BURN SURGERY AND RESEARCH
Topical Application of Aloe vera Accelerated Wound Healing, Modeling, and Remodeling An Experimental Study Ahmad Oryan, DVM, PhD,* Adel Mohammadalipour, DVM, PhD,Þ Ali Moshiri, DVM, PhD,þ and Mohammad Reza Tabandeh, DVM, PhD§ Objective: Treatment of large wounds is technically demanding and several attempts have been taken to improve wound healing. Aloe vera has been shown to have some beneficial roles on wound healing but its mechanism on various stages of the healing process is not clear. This study was designed to investigate the effect of topical application of A. vera on cutaneous wound healing in rats. Methods: A rectangular 2 2-cm cutaneous wound was created in the dorsum back of rats. The animals were randomly divided into 3 groups of control (n = 20), low-dose (n = 20), and high-dose (n = 20) A. vera. The control and treated animals were treated daily with topical application of saline, low-dose (25 mg/mL), and high-dose (50 mg/mL) A. vera gel, up to 10 days, respectively. The wound surface, wound contraction, and epithelialization were monitored. In each group, the animals were euthanized at 10 (n = 5), 20 (n = 5), and 30 (n = 10) days post injury (DPI). At 10, 20, and 30 DPI, the skin samples were used for histopathological and biochemical investigations; and at 30 DPI, the skin samples were also subjected for biomechanical studies. Results: Aloe vera modulated the inf lammation, increased wound contraction and epithelialization, decreased scar tissue size, and increased alignment and organization of the regenerated scar tissue. A dose-dependent increase in the tissue level of dry matter, collagen, and glycosaminoglycans’ content was seen in the treated lesions, compared to the controls. The treated lesions also demonstrated greater maximum load, ultimate strength, and modulus of elasticity compared to the control ones (P G 0.05). Conclusions: Topical application of A. vera improved the biochemical, morphological, and biomechanical characteristics of the healing cutaneous wounds in rats. This treatment option may be valuable in clinical practice. Key Words: Aloe vera, hydroxyproline, hexosamine, wound healing, histopathology, biomechanics (Ann Plast Surg 2016;77: 37Y46)
W
ound healing is a complicated process and is composed of 3 overlapping phases including hemostasis and inf lammation, proliferation or fibroplasia, and remodeling.1Y3 Wound healing is often associated with the development of scar tissue formation that is not cosmetically pleasant and also the biomechanical characteristics Received December 27, 2013, and accepted for publication, after revision, March 26, 2014. From the *Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz; †Department of Clinical, Biochemistry, Medical School, Hamadan University of Medical Sciences, Hamadan; ‡Division of Surgery, Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz; and §Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Conflicts of interest and sources of funding: none declared. This study was funded by grants from the Shiraz University and Shahid Chamran University. Reprints: Ahmad Oryan, DVM, PhD, Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran. E-mail: [email protected]. Copyright * 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/16/7701-0037 DOI: 10.1097/SAP.0000000000000239
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of the healing wound is inferior to the normal skin which is important for those wounds heal under tensional forces.1,2 Therefore, a good wound healing agent is that to be effective to modulate the inf lammation, accelerate fibroplasia, and enhance remodeling of the healing tissue in minimum time with least adverse effects. The healed wound should have least scar tissue size and highest biomechanical performance to be pleasant clinically.4,5 Aloe vera (Aloe barbadensis Miller) is obtained from the parenchymatous cells in the fresh leaves of A. barbadensis. Aloe vera is one of the best healer plants that has been used widely in the traditional herbal medicine since many years ago.5,6 The immune modulatory, anti-inflammatory, antiprotozoal, ultraviolet protective, and wound and burn healing promoting properties of this plant has been previously reported.7Y15 The A. vera leaves contain phytochemical components such as acemannan, maloyl glucans, aloine, emodin, anthrones, asarabinan, arabinorhamnogalactan, galactan, galactogalacturan, lucogalactomannan, galacto glucoarabinomannan, glucuronic acid, and various lectins.5,16Y19 In addition, A. vera consists of monosaccharides (eg, glucose and fructose) and polysaccharides (eg, glucomannans and acetylated mannan). These components have anti-inflammatory and immune modulating activities.11,14,20 Acetylated polymannan, acemannan, mannose monomer/acetyl, and aloeride have been shown to stimulate macrophages, killer T cells, and cytokine production. They increase blood CD8+, IL-2, and IFN-c levels; decrease CD4+/CD8+; and induce the expression of the mRNAs encoding IL-1A and TNF->. They also have anti-inflammatory effects because they decrease the IL-5 and IL-10 and down-regulate the MMP-9.12,13,20Y24 The acetylated glucomannan, located within the protoplast of the parenchyma cells, is a mannose-rich polysaccharide with gibberellin, a growth factor, interacts with growth factor receptors on the fibroblast, stimulates its activity and proliferation, and results in significant increase of collagen synthesis.6,16 Three malic acid acylated carbohydrates have been isolated from the A. vera gel and characterized as veracylglucan A to C.17 Veracylglucan B has potent anti-inf lammatory and antiproliferative effects; veracylglucan C, on the other hand, has significant cell proliferative and antiinf lammatory activities. Veracylglucan B and C have been reported to be antagonistic and competitive in their effects on cell proliferation.17 This plant also dose dependently stimulates the macrophages to release IL-1, IL-6, TNF->, and IFN-F.13,25 It has been shown that A. vera gel not only increases collagen content of the healing wound but also changes the collagen composition and increases the degree of collagen cross-linking.15,26 In addition, increased synthesis of hyaluronic acid and dermatan sulfate in the granulation tissue of a healing wound after oral or topical administration of A. vera has been reported.27 The angiogenesis and oxygen access are other factors enhanced by the A. vera gel.28,29 It has been shown that superoxide dismutase and glutathione peroxidase are present in A. vera gel, which may be responsible for its antioxidant effects.9 Moreover, A. vera has been shown to inhibit prostaglandin E2 production by inhibiting cyclooxygenase.23 Although most of the investigations have reported the beneficial effects www.annalsplasticsurgery.com
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of A. vera,6,15,26,27 some others suggest that A. vera may retard wound healing.30Y32 Given the previously mentioned explanations, this study was designed to investigate the effect of topical application of A. vera gel on the healing of an experimentally induced large cutaneous wound defect in rats. We hypothesized that (1) A. vera may enhance the rate and quality of inf lammation by increasing the macrophages, thus may reduce its duration. (2) Aloe vera may increase the quality of fibroplasia at midterm and this may enhance the quality of remodeling at long-term. (3) If A. vera was effective on different stages of wound healing and improves the structure of the healing tissue, the healed wound is expected to have higher biomechanical performance than the controls. (4) Aloe vera may exert its beneficial effects on wound healing dose dependently.
MATERIALS AND METHODS Ethics The investigators who undertook the measurements and analyses of the results were unaware of the experimental design and grouping details. The same surgeon performed all the operative procedures. All animals received humane care in compliance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23). The local Ethics Committee of our faculty approved the study.
Experimental Animals Sixty adult mature male Wistar rats weighing 250 (50) g were purchased from a certified laboratory animal house. The animals were kept individually in standard rat cages and had free access to commercial pellet food and water. All the standard temperature, humidity, and light cycle were provided for the experimental animals.33
Preparation of A. vera Gel Full-sized mature leaves of A. vera, cultivated in botany garden, were cut and the rind was removed. The colorless parenchyma was ground in a blender and centrifuged at 10,000g for 30 minutes at 4-C to remove the fibers. The A. vera was freeze-dried and transformed to powder. This increased the shelf life of the A. vera.34 Before in vivo experimentation and topical application of the A. vera on rat cutaneous wounds, the A. vera powder was dissolved in sterile saline 0.9% to produce a solution with the concentrations of 25 and 50 mg of A. vera per milliliter of saline. The biologic activity of the A. vera solution with the doses of 25 and 50 mg/kg on molecular aspects of wound healing has been previously shown and confirmed.34
Injury Induction The animals were anesthetized by intramuscular injection of 2 mg/kg xylazine HCl as premedication and 60 mg/kg ketamine HCl (both from Alfasan, Woerden, the Netherlands) for anesthesia.33 This type of anesthesia prevents movement of the animals at least for 10 minutes after administration of the anesthetic solution; therefore, the animals were left without being restrained. Before making incisions, hairs of the dorsum back were clipped and scrubbed. Under aseptic condition, a rectangular full-thickness skin (2 2 cm) including epidermis, dermis, and subcutaneous fat was incised, dissected, and removed to create a wound defect on the dorsum back of the rats.
Study Design and Therapeutic Regimens The animals were randomly divided into 3 groups of control (n = 20), treated with low-dose A. vera (n = 20) and treated with high-dose A. vera (n = 20). The animals of the control group were treated daily by topical application of isotonic 0.9% saline for 10 days. The treated animals were treated daily by topical application of low dose of A. vera (25 mg A. vera in 1 mL saline) and high dose of A. 38
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vera (50 mg A. vera in 1 mL saline) for 10 days, respectively. Gross morphology of the wound area of all the animals was assessed daily. Each group was then divided into 3 groups of 10 (n = 5), 20 (n = 5), and 30 (n = 10) days post injury (DPI). The biochemical and histological examinations of the lesions of animals of all the groups were conducted at 10, 20, and 30 DPI. In addition, biomechanical characteristics of the skin samples of the 30 DPI was also tested (n = 5 for each group).
Gross Morphology of the Wounds The wound area was observed and photographed daily. The photographs were transferred to Scion image software for morphometric analysis including measurement of the wound diameter and calculation of the wound surface area. Percentage of wound contraction was then calculated by the following equations: Wound area (%) = (Wound area at X DPI/wound area at 0 DPI) 100 Percent of contraction at X DPI = 100 j percent wound area at X DPI
Euthanasia The animals were euthanized by intracardiac administration of 50-mg/kg sodium thiopental and 1-mg/kg pancuronium (Pavulon Ink Co, USA) to induce coma and stop breathing, respectively.
Sample Collection The full-thickness skin samples were dissected and harvested from the wound site and were assessed for histological studies and biochemical analyses (n = 5 at each time interval in each group). The samples were longitudinally divided into 2 equal parts. A half was used for histopathological evaluations (including the intact healthy skin at periphery of the wound site and the injured healing area just in the wound site) and another half (only the injured healing area) was used for determination of the dry matter content and biochemical analyses. At 30 DPI, a rectangular skin sample of 3 10 cm including the injured area was harvested for biomechanical evaluation (n = 5 for each group at 30 DPI).33 Another 5 intact skin samples were harvested from the contralateral part and were tested for biomechanical testing as an index of normal healthy skin.
