The Efficacy of Platelet-rich Plasma Gel and Topical Estradiol Alone or in Combination on Healing of Full-thickness Wounds

This randomized study was designed to establish the efficacy of platelet-rich plasma (PRP) gel and topical estradiol alone, or in combination, on healing of full-thickness wounds created on the trunk of rabbits. Fifteen New Zealand white female rabbits and 60 wounds were studied. Four 2.25-cm² full-thickness wounds were created using a template and treatments including a sterile saline solution that were assigned randomly to the wounds. Wounds were bandaged, dressed intermittently, and assessed by wound measurements and a collection of samples at 7, 14, and 21 days to evaluate healing. Variables of interest were hydroxyproline concentration and scored gross and microscopic morphologic characteristics reflective of wound healing. PRP gel + estradiol-treated wounds completely healed faster than wounds given other treatments. Granulation tissue growth to the skin level was faster (P < 0.05) in the PRP gel + estradiol group than the other groups. Only the PRP gel + estradiol group had a significantly lower collagen level than the sterile saline solutions on day 7 (P < 0.05). The PRP gel + estradiol group and estradiol group led to an increase in angiogenesis on day 14 (P < 0.05). Complete epithelization was observed only in the PRP gel + estradiol group compared with the other groups on day 7. PRP gel + estradiol-treatment enhanced healing in full-thickness wounds by reducing the contraction rate with a trend toward accelerating both epithelial migration and the angiogenic response.

The primary goal of wound care is to achieve rapid and functional healing with a cosmetic scar.¹ Wound healing involves a series of events (ie, clotting, inflammation, organization of granulation tissue, epithelization, and tissue restoration). These events are ensued by stimulation and organization of cellular activities through the interaction of the signals formed by cytokines and growth factors.^1–3 Following injury, various growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta and alpha (TGF-β and TGF-α), platelet-derived endothelial cell growth hormone (PDEGH), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), interleukin l (IL-1), interleukin 2 (IL-2), and platelet-activating factor 4 (PAF-4), are released.^1,3–8 These factors play a particular role in restoring tissue (re-epithelization and neovascularization), synthesizing the extracellular matrix, and recruiting mesenchymal cells.³ PRP is a concentrated thrombocyte product that has been used in clinical trials to hasten wound healing.^2,5

Estradiol receptors are known to exist in cells involved in the healing process such as macrophages, fibroblasts, endothelium, and in the cytoplasm and/or nucleus of various cells in the dermis,⁹ and are reported to indirectly influence the proliferative phase of the wound healing by increasing the production of growth factors.^10–12

To our knowledge, there was no study on the effect of PRP gel and estradiol combination on wound healing in the literature.

The purpose of the present study was to investigate the clinical, biochemical, and histopathological effects of these substances used either alone or in combination on wounds with substantial loss.

Study population
A total of 15, 6-month-old, New Zealand white female rabbits (2500 ± 300 g body weight), which had been supplied by the Surgical Research Center at Ondokuz Mayis University (Samsun, Turkey) were used in the study. The room temperature and humidity were maintained at 19˚C ± 1˚C and 55˚C ± 10%, respectively. All rabbits were fed a 160-g pelleted rabbit diet (Ankara Feedstuff Industry; Ankara, Turkey) daily and water was available ad libitum. Care was taken to avoid unnecessary stress and discomfort to the animals throughout the experimental period. A complete blood cell count was performed for each rabbit on days 0, 7, 14, and 21 to evaluate the animals’ health. The Ondokuz Mayis University Animal Care and Use Committee approved the study protocol.

Wound creation
The animals were medicated before surgery xylazine (7 mg/kg intramuscularly [IM]) (Rompun®, Bayer, Istanbul, Turkey) and anesthetized with ketamine (40 mg/kg IM) (Ketasol®, Richterpharma, Interhas, Ankara, Turkey). A single dose of cephazolin sodium (30 mg/kg IM) (Cefozin® Bilim, Istanbul, Turkey) was administered for antibiotic therapy immediately preoperatively. Carprofen (4 mg/kg SC) (Rimadyl®, Pfizer Inc, Zaventem, Belgium) was injected to all animals once just before the operation and every 24 hours for 3 days postoperatively.

