Oestrogens and wound healing

Maturitas 34 (2000) 195 – 210 www.elsevier.com/locate/maturitas

Invited Review

Oestrogens and wound healing Melissa Calvin * Tissue Repair Research Unit, Department of Anatomy and Cell Biology, Guy’s Hospital, London SE1 9RT, UK Received 16 March 1999; received in revised form 1 September 1999; accepted 6 September 1999

Abstract During the past few decades several studies have documented the deleterious impact of the menopause on bone mass and cardiovascular disease, and the reduction of risk in this area by HRT. However, the possible effects of the postmenopausal deficiency in ovarian hormones on skin and its repair post-injury, are less well documented. This review provides a survey of the literature that is available regarding the involvement and influence of oestrogens on the various phases of cutaneous repair — inflammation, proliferation and remodelling. Research carried out on the effects of oestrogens, both in terms of deficiency and replacement, on the process of wound healing in various animal models is described and discussed, together with the very limited work undertaken in humans. This area of research is of paramount clinical importance both in terms of financial cost and human suffering, since many chronic wounds such as venous ulcers, pressure sores and burns afflict the elderly population, of whom postmenopausal women comprise the majority. Clinically our aim should be to restore the integrity and function of wounded tissue as rapidly as possible after injury and it is generally believed that a better understanding of the effects of oestrogens on wound healing could lead to improved care of cutaneous wounds, and the treatment of not only the wound but of the postmenopausal woman as a whole. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Oestrogens; Wound healing; HRT

1. Introduction With the declining trends in birth rate and increases in longevity in the Western world, postmenopausal women are representing a greater percentage of the population. The life expectancy * Corresponding author. Present address: Tissue Viability Unit, Guy’s Nuffield House, Newcomen Street, London SE1 1YR, UK. Tel./fax: +44-171-9554667. E-mail address: [email protected] (M. Calvin)

of women has steadily increased so that today most spend more than one-third of their life-time postmenopausally in a state of profound oestrogen deprivation [1,2] and there are increasing medical concerns for their health. The menopause has come to signify much more than just the loss of reproductive capacity; the declining ovarian function and consequent oestrogen deficiency, rapid for some experiencing surgical removal of the ovaries, slower for others experiencing the

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natural menopause, is reflected physiologically in oestrogen-dependent tissues, provoking a variety of symptomatic consequences in 80% of women [3,4]. During the past few decades several studies have documented the deleterious impact of the menopause on bone mass [5,6] and cardiovascular disease, and the reduction of risk in these areas by hormone replacement therapy (HRT) [7]. This may be attributed to the fact that these degenerative disorders are responsible for many fatalities in people over 50 years of age [8]. However, the possible effects of the postmenopausal deficiency in ovarian hormones on skin are less well documented, despite skin being the largest organ of the body and the primary barrier against microbial invasion, dehydration, and mechanical, chemical, osmotic, thermal and photic damage. After the menopause women start to complain of dry, flaky skin and easy bruising. These symptoms are often reversible with HRT, usually within the first 6 months of administration [9]. It is evident therefore that the sex hormones and in particular oestrogens play an important part in the maintenance of skin quality in women, and thus may also play a pivotal role in the healing of skin post-injury. This is of paramount clinical importance both in terms of financial cost and human suffering, since many of the chronic wounds such as venous ulcers, pressure sores [10] and burns [11] afflict the elderly population, of whom postmenopausal women comprise the majority. Oestrogen receptors have been demonstrated in the nucleus and/or cytoplasm of various cells in human skin [12–14] and in cells such as macrophages [15], fibroblasts [16], and endothelial cells [17] all of which play vital roles in the healing process, suggesting a direct effect of oestrogens on both intact and wounded skin. However, there is a paucity of information regarding the effects of oestrogens on these targets. There have been a number of studies on epidermal [18] and dermal thickness [19], skin mitotic figures [20], elastic properties [21] and collagen content of skin [22]; however, collectively the results have been inconclusive. Furthermore, few and contradictory observations have been reported on the influence of

oestrogens on wound healing in extragenital tissues. The aim of this review is to provide a survey of the literature that is available regarding the physiological effect of oestrogens on the various phases of cutaneous repair described by Clark [23]: (1) inflammation; (2) new tissue formation (proliferation); and (3) matrix formation and remodelling, although many of the studies were performed in the 1960s and 1970s before much of our current knowledge on wound repair was acquired. Research carried out on the effects of oestrogens, both in terms of their deficiency and replacement, on the process of tissue repair in various animal models is described and discussed, together with the very limited work undertaken in humans. Some of the earliest work was investigated in gingival tissue in the field of periodontology, prompted by the clinical symptoms of chronic gingival inflammation in pregnant women [24,25]. The studies that followed examined, in laboratory animals, the influence of female sex hormones on healing using subcutaneous implantation of cellulose sponges, stainless steel and Teflon® cylinders, the production of sterile abscesses etc in addition to cutaneous wounds. In recent years there has been negligible work on the effects of oestrogens on the repair of wounded cutaneous tissue, emphasising the need for further experimentation in this area. However, some of the more up-to-date research on the influence of oestrogens on the individual cell types e.g. neutrophils, macrophages, fibroblasts, and components of the extracellular matrix e.g. collagen, elastin, glycosaminoglycans that play a fundamental role in the healing wound is included.