Histopathologic and Histomorphometric Analysis After fixation in 10% neutral buffered formalin, blocks of the skin were washed, dehydrated in a graded series of ethanol, cleared and embedded in paraffin wax, sectioned at 5 Km in thickness, stained with hematoxylin and eosin, and examined by a light microscope (Olympus, Tokyo, Japan). The photomicrographs were then recorded by a digital camera (Sony T-700, Tokyo, Japan) and transferred to the computer software (Adobe Photoshop CS-5, Calif ) for digital analyses. Five photomicrographs equivalent to 5 microscopic fields from each tissue section were used for histopathologic and histomorphometric analysis.35 Total cellularity (200), and number of fibroblasts, fibrocytes, neutrophils, lymphocytes, macrophages, and blood vessels of the injured area were counted (800). The mesenchymal cells in the injured area were divided into 2 categories based on their diameter, cytoplasmic granules, and cell staining capacities. The largest elliptical cells with high granular and basophilic cytoplasm were assessed as fibroblasts. The long, cigar-shaped cells with less granulated but more eosinophilic cytoplasm and having small nucleus/cytoplasm ratio were estimated as fibrocyte.35 The caliber of the blood vessels was measured in digital photomicrographs, using computer software measuring system (Adobe Photoshop CS-5 extended, Calif ). In addition, the wound surface, tissue alignment, tissue maturation, vascularity, and perivascular edema were assessed and scored according to Tables 1 and 2. * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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TABLE 1. Scoring Criteria Used for Defining the Wound Surface, Tissue Alignment, and Tissue Maturation Score 1. Wound surface 0, Near normal 1, Highly mature 2, Mature 3, Immature 4, Highly immature Score 2. Tissue alignment 0, Near normal 1, Highly mature 2, Moderately mature 3, Immature
4, Highly immature Score 3. Tissue maturation 0, Near normal 1, Highly mature 2, Moderately mature 3, Immature 4, Highly immature
Morphology of Epidermis
Distance Between Wound Edge, Km
Completely regenerated G80% regenerated G50% regenerated G20% regenerated No epidermis formed
9500 91000 92000 9500 G2000
Direction of Collagen
Direction of Fibroblast
Almost in 1 direction Mostly in 1 longitudinal direction, but with few areas of unrecognized collagen fibers More than 1 longitudinal direction Irregular orientation: collagen fibers have some orientation but orientation is not longitudinal (eg, circular pattern) No diagnostic orientation
Parallel collagen fiber G75% in direction of collagen fibers G50% in direction of collagen fibers G50% in direction of collagen fibers
No orientation
Cellular Populations
Appearance of Collagen Fibers
G75% fibrocyte G50% fibrocyte G25% fibrocyte G75% fibroblasts Inflammatory cells predominant
G75% of collagen fibers are dense and have large size G50% of collagen fibers are dense and have large size G75% of collagen fibers are dense and have medium size Collagen fibers are not dense, but are medium sized Collagen fibers are not dense, but are small sized
Biomechanical Testing
Percentage Dry Weight
The method has been previously described by Oryan and colleagues.35 Brief ly; the injured skin and their related contralateral skin from 5 animals in each group were dissected and stored for 3 days at j20-C. Before the tensile testing, the specimens were defrosted at room temperature. Each skin sample was mounted between the 2 cryoclamps of a tensile testing machine (Instron Tensile Testing Machine, London, UK). Testing to failure was then conducted. Each sample was loaded by elongating it at a displacement rate of 20 mmsj1. Load and crosshead displacement data were recorded at 1500 Hz, and the load-deformation and stress-strain curves were generated for each specimen, using Test Works 4 software (SUME Systems Corporation).35 The maximum load, yield load, ultimate strain, yield strain, maximum stress, and modulus of elasticity of the samples were extracted from the curves and statistically analyzed.
The percentage dry matter of the harvested skin samples was calculated according to the following equation: Percentage dry matter content = (dry weight/wet weight) 100.36
Biochemical Analyses Estimation of hydroxyproline and collagen content was done as described previously.37 Brief ly, the excised granulation tissues were weighed, defatted in chloroform/methanol mixture (2:1 vol/vol), hydrolyzed in 6.0 N HCl for 18 hours at 110-C, and evaporated to dryness. The hydrolysate was neutralized to pH 7.0 and subjected to chloramine-T oxidation; all the test tubes were placed in a water bath at 60-C for 20 minutes. The reaction was terminated by addition of 1 mL (0.4 M) perchloric acid; the color was developed by adding 1-mL paradimethylaminobenzaldehyde and was read spectrophotometrically
TABLE 2. Scoring Criteria Used for Defining Vascularity and Perivascular Edema Score
Description
1. No. vessels and their diameter (vascularity) 0, Near normal 1, Highly mature 2, Mature 3, Immature 4, Highly immature 2. Perivascular edema (observed in an 10 objective field) 0, Normal 1, Mild 2, Moderate 3, Severe
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Up to 4, all with and diameter 5Y10 vessels; 70% are large 11Y20 vessels; 50% are large 21Y30 vessels; G20% are large, with the rest of small size 930 vessels, almost of small size No edema is seen around the vessels Presence of edema around small vessels Presence of edema around medium vessels Presence of edema around large vessels
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at 557 nm. The amount of hydroxyproline in each sample was measured, using the regression curve from the hydroxyproline standards (0.0, 0.5, 1.0, 2.0, 4.0, and 6.0 mg hydroxyproline) and reported as mg/ 100 mg tissue. The collagen content was estimated from the hydroxyproline concentration of each tissue sample by multiplication in the factor 7.46.38 n-Acetyl glucosamine (NAGLA) and n-acetyl galactosamine (NAGA) concentrations of the dry granulation tissues were measured by the method of Reissig et al.39 Briefly, alkaline hydrolysate was converted into pyrrole derivatives by acetyl-acetone and subsequently treated with paradimethylaminobenzaldehyde to a red solution which was measured at 585 nm by spectrophotometry.
Statistical Analysis To statistically analyze differences between multiple groups at 1 time point, 1-way analysis of variance (ANOVA) with its subsequent post hoc Tukey tests were used; and between multiple groups at 2 time points, 2-way ANOVA with its subsequent post hoc Tukey tests were used. The scoring values were statistically analyzed, using the Kruskal-Wallis nonparametric test and Mann-Whitney U tests were performed to test the significant differences between the groups. Statistics were performed using the computer software SPSS version 21 for windows (SPSS Inc, Chicago, Ill). A value of P of less than 0.05 was considered statistically significant. The quantitative results were expressed as mean (SD) and the scoring results were expressed as median (min-max).
RESULTS Macroscopic Findings At 15 DPI, treatment with high dose of A. vera almost closed the wounds completely, whereas the lower dose of A. vera was able to close the wounds at 20 DPI. In the control lesions, the wound was completely closed at 26 DPI (Fig. 1). Treatment with high dose of A. vera significantly increased the percentage of wound contraction at 10, 15, and 20 DPI (P = 0.001 for all) compared to the control lesions. Also, those lesions treated with high dose of A. vera showed
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significantly higher percentage of wound contraction at 15 and 20 DPI compared to the treated rats with low dose of A. vera (P = 0.001 for both). Treatment with low dose of A. vera significantly increased the percentage of wound contraction compared to the control lesions at 10, 15, and 20 DPI (P = 0.001 for all) (Fig. 2A). In general, the treated wounds with the low and high doses of A. vera had higher granulation tissue during the first 10 days after injury; and compared to the controls, the granulation tissue developed earlier and organized as scar tissue at later stages of wound healing. In the treated groups, the size of scar tissue gradually decreased time dependently (at 20 and 30 DPI) so that wound contraction and epithelialization had occurred better and faster than the controls. Therefore, the healing treated wounds had better cosmetic appearance at 30 DPI than the control lesions and these beneficial characteristics of the A. vera was found to be dose dependent because those wounds treated with the higher dose of A. vera had better cosmetic appearance and less scar tissue size than the treated wounds with low dose of A. vera. No wounds became infected and normal healing response occurred in all the wounds.
Histopathological Findings The qualitative report has been expressed in Figures 3 and 4. Treatment with high dose of A. vera significantly reduced total cellularity and significantly increased number of fibrocytes (as the mature mesenchymal cells) and macrophages (as the most important inf lammatory cells) compared to the lesions treated with low dose of A. vera and the control lesions at 10 DPI (P = 0.001 for all). This dose also significantly increased the collagen mass density and the number and diameter of the blood vessels and decreased the number of the lymphocytes compared to the control lesions at this stage (P = 0.001 for all). At this stage, treatment with a low dose of A. vera significantly increased the diameter and number of the blood vessels, number of the fibrocytes and macrophages, and collagen mass density (P = 0.001 for all) and significantly decreased the total cellularity and number of the lymphocytes compared to the control lesions (P = 0.001 for all; Table 3; Figs. 3A, D, G and 4).
FIGURE 1. Wound surface of the control (A, D), low-dose A. vera (25 mg/mL) (B, E), and high-dose A. vera (50 mg/mL) (C, F), at 15 DPI. The A. vera dose-dependently increased wound contraction and closure. The margins of the wound are indicated by the arrows in the inverted images (DYE). The wound shape is trapezium, elliptic, and circular in the control, low-, and high-dose A. vera, respectively, suggesting the groups treated with A. vera are in a more advanced stage of wound contraction. 40
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FIGURE 2. Aloe vera dose-dependently increased the percentage of wound contraction (A) and dry matter content of the healing tissue (B). A dose-dependent increase in the tissue level of the collagen (C), NAGLA (D), and NAGA (E) was seen in the treated lesions compared to the controls.
High dose of A. vera significantly decreased the total cellularity and increased the diameter of blood vessels at 20 DPI compared to the lesions treated with the low dose of A. vera (P = 0.001 for all). This treatment regimen also significantly decreased total cellularity, number of fibroblasts (as the immature mesenchymal cells), lymphocytes, and macrophages and significantly increased the diameter of the blood vessels, number of fibrocytes, and collagen mass density compared to the control ones. Low dose of A. vera also significantly decreased total cellularity, number of fibroblasts and macrophages, and it also significantly increased diameter of the blood vessels, number of fibrocytes, and collagen mass density compared to those in the control ones (P = 0.001 for all; Table 3; Figs. 3B, E, H and 4) at this stage. At 30 DPI, treatment with high dose of A. vera significantly decreased total cellularity, number of fibroblasts and significantly increased the diameter of blood vessels and number of fibrocytes compared to the lesions treated with the low dose of A. vera (P = 0.001 for all). High dose of A. vera also significantly decreased total cellularity, number of blood vessels, fibroblasts, lymphocytes, and macrophages and significantly increased the diameter of blood vessels, number of fibrocytes, and collagen mass density compared to the control lesions (P = 0.001 for all). At this stage, treatment with low dose of A. vera significantly decreased number of immature blood vessels, fibroblasts, and macrophages and significantly increased the diameter of blood vessels, number of fibrocytes, and collagen mass density compared to the control lesions (P = 0.001 for all; Table 3; Figs. 3C, F, I and 4). Treated lesions with high dose of A. vera showed significantly superior scored values for the wound surface area (Table 1) compared to the treated lesions with low dose of A. vera and the control lesions at 10 DPI [0.5 (0Y2)A. vera high dose vs 1.5 (1Y3)A. vera low dose vs 3 (2Y4)Control, P = 0.001 for both]. At this stage, the treated lesions with high dose of * 2014 Wolters Kluwer Health, Inc. All rights reserved.
A. vera also significantly gained superior scored values for the vascularity [0.5 (0Y2)A. vera high dose vs 1.5 (1Y4)A. vera low dose vs 3.5 (3Y4)Control, P = 0.001 for both] and perivascular edema [0 (0Y1)A. vera high dose vs 1.5 (1Y3)A. vera low dose vs 2.5 (2Y3)Control, P = 0.001 for both] compared to the lesions treated with low dose of A. vera and the control lesions (Table 2). At 30 DPI, the newly formed scar tissue in the treated lesions with high dose of A. vera showed significantly superior scored values for the alignment (Table 1) compared to the treated lesions with low dose of A. vera and the control lesions [0.5 (0Y1)A. vera high dose vs 2.5 (1Y3)A. vera low dose vs 3.5 (1Y4)Control, P = 0.001 for both]. At this stage, the treated lesions with high dose of A. vera also showed significantly superior scored values for the tissue maturation (Table 1) compared to the lesions treated with low dose of A. vera and the control lesions [0.5 (0Y1)A. vera high dose vs 1.5 (1Y2)A. vera low dose vs 3.5 (2Y4)Control, P = 0. 001 for both]. In general, treatment with high dose of A. vera accelerated the rate and quality of inf lammation and fibroplasia so that at short-term (day 10) more granulation tissue developed and filled the wound area when compared to the controls. Compared to controls, A. vera organized the granulation tissue and produced a scar tissue faster than the controls at 20 DPI. In addition, A. vera decreased the size of the scar tissue by aligning the newly developed collagen fibers and cellular structures at 30 DPI. The treated lesions with either the low or high doses of A. vera showed superior quality of wound contraction and epithelialization when compared to the control lesions at various stages of wound healing. These effects were dose dependent.