Each rabbit was positioned in sternal recumbency. After clipping the hair on the back of the rabbits, the skin was sterilized with polyvidone-iodine (Betadine®, Kansuk, Istanbul, Turkey). Four 1.5 cm x 1.5 cm full-thickness skin wounds, having 4-cm intervals, not extending past the panniculus carnosum muscles, were created on the back of each animal with a scalpel. One wound from each animal (test wounds) was assigned randomly to receive PRP gel (500 µL), estradiol ([estradiol hemihydrate], 0.5-mg Estreva® Gel Transdermal 0.1%, 50 g, Theramex, France) + PRP gel (500 µL), or estradiol (0.5-mg estradiol) with the fourth wound serving as the untreated control (sterile saline solution). All wounds were covered with an occlusive dressing. The dressing was secured in place with surgical tape and rabbit jackets to prevent the rabbits from irritating the wounds and/or removing the dressing. A superficial scab (Figure 1A) or layer of medication (Figure 1B) on wound surface cleaned with sterile saline solution (isotonic NaCl 0.9% solution) and the applications were done on every other day until they were completely epithelialized; thus, the scab was not a real scab. It was more like a film layer and when it was washed with saline, the wound was cleaned easily. This did not cause re-injury. Five rabbits were evaluated on day 7, 14, and 21, respectively. A 5-mm punch biopsy instrument was used to take two skin specimens from the left corners of the wound of each rabbit immediately after measurement was performed using the ruler method. The skin specimens were sent to a laboratory for pathological and biochemical analysis. After sampling, the biopsy sites of the full-thickness wounds were sutured.

PRP preparation
Platelet counts were performed on each rabbit to confirm the concentration of platelets in the blood. The average peripheral blood platelet count was 307,000/mm^³ with a range from 217,000 to 405,000.

A total of 10-mL of autologous blood was drawn from each rabbit by venipuncture of the ramus intermedius of v. auricularis caudalis before surgery, and on alternate days, it was combined with 1-mL of anticoagulant 3.8% Na citrate solution to prevent coagulation.

The blood was centrifuged at 2100 rpm for 10 minutes to separate the plasma containing the platelets from the red cells. After discarding the platelet-poor plasma, the centrifugation speed was increased to 4000 rpm to separate the PRP from the red blood cells. The platelet-poor plasma was separated from the PRP along with the buffy coat. The buffy coat and PRP, approximately 1 mL–1.5 mL, was resuspended. Platelet counts were performed on each rabbit to confirm the concentration of platelets in the PRP. The mean platelet count of the PRP was 1,206,000/mm^³ with a range from 644,000 to 2,518,000. Platelet counts were on average 3.92-fold greater compared to whole blood.

To form a gel and to activate the PRP, a mixture was added to the autologous platelet concentrate (APC) of equal volumes of 10% calcium chloride and the patient’s serum, as a source of autologous thrombin (1.0-mL APC/115-⎧L mixture).

Gross evaluation
On day 7, 14, and 21 after removing the crusts, the unhealed wound area was measured using the ruler method.¹³ The area was expressed in centimeters squared. The following wound parameters were evaluated: exudate, scab formation, wound bed appearance, color, and odor.

Ruler method
The wound area of each lesion on each evaluation day was obtained by tracing the perimeter of the wound onto a sterile piece of clear acetate film with a special marking pen. The acetate was laid on the wound surface, smoothed, and held flat and immobile by an assistant while the examiner traced the wound area. The outlined area was defined as “total wound area.” Thereafter, the examiner traced the margin at the leading edge of the advancing epithelium. The area within the margin of the advancing epithelium was defined as “unhealed wound area.” When the unhealed wound area was removed from the total measured wound area value, the total wound healing was found.

Biochemical evaluation
The biopsy specimens were taken from the wounds on day 7, 14, and 21. The samples were freeze-dried and stored at -80˚C until use. Hydroxyproline was measured using Bergman’s spectrophotometric method.¹⁴

Histopathological evaluation
A 5-mm punch biopsy instrument was used to take skin specimens from the left corner of the wound of each rabbit on days 7, 14, and 21 immediately after the measurement was performed using the ruler method. The specimens were fixed in 10% neutral buffered formalin and processed routinely for histopathological examination. Five-micrometer sections were stained with hematoxylin and eosin (H&E). Although several histopathological parameters could be used to assess the progression of healing from the inflammatory into the repair stage, progressive decrease in macrophage numbers and progressive increase in angiogenesis, fibrosis, epithelization, and collagen levels were selected. In every skin section, an area just beneath the epidermis or crust formation was randomly selected.

