Topical Growth Hormone Accelerates Wound Healing in Mice

Objective. The aim of this study is to investigate the effects of topical growth hormone (GH) treatment on skin wound healing in mice. Materials and Methods. An excisional wound healing model was established on male Swiss mice, and wound healing ability was evaluated by macroscopic and histologic analyses of mice treated with topical 10^-8 M and 10^-7 M of GH versus the mice receiving vehicle alone. Wound tissues were collected on post treatment days 3, 7, and 14. Skin fragments were subjected to hematoxylin and eosin and Masson’s trichrome staining for morphological analyses. The expression of type I collagen and platelet endothelial cell adhesion molecule 1 (CD31) was detected by immunohistochemical analysis. Results. Topical treatment with GH resulted in faster wound closure rates at all time points analyzed versus those observed in the control group (day 3: 18.3 ± 3.1 vs. 44.4 ± 7.4, 43.6 ± 0.6; day 7: 41.7 ± 6.3 vs. 73.8 ± 6.6, 71.3 ± 5.8; day 12: 94.3 ± 3.9 vs. 100 ± 0, 100 ± 0). Histological analysis of the wound on post treatment day 3 revealed a more diffused infiltration of inflammatory cells in the group treated with GH. After day 7, GH-treated animals began forming granulation tissue, and there was an increase in inflammatory cell infiltration. The GH significantly increased the expression of type I collagen (day 7: 57.4 ± 4.0 vs. 120.2 ± 9.7, 79.3 ± 7.9; day 14: 218.2 ± 10.4 vs. 301.5 ± 9.1, 235.0 ± 7.5) as well as the number of blood vessels (day 7: 10.0 ± 2.4 vs. 15.3 ± 2.0, 10.1 ± 2.2; day 14: 3.2 ± 0.8 vs. 5.6 ± 2.0, 6.2 ± 2.2) in the injured area. Conclusions. The GH accelerates the closure of skin wounds by resolving the inflammatory phase faster, accelerating reepithelialization and collagen deposition, and stimulating angiogenesis.

Wound healing is a complex, sequential process that requires the participation of many tissues and cell types. The process involves proliferation and migration of keratinocytes at the wound edges as well as the formation of granulation tissue that will originate a mature dermal tissue.^1,2 In recent years, several studies^1-4 have shown that cytokines and growth factors are capable of modulating the communication between cells and regulating the wound healing process. These growth factors are intrinsically released from blood to the wound site, or are locally produced via paracrine or autocrine mechanisms, and thus exert local effects. Since these growth factors are rapidly eliminated from the body, they need to be administered in large amounts for systemic use. Furthermore, these factors exert a variety of effects not only locally at the wound site, but also systemically.³

Among various growth factors, studies have investigated the effects of systemic growth hormone (GH) therapy on wound healing. The GH has been implicated as a modulator that may influence the biology of wound healing.^5,6 For example, patients with severe burns have been treated with systemic GH, and, in most cases, treatment improved healing and patient survival.^7,8 In GH transgenic mice, high levels of circulating GH stimulated reepithelialization and the formation of granulation tissue.⁹ Furthermore, another study¹⁰ showed that systemic application of GH stimulated the formation of granulation tissue in wounds of malnourished rats. 

Although GH has shown a positive effect on wound healing, it has been reported that high levels of circulating GH may cause changes in various organs of the body, as seen in a subject with acromegaly.^11,12 Moreover, circulating GH has been shown to increase tumor aggressiveness.¹³ Given the adverse side effects of systemic GH administration, the topical application of GH in cutaneous tissue repair processes was investigated with the hope that topical use of this hormone could be an additional therapeutic strategy for treatment of traumatic injuries, surgical injuries, or illnesses.

Mice and wound-healing assay. Male Swiss mice (n = 45), weighing 38 g to 40 g, aged ≥ 6 months were randomly allocated to the different treatment groups and were housed individually during the healing period. An in vivo wound-healing assay was performed after an intraperitoneal injection of an anesthetic (100 mg/kg ketamine, 10 mg/kg xylazine diluted in phosphate-buffered saline [PBS]). A 1-cm punch biopsy tool was used to create 1 circular full-thickness cutaneous wound on the shaved dorsal skin of the mice. During the 14 days of observation, 10^-8 M of GH (n = 15), 10^-7 M of GH (n = 15), or vehicle control (PBS; n = 15) were applied once daily in a volume of 200 µL. To evaluate the effects of GH in tissue repair, at least 5 animals were used at each treatment time for each group. For the wound-healing assay, a single incision was made on the dorsum of each mouse. 