Biomechanical Performance As it has been shown in Table 4, treatment with high dose of A. vera significantly increased the maximum load of the treated lesions compared to the control lesions (P = 0.013), at 30 DPI. www.annalsplasticsurgery.com
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FIGURE 3. Longitudinal sections of the control lesions (A, day 10; D, day 20; G, day 30), lesions treated with 25 mg/mL of A. vera (B, day 10; E, day 20; H, day 30), and those treated with 50 mg/mL of A. vera (C, day 10; F, day 20; I, day 30). Scale bars of (A) to (I) are equal to 50 Km. During wound healing, A. vera dose-dependently reduced cellularity and inf lammation at 10 DPI but also increased epithelialization and granulation tissue formation at 20 DPI; and finally, it enhanced remodeling of the scar tissue by aligning the collagen fibers and cells at 30 DPI. Therefore, A. vera improved regeneration and maturation of the epithelial tissue and reduced size of the scar tissue at 30 DPI. Stained by hematoxylin and eosin.
However, the maximum load of all the lesions were significantly lower than the intact healthy skin samples at this stage (P = 0.001 for all). Treatment with high dose of A. vera significantly increased the maximum stress of the healing treated tissue compared to the control lesions (P = 0.018), but there was no significant difference in the maximum stress of the lesions treated with low dose of A. vera and the control lesions at this stage (P 9 0.05). Although treatment with high dose of A. vera significantly increased the maximum stress; the measured value was still significantly lower than that of the intact healthy skin samples at this stage (P = 0.001). Treatment with high dose of A. vera also significantly increased the modulus of elasticity of the treated lesions when compared with the control lesions at 30 DPI (P = 0.006). There was no significant difference between the modulus of elasticity of the lesions treated with high dose of A. vera and those treated with low dose of A. vera at this stage (P 9 0.05). The modulus of elasticity of the lesions treated with high dose of A. vera was also significantly lower than those of the intact healthy skin samples (P = 0.001) at this stage.
Dry Matter Content Treatment with high dose of A. vera significantly increased the percentage dry matter content compared to the control ones at 20 and 30 DPI (P = 0.001 for both). Although the high dose of A. vera 42
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significantly improved the dry matter content, the measured value for the treated lesions was still significantly lower than those of the intact normal skin samples (P = 0.001) (Fig. 2B) at this stage.
Biochemical Findings A dose-dependent increase in the tissue level of collagen (Fig. 2C), NAGLA (Fig. 2D), and NAGA (Fig. 2E) was seen in the treated lesions so that topical application of low-dose A. vera significantly increased the concentration of collagen and glycosaminoglycans compared to the control lesions at 10, 20, and 30 DPI (P = 0.001 for all) and treatment with high dose of A. vera significantly increased these items compared to those treated with low dose of A. vera at 10, 20, and 30 DPI (P = 0.001 for all).
DISCUSSION Several studies have reported the anti-inf lammatory, cell proliferative, immune modulating, collagen stimulatory, antioxidative, and angiogenic activity of A. vera; however, some controversies exist between these studies that make the real efficacy of A. vera unclear.5,6,8,9,11Y18,20,21,24Y27,30Y32,40 These controversies could initially be due to the design of the studies. For example, Oryan and colleague26 studied the effect of A. vera on wound healing by brief histopathologic and tensile testing methods. Some others studied the * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 4. Longitudinal sections of the control (A, D), 25 mg/mL of A. vera (B, E), and 50 mg/mL of A. vera (C, F) at 30 DPI. A to C are stained by hematoxylin and eosin. D to F are inverted figures for better clarification of the structures. Scale bars of (A) to (C) are equal to 12.5 Km and (D) to (E) are equal to 6.25 Km. Aloe vera dose-dependently reduced the immature fibroblasts and inf lammatory cells but also enhanced maturation and alignment of the fibroblasts at 30 DPI so that more mature fibrocytes were present in the treated lesions compared to those of the control ones.
role of A. vera on tissue level of hyaluronic acid and dermatan sulfate27 and some others focused on the angiogenic and oxygen access effects of A. vera.29,40 The major merit of the present study was that various methods were used to study the different roles of A. vera on cutaneous wound healing in rats to test whether there is a correlation between the results and to show whether A. vera has dose-dependent effect on wound healing. We showed that A. vera significantly increases the rate of wound contraction, epithelialization, and maturation. Higher dose of A. vera was also effective in increasing the maximum load and ultimate strength of the healing tissues compared to other groups. These beneficial changes could be initially due to the modulatory effects of A. vera on the inf lammatory phase of wound healing.23 At the earlier stages of wound healing, the reduced total cellularity, edema, and fibrin clot together with the elevated number of macrophages, fibroblasts, and large blood vessels observed in the treated lesions, compared to the controls, suggest that A. vera enhances the rate and quality of the inf lammatory phase of wound healing.23 Increase in the number of blood vessels in the treated lesions could indicate the angiogenic activity of A. vera at earlier stages of wound healing which established a better perfusion and appropriate circulation in the injured area. Angiogenic activity of A. vera has previously been reported.40 Introduction of sufficient blood f low in the injured area increased the number of the endothelial cells, macrophages, and fibroblasts and further enhanced maturation of the fibroblasts which resulted in increased collagen production by these cells.23 Compared to the control group, lower number of lymphocytes and higher number of macrophages observed in the treated group at earlier stages of wound healing indicate that this reagent has selective and modulatory effects on inf lammation during wound healing.4 Lymphocytes are chronic inf lammatory cells and have fewer roles on wound healing than the macrophages. In contrast, the macrophages regulate several beneficial mechanisms during wound healing such as phagocytosis of the fibrin clot and necrotic tissues in the injured area.4,13,23 They also deliver the angiogenic and growth factors and * 2014 Wolters Kluwer Health, Inc. All rights reserved.
increase migration and proliferation of the endothelial cells and fibroblasts, thus facilitate the transition between the inf lammation to fibroplasia.28,41 Possibly A. vera increased the rate and quality of inf lammation by increasing the macrophages and reduced its duration by decreasing the lymphocytes.33 The extracellular matrix that is produced by fibroblasts is made up of collagen, glycosaminoglycans, and elastin.28 Our results indicated that higher number of fibroblasts and fibrocytes at different stages of wound healing had a strong positive correlation with the tissue level of collagen and glycosaminoglycans and A. vera increased the amount of these items in the healing treated tissues compared to the unassisted control lesions. At earlier stages of fibroplasia the newly deposited extracellular matrix mainly consists of glycosaminoglycans with lower proportion of collagen type III.4,28 Collagen type III is an immature form of collagen and its collagen fibrils have smaller diameter than those of the mature type 1 collagen fibrils. The cross-link bonds between these collagen molecules are weak, and they are polymerized at multidirections and represent an amorphous appearance in the regenerated tissue. The glycosaminoglycans have crucial roles on wound healing and act as a scaffold for collagen fiber polymerization and also incorporate in collagen maturation.41 Compared to the controls, A. vera dose-dependently increased the tissue level of glycosaminoglysans specially at the fibroplasia stage of wound healing. These findings are in line with those of Chithra et al,27 who showed that A. vera increases the amount of glycosaminoglycans in the injured area. Unlike the treated lesions, presence of neutrophils and numerous macrophages and lymphocytes in the untreated tissues at fibroplasia stage suggest that the unassisted wound healing was at the earlier stages of healing and the chronic inf lammation was still in progress, whereas the inf lammation had subsided earlier in the treated lesions.14 Once the tissue level of glycosaminoglycans increased and the fibroblasts proliferated, these cells become mature and their behavior would change.4,28 At mid to late stages of fibroplasia, their matrix www.annalsplasticsurgery.com
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For the statistical analyses, 1-way and 2-way ANOVA were used. Post hoc Tukey tests were used for comparison between the groups. Statistics were considered statistically significant when P G 0.05. For each evaluation, 5 histopathologic fields of each histopathologic section (n = 5) were used in each group. There are 5 animals in each group. Lymphocytes, macrophages, and neutrophils were counted as an index of inf lammation. Total cellularity was counted at 200. The other variables were counted at 800.
118.80 (20.16) 1.01 (1.22) 87.62 (4.50) 49.69 (2.96) 5.62 (1.51) 5.80 (2.58) 2.66 (1.14) 0 88.51 (4.18) 157.28 (15.83) 2.12 (1.58) 46.36 (4.25) 26.48 (2.96) 8.81 (1.12) 9.48 (2.30) 3.68 (1.14) 0 80.18 (7.99) 196.68 (23.89) 8.15 (1.68) 31.66 (1.49) 11.12 (3.48) 15.49 (1.51) 14.47 (2.35) 7 (2.39) 0 62.01 (5.42) 202.60 (9.01) 10.20 (1.64) 57.88 (2.46) 28.80 (1.48) 11.29 (1.64) 7.47 (1.14) 7.40 (1.81) 0 80.32 (6.18) 241.29 (25.35) 12.61 (1.67) 38.83 (5.20) 15.29 (3.56) 14.27 (3.27) 9.41 (2.60) 8.88 (2.16) 0 74.92 (6.71) 288.64 (12.35) 11.65 (1.88) 23.42 (1.83) 6.11 (2.76) 24.28 (1.35) 11.22 (2.34) 21.65 (1.34) 0 51.29 (3.18) 296.60 (24.65) 21.6 (3.28) 26.55 (5.88) 17.00 (1.58) 19.23 (2.12) 12.20 (4.32) 28.08 (4.33) 0.89 (0.44) 75.16 (6.24) 471.22 (16.14) 7.62 (1.53) 12.33 (2.79) 3.49 (1.16) 21.04 (5.56) 25.43 (5.78) 8.56 (2.79) 0.85 (0.27) 45.27 (5.18)
A. vera, 50 mg
Total cellularity Vascular number Diameter of vessel, Km Fibrocyte number Fibroblast number Lymphocyte number Macrophage number Neutrophil number Collagen mass density,%
384.21 (13.88) 18.25 (3.49) 21.34 (2.86) 9.81 (0.83) 15.49 (3.64) 18.43 (2.96) 17.66 (2.88) 0.21 (0.44) 62.18 (8.91)
A. vera, 25 mg Control Saline A. vera, 25 mg
A. vera, 50 mg Day 20 After Injury
A. vera, 25 mg
Control Saline Day 10 After Injury
Control Saline Variables/Groups
TABLE 3. Histopathologic and Histomorphometric Analysis: Comparison Between the Control, Low Dose, and High Dose of A. vera
Day 30 After Injury
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production is shifted from glycosaminoglycans to collagen production.4,28 At this time, the tissue level of the glycosaminoglycans decreases and the collagen content increases.4,27,41 By increasing the collagen content of the healing tissue, a scar tissue forms and the tissue becomes mature.4,41 The total cellularity decreases and the fibroblasts transform to fibrocytes. In addition, several immature blood vessels disappear and the few preserved blood vessels are canalized, receive blood supply, their caliber increases and approximate to the normal blood vessels.4 The scar tissue then becomes more organized by time and its unorganized structure changes to a highly aligned pattern. In such circumstance, the collagen fibers become thicker, the quantity and quality of their cross-links improve, and the amount of collagen type III diminishes and shifts to formation of higher amounts of collagen type I concentration.10,41 The matured collagen fibrils are aligned unidirectionally in accordance to the forces transmitted through the wound, during healing. Therefore, in the remodeling phase of wound healing, size of the scar tissue decreases because the collagen fibers are aligned and the free spaces between them decrease.4,28 At 30 DPI, our results suggest that A. vera significantly reduced the total cellularity and immature blood vessels. It also increased fibrocytes and caliber of the blood vessels and enhanced alignment of the collagen fibers in the injured area. Aloe vera also significantly increased the tissue level of collagen and dry matter content compared to the controls. In addition, it interestingly decreased the scar size at this stage. The remodeling phase could be divided into 3 stages of alignment, maturation, and consolidation. On the basis of the results of the present study, the treated wounds at 30 DPI were in the alignment and maturation stages of wound healing because the epidermis was fully regenerated, the wound contraction completely occurred, and the wounds closed. In addition, the scar tissue formed in the injured area was aligned and mature. The consolidation stage of wound remodeling lasted up for months. In this stage, the healing tissue may gain almost its normal mechanical strength.42 All these results suggest that A. vera improved the structural organization of the healing tissue and this enhanced hierarchical organization was responsible for the superior biomechanical performance of the healing treated tissues compared to the controls. Increase in the maximum load, maximum stress, and modulus of elasticity of the treated wounds indicated that A. vera not only increased collagen synthesis per cell but also aided in cross-linking and maturation of the collagen fibrils and fibers. The effect of A. vera in increasing the mechanical strength of the healing tissue has major clinical relevance to those clinically injured wounds which heal under tensional forces. Under tension, there is a risk of dehiscence and healing failure and the role of A. vera in increasing the mechanical strength of the healing tissue should be highlighted.33 At 30 DPI, A. vera enhanced the cosmetic appearance of the healing wounds dose dependently. The A. vera exerted this beneficial effect by increasing the wound contraction and epithelialization, which resulted in a faster wound closure, and by decreasing the size of scar tissue through accelerating its organization and remodeling. The role of A. vera in improving the cosmetic appearance of the experimental wounds has major clinical relevance. It should be highlighted that, in this study, the experimentally induced wounds were surgically created under aseptic condition in a rat model. This model of wound has least postsurgical complication and is best suited to compare the effectiveness of biomaterials and healing factors on normal wound healing. The clinical wounds are often more complicated and are presented in different conditions. They may be larger, infected and gangrenous, burned, or may have chronic nature. In addition, the rate of wound healing is faster in laboratory animal models due to their faster maturation. Therefore, it may take months to judge about the cosmetic appearance of the clinical wounds in * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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Topical application of Aloe vera
TABLE 4. Biomechanical Characteristics of the Wound Samples Control
A. vera, 25 mg
A. vera, 50 mg
Normal
20.60 (2.70) 36.97 (14.26) 29.8 (9.69) 14.92 (3.56) 1.10 (0.48) 0.03 (0.01)
37.96 (3.20) 28.89 (4.49) 23 (4.47) 31.76 (3.35) 1.82 (0.05) 0.064 (0.01)
46.76 (14.58) 29.17 (1.64) 20.85 (3.89) 31.92 (11.14) 2.30 (0.65) 0.079 (0.022)
77.90 (17.72) 22.91 (1.42) 17.95 (2.16) 61.68 (14.60) 3.79 (0.75) 0.16 (0.029)
Biomechanical Factors Maximum load, N Ultimate strain, % Yield strain, % Yield load, N Ultimate strength, N/mm2 Modulus of elasticity, N/mm2
For the statistical analyses, 1-way and 2-way ANOVA were used. Post hoc Tukey tests were used for comparison between the groups. Statistics were considered significant when P G 0.05. There are 5 samples in each group.
