Sex Hormones and Wound Healing

Desiree May Oh, MD; Tania J. Phillips, MD


Wounds. 2006;18(1):8-18. 

In This Article

Estrogens and Wound Healing

Animal Studies

Topical estrogen application has been demonstrated to accelerate wound repair.[12] Six-week-old female rats were randomized to ovariectomized and control groups. Predefined sites were marked and injected intradermally with 100 μL of 50 μM estradiol, 5 μM estradiol, phosphate-buffered saline (PBS), or the carrier (2-hydroxypropyl-beta-cyclodextrin). Four dorsal 1-cm full-thickness incisions were then made on these sites. Wounds were assessed for reepithelization, new collagen deposition, and wound width 7 days after wounding. Wounds in ovariectomized rats exhibited a marked delay in wound healing compared with intact control animals. There was a delay in reepithelization, an increase in wound width, and a reduction in collagen deposition in the ovariectomized group. Application of 50 μM and 5 μM estradiol to the wounds of ovariectomized rats increased the rate of wound healing. Estrogen administration accelerated wound healing in the intact animals as well.

Recent animal studies on the influence of estrogens on wound healing examine the role of specific mediators and signal molecules, such as Smad3, macrophage migration inhibitory factor, and laminin-5 gamma 2-chain.

Estrogen and Smad3. The role of Smad3 in the hormonal modulation of wound healing by TGF-b was examined by Ashcroft et al.[13] Smad is a known intracellular mediator of TGF-β function that has been previously demonstrated to inhibit reepithelization in mice subjected to full-thickness incisional wounds. Smad3-null mice had markedly accelerated wound-healing rates compared to wild-type mice.[14] In another study comparing ovariectomized wild-type female mice and Smad3-null female mice,13 delayed healing was noted in both groups. Estrogen administration resulted in reversal of the delay in both groups, suggesting that Smad3 has no role in mediating estrogen signaling during the wound healing process.

Estrogen and Macrophage Migration Inhibitory Factor. Another cytokine that may possibly be involved in modulating the effects of estrogen on wound healing is macrophage migration inhibitory factor (MIF). This cytokine is a T-cell-derived protein that is an important regulator of inflammation, influencing both the innate and antigen-specific functions of the immune system. A recent study showed that a variety of cells, including monocytes, endothelial cells, and keratinocytes, produce MIF.[15]

Clinical trials on normal female mice (intact group) and ovariectomized female mice (ovx) were performed to determine the interactions of estrogen and MIF in the regulation of wound healing.[16] Full-thickness incisional wounds were artificially produced on the backs of the mice, and healing patterns and MIF levels were noted on Days 3, 7, and 14 post-wounding. Increased wound tissue MIF levels were noted on Days 3 through 7 after wounding for both the intact and ovx mice with normalization to baseline by Day 14. The ovx mice, however, had markedly higher MIF levels compared to the intact mice. The authors concluded that a specific spatial and temporal regulation of MIF occurs in wound repair, localizing to keratinocytes and macrophages. To further determine the role of MIF on wound repair and its relation to estrogen, some mice from both the intact group and ovx group were rendered incapable of expressing MIF by MIF gene inactivation. An important and interesting observation was that when the rate of healing of the ovx, MIF gene-negative group was compared to the intact, MIF gene-positive mice, no significant differences were noted. In contrast, ovx, MIF gene-positive mice exhibited delayed wound healing. This implies that estrogen has a direct or indirect regulatory role in MIF expression and that in the absence of MIF, the presence or absence of estrogen is irrelevant. When MIF is lacking, the excessive inflammation and delayed healing phenotype associated with reduced amounts of estrogen is reversed. Therefore, local MIF inhibition may be a specific target in accelerating cutaneous healing in impaired age-related wound-healing states.