To measure wound area, digital photographs were taken on days 1, 3, 7, 12, and 14 after wounding and were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD). In each group, the animals were randomly euthanized and tissues were collected 24 hours after days 3, 7, and 14 of treatment.

All animal experimentation described was approved by the ethics committee on research of the Federal University of Alagoas, (Maceió, Alagoas, Brazil; protocol number: 028370/2010-07). The GH was purchased from Merck Serono S.A. (Geneva, Switzerland).

Histological analysis. Excised wound specimens were fixed with 10% neutral buffered formalin and embedded in paraffin. Tissue samples from mice wounds were deparaffinized by xylene and rehydrated in increasing concentrations of ethyl alcohol and distilled water. These samples were then stained with hematoxylin and eosin for morphological assessment and with Masson’s trichrome for collagen analysis. 

Immunohistochemistry. Frozen wound samples were subjected to indirect immunofluorescence or immunoperoxidase assay as previously described.¹⁴ For immunofluorescence, the samples were incubated with the primary antibody anticollagen I (rabbit antibody, 1:100; Sigma-Aldrich, St Louis, MO) for 1 hour at room temperature. After washing, cells were incubated with the secondary antibody, goat anti-rabbit-FITC conjugated (1:200, Sigma-Aldrich), for 45 minutes at room temperature. Immunostained samples were analyzed by fluorescence microscopy (Nikon Eclipse 50i; Nikon Instruments Inc, Melville, NY). Negative controls in which the indicated primary antibody was replaced by an unrelated immunoglobulin or in which the secondary antibody was used alone were also assessed. Quantitative analysis of immunofluorescence data was performed with the imaging software, using appropriate thresholding to eliminate background signal before histogram analysis, as previously described by De Felice et al.¹⁵ 

For immunoperoxidase, samples were hydrated and endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes. Subsequently, nonspecific binding was blocked with 1% bovine serum albumin for 40 minutes. Samples were incubated with the anti-mouse CD31 primary antibody (rat antibody, 1:100; BD Pharmingen, San Diego, CA) for 1 hour at room temperature. Biotinylated secondary antibodies (biotin goat anti-rat [BD Pharmingen, San Diego, CA]) were then applied at 1:200 for 30 minutes followed by incubation with streptavidin conjugated with horseradish peroxidase at 1:200 for 30 minutes. Color development was performed with 3’-diaminobenzidine for 5 minutes for all samples followed by hematoxylin counter staining, dehydration, and coverslipping. The immunopositive cells in 10 fields per section were counted using the imaging software.

Statistical analysis. Data are shown as mean ± standard error of the mean. Results were statistically analyzed by unpaired Student’s test and one-way analysis of variance followed by Dunnett’s test using GraphPad Prism (GraphPad Software Inc, La Jolla, CA). Data were considered statistically different at P ≤ .05.

Growth hormone therapy accelerated closure of skin wounds in vivo
Wound healing was evaluated macroscopically in mice treated topically with GH for 14 days. To evaluate the action of this hormone, the investigators created a comparison with the control group. Macroscopic examination of the wounds revealed edema, hyperemia, and formation of a crust in all groups, and these were maintained during the first week of treatment. All animals remained healthy without evidence of infection. It was observed that the GH in both concentrations resulted in quicker closure times for skin wounds in comparison with the controln (Figure 1A).  As shown in Figure 1B, the presence of 10^-7 M or 10^-8 M of GH showed faster wound healing when compared with the control group. Signs of healing were observed as early as day 3, and wounds were almost completely healed 12 days after incision. It should be noted that by the end of day 14, the control group that received PBS showed a regenerated epidermis, indicating that the wound-healing process used in the experimental model does not exceed 14 days. 

Growth hormone therapy stimulated recruitment of inflammatory cells in mice skin wounds after day 3 of topical treatment
Considering that GH treatment accelerated the closure of skin lesions, the investigators examined the effect of GH during the 3 phases of wound healing. 

During the initial events of tissue repair (ie, the inflammatory phase) in mice (post treatment day 3 [Figure 2]), there was tissue destruction with moderate infiltration of inflammatory cells and the presence of a tissue with very loose consistency. Moreover, a crust on the surface of the granulation tissue was noted. 

In the PBS-treated wounds, the area corresponding with the granulation tissue showed the presence of inflammatory cells and fibroblasts (Figure 2). The same was observed for the GH-treated wounds (Figure 2). However, through the qualitative assessment of tissue samples, it was apparent that treatment with GH resulted in a faster influx of inflammatory cells on the third day than that observed in the control group. In addition, blood vessels, fibroblasts, and a crust on the surface of the injury were apparent after 3 days of GH treatment. 