response to treatment with A. vera because the healing rates of these complicated wounds are possibly slower than the experimental wounds used in the present study.42 Although this study comprehensively investigated the role of A. vera on experimental cutaneous wound healing and provided new insights in this regard, the potential physiologic variations between human and animals should be considered as one of the limitations of this study. In addition, this study used 3 time points to cover all the 3 phases of wound healing but it should be remembered that the remodeling phase of wound healing takes longer time to be completed; therefore, longer observational times (eg, 60 and 120 DPI) are needed to show the final outcome of the A. vera on remodeling phase of wound healing and also the cosmetic appearance of the healing wounds. Finally, we used A. vera with the concentrations of 25 and 50 mg/mL that were based on the molecular effects of A. vera during wound healing.34 It would be beneficial to compare the effectiveness of higher doses of A. vera on cutaneous wound healing with the results of the present study in future investigations.
CONCLUSIONS Topical application of A. vera modulated the inf lammation, increased the rate and quality of fibroplasia by enhanced collagen and glycosaminoglycans production, and improved the remodeling stage of the healing tissue. The lesions treated by A. vera showed dose-dependent improvement at various stages of wound healing so that the treated lesions showed faster wound contraction, enhanced epithelialization, smaller scar tissue formation, and higher tissue alignment. A dose-dependent effect of A. vera was shown in this study. The role of A. vera in increasing the cosmetic appearance and biomechanical characteristics of the healing cutaneous wounds has important clinical relevance and should be highlighted. These findings introduce A. vera as one of the best therapeutic agents in wound healing and it may be valuable in clinical practice. ACKNOWLEDGMENTS The authors thank Mr L. Shirvani and Mr G. Yousefi for their technical assistance and Dr N. Tanideh, Dr M. Hashemnia, and Dr A.R. Hamidi for providing the facilities. REFERENCES 1. Oryan A, Khalafinezad A, Toloo N, et al. Effects of 4-chloro-2,6Ybis (2-hydroxyl-benzyl) phenol on healing of skin wounds and growth of bacteria. J Vet Med A Physiol Pathol Clin Med 2007;54:585Y591. 2. Oryan A, Zaker R. Effects of topical application of honey on cutaneous wound healing in rabbits. Zentralbl Veterinarmed A. 1998;45:181Y188. 3. Kondo T, Ishida Y. Molecular pathology of wound healing. Forensic Sci Int. 2010;203:93Y98. 4. Monaco JAL, Lawrence T. Acute wound healing: an overview. Clin Plast Surg 2003;30:1Y12.
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5. Eshun K, Qian H. Aloe vera: a valuable ingredient for the food, pharmaceutical and cosmetic industriesVa review. Crit Rev Food Sci Nutr. 2004;44:91Y96. 6. Chithra P, Sajithlal G, Chandrakasan G. Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats. Mol Cell Biochem. 1998;181: 71Y76. 7. Jia Y, Zhao G, Jia J. Preliminary evaluation: the effects of Aloe ferox Miller and Aloe arborescens Miller on wound healing. J Ethnopharmacol 2008;120: 181Y189. 8. Langmead L, Feakins R, Goldthorpe S, et al. Randomized, double blind, placebo controlled trial of oral aloe vera gel for active ulcerative colitis. Aliment Pharmacol Ther 2004;19:739Y747. 9. Langmead L, Makins R, Rampton D. Anti-inflammatory effects of aloe vera gel in human colorectal mucosa in vitro. Aliment Pharmacol Ther 2004;19:521Y527. 10. Maenthaisong R, Chaiyakunapruk N, Niruntraporn S, et al. The efficacy of aloe vera used for burn wound healing: a systematic review. Burns. 2007;33: 713Y718. 11. Tai-Nin Chow J, Williamson DA, Yates KM, et al. Chemical characterization of the immunomodulating polysaccharide of Aloe vera L. Carbohydr Res. 2005;340:1131Y1142. 12. Vijayalakshmia D, Dhandapanib R, Jayavenia S, et al. In vitro anti inflammatory activity of Aloe vera by down regulation of MMP-9 in peripheral blood mononuclear cells. J Ethnopharmacol 2012;141:542Y546. 13. Zhang L, Tizard IR. Activation of a mouse macrophage cell line by acemannan: the major carbohydrate fraction from Aloe vera gel. Immunopharmacology. 1996;35:119Y128. 14. Im SA, Oh ST, Song S, et al. Identification of optimal molecular size of modified Aloe polysaccharides with maximum immunomodulatory activity. Int Immunopharmacol. 2005;5:271Y279. 15. Chithra P, Sajithlal G, Chandrakasan G. Influence of Aloe vera on collagen turnover in healing of dermal wounds in rats. Indian J Exp Biol. 1998;36:896. 16. Boudreau MD, Fredrick AB. An evaluation of the biological and toxicological properties of Aloe barbadensis (Miller), Aloe vera. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2006;24:103Y154. 17. Esua MF, Rauwald JW. Novel bioactive maloyl glucans from Aloe vera gel: isolation, structure elucidation and in vitro bioassays. Carbohydr Res. 2006;341: 355Y364. 18. Ramamoorthy L, Kemp MC, Tizard IR. Acemannan, a beta-(1, 4)-acetylated mannan, induces nitric oxide production in macrophage cell line RAW 264.7. Mol Pharmacol. 1996;50:878Y884. 19. Talmadge J, Chavez J, Jacobs L, et al. Fractionation of Aloe vera L. inner gel, purification and molecular profiling of activity. Int Immunopharmacol. 2004;4:1757Y1773. 20. Kima J, Leea IS, Parkb S, et al. Effects of Scutellariae radix and Aloe vera gel extracts on immunoglobulin E and cytokine levels in atopic dermatitis NC/Nga mice. J Ethnopharmacol. 2010;132:529Y532. 21. Marshall G, Druck J. In vitro stimulation of NK activity by acemannan (ACM). J Immunol. 1993;150:1381. 22. Pugh N, Ross SA, ElSohly MA, et al. Characterization of Aloeride, a new highmolecular-weight polysaccharide from Aloe vera with potent immunostimulatory activity. J Agric Food Chem. 2001;49:1030Y1034. 23. Va´zquez B, Avila G, Segura D, et al. Antiinflammatory activity of extracts from Aloe vera gel. J Ethnopharmacol. 1996;55:69Y75. 24. Womble D, Helderman J. The impact of acemannan on the generation and function of cytotoxic T-lymphocytes. Immunopharmacol Immunotoxicol. 1992;14: 63Y77.
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25. Marshall G, Gibbons A, Parnell L. Human cytokines induced by acemannan. J Agric Food Chem. 1993;91:295. 26. Oryan A, Tabatabaeinaeini A, Nikahval B, et al. Effects of aqueous extract of Aloe vera on experimental cutaneous wound healing in rat. Vet Arh. 2010;80: 509Y522. 27. Chithra P, Sajithlal G, Chandrakasan G. Influence of Aloe vera on the glycosaminoglycans in the matrix of healing dermal wounds in rats. J Ethnopharmacol. 1998;59:179Y186. 28. Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol. 2007;25:9Y18. 29. Moon EJ, Lee YM, Lee OH, et al. A novel angiogenic factor derived from Aloe vera gel: beta-sitosterol, a plant sterol. Angiogenesis. 1999;3:117Y123. 30. Gallagher J, Gray M. Is aloe vera effective for healing chronic wounds? J Wound Ostomy Continence Nurs. 2003;30:68Y71. 31. Kaufman T, Kalderon N, Ullmann Y, et al. Aloe vera gel hindered wound healing of experimental second-degree burns: a quantitative controlled study. J Burn Care Rehabil. 1988;9:156Y189. 32. Schmidt JM, Greenspoon JS. Aloe vera dermal wound gel is associated with a delay in wound healing. Obstet Gynecol. 1991;78:115Y117. 33. Oryan A, Tabatabaei-Naieni A, Moshiri A, et al. Modulation of cutaneous wound healing by silymarin in rats. J Wound Care. 2012;21:457Y464.
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34. Tabandeh MR, Oryan A, Mohammadalipour A. Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat. Int J Biol Macromol. 2014;65C:424Y430. 35. Oryan A, Moshiri A, Meimandiparizi AH. Effects of sodium-hyaluronate and glucosamine-chondroitin sulfate on remodeling stage of tenotomized superficial digital flexor tendon in rabbits: a clinical, histopathological, ultrastructural, and biomechanical study. Connect Tissue Res. 2011;52:329Y339. 36. Oryan A, Silver IA, Goodship AE. Effects of a serotonin S2-receptor blocker on healing of acute and chronic tendon injuries. J Invest Surg. 2009;22: 246Y255. 37. Switzer BR, Summer GK. Improved method for hydroxyproline analysis in tissue hydrolyzates. Anal Biochem. 1971;39:487. 38. Neuman RE, Logan MA. The determination of collagen and elastin in tissues. J Biol Chem. 1950;186:549Y556. 39. Reissig JL, Strominger JL, Leloir LF. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem. 1955;217:959Y966. 40. Lee MJ, Yoon SH, Lee SK, et al. In vivo angiogenic activity of dichloromethane extracts of Aloe vera gel. Arch Pharm Res. 1995;18:332Y335. 41. Enoch S, Leaper DJ. Basic science of wound healing. Surgery. 2008;26:31Y37. 42. Oryan A, Mohammadalipour A, Moshiri A, et al. Avocado/soybean unsaponifiables: a novel regulator of cutaneous wound healing, modelling and remodelling. Int Wound J. 2015;12:674Y685.