2. The effects of oestrogens on the inflammatory phase of wound repair The inflammatory phase of repair is marked by platelet accumulation, coagulation, an increased permeability of the vessels adjacent to the wound and leucocyte migration into the wound bed [26]. Studies investigating the effects of oestrogens on these processes are addressed.

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One of the earliest experiments [27] examined the influence of excessive amounts of oestradiol dipropionate on the tissue in the walls of sterile turpentine abscesses produced subcutaneously in both male and female rats. A histological comparison was made of the inflammatory response and the granulation tissue in the oestrogen-treated animals, a group rendered virtually free of circulating steroid hormones by the removal of the adrenals and gonads, and an intact group not receiving hormonal therapy. The investigators found that in the animals receiving oestradiol the walls of the abscesses were thin, very poorly demarcated from the surrounding oedematous tissue, and almost completely void of granulation tissue and fibroblastic response, consisting almost entirely of polymorphonuclear leucocytes (PMNLs) suggesting a lack of progression in healing from the early inflammatory phase of repair. Baker [28] investigated the tissue reaction surrounding implanted cholesterol crystals and found that the administration of oestradiol tended to suppress the infiltration and activity of macrophages, indicative of a reduced inflammatory response. In contrast, earlier work by Rigdon and Chrisman [29] who examined local areas of inflammation in the skin of rabbits found that intramuscular injections of oestradiol benzoate did not influence the inflammatory phase of healing in terms of vascular permeability, measured by the leakage of injected trypan blue, nor the emigration of PMNLs from the vessels into the wound site. However, all these studies involved the administration of physiologically excessive amounts of oestradiol to the experimental animals, and there seems to be an optimum physiological oestrogen dose for their beneficial effects to be demonstrated on epidermal thickness and collagen content in intact skin [30 – 32]; this may also apply to the process of healing, resulting in the occurrence of detrimental effects if the oestrogen dose is too high. Following the clinical observations of chronic gingival inflammation in pregnant women [24,25] and those using hormonal contraceptives [33], there have been a number of studies on the effects of oestrogens and progesterone on the inflammatory process in gingival tissue. Lindhe et al. [34]

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reported that pregnant rats, and non-pregnant rats treated with a combination of oestrogen and progesterone, showed an increased permeability of the gingival vasculature to intravenously injected trypan blue; that there was an enhanced inflammatory response was further substantiated from the findings of increased exudate in pregnant women [35], and hormone-treated dogs [36]. In a study of the gingival condition of pregnant women, Hugoson [35] observed that the hormonal alterations which occur during pregnancy did not influence normal gingiva, whereas the reactions in areas exhibiting gingivitis (i.e. an inflammatory response), gradually increased throughout pregnancy. Subsequent to these findings Nyman and his colleagues carried out a series of investigations [37–41] studying the effects of oestrogens and progesterone on granulation tissue outside the oral cavity, including that developing at the site of wounds to the skin. This was some of the first work carried out on the effects of oestrogens on cutaneous healing. In one of these experiments, Nyman et al. [40] investigated vascular permeability in the inflammatory phase of healing in wounded ovariectomised rabbits treated with daily intramuscular injections of oestradiol benzoate and progesterone, compared with a control group receiving a vehicle only. This was achieved by measuring the penetration of intravenously injected I125 -labelled human serum albumin (RIHSA) into exudates formed in stainless steel cylinders implanted subcutaneously in the backs of rabbits. A significantly lower amount of exudate was formed in the oestradiol and progesterone treated rabbits than in the controls up to, but not on, day 4 after implantation of the cylinders, but no differences were observed in the penetration of RIHSA into the exudates. These results are in accordance with those of Hugoson and Lindhe [36] who reported that after gingival wounding, progesterone and stilboestrol treated female dogs exhibited a significantly lower exudation than controls. The observation by Nyman et al. [40] of differences in vascular exudation between the groups up to, but not on day 4, suggests that the influence of the hormones on vascular permeability in wounded areas is limited to the first few days (i.e. the inflammatory phase) of the

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healing process. It is possible that it was the oestradiol component of the hormonal administration that suppressed exudation in the inflammatory response since treatment with progesterone alone was found to enhance vascular exudation during the inflammatory phase of healing in the ovariectomised rabbits. It was also demonstrated in the same study that the total protein and the concentration and relative percentage of albumin in plasma and the exudates did not differ between the oestradiol and progesterone treated rats and the control group, resulting in the authors suggesting that in the treated animals there may be a reduced total surface area of vessels (fewer vessels) rather than a specific hormone dependent alteration of the vessel walls. This inference was supported by the observations of another study performed by Nyman et al. [38] of a reduced angiogenesis following wounding in rabbits treated with a combination of oestradiol and progesterone, compared to the controls. In a large study involving 300 ovariectomised rats, Lundgren [42] analysed the influence of daily oestradiol benzoate intramuscular injections, on the amount of exudation and inflammatory cell migration following the subcutaneous implantation of Teflon® cylinders in the backs of the animals. The authors found that the degree of exudate accumulation in the cylinder was markedly decreased in the oestrogen-treated rats at 12 h, 4 days and 1 week after implantation compared to the control group. In both groups there was an early predominance of PMNLs followed by a preponderance of mononuclear cells, but the oestrogen-treated animals had a smaller number of PMNLs within the cylinder at all the examination periods (12 h, and 4, 7, 14 and 21 days). Furthermore, oestrogen treatment alone or combined with progesterone resulted in a reduction of the total amount of both types of inflammatory cell from day 7 inclusive, in accordance with the findings reported by Deasy et al. [43] who observed a significant decrease in the gingival connective tissue area occupied by inflammatory cells in ovariectomised monkeys treated with oestrogen and progesterone. In contrast to the observations by Nyman et al. [40] who found a decreased amount of exudate in