Cell recruitment and collagen deposition were accelerated in response to GH in the formation of granulation tissue
On post incision day 7, the investigators found high mitotic activity and keratinocyte migration as well as the formation of a granular consistency of tissue, comprising cells and extracellular matrix (ECM; eFigure 3). 

In the region of the epidermis, the control group (eFigure 3A) revealed a narrow layer of epithelial cells in the region of the lesion. In the GH-treated groups, there was a slightly more stratified epidermis than in the control. In the region of the dermis, there were characteristics of granulation tissue in the 3 groups (eFigure 3A, 3D, 3G). In the treatment groups, the cellular infiltrate of inflammatory cells and fibroblasts were observed to be diffused throughout the granulation region (Figure 3D, 3G). However, in the control group (eFigure 3A), this infiltrate was found to be concentrated in the peripheral region of the injury. From these results, it was noticed that the GH accelerated the migration and proliferation of these cells in the first week of treatment. Furthermore, GH treatment increased the total collagen deposition during the formation of granulation tissue (eFigure 3E, 3H). Collagen deposition was highest in the group receiving GH at a concentration of 10^-8 M followed by that observed in the group treated with 10^-7 M of GH (see blue collagen stain in eFigure 3E, 3H). The immunofluorescence assay was used to show the expression of type I collagen. It was found that topical treatment with GH increased the deposition of type I collagen in the skin wounds of the mice in the first week of treatment (eFigure 3F, 3I). The quantification of fluorescence intensity (eFigure 3J) confirmed that GH treatment was able to significantly increase the deposition of type I collagen at both concentrations.

Growth hormone therapy accelerated remodeling of granulation tissue and reepithelialization
On post treatment day 14, all animals analyzed (n = 5/group) showed a regenerated epidermis (eFigure 4). Photomicrographs of the control group demonstrated that the epidermis was regenerated and consisted of a thick stratified epithelium (eFigure 4A) with active epithelial cells. The dermis is composed of a diffuse and dense cellular infiltrate, indicating an inflammatory response and remodeling occurred in the newly formed area. Similar results were observed in the GH-treated animals. eFigures 4D and 4G show a stratified epithelium with few keratinocyte layers that is very similar to that of the skin region which was not affected. The dermis contained a diffuse infiltration of inflammatory cells, indicating that the inflammatory process had been attenuated. 

Treatment with GH resulted in faster collagen deposition after day 14 of treatment (see blue collagen stain in eFigure 4E, 4H) when compared with the control group (eFigure 4B). Immunofluorescence using the antibody specific for type I collagen revealed that regardless of the concentration used, GH increases collagen deposition (eFigure 4C, 4F, 4I, 4J). 

Growth hormone therapy promoted angiogenesis during the granulation phase and tissue remodeling
The formation of new blood vessels is required for the progression of wound healing. In this study, the formation of blood vessels was observed after days 7 and 14 (eFigure 5A). 

The 10^-7 M of GH (after day 7 of treatment) resulted in a higher number of blood vessels in the region of granulation tissue when compared with the control group. However, in the same treatment period, 10^-8 M of GH was not able to change the number of vessels. Also, a similar effect was observed after day 14 of treatment. Thus, 10^-7 M of GH maintained its proangiogenic effect throughout the 2-week healing period, significantly increasing the number of blood vessels (eFigure 5B).

The present study showed positive effects of topical GH treatment on the healing of skin wounds in adult mice. Healing is dependent on a complex set of molecular and cellular events, such as proliferation, cell migration, apoptosis, and angiogenesis. These events, driven largely by cytokines and growth factors, lead resident cells in the dermis to undergo proliferation and interact with other cells in the affected area, resulting in wound healing in the skin.^16,17 The investigators found that GH accelerated wound closure in mice with skin wounds. In addition, after 12 days of GH treatment, mice were already showing signs of complete tissue repair. This result pointed to a physiological role of GH, indicating it to be a member of the group of molecules that have pleiotropic actions on skin cells. This finding corroborates with previous research,⁹ which demonstrated that after injury to the skin, the process of wound healing is accelerated in GH-transgenic mice. Furthermore, another study¹⁰ showed that the systemic application of GH stimulated the formation of granulation tissue in wounds of malnourished rats.