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Topical Application of Aloe vera Accelerated Wound Healing, Modeling, and Remodeling An Experimental Study Ahmad Oryan, DVM, PhD,* Adel Mohammadalipour, DVM, PhD,Þ Ali Moshiri, DVM, PhD,þ and Mohammad Reza Tabandeh, DVM, PhD§ Objective: Treatment of large wounds is technically demanding and several attempts have been taken to improve wound healing. Aloe vera has been shown to have some beneficial roles on wound healing but its mechanism on various stages of the healing process is not clear. This study was designed to investigate the effect of topical application of A. vera on cutaneous wound healing in rats. Methods: A rectangular 2 2-cm cutaneous wound was created in the dorsum back of rats. The animals were randomly divided into 3 groups of control (n = 20), low-dose (n = 20), and high-dose (n = 20) A. vera. The control and treated animals were treated daily with topical application of saline, low-dose (25 mg/mL), and high-dose (50 mg/mL) A. vera gel, up to 10 days, respectively. The wound surface, wound contraction, and epithelialization were monitored. In each group, the animals were euthanized at 10 (n = 5), 20 (n = 5), and 30 (n = 10) days post injury (DPI). At 10, 20, and 30 DPI, the skin samples were used for histopathological and biochemical investigations; and at 30 DPI, the skin samples were also subjected for biomechanical studies. Results: Aloe vera modulated the inf lammation, increased wound contraction and epithelialization, decreased scar tissue size, and increased alignment and organization of the regenerated scar tissue. A dose-dependent increase in the tissue level of dry matter, collagen, and glycosaminoglycans’ content was seen in the treated lesions, compared to the controls. The treated lesions also demonstrated greater maximum load, ultimate strength, and modulus of elasticity compared to the control ones (P G 0.05). Conclusions: Topical application of A. vera improved the biochemical, morphological, and biomechanical characteristics of the healing cutaneous wounds in rats. This treatment option may be valuable in clinical practice. Key Words: Aloe vera, hydroxyproline, hexosamine, wound healing, histopathology, biomechanics (Ann Plast Surg 2016;77: 37Y46)
W
ound healing is a complicated process and is composed of 3 overlapping phases including hemostasis and inf lammation, proliferation or fibroplasia, and remodeling.1Y3 Wound healing is often associated with the development of scar tissue formation that is not cosmetically pleasant and also the biomechanical characteristics Received December 27, 2013, and accepted for publication, after revision, March 26, 2014. From the *Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz; †Department of Clinical, Biochemistry, Medical School, Hamadan University of Medical Sciences, Hamadan; ‡Division of Surgery, Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz; and §Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Conflicts of interest and sources of funding: none declared. This study was funded by grants from the Shiraz University and Shahid Chamran University. Reprints: Ahmad Oryan, DVM, PhD, Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran. E-mail: [email protected]. Copyright * 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/16/7701-0037 DOI: 10.1097/SAP.0000000000000239
Annals of Plastic Surgery
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of the healing wound is inferior to the normal skin which is important for those wounds heal under tensional forces.1,2 Therefore, a good wound healing agent is that to be effective to modulate the inf lammation, accelerate fibroplasia, and enhance remodeling of the healing tissue in minimum time with least adverse effects. The healed wound should have least scar tissue size and highest biomechanical performance to be pleasant clinically.4,5 Aloe vera (Aloe barbadensis Miller) is obtained from the parenchymatous cells in the fresh leaves of A. barbadensis. Aloe vera is one of the best healer plants that has been used widely in the traditional herbal medicine since many years ago.5,6 The immune modulatory, anti-inflammatory, antiprotozoal, ultraviolet protective, and wound and burn healing promoting properties of this plant has been previously reported.7Y15 The A. vera leaves contain phytochemical components such as acemannan, maloyl glucans, aloine, emodin, anthrones, asarabinan, arabinorhamnogalactan, galactan, galactogalacturan, lucogalactomannan, galacto glucoarabinomannan, glucuronic acid, and various lectins.5,16Y19 In addition, A. vera consists of monosaccharides (eg, glucose and fructose) and polysaccharides (eg, glucomannans and acetylated mannan). These components have anti-inflammatory and immune modulating activities.11,14,20 Acetylated polymannan, acemannan, mannose monomer/acetyl, and aloeride have been shown to stimulate macrophages, killer T cells, and cytokine production. They increase blood CD8+, IL-2, and IFN-c levels; decrease CD4+/CD8+; and induce the expression of the mRNAs encoding IL-1A and TNF->. They also have anti-inflammatory effects because they decrease the IL-5 and IL-10 and down-regulate the MMP-9.12,13,20Y24 The acetylated glucomannan, located within the protoplast of the parenchyma cells, is a mannose-rich polysaccharide with gibberellin, a growth factor, interacts with growth factor receptors on the fibroblast, stimulates its activity and proliferation, and results in significant increase of collagen synthesis.6,16 Three malic acid acylated carbohydrates have been isolated from the A. vera gel and characterized as veracylglucan A to C.17 Veracylglucan B has potent anti-inf lammatory and antiproliferative effects; veracylglucan C, on the other hand, has significant cell proliferative and antiinf lammatory activities. Veracylglucan B and C have been reported to be antagonistic and competitive in their effects on cell proliferation.17 This plant also dose dependently stimulates the macrophages to release IL-1, IL-6, TNF->, and IFN-F.13,25 It has been shown that A. vera gel not only increases collagen content of the healing wound but also changes the collagen composition and increases the degree of collagen cross-linking.15,26 In addition, increased synthesis of hyaluronic acid and dermatan sulfate in the granulation tissue of a healing wound after oral or topical administration of A. vera has been reported.27 The angiogenesis and oxygen access are other factors enhanced by the A. vera gel.28,29 It has been shown that superoxide dismutase and glutathione peroxidase are present in A. vera gel, which may be responsible for its antioxidant effects.9 Moreover, A. vera has been shown to inhibit prostaglandin E2 production by inhibiting cyclooxygenase.23 Although most of the investigations have reported the beneficial effects www.annalsplasticsurgery.com
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of A. vera,6,15,26,27 some others suggest that A. vera may retard wound healing.30Y32 Given the previously mentioned explanations, this study was designed to investigate the effect of topical application of A. vera gel on the healing of an experimentally induced large cutaneous wound defect in rats. We hypothesized that (1) A. vera may enhance the rate and quality of inf lammation by increasing the macrophages, thus may reduce its duration. (2) Aloe vera may increase the quality of fibroplasia at midterm and this may enhance the quality of remodeling at long-term. (3) If A. vera was effective on different stages of wound healing and improves the structure of the healing tissue, the healed wound is expected to have higher biomechanical performance than the controls. (4) Aloe vera may exert its beneficial effects on wound healing dose dependently.
MATERIALS AND METHODS Ethics The investigators who undertook the measurements and analyses of the results were unaware of the experimental design and grouping details. The same surgeon performed all the operative procedures. All animals received humane care in compliance with the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23). The local Ethics Committee of our faculty approved the study.
Experimental Animals Sixty adult mature male Wistar rats weighing 250 (50) g were purchased from a certified laboratory animal house. The animals were kept individually in standard rat cages and had free access to commercial pellet food and water. All the standard temperature, humidity, and light cycle were provided for the experimental animals.33
Preparation of A. vera Gel Full-sized mature leaves of A. vera, cultivated in botany garden, were cut and the rind was removed. The colorless parenchyma was ground in a blender and centrifuged at 10,000g for 30 minutes at 4-C to remove the fibers. The A. vera was freeze-dried and transformed to powder. This increased the shelf life of the A. vera.34 Before in vivo experimentation and topical application of the A. vera on rat cutaneous wounds, the A. vera powder was dissolved in sterile saline 0.9% to produce a solution with the concentrations of 25 and 50 mg of A. vera per milliliter of saline. The biologic activity of the A. vera solution with the doses of 25 and 50 mg/kg on molecular aspects of wound healing has been previously shown and confirmed.34
Injury Induction The animals were anesthetized by intramuscular injection of 2 mg/kg xylazine HCl as premedication and 60 mg/kg ketamine HCl (both from Alfasan, Woerden, the Netherlands) for anesthesia.33 This type of anesthesia prevents movement of the animals at least for 10 minutes after administration of the anesthetic solution; therefore, the animals were left without being restrained. Before making incisions, hairs of the dorsum back were clipped and scrubbed. Under aseptic condition, a rectangular full-thickness skin (2 2 cm) including epidermis, dermis, and subcutaneous fat was incised, dissected, and removed to create a wound defect on the dorsum back of the rats.
Study Design and Therapeutic Regimens The animals were randomly divided into 3 groups of control (n = 20), treated with low-dose A. vera (n = 20) and treated with high-dose A. vera (n = 20). The animals of the control group were treated daily by topical application of isotonic 0.9% saline for 10 days. The treated animals were treated daily by topical application of low dose of A. vera (25 mg A. vera in 1 mL saline) and high dose of A. 38
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vera (50 mg A. vera in 1 mL saline) for 10 days, respectively. Gross morphology of the wound area of all the animals was assessed daily. Each group was then divided into 3 groups of 10 (n = 5), 20 (n = 5), and 30 (n = 10) days post injury (DPI). The biochemical and histological examinations of the lesions of animals of all the groups were conducted at 10, 20, and 30 DPI. In addition, biomechanical characteristics of the skin samples of the 30 DPI was also tested (n = 5 for each group).
Gross Morphology of the Wounds The wound area was observed and photographed daily. The photographs were transferred to Scion image software for morphometric analysis including measurement of the wound diameter and calculation of the wound surface area. Percentage of wound contraction was then calculated by the following equations: Wound area (%) = (Wound area at X DPI/wound area at 0 DPI) 100 Percent of contraction at X DPI = 100 j percent wound area at X DPI
Euthanasia The animals were euthanized by intracardiac administration of 50-mg/kg sodium thiopental and 1-mg/kg pancuronium (Pavulon Ink Co, USA) to induce coma and stop breathing, respectively.
Sample Collection The full-thickness skin samples were dissected and harvested from the wound site and were assessed for histological studies and biochemical analyses (n = 5 at each time interval in each group). The samples were longitudinally divided into 2 equal parts. A half was used for histopathological evaluations (including the intact healthy skin at periphery of the wound site and the injured healing area just in the wound site) and another half (only the injured healing area) was used for determination of the dry matter content and biochemical analyses. At 30 DPI, a rectangular skin sample of 3 10 cm including the injured area was harvested for biomechanical evaluation (n = 5 for each group at 30 DPI).33 Another 5 intact skin samples were harvested from the contralateral part and were tested for biomechanical testing as an index of normal healthy skin.