Estrogen and Chemically Modified Nonantimicrobial Tetracycline-8. Pirila et al.[17] investigated the effects of estrogen and chemically modified nonantimicrobial tetracycline (CMT)-8 on molecular processes in wound healing in an animal model. CMT-8 is a potent matrix metalloproteinase (MMP) inhibitor. MMPs function in extracellular matrix and basement membrane remodeling during wound healing.[18] They include secreted and membrane-associated zinc-requiring peptidases with a diverse spectrum of substrate specificities. Elevated levels of MMPs are associated with intrinsic aging as well as chronic wound states. An earlier study had shown that estrogen and CMT-8 may improve wound healing by reducing MMP-mediated collagenolysis.[19] Pirila et al.[17] focused on the wound healing mediator laminin-5 (Ln-5) gamma 2-chain. Ln-5 gamma 2-chain is a key molecule in wound repair, functioning in epithelial attachment and migration. It is an integral part of the anchoring filaments in hemidesmosomes and is localized to the basement membrane of the skin. The authors demonstrated that estrogen can promote wound healing by recovering the expression and processing of the Ln-5 gamma 2-chain.

In ovx animals, reepithelized wounds exhibited faulty attachment of the basal lamina and a split in the epidermal-dermal junction. Detachment was co-localized with diffuse and discontinuous expression of Ln-5 gamma 2-chain immunoreactivity. In contrast, estrogen-positive animals and ovx rats treated with CMT displayed similar epidermal attachment patterns. It is interesting to note that both estrogen-treated and CMT-8-treated ovx rats had higher levels of Ln-5 gamma 2-chain immunoreactivity compared to sham and untreated ovx rats. Thus, although Ln-5 chain protein expression was decreased in ovx placebo-treated animals, estrogen and CMT-8 were both able to increase its expression. This study showed for the first time the role of Ln-5 chain modulation in vivo.[17]

Human Studies

The effects of topical estrogen on wound healing have been studied in elderly men and women. Ashcroft et al.[20] randomized 18 men (mean age 70.7 years) and 18 women (mean age 74.4 years) to receive either placebo or estrogen. An area of skin in the left upper inner arm of each subject was covered with either a 5 cm x 4 cm patch containing 25 μg estradiol or a placebo pad and biopsied immediately through these pads. The patch was then covered with gauze, which was left in place for 24 hours. Wound size was then measured by planimetery and image analysis, and collagen levels were assessed using a hydroxyproline assay. Results indicated that topical estrogen applied to normal skin immediately before wounding and to wound margins for 24 hours post-wounding increased the rate of wound healing in both sexes. Wound size was noted to be significantly smaller (approximately 18% measured by planimetry) 7 days after wounding in estrogen-treated wounds (Figure 1). Collagen levels were consistently higher in estrogen-treated patients of both sexes versus placebo. Women tended to deposit more collagen than men. The data suggest that topical estrogen facilitates wound healing in elderly men as well as women by reducing wound size and stimulating matrix deposition.[20]

Figure 1.

Wound area results on Day 7 post-wounding. A) Macroscopic area as determined by planimetry were significantly reduced with estrogen treatments. B) Cross-sectional areas assessed using image analysis were significantly reduced with estrogen treatment.

Ashcroft et al.[12] reported that systemic administration of estrogen reverses the age-related impairment of cutaneous wound healing in women. They studied the effects of age and estrogen therapy on wound-healing rate and quality (both macroscopic and microscopic) by comparing these parameters in 3 groups: young women aged 20 to 39, post-menopausal women receiving hormonal replacement therapy, and post-menopausal women of the same age not receiving hormonal therapy. The authors artificially created wounds by performing 4-mm punch biopsies on the patients' upper inner arms and measured the rate of reepithelization on Day 7 by image analysis. They noted that in the aged group not receiving hormone replacement therapy, there was a delay in the rate of reepithelization. By Day 7, wounds were only 20% reepithelized compared to 80% in the aged group receiving hormone replacement therapy. In fact, the reepithelization rate of the aged hormone replacement therapy-treated group approached that of the young group (85%). Similar results were observed with collagen deposition. Both the young group and hormone replacement therapy group had markedly increased levels of collagen deposition compared with the aged, no hormone replacement therapy group.

Postmenopausal women taking hormone replacement therapy are significantly less likely to develop venous and pressure ulcers than older women who do not take hormone replacement therapy. A case-cohort study[21] examined the data taken from 44,195 women in the United Kingdom. It assessed the tendency for formation of these 2 types of chronic ulcers in women older than 65 years. The age adjusted relative-risk estimate for the development of venous leg ulcers was 0.65 (95% CI, 0.61 to 0.69), while that for pressure ulcers was 0.68 (95% CI, 0.62 to 0.76) in patients receiving hormone replacement therapy. The authors concluded that patients who received hormone replacement therapy were 30%-40% less likely to develop these ulcer types compared to age-matched patients who were not on hormone replacement therapy. They viewed this finding as early evidence that hormone replacement therapy could have a beneficial effect in the prevention of chronic wounds.