Thereafter, three consecutive areas moving toward the deep dermis were selected. The four selected areas were examined under x400 magnification. The number of macrophages was scored as 0–25 = 1, 26–50 = 2, 51–75 = 3, > 75 = 4. The same areas were also examined for the number of vessels; the actual count was noted. Fibrosis, epithelization, and collagen levels were assessed individually with the following scale: 0 = no alteration; 1 = mild; 2 = moderate; 3 = dense. The following criteria were used to evaluate and compare the normal tissue morphology in different regions of the trunk. Abramov’s histologic scoring system (modified Greenhalgh’s scoring system) was used for scoring epithelization, fibrosis, and collagen levels.^15,16 While the Greenhalgh’s scoring system compiled several histological parameters simultaneously to create a single score, Abramov’s system assessed each parameter independently and gave a score of 0–3. The same blinded investigator evaluated all histological sections.

Statistical analysis
Gross, biochemical, and histopathological data analysis were carried out using SPSS 13.0 statistical package programs. The results were evaluated using one-way ANOVA, and any significant differences were further evaluated using Tukey’s multiple-comparison test. The level of significance was P < 0.05.

Observations during daily wound care
A superficial scab or layer of medication often hid the wounds. Although there was no significant difference in gross appearance during the first week, a film layer was present in the wound bed in PRP gel group and PRP gel + estradiol group on day 14. A yellow-brown scar was observed in the estradiol and sterile saline solution groups.

On day 21, complete coverage of the wounds with granulation tissue and epithelization was visible in some animals in the PRP gel + estradiol-treated wounds (n = 4), whereas wounds in the other two groups were not completely epithelialized. All wounds continued to reduce in size parallel to wound healing.

Exuberated or deficient granulation tissue was not observed in any group.

The extent and nature of wound contraction and scab formation varied considerably. The wounds were significantly contracted in all groups in relation to days (P < 0.01) despite the fact that a significant difference was not determined between groups.

Gross evaluation
A significant decrease in wound area was measured in the PRP gel + estradiol-treated group and PRP gel-treated group when compared with the other two groups on day 7 (P < 0.05, Table 1). The mean unhealed wound area in the PRP gel + estradiol-treated and PRP gel group-treated groups was significantly smaller than in the other two groups on day 7 and 21. The smallest unhealed wound area was observed in PRP gel + estradiol-treated group on day 14 (P < 0.05, Table 1). The mean unhealed wound area in the sterile saline solution group and in the estradiol-treated group on day 7 and 21 was significantly greater than in the other two groups (P < 0.01, Table 1).

The mean percentage of total wound healing in the PRP gel + estradiol-treated group (P < 0.05) was significantly higher than in the other groups on day 7 and 14.

No significant differences were observed between the estradiol-treated-group and the sterile saline solution group regarding total wound healing rates on day 7 nor between the PRP gel-treated group and the estradiol-treated group on day 14 (P > 0.05, Table 1).

At the end of the study (day 21), mean percentage of total wound healing in the PRP gel + estradiol-treated group (100%) and PRP gel-treated (100%) groups was better than the other groups (P < 0.05, Table 1).

Biochemical evaluation
Hydroxyproline levels of all groups are presented in Table 2. Although statistically insignificant, hydroxyproline content was higher in sterile saline solution and PRP gel + estradiol-treated groups compared with other groups on day 7. On day 14, the hydroxyproline level was the highest in PRP gel + estradiol group, but it showed a steady increase in all groups with time and reached approximately the same level on day 21.

Histopathological evaluation
Collagen level, fibrosis, macrophage count, angiogenesis, and epithelization are illustrated after treatment with normal saline, PRP gel, PRP gel + estradiol, and estradiol for the three time periods (Table 3). On day 7, the collagen level was significantly lower in PRP gel + estradiol group (P < 0.05) than in the sterile saline solution group. There was no significant difference between the treatment groups on day 14 though the collagen level was the highest in PRP gel + estradiol group. Intense collagen formation was observed in all groups on day 21.

Fibrosis was nearly the same on day 7 in all groups and an increased tendency toward fibrosis was observed afterward. There was no significant difference between the treatment groups and sterile saline solution group noted during the remainder of the study.