Histopathologic and Histomorphometric Analysis After fixation in 10% neutral buffered formalin, blocks of the skin were washed, dehydrated in a graded series of ethanol, cleared and embedded in paraffin wax, sectioned at 5 Km in thickness, stained with hematoxylin and eosin, and examined by a light microscope (Olympus, Tokyo, Japan). The photomicrographs were then recorded by a digital camera (Sony T-700, Tokyo, Japan) and transferred to the computer software (Adobe Photoshop CS-5, Calif ) for digital analyses. Five photomicrographs equivalent to 5 microscopic fields from each tissue section were used for histopathologic and histomorphometric analysis.35 Total cellularity (200), and number of fibroblasts, fibrocytes, neutrophils, lymphocytes, macrophages, and blood vessels of the injured area were counted (800). The mesenchymal cells in the injured area were divided into 2 categories based on their diameter, cytoplasmic granules, and cell staining capacities. The largest elliptical cells with high granular and basophilic cytoplasm were assessed as fibroblasts. The long, cigar-shaped cells with less granulated but more eosinophilic cytoplasm and having small nucleus/cytoplasm ratio were estimated as fibrocyte.35 The caliber of the blood vessels was measured in digital photomicrographs, using computer software measuring system (Adobe Photoshop CS-5 extended, Calif ). In addition, the wound surface, tissue alignment, tissue maturation, vascularity, and perivascular edema were assessed and scored according to Tables 1 and 2. * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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TABLE 1. Scoring Criteria Used for Defining the Wound Surface, Tissue Alignment, and Tissue Maturation Score 1. Wound surface 0, Near normal 1, Highly mature 2, Mature 3, Immature 4, Highly immature Score 2. Tissue alignment 0, Near normal 1, Highly mature 2, Moderately mature 3, Immature
4, Highly immature Score 3. Tissue maturation 0, Near normal 1, Highly mature 2, Moderately mature 3, Immature 4, Highly immature
Morphology of Epidermis
Distance Between Wound Edge, Km
Completely regenerated G80% regenerated G50% regenerated G20% regenerated No epidermis formed
9500 91000 92000 9500 G2000
Direction of Collagen
Direction of Fibroblast
Almost in 1 direction Mostly in 1 longitudinal direction, but with few areas of unrecognized collagen fibers More than 1 longitudinal direction Irregular orientation: collagen fibers have some orientation but orientation is not longitudinal (eg, circular pattern) No diagnostic orientation
Parallel collagen fiber G75% in direction of collagen fibers G50% in direction of collagen fibers G50% in direction of collagen fibers
No orientation
Cellular Populations
Appearance of Collagen Fibers
G75% fibrocyte G50% fibrocyte G25% fibrocyte G75% fibroblasts Inflammatory cells predominant
G75% of collagen fibers are dense and have large size G50% of collagen fibers are dense and have large size G75% of collagen fibers are dense and have medium size Collagen fibers are not dense, but are medium sized Collagen fibers are not dense, but are small sized
Biomechanical Testing
Percentage Dry Weight
The method has been previously described by Oryan and colleagues.35 Brief ly; the injured skin and their related contralateral skin from 5 animals in each group were dissected and stored for 3 days at j20-C. Before the tensile testing, the specimens were defrosted at room temperature. Each skin sample was mounted between the 2 cryoclamps of a tensile testing machine (Instron Tensile Testing Machine, London, UK). Testing to failure was then conducted. Each sample was loaded by elongating it at a displacement rate of 20 mmsj1. Load and crosshead displacement data were recorded at 1500 Hz, and the load-deformation and stress-strain curves were generated for each specimen, using Test Works 4 software (SUME Systems Corporation).35 The maximum load, yield load, ultimate strain, yield strain, maximum stress, and modulus of elasticity of the samples were extracted from the curves and statistically analyzed.
The percentage dry matter of the harvested skin samples was calculated according to the following equation: Percentage dry matter content = (dry weight/wet weight) 100.36
Biochemical Analyses Estimation of hydroxyproline and collagen content was done as described previously.37 Brief ly, the excised granulation tissues were weighed, defatted in chloroform/methanol mixture (2:1 vol/vol), hydrolyzed in 6.0 N HCl for 18 hours at 110-C, and evaporated to dryness. The hydrolysate was neutralized to pH 7.0 and subjected to chloramine-T oxidation; all the test tubes were placed in a water bath at 60-C for 20 minutes. The reaction was terminated by addition of 1 mL (0.4 M) perchloric acid; the color was developed by adding 1-mL paradimethylaminobenzaldehyde and was read spectrophotometrically
TABLE 2. Scoring Criteria Used for Defining Vascularity and Perivascular Edema Score
Description
1. No. vessels and their diameter (vascularity) 0, Near normal 1, Highly mature 2, Mature 3, Immature 4, Highly immature 2. Perivascular edema (observed in an 10 objective field) 0, Normal 1, Mild 2, Moderate 3, Severe
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Up to 4, all with and diameter 5Y10 vessels; 70% are large 11Y20 vessels; 50% are large 21Y30 vessels; G20% are large, with the rest of small size 930 vessels, almost of small size No edema is seen around the vessels Presence of edema around small vessels Presence of edema around medium vessels Presence of edema around large vessels
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at 557 nm. The amount of hydroxyproline in each sample was measured, using the regression curve from the hydroxyproline standards (0.0, 0.5, 1.0, 2.0, 4.0, and 6.0 mg hydroxyproline) and reported as mg/ 100 mg tissue. The collagen content was estimated from the hydroxyproline concentration of each tissue sample by multiplication in the factor 7.46.38 n-Acetyl glucosamine (NAGLA) and n-acetyl galactosamine (NAGA) concentrations of the dry granulation tissues were measured by the method of Reissig et al.39 Briefly, alkaline hydrolysate was converted into pyrrole derivatives by acetyl-acetone and subsequently treated with paradimethylaminobenzaldehyde to a red solution which was measured at 585 nm by spectrophotometry.
Statistical Analysis To statistically analyze differences between multiple groups at 1 time point, 1-way analysis of variance (ANOVA) with its subsequent post hoc Tukey tests were used; and between multiple groups at 2 time points, 2-way ANOVA with its subsequent post hoc Tukey tests were used. The scoring values were statistically analyzed, using the Kruskal-Wallis nonparametric test and Mann-Whitney U tests were performed to test the significant differences between the groups. Statistics were performed using the computer software SPSS version 21 for windows (SPSS Inc, Chicago, Ill). A value of P of less than 0.05 was considered statistically significant. The quantitative results were expressed as mean (SD) and the scoring results were expressed as median (min-max).
RESULTS Macroscopic Findings At 15 DPI, treatment with high dose of A. vera almost closed the wounds completely, whereas the lower dose of A. vera was able to close the wounds at 20 DPI. In the control lesions, the wound was completely closed at 26 DPI (Fig. 1). Treatment with high dose of A. vera significantly increased the percentage of wound contraction at 10, 15, and 20 DPI (P = 0.001 for all) compared to the control lesions. Also, those lesions treated with high dose of A. vera showed
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significantly higher percentage of wound contraction at 15 and 20 DPI compared to the treated rats with low dose of A. vera (P = 0.001 for both). Treatment with low dose of A. vera significantly increased the percentage of wound contraction compared to the control lesions at 10, 15, and 20 DPI (P = 0.001 for all) (Fig. 2A). In general, the treated wounds with the low and high doses of A. vera had higher granulation tissue during the first 10 days after injury; and compared to the controls, the granulation tissue developed earlier and organized as scar tissue at later stages of wound healing. In the treated groups, the size of scar tissue gradually decreased time dependently (at 20 and 30 DPI) so that wound contraction and epithelialization had occurred better and faster than the controls. Therefore, the healing treated wounds had better cosmetic appearance at 30 DPI than the control lesions and these beneficial characteristics of the A. vera was found to be dose dependent because those wounds treated with the higher dose of A. vera had better cosmetic appearance and less scar tissue size than the treated wounds with low dose of A. vera. No wounds became infected and normal healing response occurred in all the wounds.
Histopathological Findings The qualitative report has been expressed in Figures 3 and 4. Treatment with high dose of A. vera significantly reduced total cellularity and significantly increased number of fibrocytes (as the mature mesenchymal cells) and macrophages (as the most important inf lammatory cells) compared to the lesions treated with low dose of A. vera and the control lesions at 10 DPI (P = 0.001 for all). This dose also significantly increased the collagen mass density and the number and diameter of the blood vessels and decreased the number of the lymphocytes compared to the control lesions at this stage (P = 0.001 for all). At this stage, treatment with a low dose of A. vera significantly increased the diameter and number of the blood vessels, number of the fibrocytes and macrophages, and collagen mass density (P = 0.001 for all) and significantly decreased the total cellularity and number of the lymphocytes compared to the control lesions (P = 0.001 for all; Table 3; Figs. 3A, D, G and 4).
FIGURE 1. Wound surface of the control (A, D), low-dose A. vera (25 mg/mL) (B, E), and high-dose A. vera (50 mg/mL) (C, F), at 15 DPI. The A. vera dose-dependently increased wound contraction and closure. The margins of the wound are indicated by the arrows in the inverted images (DYE). The wound shape is trapezium, elliptic, and circular in the control, low-, and high-dose A. vera, respectively, suggesting the groups treated with A. vera are in a more advanced stage of wound contraction. 40
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FIGURE 2. Aloe vera dose-dependently increased the percentage of wound contraction (A) and dry matter content of the healing tissue (B). A dose-dependent increase in the tissue level of the collagen (C), NAGLA (D), and NAGA (E) was seen in the treated lesions compared to the controls.
High dose of A. vera significantly decreased the total cellularity and increased the diameter of blood vessels at 20 DPI compared to the lesions treated with the low dose of A. vera (P = 0.001 for all). This treatment regimen also significantly decreased total cellularity, number of fibroblasts (as the immature mesenchymal cells), lymphocytes, and macrophages and significantly increased the diameter of the blood vessels, number of fibrocytes, and collagen mass density compared to the control ones. Low dose of A. vera also significantly decreased total cellularity, number of fibroblasts and macrophages, and it also significantly increased diameter of the blood vessels, number of fibrocytes, and collagen mass density compared to those in the control ones (P = 0.001 for all; Table 3; Figs. 3B, E, H and 4) at this stage. At 30 DPI, treatment with high dose of A. vera significantly decreased total cellularity, number of fibroblasts and significantly increased the diameter of blood vessels and number of fibrocytes compared to the lesions treated with the low dose of A. vera (P = 0.001 for all). High dose of A. vera also significantly decreased total cellularity, number of blood vessels, fibroblasts, lymphocytes, and macrophages and significantly increased the diameter of blood vessels, number of fibrocytes, and collagen mass density compared to the control lesions (P = 0.001 for all). At this stage, treatment with low dose of A. vera significantly decreased number of immature blood vessels, fibroblasts, and macrophages and significantly increased the diameter of blood vessels, number of fibrocytes, and collagen mass density compared to the control lesions (P = 0.001 for all; Table 3; Figs. 3C, F, I and 4). Treated lesions with high dose of A. vera showed significantly superior scored values for the wound surface area (Table 1) compared to the treated lesions with low dose of A. vera and the control lesions at 10 DPI [0.5 (0Y2)A. vera high dose vs 1.5 (1Y3)A. vera low dose vs 3 (2Y4)Control, P = 0.001 for both]. At this stage, the treated lesions with high dose of * 2014 Wolters Kluwer Health, Inc. All rights reserved.
A. vera also significantly gained superior scored values for the vascularity [0.5 (0Y2)A. vera high dose vs 1.5 (1Y4)A. vera low dose vs 3.5 (3Y4)Control, P = 0.001 for both] and perivascular edema [0 (0Y1)A. vera high dose vs 1.5 (1Y3)A. vera low dose vs 2.5 (2Y3)Control, P = 0.001 for both] compared to the lesions treated with low dose of A. vera and the control lesions (Table 2). At 30 DPI, the newly formed scar tissue in the treated lesions with high dose of A. vera showed significantly superior scored values for the alignment (Table 1) compared to the treated lesions with low dose of A. vera and the control lesions [0.5 (0Y1)A. vera high dose vs 2.5 (1Y3)A. vera low dose vs 3.5 (1Y4)Control, P = 0.001 for both]. At this stage, the treated lesions with high dose of A. vera also showed significantly superior scored values for the tissue maturation (Table 1) compared to the lesions treated with low dose of A. vera and the control lesions [0.5 (0Y1)A. vera high dose vs 1.5 (1Y2)A. vera low dose vs 3.5 (2Y4)Control, P = 0. 001 for both]. In general, treatment with high dose of A. vera accelerated the rate and quality of inf lammation and fibroplasia so that at short-term (day 10) more granulation tissue developed and filled the wound area when compared to the controls. Compared to controls, A. vera organized the granulation tissue and produced a scar tissue faster than the controls at 20 DPI. In addition, A. vera decreased the size of the scar tissue by aligning the newly developed collagen fibers and cellular structures at 30 DPI. The treated lesions with either the low or high doses of A. vera showed superior quality of wound contraction and epithelialization when compared to the control lesions at various stages of wound healing. These effects were dose dependent.
Biomechanical Performance As it has been shown in Table 4, treatment with high dose of A. vera significantly increased the maximum load of the treated lesions compared to the control lesions (P = 0.013), at 30 DPI. www.annalsplasticsurgery.com
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FIGURE 3. Longitudinal sections of the control lesions (A, day 10; D, day 20; G, day 30), lesions treated with 25 mg/mL of A. vera (B, day 10; E, day 20; H, day 30), and those treated with 50 mg/mL of A. vera (C, day 10; F, day 20; I, day 30). Scale bars of (A) to (I) are equal to 50 Km. During wound healing, A. vera dose-dependently reduced cellularity and inf lammation at 10 DPI but also increased epithelialization and granulation tissue formation at 20 DPI; and finally, it enhanced remodeling of the scar tissue by aligning the collagen fibers and cells at 30 DPI. Therefore, A. vera improved regeneration and maturation of the epithelial tissue and reduced size of the scar tissue at 30 DPI. Stained by hematoxylin and eosin.
