histological analysis without the previous ethical considerations.

As in most studies, this article has its limitations. A limitation of this study is the animal model. It is not clear how mouse cells could influence the ulcer healing process. Special care was taken to keep healthy human skin around the ulcer. The clip was placed in the center of the skin graft, maintaining healthy human skin around the ulcer, and injecting the rhGH into human skin, so that the healing and re-epithelialization were carried out by surrounding human cells (without the involvement of mouse skin). The human tissue over the mouse was assessed using fluorescence in situ hybridization (FISH) for chromosomes XX and XY, as previously described in *Maldonado* et al. [59]. We took advantage of the condition that the model was performed on male mice (XY chromosomes), and the human skin graft came from human female donors (chromosomes XX). Chromosomes XX were found in the areas of human skin graft. We think this model provides the opportunity to test di fferent therapeutic strategies directly on human skin in the context of a living organism, without the ethical considerations involved in human research. A second bias is the initial pressure ulcer sizes: the control group presented smaller ulcers compared to the rhGH group. However, it is reported that wound contraction is 0.6–0.75 mm/day and keratinocyte migration up to 0.5 mm/day, regardless of the wound size [60], and therefore the initial size of the ulcer was not considered in the process of randomization. Finally, there are other histological techniques that could have provided useful information (i.e., the Fontana Masson Picrosirius technique [61] for identification of pigmented melanocytic lesions and the correlation between normal and neoplastic pigmented cells). Future studies will have to developed new observations and ideas. In spite of these limitations, we think our model opens up prospects for expanding knowledge about multiple fields such as skin wound healing, mechanisms of tolerance and immunological rejection, skin diseases, and carcinogenesis, among others. As we did in this study, cell therapy, growth factors, and other therapeutic strategies can be tested directly on damaged human skin through our ulcer model, without the ethical considerations involved in human research. Advances in skin healing and skin regeneration performed on our model could be potentially applied in clinical practice.