Estrogen Mechanism of Action

Mechanisms underlying the effects of estrogen on the complex process of wound healing have not been fully established.

Wound healing traditionally has been subdivided into 3 phases: the inflammatory phase, the proliferative-granulation phase, and the remodeling phase. The inflammatory phase involves platelet accumulation, coagulation, and neutrophil and macrophage infiltration into the wound bed. In the proliferative-granulation phase, reepithelization occurs by proliferation of epidermal keratinocytes at the wound edges and their migration through a provisional matrix rich in fibrin and fibronectin. This phase is also characterized by formation of granulation tissue, which contains newly developed vessels and migrating inflammatory cells and fibroblasts, and wound contraction. A dynamic continuum of collagen synthesis and degradation is present during the remodeling phase. Collagen changes from type III to type I with an increase in tensile strength and maturation of extracellular tissue. The once highly vascularized granulation tissue also matures into less vascularized scar tissue. In all phases of wound repair, there is a complex interplay of growth factors and cytokines, matrix signals, soluble mediators, receptors, proteases, and many other biomolecules exerting their influence on a wide variety of cell types including keratinocytes, fibroblasts, Langerhans cells, melanocytes, dermal dendritic cells, endothelial cells, and mast cells.

Estrogen and Polymorphonuclear Cell Chemotaxis

The primary modulatory effects of estrogen on wound repair may occur during the inflammatory phase of healing. Data from animals suggest that estrogen has anti-inflammatory properties. A clinical trial[22] was performed on castrated mice in which local inflammation was induced by intradermal injection of olive oil and cholera toxin. Estradiol administration significantly suppressed the inflammatory response as measured by footpad swelling and documented by histologic examination. Although the manner by which estrogen exerts its anti-inflammatory effect is not well understood, it is postulated that it mainly affects chemotaxis of polymorphonuclear leukocytes (PMN) via a receptor-mediated mechanism. Beta-estradiol has been shown to suppress chemotaxis of PMN in a dose-dependent manner,[21] and this suppressive effect on PMN chemotaxis has been shown to be inhibited by use of anti-estrogens.[22] Estrogens may also alter expression of neutrophil adhesion molecules, such as L-selectin.[20]

The suppressive effect of estrogen on PMN chemotaxis may have cascading effects and may significantly influence the second phase of healing, the proliferative-granulation phase.[23,24] In a proposed model by Ashcroft et al.,[20] direct inhibition of chemotaxis by estrogen caused an early decrease in wound neutrophil numbers. The suppression in neutrophil accumulation resulted in reduced neutrophil elastase activity. Elastase is a serine protease derived from neutrophils that plays an important role in wound repair. It is capable of degrading a wide variety of structural and functional proteins deposited in wounds, such as proteoglycans and collagen, and is the protease mainly responsible for fibronectin degradation.[25,26]

Fibronectin, an essential component of the early wound environment, directs the migration of keratinocytes, increases fibroblast influx and collagen deposition, and stimulates wound contraction.[27] Previous studies[28,29] have indicated that impaired age-related wound healing states are characterized by reduced local amounts of fibronectin secondary to higher elastase levels in the wounds of the elderly. Neutrophil elastase in early acute wounds tended to be present for longer periods of time and in greater numbers in wounds of aged compared to younger subjects. In venous ulcers, persistent and chronically high levels of elastase were likewise noted. This suggests that the process of aging in healthy human subjects is associated with an up-regulation of elastase, and an abnormality in its down-regulation may be partially responsible for transition of wounds into chronicity in the elderly.[28]

Use of estrogen may counteract this age-related increase in elastase levels. In the study by Ashcroft et al.[20] where biopsy wounds in elderly men and women were treated with either placebo or estrogen, the estrogen-treated group exhibited markedly decreased wound tissue elastase activity (Figure 2), decreased in-vitro degradation of fibronectin by elastase, and a parallel increase in fibronectin levels in vivo in comparison to the placebo group. Based on the data, the authors proposed that estrogen may accelerate wound healing by down-regulation of wound elastase activity and consequently decrease fibronectin degradation.