In the inflammatory stage of the wound-healing process, topical GH treatment positively accelerated the recruitment of inflammatory cells. In addition, the investigators analyzed the areas of wounds in the inflammatory phase and counted the cells in 20 random fields in each group. This revealed that treatment with GH increased the number of macrophages by about 15% and the number of lymphocytes by 50% without changing neutrophil recruitment. The effects of GH on the immune system have been extensively characterized. For instance, Inoue et al¹⁸ reported that the treatment of animals with recombinant human GH in a model of peritonitis reduced bacterial counts in the peritoneal layer and increased the number of exudative neutrophils. Furthermore, Napolitano et al¹⁹ demonstrated that GH increased the thymic mass in patients infected with human immunodeficiency virus, and this increase was accompanied by an increase in the number of CD4⁺ T lymphocytes. In these cases, GH acted as an immunomodulator able to restore and/or enhance immune function. 

After 7 days, the GH treatment resulted in higher cellular infiltration in the region of tissue injury than that observed in the control group. In these animals, the region of injury is occupied by inflammatory cells, fibroblasts, and myofibroblasts in a diffused manner, whereas in the control group, this infiltration was observed solely at the wound edges. This finding indicates that GH, directly or indirectly, accelerated the migration and recruitment of cells, such as fibroblasts, to the site of injury. Growth hormone therapy exerts various effects directly or via insulin-like growth factor 1 (IGF-1), which is produced mainly in the liver in response to GH.¹¹ Insulin-like growth factor 1 is a mitogen for keratinocytes of the epidermis²⁰; in addition, this peptide is an important stimulator of protein synthesis in dermal fibroblast ECM.²¹ Moreover, IGF-1 stimulates the proliferation of these cells.²² Given this information, along with the experimental results, it is hypothesized that this may be a mechanism of GH action on the skin.

In this study, the effect of GH on collagen deposition during the process of tissue repair was evaluated. Analysis of the samples showed that topical treatment with GH, independent of the concentration used, increases the deposition of total collagen after 7 and 14 days of treatment. Previous work²³ showed that skin-injured rodents treated with GH had increased granulation tissue. Furthermore, it was demonstrated that topical treatment with growth hormone-releasing hormone (GHRH) stimulated the proliferation of fibroblasts, resulting in an 80% higher number than that observed in the control group. In addition, GHRH accelerated the closure of the epidermis, stimulated the differentiation of fibroblasts into myofibroblasts, and positively modulated collagen deposition.²⁴ 

One essential aspect for adequate tissue repair is the creation of a new vasculature.²⁵ After 7 days of treatment in the present study, GH stimulated the formation of capillaries, and this effect persisted until day 14 of treatment. Endothelial cells express the receptor for GH,²⁶ and the involvement of GH in endothelial function has been demonstrated previously.²⁶ Growth hormone therapy has been shown²⁷ to mediate the production of tissue growth modulators, such as vascular growth factors, and mediate changes in the morphology of blood vessels. Growth hormone-transgenic mice showed an increase in blood vessels during tissue repair.⁹ Furthermore, GH improves intestinal wound healing by stimulating collagen deposition, angiogenesis, and cell proliferation.²⁸ Thus, the results of this study suggest that topical GH treatment modulates the activity of endothelial cells and stimulates angiogenesis.

This study presents interesting results regarding the acceleration of wound healing in mice by topical GH treatment. However, the authors do not know if this effect is due to a direct action of GH over the scar tissue or if it depends on IGF-1, a GH-stimulated factor. Future research will evaluate if GH acts directly or through IGF-1. Further, additional research is needed to elucidate the topical effects of GH on the molecular and cellular mechanisms involved in the inflammatory phase.

In this study, the investigators found that topical GH treatment accelerates the closure of skin wounds in a mouse model by resolving the inflammatory phase of tissue repair quicker. In addition, GH acts on keratinocytes and fibroblasts, accelerating reepithelialization and collagen deposition, as well as stimulates angiogenesis during the formation of granulation tissue and tissue remodeling. These results suggest the possibility of the topical use of GH as a therapeutic strategy for accelerating wound healing after traumatic injury, surgery, or disease.

Affiliation: Laboratory of Cell Biology, Institute of Biology and Health Science, Federal University of Alagoas, Maceió, Alagoas, Brazil

Correspondence:
Salete Smaniotto, PhD
Laboratory of Cell Biology
Institute of Biology and Health Science
Federal University of Alagoas
57072-970, Maceió – Alagoas, Brazil
smaniotto@icbs.ufal.br

Disclosure: The authors have no financial or other conflicts of interest to disclose. The study was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (Brazil) and Fundação de Amparo à Pesquisa do Estado de Alagoas (Alagoas, Brazil).