inflamed areas in oestrogen and progesterone treated ovariectomised rabbits, Lundgren [42] reported that in his study the group of rats treated with a combination of oestrogen and progesterone retained an amount of exudate similar to that of the control group. This discrepancy may however, be explained by the use of a different species of experimental animal or the difference in the ratio between the doses of oestrogen and progesterone administered. A study by Murthy et al. [44] investigated the effect of oestrogens on the healing of cutaneous wounds in 136 albino rats, randomly divided into (1) a control group receiving no hormonal treatment; (2) a group receiving 0.5 mg/kg body weight of oestrogen injected on alternate days; (3) a group receiving 2 mg/kg body weight of oestrogen injected on alternate days; and (4) a group of ovariectomised rats. The only comments by the authors regarding the inflammatory phase of repair was that the animals receiving the highest dose of oestrogen (group 3) showed a decreased inflammatory exudate compared to the controls, supporting the findings discussed above [36,40,42,43], and that similar results in the ovariectomised group (group 4) and the controls (group 1) suggested that bilateral ovariectomy did not have any influence on the inflammatory phase of wound healing. Contrary to the results observed by Lundgren [42], Pallin et al. [45] who examined granulation tissue formation in cellulose sponges implanted subcutaneously in the backs of 98 ovariectomised rats, found that daily intramuscular injections of oestrogen resulted in similar histological findings to the control group in terms of infiltration of PMNLs and monocytes, 7 days after implantation of the sponges. The group treated with a combination of oestrogen and progesterone showed a lower number of inflammatory cells than did the controls, a finding that correlates well with the reduced number of functioning vessels which develop in the wound demonstrated after treatment with oestrogen and progesterone [38]. Some of the more recent research, although not specifically on the effects of oestrogens on the inflammatory phase of tissue repair following the creation of a wound or inflammatory area, has

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investigated the influence of these hormones on some of the properties of the cells and vasculature that are involved in this phase of healing. Gouveia et al. [46] in a study examining the significance of the effects of female sex steroids on the vascular permeability of the uterine horn in virgin rats found that the administration of conjugated oestrogens inhibited vascular permeability in inflammatory conditions. The findings are in accordance with those of Murthy et al. [44] who found that administration of high doses of oestrogen decreased the inflammatory exudate present in cutaneous rat wounds suggesting that oestrogens may influence vascular permeability and the resulting degree of inflammatory exudate accumulation. The demonstration of oestrogen receptors in human megakaryocytes [47] and a relationship between an increase in intravascular blood coagulation and thrombus formation and increased levels of oestrogens from the oral contraceptive pill [48] and to a lesser extent HRT [49], suggests that platelet aggregation during the inflammatory phase of wound healing may be affected by oestrogens. This theory is supported by a study by Rosenblum et al. [50] who have reported that treatment with oestradiol implants enhances platelet aggregation in mice at the site of microvascular injury in the pial arterioles of the brain, compared to placebo-treated controls. However, in contrast to this enhancement of aggregation, when platelets were tested in vitro in platelet rich plasma, aggregation in response to sodium arachidonate or to adenosine diphosphate (ADP) was not altered in the oestradiol treated mice. This finding led the authors to conclude that the enhanced aggregation observed in injured pial arterioles of oestradiol treated mice may not reflect direct effects of oestradiol on the platelet itself, but may reflect an effect of oestradiol on endothelium or adjacent tissue. Contrary to the above findings, Aune et al. [51] investigating the effects of 12 months treatment with topical and oral combined HRT on the reactivity of platelets in whole blood in 32 postmenopausal women found significant reductions in the formation of thromboxane B2. From these findings the authors concluded that HRT reduces

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the cellular activation of blood platelets, which may account for some of the beneficial effects in reducing the risk of cardiovascular disease, although it is not known that oestrogens would have the same effect on platelets in the wound environment. Neutrophils are the first cells to arrive at the wound site in substantial numbers during the inflammatory phase of repair, and they play an important role in the phagocytosis of pathogenic bacteria and debris [52]. Micromolar concentrations of oestrogens have been found to enhance the oxidative metabolism of activated human PMNLs possibly by an increase in their myeloperoxidase activity [53]. This author reported a 10fold increase in enzyme activity in the presence of 5 mm b-oestradiol or oestriol; the activity of the released enzyme (as well as purified myeloperoxidase) was effectively inhibited by small amounts of anti-myeloperoxidase antibodies. This observation was taken as evidence by the investigators that the released enzyme was identical with myeloperoxidase and suggests that either postmenopausal oestrogen deficiency or replacement with hormonal therapy may have an effect on the phagocytic activity of PMNLs in the inflammatory phase of wound healing, via an action on the myeloperoxidase enzyme system. Experiments on the effects of exogenous 17boestradiol on the phagocytic capacity of PMNLs, determined in chemiluminescence assays, in ovariectomised sows [54], demonstrated a significant increase in phagocytic capacity in vivo after a 6 week treatment period with physiological levels of oestradiol, whereas administration of a higher concentration resulted in the maximal rate of phagocytosis being significantly reduced (by 12%) in vitro [55]. These findings appear to support those of Shahrad and Marks [30] on epidermal thickness and Brincat et al. [31,32] on skin collagen content, suggesting that there may be an optimum physiological oestrogen dose for the beneficial effects of the hormone to be demonstrated on these components of skin structure; the same may be true for the influence of oestrogens on the phagocytic capacity of PMNLs. Josefsson et al. [56] reported that physiological and pharmacological doses of oestradiol signifi-