However, the maximum load of all the lesions were significantly lower than the intact healthy skin samples at this stage (P = 0.001 for all). Treatment with high dose of A. vera significantly increased the maximum stress of the healing treated tissue compared to the control lesions (P = 0.018), but there was no significant difference in the maximum stress of the lesions treated with low dose of A. vera and the control lesions at this stage (P 9 0.05). Although treatment with high dose of A. vera significantly increased the maximum stress; the measured value was still significantly lower than that of the intact healthy skin samples at this stage (P = 0.001). Treatment with high dose of A. vera also significantly increased the modulus of elasticity of the treated lesions when compared with the control lesions at 30 DPI (P = 0.006). There was no significant difference between the modulus of elasticity of the lesions treated with high dose of A. vera and those treated with low dose of A. vera at this stage (P 9 0.05). The modulus of elasticity of the lesions treated with high dose of A. vera was also significantly lower than those of the intact healthy skin samples (P = 0.001) at this stage.
Dry Matter Content Treatment with high dose of A. vera significantly increased the percentage dry matter content compared to the control ones at 20 and 30 DPI (P = 0.001 for both). Although the high dose of A. vera 42
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significantly improved the dry matter content, the measured value for the treated lesions was still significantly lower than those of the intact normal skin samples (P = 0.001) (Fig. 2B) at this stage.
Biochemical Findings A dose-dependent increase in the tissue level of collagen (Fig. 2C), NAGLA (Fig. 2D), and NAGA (Fig. 2E) was seen in the treated lesions so that topical application of low-dose A. vera significantly increased the concentration of collagen and glycosaminoglycans compared to the control lesions at 10, 20, and 30 DPI (P = 0.001 for all) and treatment with high dose of A. vera significantly increased these items compared to those treated with low dose of A. vera at 10, 20, and 30 DPI (P = 0.001 for all).
DISCUSSION Several studies have reported the anti-inf lammatory, cell proliferative, immune modulating, collagen stimulatory, antioxidative, and angiogenic activity of A. vera; however, some controversies exist between these studies that make the real efficacy of A. vera unclear.5,6,8,9,11Y18,20,21,24Y27,30Y32,40 These controversies could initially be due to the design of the studies. For example, Oryan and colleague26 studied the effect of A. vera on wound healing by brief histopathologic and tensile testing methods. Some others studied the * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 4. Longitudinal sections of the control (A, D), 25 mg/mL of A. vera (B, E), and 50 mg/mL of A. vera (C, F) at 30 DPI. A to C are stained by hematoxylin and eosin. D to F are inverted figures for better clarification of the structures. Scale bars of (A) to (C) are equal to 12.5 Km and (D) to (E) are equal to 6.25 Km. Aloe vera dose-dependently reduced the immature fibroblasts and inf lammatory cells but also enhanced maturation and alignment of the fibroblasts at 30 DPI so that more mature fibrocytes were present in the treated lesions compared to those of the control ones.
role of A. vera on tissue level of hyaluronic acid and dermatan sulfate27 and some others focused on the angiogenic and oxygen access effects of A. vera.29,40 The major merit of the present study was that various methods were used to study the different roles of A. vera on cutaneous wound healing in rats to test whether there is a correlation between the results and to show whether A. vera has dose-dependent effect on wound healing. We showed that A. vera significantly increases the rate of wound contraction, epithelialization, and maturation. Higher dose of A. vera was also effective in increasing the maximum load and ultimate strength of the healing tissues compared to other groups. These beneficial changes could be initially due to the modulatory effects of A. vera on the inf lammatory phase of wound healing.23 At the earlier stages of wound healing, the reduced total cellularity, edema, and fibrin clot together with the elevated number of macrophages, fibroblasts, and large blood vessels observed in the treated lesions, compared to the controls, suggest that A. vera enhances the rate and quality of the inf lammatory phase of wound healing.23 Increase in the number of blood vessels in the treated lesions could indicate the angiogenic activity of A. vera at earlier stages of wound healing which established a better perfusion and appropriate circulation in the injured area. Angiogenic activity of A. vera has previously been reported.40 Introduction of sufficient blood f low in the injured area increased the number of the endothelial cells, macrophages, and fibroblasts and further enhanced maturation of the fibroblasts which resulted in increased collagen production by these cells.23 Compared to the control group, lower number of lymphocytes and higher number of macrophages observed in the treated group at earlier stages of wound healing indicate that this reagent has selective and modulatory effects on inf lammation during wound healing.4 Lymphocytes are chronic inf lammatory cells and have fewer roles on wound healing than the macrophages. In contrast, the macrophages regulate several beneficial mechanisms during wound healing such as phagocytosis of the fibrin clot and necrotic tissues in the injured area.4,13,23 They also deliver the angiogenic and growth factors and * 2014 Wolters Kluwer Health, Inc. All rights reserved.
increase migration and proliferation of the endothelial cells and fibroblasts, thus facilitate the transition between the inf lammation to fibroplasia.28,41 Possibly A. vera increased the rate and quality of inf lammation by increasing the macrophages and reduced its duration by decreasing the lymphocytes.33 The extracellular matrix that is produced by fibroblasts is made up of collagen, glycosaminoglycans, and elastin.28 Our results indicated that higher number of fibroblasts and fibrocytes at different stages of wound healing had a strong positive correlation with the tissue level of collagen and glycosaminoglycans and A. vera increased the amount of these items in the healing treated tissues compared to the unassisted control lesions. At earlier stages of fibroplasia the newly deposited extracellular matrix mainly consists of glycosaminoglycans with lower proportion of collagen type III.4,28 Collagen type III is an immature form of collagen and its collagen fibrils have smaller diameter than those of the mature type 1 collagen fibrils. The cross-link bonds between these collagen molecules are weak, and they are polymerized at multidirections and represent an amorphous appearance in the regenerated tissue. The glycosaminoglycans have crucial roles on wound healing and act as a scaffold for collagen fiber polymerization and also incorporate in collagen maturation.41 Compared to the controls, A. vera dose-dependently increased the tissue level of glycosaminoglysans specially at the fibroplasia stage of wound healing. These findings are in line with those of Chithra et al,27 who showed that A. vera increases the amount of glycosaminoglycans in the injured area. Unlike the treated lesions, presence of neutrophils and numerous macrophages and lymphocytes in the untreated tissues at fibroplasia stage suggest that the unassisted wound healing was at the earlier stages of healing and the chronic inf lammation was still in progress, whereas the inf lammation had subsided earlier in the treated lesions.14 Once the tissue level of glycosaminoglycans increased and the fibroblasts proliferated, these cells become mature and their behavior would change.4,28 At mid to late stages of fibroplasia, their matrix www.annalsplasticsurgery.com
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For the statistical analyses, 1-way and 2-way ANOVA were used. Post hoc Tukey tests were used for comparison between the groups. Statistics were considered statistically significant when P G 0.05. For each evaluation, 5 histopathologic fields of each histopathologic section (n = 5) were used in each group. There are 5 animals in each group. Lymphocytes, macrophages, and neutrophils were counted as an index of inf lammation. Total cellularity was counted at 200. The other variables were counted at 800.
118.80 (20.16) 1.01 (1.22) 87.62 (4.50) 49.69 (2.96) 5.62 (1.51) 5.80 (2.58) 2.66 (1.14) 0 88.51 (4.18) 157.28 (15.83) 2.12 (1.58) 46.36 (4.25) 26.48 (2.96) 8.81 (1.12) 9.48 (2.30) 3.68 (1.14) 0 80.18 (7.99) 196.68 (23.89) 8.15 (1.68) 31.66 (1.49) 11.12 (3.48) 15.49 (1.51) 14.47 (2.35) 7 (2.39) 0 62.01 (5.42) 202.60 (9.01) 10.20 (1.64) 57.88 (2.46) 28.80 (1.48) 11.29 (1.64) 7.47 (1.14) 7.40 (1.81) 0 80.32 (6.18) 241.29 (25.35) 12.61 (1.67) 38.83 (5.20) 15.29 (3.56) 14.27 (3.27) 9.41 (2.60) 8.88 (2.16) 0 74.92 (6.71) 288.64 (12.35) 11.65 (1.88) 23.42 (1.83) 6.11 (2.76) 24.28 (1.35) 11.22 (2.34) 21.65 (1.34) 0 51.29 (3.18) 296.60 (24.65) 21.6 (3.28) 26.55 (5.88) 17.00 (1.58) 19.23 (2.12) 12.20 (4.32) 28.08 (4.33) 0.89 (0.44) 75.16 (6.24) 471.22 (16.14) 7.62 (1.53) 12.33 (2.79) 3.49 (1.16) 21.04 (5.56) 25.43 (5.78) 8.56 (2.79) 0.85 (0.27) 45.27 (5.18)
A. vera, 50 mg
Total cellularity Vascular number Diameter of vessel, Km Fibrocyte number Fibroblast number Lymphocyte number Macrophage number Neutrophil number Collagen mass density,%
384.21 (13.88) 18.25 (3.49) 21.34 (2.86) 9.81 (0.83) 15.49 (3.64) 18.43 (2.96) 17.66 (2.88) 0.21 (0.44) 62.18 (8.91)
A. vera, 25 mg Control Saline A. vera, 25 mg
A. vera, 50 mg Day 20 After Injury
A. vera, 25 mg
Control Saline Day 10 After Injury
Control Saline Variables/Groups
TABLE 3. Histopathologic and Histomorphometric Analysis: Comparison Between the Control, Low Dose, and High Dose of A. vera
Day 30 After Injury
A. vera, 50 mg
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production is shifted from glycosaminoglycans to collagen production.4,28 At this time, the tissue level of the glycosaminoglycans decreases and the collagen content increases.4,27,41 By increasing the collagen content of the healing tissue, a scar tissue forms and the tissue becomes mature.4,41 The total cellularity decreases and the fibroblasts transform to fibrocytes. In addition, several immature blood vessels disappear and the few preserved blood vessels are canalized, receive blood supply, their caliber increases and approximate to the normal blood vessels.4 The scar tissue then becomes more organized by time and its unorganized structure changes to a highly aligned pattern. In such circumstance, the collagen fibers become thicker, the quantity and quality of their cross-links improve, and the amount of collagen type III diminishes and shifts to formation of higher amounts of collagen type I concentration.10,41 The matured collagen fibrils are aligned unidirectionally in accordance to the forces transmitted through the wound, during healing. Therefore, in the remodeling phase of wound healing, size of the scar tissue decreases because the collagen fibers are aligned and the free spaces between them decrease.4,28 At 30 DPI, our results suggest that A. vera significantly reduced the total cellularity and immature blood vessels. It also increased fibrocytes and caliber of the blood vessels and enhanced alignment of the collagen fibers in the injured area. Aloe vera also significantly increased the tissue level of collagen and dry matter content compared to the controls. In addition, it interestingly decreased the scar size at this stage. The remodeling phase could be divided into 3 stages of alignment, maturation, and consolidation. On the basis of the results of the present study, the treated wounds at 30 DPI were in the alignment and maturation stages of wound healing because the epidermis was fully regenerated, the wound contraction completely occurred, and the wounds closed. In addition, the scar tissue formed in the injured area was aligned and mature. The consolidation stage of wound remodeling lasted up for months. In this stage, the healing tissue may gain almost its normal mechanical strength.42 All these results suggest that A. vera improved the structural organization of the healing tissue and this enhanced hierarchical organization was responsible for the superior biomechanical performance of the healing treated tissues compared to the controls. Increase in the maximum load, maximum stress, and modulus of elasticity of the treated wounds indicated that A. vera not only increased collagen synthesis per cell but also aided in cross-linking and maturation of the collagen fibrils and fibers. The effect of A. vera in increasing the mechanical strength of the healing tissue has major clinical relevance to those clinically injured wounds which heal under tensional forces. Under tension, there is a risk of dehiscence and healing failure and the role of A. vera in increasing the mechanical strength of the healing tissue should be highlighted.33 At 30 DPI, A. vera enhanced the cosmetic appearance of the healing wounds dose dependently. The A. vera exerted this beneficial effect by increasing the wound contraction and epithelialization, which resulted in a faster wound closure, and by decreasing the size of scar tissue through accelerating its organization and remodeling. The role of A. vera in improving the cosmetic appearance of the experimental wounds has major clinical relevance. It should be highlighted that, in this study, the experimentally induced wounds were surgically created under aseptic condition in a rat model. This model of wound has least postsurgical complication and is best suited to compare the effectiveness of biomaterials and healing factors on normal wound healing. The clinical wounds are often more complicated and are presented in different conditions. They may be larger, infected and gangrenous, burned, or may have chronic nature. In addition, the rate of wound healing is faster in laboratory animal models due to their faster maturation. Therefore, it may take months to judge about the cosmetic appearance of the clinical wounds in * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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Topical application of Aloe vera
TABLE 4. Biomechanical Characteristics of the Wound Samples Control
A. vera, 25 mg
A. vera, 50 mg
Normal
20.60 (2.70) 36.97 (14.26) 29.8 (9.69) 14.92 (3.56) 1.10 (0.48) 0.03 (0.01)
37.96 (3.20) 28.89 (4.49) 23 (4.47) 31.76 (3.35) 1.82 (0.05) 0.064 (0.01)
46.76 (14.58) 29.17 (1.64) 20.85 (3.89) 31.92 (11.14) 2.30 (0.65) 0.079 (0.022)
77.90 (17.72) 22.91 (1.42) 17.95 (2.16) 61.68 (14.60) 3.79 (0.75) 0.16 (0.029)
Biomechanical Factors Maximum load, N Ultimate strain, % Yield strain, % Yield load, N Ultimate strength, N/mm2 Modulus of elasticity, N/mm2
For the statistical analyses, 1-way and 2-way ANOVA were used. Post hoc Tukey tests were used for comparison between the groups. Statistics were considered significant when P G 0.05. There are 5 samples in each group.