Macrophage counts in the wounds were not significantly different between the groups on days 7, 14, and 21.

Angiogenesis significantly increased in the PRP gel + estradiol group and estradiol group compared to the sterile saline solution group and PRP gel group on day 14 (P < 0.05). By day 21, although angiogenesis had increased in all groups, the sterile saline solution group had a lower value in comparison with the other groups.

Minimal epithelization was observed on day 7 only in the PRP gel + estradiol group as compared to the other groups. Epithelization was the highest in the PRP gel + estradiol group on day 14, but it was not statistically significant. Intense epithelization was observed in all groups on day 21. No significant differences were found between the treatment groups and sterile saline solutions on day 21.

Various methods have been used for wounds with substantial skin loss. The present study aimed to develop a new topical agent for dermal wounds combining the positive effects of PRP gel and estradiol on wound healing.

Platelet counts in PRP were an average of 3.92-fold greater than those in whole blood. This result is consistent with previously published results using the Harvest Technologies SmartPReP2 system and is greater than that reported with other devices used to prepare PRP, where 1.3- to 1.9-fold platelet concentrations were achieved.¹⁷ There are numerous commercially available machines, all of which yield slightly different increases in platelets.¹⁸ Greater fold concentrations of platelets compared to whole blood have been obtained using laboratory buffy coat and apheresis methods with up to a 13.1-fold increase reported in the concentration of platelets,¹⁹ but these methods are not widely available for clinical use.

Most of the studies on PRP gel used 10% calcium chloride and topical bovine thrombin (TBT) as an activator to facilitate the release of growth factors and to form a gel.^6,20–23 However, fatal coagulopathies have been reported after TBT use due to formation of antibodies against factor V, factor XI, and thrombin.²⁴ Considering this risk, the present study used serum²⁵ with 10% calcium chloride and no fatalities occurred.

Clinical studies on growth factors have not reported hypertrophic scar formation, malignant transformation, or metaplasia.^3,7 Adverse effects also have not been observed.^3,7 Exuberated or deficient granulation tissue was not observed in any group of the study, which seems to support the results obtained in other studies.

In-vitro and in-vivo studies on estradiol have reported that estradiol increases endothelial cell activity, which is crucial for neovascularization. Estrogens accelerate angiogenesis, thus it is effective in the proliferative phase of wound healing.²⁶ Estrogens enhance modulation of PDGF by monocyte/macrophages. PDGF is known to be mitogenic and chemotactic for fibroblasts and plays a role in angiogenesis. Estrogens can indirectly influence the proliferative phase of wound healing.^27–29

In normal wounds, synthesis of heat-shock protein, PDGF, and FGF is stimulated within 24 hours after dermal injury, then VEGF, PGDF, FGF, and EGF are synthesized. Increased synthesis returns to normal levels between days 7 and 14 of injury parallel to wound healing.⁸ In the present study, angiogenesis was determined a significant increase in PRP gel + estradiol group and estradiol group on the 14th day (P < 0.05). Estrogen is assumed to be responsible for the rapid increase of angiogenesis on day 14. The application of PRP to the full-thickness skin wounds improved overall healing by reducing contraction as well as showing a trend toward increasing epithelization rate and stimulating angiogenesis. However, these differences did not seem to occur in the low platelet count (PRP [3.92-fold]) treatment. This was likely due to the small sample size with insufficient power to detect the difference. Two other possible explanations are the inability of this volume to cover the wound serving as a tissue scaffold completely and possible reduction in the amount of growth factors released.

Studies on ovariectomized rat models have shown that the number of macrophages in full-thickness wounds (with substantial loss) significantly decrease on days 3 and 5, and that the inflammatory phase of the healing was delayed in the absence of estrogen.^30,31 This also shows that the presence of estrogen shapes the inflammatory phase and increases the number of macrophages. Macrophages, in addition to their known functions, take part in the synthesis of growth factors such as interleukin-1 (IL-1), PDGF, TGF-β, epidermal growth factor, and insulin-like growth factor, thus contributing to the proliferation phase.³⁰ In the present study, macrophage counts were not significantly different between the groups on days 7, 14, and 21. Considering these results, one can hypothesize that the substances used did not exert a notable effect on macrophage counts.