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cantly suppressed the inflammatory response in ovariectomised mice, measured by footpad swelling and histological examination, after intradermal injection of olive oil and cholera toxin to induce local inflammation. The authors concluded that the oestradiol significantly suppressed the bone marrow production of leucocytes and affected the distribution of PMNLs in peripheral blood. Recently Ito et al. [57] in accordance with similar findings by Miyagi et al. [58] demonstrated that physiological concentrations of 17b-oestradiol in humans significantly reduced the chemotaxis of PMNLs. Pre-incubation with clomiphene or tamoxifen (oestrogen receptor antagonists), eliminated the inhibitory effect restoring it to the control levels, suggesting that 17b-oestradiol may suppress the chemotaxis of PMNLs by a receptordependent mechanism. Whether these anti-inflammatory effects exerted by oestrogens are reduced following the reduction of oestrogens after the menopause, or have any effect on the inflammatory phase of healing following injury remains to be investigated. Monocytes and the macrophages they differentiate into, also play a crucial role in the healing wound, not only eliminating deleterious materials e.g. micro-organisms and wound debris by phagocytosis in the inflammatory phase, but also generating chemotactic and growth factors critical to the co-ordination of granulation tissue formation in the proliferative phase of repair. The demonstration of oestrogen receptors in both the human monocytic leukaemia cell line J111 and rat peritoneal macrophages by Gulshan et al. [15], suggests that oestrogens may have a direct effect on this cell type, which plays a role throughout the process of wound repair. Indeed, one of our recent studies investigating the effects of ovarian hormone deficiency on the infiltration of neutrophils and monocytes into the wound bed postinjury, in a rat model, supports this [59,60]. Following the creation of full-thickness excised lesions at either 2 weeks or 4 months post-ovariectomy, the 48 ovariectomised rats showed markedly reduced numbers of PMNLs, monocytes and macrophages in the wound bed on days 3 and 5 post-injury, but by day 10 these cells were

frequently more numerous in the ovariectomised groups than in the sham-ovariectomised controls, where the numbers had reduced as occurs typically in the proliferative and remodelling phases of healing. This suggests that the inflammatory phase of repair may be delayed as well as depressed and that this may be related to a detrimental effect of oestrogen and/or progesterone deficiency on the early stages of healing. A significantly reduced number of mature tissue macrophages was noted in the ovariectomised groups compared to the controls on days 3, 5, 10 and 22 post-injury in the tissue wounded at either 2 weeks or 4 months post-ovariectomy. Since the total monocyte and macrophage number was shown to be higher in the ovariectomised groups than in the control groups by 10 days post-injury, it is possible that the reduced levels of ovarian hormones resulting from ovariectomy, had an effect on the ability of the monocyte or immature macrophage to differentiate into its mature form [59].

3. The effects of oestrogens on the proliferative phase of wound repair The proliferative phase of repair is characterised by (1) re-epithelialisation restoring the cutaneous barrier; (2) angiogenesis, the neovasculature supplying much of the nutrition required for healing; (3) fibroplasia, during which the matrix of the granulation tissue and scar tissue is formed; and (4) wound contraction, reducing wound size and thus the need for scar tissue [26]. Studies investigating the effects of oestrogens on these processes are addressed. Taylor et al. [61] investigated the effects of daily oestradiol dipropionate injections on the healing of sutured full-thickness cutaneous wounds in male rats and found that on histological analysis both the oestradiol treated rats and the control group demonstrated fibroblastic proliferation 4 days post-wounding. There was often a reduced degree of fibroplasia in the rats receiving oestrogen treatment compared to the controls, but the authors reported no clear-cut group demarcation and concluded that the rate of healing in terms of

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fibroblastic infiltration and proliferation, was not altered by the administration of oestradiol. In contrast, a later study by Murthy et al. [44] examining the effects of subcutaneous oestrogen injections following cutaneous wounding in male and female rats reported a significantly reduced fibroblastic response and retarded collagen synthesis and maturity in the oestrogen treated groups. The influence of oestrogens on granulation tissue formation, provoked by the subcutaneous insertion of a plastic ring in the back of female rats, was examined by Jorgensen and Schmidt [62]. The authors found no significant differences in the dry weight of granulation tissue in the rings, nor in the content of hydroxyproline taken to indicate collagen content, in the oestradiol treated group compared to the controls, and concluded from these findings that oestrogens had no effect on these aspects of granulation tissue. These results are in conflict with those of Portugal et al. [63] who observed an inhibited fibroblast reaction in the walls of turpentine-provoked abscesses following the combined administration of testosterone and oestradiol, and those of Robertson and Sanborn [64] and Fisher and Paar [65] who, having provoked carrageenin granulomas in guinea-pigs and rats, showed that injection of stilboestrol resulted in increased fibroblast infiltration and collagen production. Possible explanations for these discrepancies may be the utilisation of different techniques to stimulate granulation tissue formation as the tissues may not react in a similar manner to the turpentine, carrageenin and plastic and also the administration of different forms and doses of the oestrogen with or without the addition of other sex hormones, such as testosterone. In a large study involving 300 ovariectomised rats, Lundgren [42] analysing the influence of daily intramuscular injections of oestradiol benzoate on the amount of granulation tissue formed following the subcutaneous implantation of Teflon® cylinders in the backs of the animals, found that the oestrogen-treated rats developed a significantly smaller amount of granulation tissue than the controls. These findings are in accordance with those of Nyman et al. [39] and Lundgren [66] who have reported a markedly reduced