response to treatment with A. vera because the healing rates of these complicated wounds are possibly slower than the experimental wounds used in the present study.42 Although this study comprehensively investigated the role of A. vera on experimental cutaneous wound healing and provided new insights in this regard, the potential physiologic variations between human and animals should be considered as one of the limitations of this study. In addition, this study used 3 time points to cover all the 3 phases of wound healing but it should be remembered that the remodeling phase of wound healing takes longer time to be completed; therefore, longer observational times (eg, 60 and 120 DPI) are needed to show the final outcome of the A. vera on remodeling phase of wound healing and also the cosmetic appearance of the healing wounds. Finally, we used A. vera with the concentrations of 25 and 50 mg/mL that were based on the molecular effects of A. vera during wound healing.34 It would be beneficial to compare the effectiveness of higher doses of A. vera on cutaneous wound healing with the results of the present study in future investigations.
CONCLUSIONS Topical application of A. vera modulated the inf lammation, increased the rate and quality of fibroplasia by enhanced collagen and glycosaminoglycans production, and improved the remodeling stage of the healing tissue. The lesions treated by A. vera showed dose-dependent improvement at various stages of wound healing so that the treated lesions showed faster wound contraction, enhanced epithelialization, smaller scar tissue formation, and higher tissue alignment. A dose-dependent effect of A. vera was shown in this study. The role of A. vera in increasing the cosmetic appearance and biomechanical characteristics of the healing cutaneous wounds has important clinical relevance and should be highlighted. These findings introduce A. vera as one of the best therapeutic agents in wound healing and it may be valuable in clinical practice. ACKNOWLEDGMENTS The authors thank Mr L. Shirvani and Mr G. Yousefi for their technical assistance and Dr N. Tanideh, Dr M. Hashemnia, and Dr A.R. Hamidi for providing the facilities. REFERENCES 1. Oryan A, Khalafinezad A, Toloo N, et al. Effects of 4-chloro-2,6Ybis (2-hydroxyl-benzyl) phenol on healing of skin wounds and growth of bacteria. J Vet Med A Physiol Pathol Clin Med 2007;54:585Y591. 2. Oryan A, Zaker R. Effects of topical application of honey on cutaneous wound healing in rabbits. Zentralbl Veterinarmed A. 1998;45:181Y188. 3. Kondo T, Ishida Y. Molecular pathology of wound healing. Forensic Sci Int. 2010;203:93Y98. 4. Monaco JAL, Lawrence T. Acute wound healing: an overview. Clin Plast Surg 2003;30:1Y12.
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5. Eshun K, Qian H. Aloe vera: a valuable ingredient for the food, pharmaceutical and cosmetic industriesVa review. Crit Rev Food Sci Nutr. 2004;44:91Y96. 6. Chithra P, Sajithlal G, Chandrakasan G. Influence of Aloe vera on collagen characteristics in healing dermal wounds in rats. Mol Cell Biochem. 1998;181: 71Y76. 7. Jia Y, Zhao G, Jia J. Preliminary evaluation: the effects of Aloe ferox Miller and Aloe arborescens Miller on wound healing. J Ethnopharmacol 2008;120: 181Y189. 8. Langmead L, Feakins R, Goldthorpe S, et al. Randomized, double blind, placebo controlled trial of oral aloe vera gel for active ulcerative colitis. Aliment Pharmacol Ther 2004;19:739Y747. 9. Langmead L, Makins R, Rampton D. Anti-inflammatory effects of aloe vera gel in human colorectal mucosa in vitro. Aliment Pharmacol Ther 2004;19:521Y527. 10. Maenthaisong R, Chaiyakunapruk N, Niruntraporn S, et al. The efficacy of aloe vera used for burn wound healing: a systematic review. Burns. 2007;33: 713Y718. 11. Tai-Nin Chow J, Williamson DA, Yates KM, et al. Chemical characterization of the immunomodulating polysaccharide of Aloe vera L. Carbohydr Res. 2005;340:1131Y1142. 12. Vijayalakshmia D, Dhandapanib R, Jayavenia S, et al. In vitro anti inflammatory activity of Aloe vera by down regulation of MMP-9 in peripheral blood mononuclear cells. J Ethnopharmacol 2012;141:542Y546. 13. Zhang L, Tizard IR. Activation of a mouse macrophage cell line by acemannan: the major carbohydrate fraction from Aloe vera gel. Immunopharmacology. 1996;35:119Y128. 14. Im SA, Oh ST, Song S, et al. Identification of optimal molecular size of modified Aloe polysaccharides with maximum immunomodulatory activity. Int Immunopharmacol. 2005;5:271Y279. 15. Chithra P, Sajithlal G, Chandrakasan G. Influence of Aloe vera on collagen turnover in healing of dermal wounds in rats. Indian J Exp Biol. 1998;36:896. 16. Boudreau MD, Fredrick AB. An evaluation of the biological and toxicological properties of Aloe barbadensis (Miller), Aloe vera. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2006;24:103Y154. 17. Esua MF, Rauwald JW. Novel bioactive maloyl glucans from Aloe vera gel: isolation, structure elucidation and in vitro bioassays. Carbohydr Res. 2006;341: 355Y364. 18. Ramamoorthy L, Kemp MC, Tizard IR. Acemannan, a beta-(1, 4)-acetylated mannan, induces nitric oxide production in macrophage cell line RAW 264.7. Mol Pharmacol. 1996;50:878Y884. 19. Talmadge J, Chavez J, Jacobs L, et al. Fractionation of Aloe vera L. inner gel, purification and molecular profiling of activity. Int Immunopharmacol. 2004;4:1757Y1773. 20. Kima J, Leea IS, Parkb S, et al. Effects of Scutellariae radix and Aloe vera gel extracts on immunoglobulin E and cytokine levels in atopic dermatitis NC/Nga mice. J Ethnopharmacol. 2010;132:529Y532. 21. Marshall G, Druck J. In vitro stimulation of NK activity by acemannan (ACM). J Immunol. 1993;150:1381. 22. Pugh N, Ross SA, ElSohly MA, et al. Characterization of Aloeride, a new highmolecular-weight polysaccharide from Aloe vera with potent immunostimulatory activity. J Agric Food Chem. 2001;49:1030Y1034. 23. Va´zquez B, Avila G, Segura D, et al. Antiinflammatory activity of extracts from Aloe vera gel. J Ethnopharmacol. 1996;55:69Y75. 24. Womble D, Helderman J. The impact of acemannan on the generation and function of cytotoxic T-lymphocytes. Immunopharmacol Immunotoxicol. 1992;14: 63Y77.
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25. Marshall G, Gibbons A, Parnell L. Human cytokines induced by acemannan. J Agric Food Chem. 1993;91:295. 26. Oryan A, Tabatabaeinaeini A, Nikahval B, et al. Effects of aqueous extract of Aloe vera on experimental cutaneous wound healing in rat. Vet Arh. 2010;80: 509Y522. 27. Chithra P, Sajithlal G, Chandrakasan G. Influence of Aloe vera on the glycosaminoglycans in the matrix of healing dermal wounds in rats. J Ethnopharmacol. 1998;59:179Y186. 28. Li J, Chen J, Kirsner R. Pathophysiology of acute wound healing. Clin Dermatol. 2007;25:9Y18. 29. Moon EJ, Lee YM, Lee OH, et al. A novel angiogenic factor derived from Aloe vera gel: beta-sitosterol, a plant sterol. Angiogenesis. 1999;3:117Y123. 30. Gallagher J, Gray M. Is aloe vera effective for healing chronic wounds? J Wound Ostomy Continence Nurs. 2003;30:68Y71. 31. Kaufman T, Kalderon N, Ullmann Y, et al. Aloe vera gel hindered wound healing of experimental second-degree burns: a quantitative controlled study. J Burn Care Rehabil. 1988;9:156Y189. 32. Schmidt JM, Greenspoon JS. Aloe vera dermal wound gel is associated with a delay in wound healing. Obstet Gynecol. 1991;78:115Y117. 33. Oryan A, Tabatabaei-Naieni A, Moshiri A, et al. Modulation of cutaneous wound healing by silymarin in rats. J Wound Care. 2012;21:457Y464.
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34. Tabandeh MR, Oryan A, Mohammadalipour A. Polysaccharides of Aloe vera induce MMP-3 and TIMP-2 gene expression during the skin wound repair of rat. Int J Biol Macromol. 2014;65C:424Y430. 35. Oryan A, Moshiri A, Meimandiparizi AH. Effects of sodium-hyaluronate and glucosamine-chondroitin sulfate on remodeling stage of tenotomized superficial digital flexor tendon in rabbits: a clinical, histopathological, ultrastructural, and biomechanical study. Connect Tissue Res. 2011;52:329Y339. 36. Oryan A, Silver IA, Goodship AE. Effects of a serotonin S2-receptor blocker on healing of acute and chronic tendon injuries. J Invest Surg. 2009;22: 246Y255. 37. Switzer BR, Summer GK. Improved method for hydroxyproline analysis in tissue hydrolyzates. Anal Biochem. 1971;39:487. 38. Neuman RE, Logan MA. The determination of collagen and elastin in tissues. J Biol Chem. 1950;186:549Y556. 39. Reissig JL, Strominger JL, Leloir LF. A modified colorimetric method for the estimation of N-acetylamino sugars. J Biol Chem. 1955;217:959Y966. 40. Lee MJ, Yoon SH, Lee SK, et al. In vivo angiogenic activity of dichloromethane extracts of Aloe vera gel. Arch Pharm Res. 1995;18:332Y335. 41. Enoch S, Leaper DJ. Basic science of wound healing. Surgery. 2008;26:31Y37. 42. Oryan A, Mohammadalipour A, Moshiri A, et al. Avocado/soybean unsaponifiables: a novel regulator of cutaneous wound healing, modelling and remodelling. Int Wound J. 2015;12:674Y685.
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