Granulation tissue is particularly important for the healing of large wounds. Fibroblasts are crucial elements of granulation tissue. The proliferation of fibroblasts is influenced by several factors such as inflammatory products, chemotactic factors, and growth factors.³³ In the present study, fibrosis was nearly the same on day 7 in all groups and an increase tendency was observed afterwards. There was no significant difference between the treatment groups and sterile saline solution group could be noted during the rest of the study. However, another study³⁴ found significant increases in fibroblast and collagen production in PRP-treated wounds because PRP contains growth factors such as PDGF and TGF-β, which can stimulate cell replication and extracellular matrix production.³⁵ Specifically, Henderson et al³⁴ showed that PRP increased fibroblast proliferation and collagen production in porcine burn wounds. The reason for such a discrepancy is not clear, but the high covariance of the present study was not sensitive enough to corroborate statistically significant differences in fibroblasts that might account for a portion of it.

A study on post-menopausal women showed that topical estradiol treatment has provided a significant increase in hydroxyproline and collagen content of the skin.³⁶ Another study on elderly men and women investigated the effect of topical estrogen on wound healing; the amount of collagen in the estrogen-treated group showed a constant increase in both genders compared to the placebo group.^28,37 Full-thickness wounds (with substantial loss) were created in the back of ovariectomized young female rats and local intradermal estradiol was injected; re-epithelization and collagen deposition was increased and wound healing was facilitated in the treatment group. Cellular mechanisms suggested to be responsible for these changes are increased secretion of latent TGF-β1 by dermal fibroblasts that were stimulated by estrogen.¹¹ In the present study, fibrosis was moderate in all groups on day 7, but became intense on days 14 and 21. Absence of a notable difference between the groups may be due to the use of young and intact females that possess high levels of estrogen. Collagen and hydroxyproline levels increased steadily in all groups over time, especially on day 14. Although statistically insignificant, the increase was greater in the PRP gel + estradiol-treated group compared to the other groups.

PRP contains EGF, which induces replication, migration, and stimulation of the basal skin cells and mucous membrane, which form the components of the basement membrane.³⁵ These increases are consistent with other studies.^17,38 Carter et al³⁸ found that treatment of lower equine limb wounds with PRP induced accelerated epithelial differentiation and produced tissue with organized, interlocking collagen bundles. Marx¹⁷ determined that patients had earlier and faster epithelization with less pain in PRP-treated split-thickness skin graft donor sites. Estrogen was shown to accelerate the wound-healing process with faster epithelization and suppressing the inflammatory response with a concurrent increase in matrix deposition.¹¹ In the present study, slight epithelization was present only in the PRP gel + estradiol-treated group on day 7 of the study, whereas, no epithelization was observed in the other groups. No significant differences were observed between the treatment groups and the sterile saline solution group regarding epithelization on day 14, but the epithelization rate was higher in PRP gel-treated and PRP gel + estradiol-treated groups than in the other groups. Epithelization started in the early period due to a shortened latent period, even though it was an open wound. Bandyopadhyay et al³⁹ report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. In this study, the PRP gel and estradiol combination seemed to have a tendency to accelerate epidermal cell migration. The estradiol may help to promote epidermal cell migration.

Clinical evaluation showed that wound healing was faster and hydroxyproline content was the highest in the group that was treated with PRP gel and estradiol combination compared to the sterile saline solution group. Histopathological evaluation showed that angiogenesis and epithelization started in the first week and advanced rapidly. This may indicate that the combined use of estradiol and PRP gel accelerates the inflammatory reaction and initiates the healing process earlier.

Two limitations of this study were the small sample size and use of female rabbits led to statistical outcomes that might not have been sensitive enough to detect the differences caused by the treatments in all cases. This might have affected the results, and therefore, the conclusions of this study. Studies with larger sample sizes and ovariectomized female rabbits or male rabbits would have increased the statistical sensitivity. Considering the results of the study, studies with larger sample sizes should also be performed to improve statistical sensitivity in evaluating the efficacy of PRP gel and estradiol combination on wound healing at even longer time intervals.

Address correspondence to: Ozlem H. Nisbet, DVM, PhD, Department of Surgery, Faculty of Veterinary Medicine, Ondokuz Mayis University Kurupelit 55139 Samsun, Turkey; E-mail: onisbet@omu.edu.tr