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amount of granulation tissue following the administration of oestrogen in ovariectomised rabbits and rats, respectively. Pallin et al. [67], examining the effects of oestradiol on granulation tissue formation following the subcutaneous implantation of cellulose sponges in ovariectomised rats, also observed a decrease in the amount of granulation tissue in terms of a reduced quantity of collagen, determined by hydroxyproline levels, in oestrogen treated rats during the early stages of the proliferative phase of healing. The amount of collagen in granulation tissue is determined by the balance of synthesis and degradation. Thus, this reported decrease in collagen quantity can be the result of decreased synthesis and/or increased breakdown. A study was undertaken by Hadberg et al. [68] to investigate the effects of a combination of oestrogen and progesterone on collagen synthesis, by studying the in vitro incorporation of 14C-proline in granulation tissue. Results showed that the incorporation of radioactive proline into hydroxyproline was decreased in granulation tissue treated with the sex hormones, indicative of reduced collagen synthesis. This effect on collagen synthesis may be mediated by the oestrogen component of the administered hormone combination directly via oestrogen receptor-proteins, which have since been demonstrated to be present in the nucleus of fibroblasts [16], the main cell type responsible for collagen synthesis. The various studies of the effects of oestrogens on vascular profiles such as the degree of vascularity, blood flow etc. during the proliferative phase of healing, also appear to show contradictory results. Lindhe et al. [69] assessing the process of wound healing following the creation of mechanical injuries to the pinnae of the ears of ovariectomised rabbits treated with oestrogens, found no effect on vascular proliferation in the blastemata which develop at the sites of such wounds, although the vessels were more dilated than those of the saline treated controls. This latter observation is consistent with data from earlier experiments in rats and guinea-pigs where it was noted that oestrogen injections promoted hyperaemia in various parts of the body by alterations of the capillary bed [70,71].

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Some of the investigations carried out by Nyman and his colleagues in the early 1970s [38,39,41] on female rabbits, examined the effects of intramuscular injections of oestradiol and progesterone on the vascularity and blood flow of granulation tissue, formed following the infliction of skin wounds and the subcutaneous implantation of stainless steel wire mesh cylinders. Nyman et al. [38] evaluated the vascularity of granulation tissue by means of microangiography following the creation of full-thickness cutaneous wounds in 40 ovariectomised rabbits. The authors reported that the daily administration of oestradiol alone for a period of 4 weeks, did not influence the number of vessels in the wounded tissue and supported the observations by Lindhe et al. [69] who found that intramuscular injections of oestrogen did not induce vascular proliferation in healing skin wounds of ovariectomised rabbits. However, limitations of the techniques employed probably result in the microangiographic representation of the vascular system of the wounded areas more accurately reflecting the in vivo functioning (open) vessels rather than the total number of existing vessels. In support of the above results, Pallin et al. [45] who examined granulation tissue formation in cellulose sponges implanted subcutaneously in the backs of 98 ovariectomised rats found that daily intramuscular injections of oestradiol benzoate resulted in similar haemoglobin levels in the granulation tissue to the control group; haemoglobin levels had been shown by previous investigators [72,73] to reflect the magnitude of the capillary bed. Pallin et al. [45] also reported a similar histological picture of vascularity between the groups. In contrast to the findings of Nyman et al. [38], Lundgren [66] in a study of the influence of oestrogen and progesterone on the vascularisation of granulation tissue in 44 ovariectomised rats, also using microangiographical techniques, observed a significant suppression in vascularisation in the oestrogen treated group compared to the controls. Discrepancies between the results of the two studies can probably be explained by the use of different species of experimental animal and the administration of oestrogens for differing du-

rations, in addition to the utilisation of different contrast mediums which may affect the degree of visualisation of the vascular system. Among its many functions, the microcirculation of the granulation tissue supplies nutrients and oxygen needed for the synthesis of the new tissue and removes carbon dioxide and other metabolites. The oxygen tension (pO2) and carbon dioxide tension (pCO2) within the wound reflect, according to Hunt et al. [74], the net affect of the ability of the local circulation to perform this function and the metabolic activity within the healing tissue. The effects of a combination of oestrogen and progesterone on these parameters were investigated in another study in ovariectomised rabbits by Nyman [39]; the effects of oestrogen alone was not studied by the authors. Wounds were created by subcutaneously implanting stainless steel wire-mesh cylinders in the rabbits backs and the pO2, pCO2 and pH of the exudate collected within the wound cylinders was determined, in addition to the amount of granulation tissue formed in the cylinders during the proliferative phase of repair. The wet- and dryweight of the granulation tissue formed in the oestrogen and progesterone treated rabbits was significantly lower than in the controls, as was the pO2, but no difference was noted in the pCO2 or pH values between the groups. Carbon dioxide is more easily diffusible than oxygen and thus a comparatively dense vascular network may be required to provide an adequate oxygen supply to the tissue, whereas within certain limits a reduced number of functioning vessels may be adequate to remove the easily diffusible carbon dioxide, which may help to explain the differences in oxygen tension of wound fluid between the groups and the lack of differences in carbon dioxide tension. From these observations the authors concluded that oxygen consumption in the oestrogen and progesterone treated animals would be comparatively low since only a small amount of granulation tissue was formed and that this low oxygen consumption, in combination with a low wound fluid pO2 indicated a decreased supply of oxygen to the proliferating cells in the rabbits treated with a combination of oestrogen and progesterone. This corroborates the observations of a reduced