Figure 2.

Elastase activity in wound tissue is reduced with estrogen treatment on Day 7 post-wounding.

Estrogen and TGF-β1

In the proliferative-granulation phase of healing, the cytokine TGF-β and its isomers have been shown to play a role in the modulatory effects of estrogen on wound healing. TGF-β and its isomers are potent stimulators of fibroblast-driven gel contraction and probably stimulate granulation tissue contraction in vivo.[30] They are multifunctional, inducing angiogenesis, fibrosis, growth inhibition, apoptosis, differentiation, and proliferation. They also contribute to immunoregulation. Several cell types at a wound site produce TGF-β, and its 3 isoforms, β1, 2, and 3, are up-regulated after skin wounding in mice. TGF-β1 is the most abundant isoform in all tissues and the only isoform stored in human platelets.[31]

Estrogen may regulate the production of TGF-β1. Ashcroft et al.[12] observed markedly decreased levels of wound TGF-β1 in an aged female group at Day 7 post-wounding compared to a young group and an estrogen-supplemented group, correlating well with the healing rates for these groups. Quantitative polymerase chain reaction with reverse transcription established that intrinsic aging in women was associated with low levels of steady-state mRNA for TGF-β1. The differences between the aged group and the young group and between the aged group and the hormone replacement therapy group were highly significant (P = 0.0006) implying that hormone replacement therapy reverses the age-related decrease in local TGF-β1 mRNA steady-state levels observed during the early stages of wound healing. This was the first report to suggest that cytokine expression during wound healing was hormonally modulated.[12]

Estrogen and Wound Remodeling

In the remodeling phase of wound healing, estrogen can affect collagen content, tensile strength, and macroscopic appearance of scar tissue. It is established that use of estrogens in intact skin increases its collagen content.[32] Studies by Ashcroft et al. suggest that estrogen administration also increases collagen content in injured skin. Postmenopausal women administered hormone replacement therapy for at least 3 months had significantly increased collagen deposition in punch biopsy wounds in comparison to age-matched controls.[12] Men and women treated with topical estrogen showed increased collagen deposition on Days 7 and 80 post-wounding versus untreated controls. Estrogen-treated women tended to deposit more collagen and to exhibit increased skin stiffness, which may correlate with wound strength.[20] Wound stiffness was measured using the nondisruptive dimensional analysis system (DAS). The system applies a multiaxial load (negative pressure) to the wound and measures the deformation caused by the load using a high-resolution camera and video-processor. Decreased MMP-mediated collagenolysis associated with estrogen use may play a role in increased wound stiffness and strength.[19]

The quality of the scar formed is also affected by estrogen. In the study on systemic estrogen by Ashcroft et al.,[12] the macroscopic appearance of mature scar tissue in the elderly group was found to be significantly superior in terms of color, texture, and contour to that of the young subjects, who tended to form hypertrophic scars. Increasing age was also a factor in determining the quality of microscopic repair with restoration of the dermal architecture in the wounds of the aged group. Hormone replacement therapy was associated with adverse scarring profiles that were microscopically and macroscopically similar to the young women. This implies that while systemic estrogen treatment may help accelerate wound healing, it also has the undesirable effect of adversely affecting the quality of scar tissue formation.

Risks and Benefits of Estrogen Therapy

In the past, estrogen was considered beneficial for treating cardiovascular disease and osteoporosis. Its use resulted in reduced incidence of coronary heart disease[33] and long-term prevention of bone loss[34] in postmenopausal women. However, controversy regarding the use of estrogens began in 1998 when results from a series of randomized, controlled trials showed that hormonal replacement therapy does not retard the progression of established coronary disease[35] nor does it prevent clinical cardiovascular events in previously healthy postmenopausal women.[36,37] The use of combination estrogen and progestin was found to increase the risk for breast cancer, coronary heart disease, stroke, and pulmonary embolisms,[38] and estrogen therapy alone has been associated with increased risks of endometrial hyperplasia and cancer. This data has led to careful evaluation and counseling of women about the risks, benefits, and uncertainties of estrogen therapy before deciding to start or continue treatment.[39]