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number of functioning vessels during the proliferative phase of healing in oestrogen and progesterone treated rabbits compared to controls [38]. The degree of blood flow was also analysed within granulation tissue formed in subcutaneously implanted stainless steel wire mesh cylinders in 15 oestradiol and progesterone treated ovariectomised rabbits [41]; again no observations were made by the authors as to the effects of solely oestrogen administration. The blood flow was determined by measuring the disappearance rate (clearance) of Xe133 injected into the cylinders, the disappearance of Xe133 from a tissue being closely related to the capillary blood flow [75]. It was noted that in both the hormone treated and control group there was a gradual increase in blood flow of the granulation tissue from day 7 after implantation to day 21, indicating a gradual improvement of the microcirculation during the observed period of granulation tissue formation, in parallel with the nutritional needs of the proliferating cells. This finding is in agreement with that of Nyman et al. [39] who showed an increase from day 7 to 21 in the oxygen tension of the wound fluid of rabbits treated with a similar combination of hormones. However, the clearance data showed no difference in blood flow between the groups in the course of granulation tissue formation during the proliferative phase of repair, and thus the low oxygen tension found in the animals given oestrogen and progesterone [39] may be mainly the result of a reduced number of vessels in this group [38], rather than a decrease in their blood flow. Some of the more recent research, although not dealing specifically with the effects of oestrogens on the proliferative phase of tissue repair following the creation of a wound or inflammatory area, does demonstrate the influence of these hormones on some of the properties of the cells and vasculature that are involved in the process. The demonstration of high affinity oestrogen receptors in monocytes and macrophages [15] suggests that oestrogens may have a direct effect on these cell types, which play a vital role in the proliferative phase of healing by the synthesis of a plethora of growth factors which have a function in regulating the co-ordination of granulation tis-

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sue development. A study by Hu et al. [76] of the effects of oestradiol on the synthesis and secretion of IL-1 by rat peritoneal macrophages found that such macrophages from adult female rats secreted greater amounts of IL-1 than those from agematched male rats or prepubescent female rats. It was also shown that ovariectomy led to reduced synthesis of IL-1 by macrophages, but oestradiol replacement therapy administered to the rats effectively increased IL-1 synthesis. These findings suggest that oestradiol may play an important role in regulating synthesis of IL-1 by macrophages and thus may indirectly affect the proliferative phase of wound healing since IL-1 is thought to be involved in the formation of granulation tissue possibly by stimulating HA synthesis [77] and collagen deposition [78]. Shanker et al. [79] demonstrated that oestrogen modulates platelet derived growth factor (PDGF)A gene expression by monocyte/macrophages, and thus may indirectly influence the proliferative phase of wound healing, since PDGF is mitogenic and chemotactic for fibroblasts [80], plays a role in angiogenesis by the attraction of cells that stimulate angiogenic factors [81] and is thought to stimulate wound contraction [82]. Thus oestrogen deficiency adversely affecting the synthesis of PDGF could be a possible explanation for the significantly slower rate of cutaneous wound contraction in ovariectomised rats compared to sham-ovariectomised controls, demonstrated in one of our recent studies [83]. In the rats wounded 4 months post-ovariectomy, the ovariectomised rat wounds were approximately 30% larger than the controls on days 3 and 5 post-injury, whereas no significant difference was observed between the sham-ovariectomised and ovariectomised groups wounded 2 weeks post-ovariectomy, indicating that the effects of ovarian hormone deficiency on this process are delayed. These findings suggest that the effects of ovariectomy on wound contraction are not only dependent on the plasma levels of oestrogens but also on the duration of the deficiency. Fibroblasts synthesise the matrix at the wound site in addition to facilitating wound contraction and releasing angiogenic factors which influence new blood vessel formation, emphasising the im-

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portant role that this cell plays in the proliferative phase of healing. Oestradiol receptors have been identified in normal breast fibroblasts [16] and thus research investigating the effects of oestrogen on fibroblasts may aid our understanding of the influence that these female sex hormones have on the repair process. Levine et al. [84] and Luo et al. [85] investigating growth factors that stimulate the proliferation of fibroblasts derived from both foetal and adult human prostates have both claimed that oestradiol did not effect fibroblast proliferation although further investigations are required to establish if this is true of the wound environment. Sato et al. [86] observed that treatment with 17b-oestradiol decreased the level of procollagenase and prostromelysin produced by cultured rabbit uterine fibroblasts, but increased the production of tissue inhibitor of metalloproteinases (TIMP) by these cells. These results suggest that collagenolysis in uterine cervical fibroblasts is negatively regulated by steroid hormones including 17b-oestradiol, via the acceleration of TIMP production and the suppression of synthesis of major metalloproteinases (MMPs). Collagenolysis is an important phenomenon in both the overlapping proliferative and remodelling phases of tissue repair since, with synthesis, it regulates the amount of collagen in the granulation tissue and ultimately in the scar; thus 17boestradiol may influence this process of wound healing indirectly by regulating the proteases involved in collagen degradation. The presence of oestrogen receptors in the nuclei of cultured human aortic and umbilical vein endothelial cells [17] and the knowledge that oestrogens and the other sexual steroids influence angiogenesis in the placenta during pregnancy, suggests that oestrogens may influence the process of angiogenesis in the proliferative phase of wound repair via a direct effect on endothelial cells. A study by Peek et al. [87] investigating the effects of natural and synthetic sex steroids on the proliferation of human decidual endothelial cells in culture, observed that oestradiol at 5.0 ng/ml stimulated culture growth, whereas lower concentrations inhibited growth and higher concentrations had no effect. These observations led the authors to suggest that oestradiol has direct ef-

fects on endothelial cells and is probably involved in the cyclical growth and regression of the endometrial blood vessels in vivo. These findings suggest that the possibility of oestradiol having an effect on the endothelial cells involved in angiogenesis is worthy of investigation. Morales et al. [88] examined the effects of 17boestradiol on human umbilical vein endothelial cell behaviour in vitro and on angiogenesis in vivo and found that the addition of 17b-oestradiol increased the attachment of the endothelial cells to laminin, collagen types I and IV and fibronectin. After a confluent monolayer of cells was ‘wounded’ by scraping, oestradiol-treated cells migrated into the wound three times faster than untreated control cells and oestradiol also enhanced the ability of the endothelial cells to organise into tubular networks when plated on a reconstituted basement membrane; both these effects were blocked by the specific oestrogen receptor antagonist ICI 182,780. In vivo vascularisation of reconstituted basement membrane plugs coinjected with bFGF was markedly decreased in ovariectomised mice, whereas with oestrogen replacement angiogenesis was increased to the levels observed in non-ovariectomised mice. These studies demonstrate that both in vitro and in vivo, oestradiol enhances endothelial cell activities important in neovascularisation, and suggests a promoting influence of oestrogens on angiogenesis, and thus an effect on the proliferative phase of healing.

4. The effects of oestrogens on matrix formation and remodelling The remodelling phase of tissue repair takes place over a period of months during which the skin responds to injury with a dynamic continuation of collagen synthesis and degradation, and the once highly vascular granulation tissue undergoes a process of devascularisation, as it matures into less vascular scar tissue [26]. Studies investigating the effects of oestrogens on this process, particularly on tensile strength of scar tissue, are addressed.

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Taylor et al. [61] testing the tensile strength of full-thickness cutaneous wounds in 60 male rats found that there was no significant difference between the oestradiol-treated and control groups from day 2 to 7 post-wounding, i.e. there was a steady increase in wound strength as it progressed from the inflammatory into the proliferative phase of repair. These findings were supported by studies performed by Nyman et al. [37] assessing the tensile strength of incised cutaneous wounds in 34 ovariectomised rabbits, and by Pallin et al. [67] examining tensile strength of granulation tissue after the insertion of subcutaneous cellulose sponges in ovariectomised rats. Both investigations demonstrated that daily intramuscular injections of oestradiol benzoate in the animals had no influence on the tensile strength measurements compared to controls. Jorgensen and Schmidt [62] also studied the effects of oestradiol on the tensile strength of cutaneous linear wounds but observed results conflicting those presented above. The authors claimed that administration of oestradiol injections to female rats significantly increased the tensile strength of the wounds on the tenth day post-wounding compared to the control group. The discrepancies may be due to the use by Jorgensen and Schmidt [62] of different animals for the control and test wounds, the administration of high pharmacological doses of oestradiol and experimentation on rats with intact ovaries, as opposed to physiological doses administered to ovariectomised rabbits, where each animal acted as its own control which was the methodology employed by Nyman et al. [37]. The findings of Jorgensen and Schmidt [62] are in accordance with a more recent study investigating the effects of oestrogens on the healing of the rat genital tract [89] which showed that the administration of oestradiol implants increased the tensile strength of anastomosed uterine horns during the first 14 days of healing, but thereafter no significant differences between the oestradiol and control group were found. If a similar pattern of healing is true for cutaneous wounds, this supports our observations of no significant difference in the breaking strength of wounded tissue between ovariectomised rats and sham-ovariec-

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tomised controls 22 days post-injury [90]. Oestrogen deficiency following ovariectomy may have resulted in a reduced breaking strength within the first 2 weeks post-injury which was not demonstrated by day 22 when the breaking strength of the tissue was assessed by Calvin [90]. In contrast to the findings of all the studies presented above, Murthy et al. [44] observed a marked reduction in tensile strength between the seventh and fifteenth day post-wounding in oestrogen treated rats compared with an ovariectomised group and an intact group not receiving hormonal therapy. The authors noted that this reduction coincided with a period during the healing process when collagen synthesis was proceeding maximally and suggested that oestrogen administration resulted in a delayed collagen synthesis and thus reduced tensile strength at these time periods. As was the case with the other phases of wound healing, some of the more recent research, although not specifically on the effects of oestrogens on the remodelling phase of tissue repair, has investigated the influence of these hormones on some of the proteins, particularly collagen, that are involved in this phase of healing when injury does occur. The remodelling of collagen is dependent upon the interplay of continued collagen synthesis and degradation [23], the degradation being controlled by a variety of collagenase enzymes which appear to be influenced by oestrogen administration. Sato et al. [86] observed that treatment with 17boestradiol decreased the level of procollagenase and prostromelysin produced by cultured rabbit uterine fibroblasts, but increased the production of TIMP by these cells. A study investigating oestrogen-mediated collagenolysis as a mechanism of cervical dilatation in the guinea-pig at parturition [91], reported that physiological concentrations of 17b-oestradiol stimulated degradation of type I collagen in the non-pregnant cervix in organ culture and collagenase degradation products were detected in the ECM and in the culture media. A similar study investigating the effects of oestrogens on collagen metabolism in human cervical tissue in relation to cervical ripening for parturition [92], noted that collagenase activity

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was significantly elevated when oestrone sulphate was added to the incubated cervical tissue. The collagenolytic effect of oestrogen-mediated nitric oxide plays an important role during parturition because nitric oxide-induced collagenolysis causes the cervical os to dilate. Kumar and Thampan [93] claimed that rat uterine collagenases which use collagen types I, III and V as the substrates are regulated by oestradiol, and Puistola et al. [94] demonstrated that oestradiol also regulates the MMP-2 gelatinase, which cleaves type IV collagen, in human granulosa-lutein cells. Collagenolysis is an important phenomenon in tissue repair since together with synthesis it determines the collagen content and thus the functional strength of the scar tissue; the findings presented above suggest that oestrogens may influence the remodelling phase of the healing wound by regulating the proteases involved in collagen degradation. Several investigations have also documented a role of oestrogens in collagen synthesis in a variety of tissues, which again may be indirectly relevant to collagen synthesis in the remodelling phase of cutaneous wound repair. Suzuki and Nakada [95] found that administration of 17boestradiol increases collagen content in the prostate of rats, and that the ratio of type III collagen to type I collagen in the gland decreases following 17b-oestradiol treatment. These findings led the authors to suggest that 17b-oestradiol increases the accumulation of collagen into the prostate with different extents of influence on the synthesis of type I and III collagens; lysis may also be affected differentially. The observations are in accordance with the results of dermal investigations by Brincat et al. [19,22,96] which demonstrated that the administration of oestrogens increased the collagen content of uninjured skin. Similarly, a more recent study by Ashcroft et al. [97] showed that postmenopausal women who had never taken HRT had reduced matrix collagen deposition 7 and 84 days post-wounding, whereas postmenopausal women who had taken HRT for at least 3 months had markedly increased levels of collagen deposition, similar to those observed in a pre-menopausal group. In vitro experiments carried out by these investigators indicated that this effect on wound healing

was due to the oestrogen component of HRT, possibly mediated by an increase in the activity of the cytokine transforming growth factor-beta 1 [97]. However, in contrast to the above, oestrogen administration resulted in a decrease in the collagen content of the temporomandibular joint disc of ovariectomised rats [98] and ovariectomy was not found to have an effect on the collagen content in the lower urinary tract smooth muscle of rabbits [99]. In accordance with these negative findings, Demirbilek et al. [100], with specific reference to the effects of oestrogens on collagen synthesis in wounds, demonstrated that combined administration of oestradiol and progesterone to rats with alkali-induced corrosive oesophageal burns resulted in significantly lower hydroxyproline levels and reduced collagen deposition in the submucosa and tunica muscularis compared to the control group. Based on these results the authors believed that oestradiol and progesterone inhibited new collagen synthesis following wounding to the oesophageal tissue, but no data was available as to the effects of the latter following the administration of oestrogen alone, or on its effects on collagenolysis.

5. Conclusions In this review the literature that is available regarding the involvement and influence of oestrogens on the various phases of cutaneous repair, has been presented and discussed. The investigations described demonstrate contradictory findings as to the effects of ovarian hormone deficiency, replacement, or excess on the process of wound healing, making it difficult to draw valid conclusions from the studies. The discrepancies in the observed results may be explained by the usage of: (1) different species and gender of animal; (2) various types of hormone administered either alone or in combination with other sex hormones; (3) assorted routes of delivery of the hormone; (4) differing frequencies, doses and durations of treatment; (5) a diversity of methods for assessing healing; and (6) a variety of tissues and cell cultures in which the healing process was

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examined. Against this background, the varying and even contradictory results described in this review regarding the effects of oestrogens on cutaneous repair are understandable and emphasise the need for further investigation in this field with a standardisation of some of the above parameters. There are estimated to be approximately 10 million postmenopausal women in the United Kingdom and 40 million in the United States, contributing to 17% of the total population [101]. Hill [102] has estimated that by 2030 the world population of postmenopausal women will be of the order of 1.2 billion. These statistics emphasise the importance of addressing the effects of ovarian hormone deficiency, and its replacement with HRT, on skin and its repair following injury, and the necessity to expand this area of research into the human population to devise methods for improving the rate and quality of wound repair in postmenopausal women. This may be particularly relevant in women with other confounding factors that may complicate the healing process such as diabetes mellitis or immunosuppressive medication, when oestrogen deficiency may be an additional factor in delaying healing in already compromised patients. Clinically our aim should be to restore the integrity and function of wounded tissue as rapidly as possible after injury and it is generally believed that a better understanding of this field could lead to improved care of cutaneous wounds, and the treatment of not only the wound but of the postmenopausal woman as a whole.

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