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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 52  |  Issue : 3  |  Page : 84-91

Enhancing rat full-thickness skin wounds with a mixed aloe/chitosan gel


1 The Persian Gulf Marine Biotechnology Research Center, Bushehr University of Medical Sciences, Bushehr, Iran
2 Department of Pharmacology, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran

Date of Submission11-Oct-2018
Date of Decision14-Nov-2018
Date of Acceptance29-Jan-2019
Date of Web Publication17-Jun-2019

Correspondence Address:
Dr. Sasan Zaeri
Department of Pharmacology, School of Medicine, Bushehr University of Medical Sciences, Imam Khomeini St. Bushehr
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/fjs.fjs_109_18

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  Abstract 


Background: Finding optimal treatment for skin wounds, especially using natural substances, has been of main focus in skin care and cosmetics. Aloe vera has been historically used in wound care, and chitosan, which is obtained from crustaceans, has a variety of biomedical applications. Accordingly, it seems that a mixture of aloe/chitosan may provide added benefits in wound healing.
Aims: The aim of the present study was to evaluate the effects of a mixture of aloe/chitosan gel in comparison to each component alone, on the healing of excisional full-thickness wounds in rats.
Materials and Methods: Round full-thickness wounds were made on the back of animal necks. Five experimental groups received either no treatment (Group 1) or daily topical treatments with 1% carboxymethyl cellulose (CMC) (Group 2), aloe (Group 3), chitosan (Group 4), or mixed aloe/chitosan gels (Group 5) for 14 days. Wound areas on days 3, 7, and 14 and histopathologic parameters on day 14 were measured for analysis.
Results: Means of wound areas showed a significant decreasing pattern in all groups through days 3–14 (P < 0.001). The topical treatment of wounds with aloe/chitosan significantly reduced wound areas compared to nontreated (P = 0.019) or 1% CMC-treated (P = 0.021) wounds. Histopathologic analysis revealed more reepithelialization, fibroblasts, collagen fibers and less polymorphonuclear leukocytes, new vessels, and granulation tissue in wounds treated with aloe/chitosan in comparison to controls (P < 0.05).
Conclusions: Aloe/chitosan-treated wounds showed convincing healing profiles affirming the benefits of using natural bioactive substances in wound healing, especially in combination therapy.

Keywords: Aloe vera, chitosan, rat, wound healing


How to cite this article:
Janahmadi Z, Motlagh MR, Zaeri S. Enhancing rat full-thickness skin wounds with a mixed aloe/chitosan gel. Formos J Surg 2019;52:84-91

How to cite this URL:
Janahmadi Z, Motlagh MR, Zaeri S. Enhancing rat full-thickness skin wounds with a mixed aloe/chitosan gel. Formos J Surg [serial online] 2019 [cited 2019 Sep 21];52:84-91. Available from: http://www.e-fjs.org/text.asp?2019/52/3/84/260436




  Introduction Top


Perfect wound healing and prevention of scar formation have been of major concern in skin care. In recent years, the approach toward the use of natural substances in the paradigm of traditional (supplemental) medicine has attained special attention regarding the skin wound care.[1] Aloe vera is a well-known medicinal herb;[2] Gel extracted from its leaves possesses healing properties on human skin wounds probably through its enhancing effects on reepithelialization of the injured skin and antibacterial activity as well. Biological benefits of aloe gel have been attributed to a wide variety of bioactive components such as amino acids, enzymes, vitamins, minerals, polysaccharides (pectin, cellulose, and glucomannan), and other low-molecular-weight substances.[3] The gel has been widely integrated into topical cosmetic pharmaceuticals for skin care purposes.[2] The ability of aloe gel in providing necessary conditions for a convincing skin wound healing (e.g., micronutrients, humidity, control of excess inflammation, fibroblast proliferation, etc.) explains why aloe-containing preparations outstand among others in skin wound care.[4],[5],[6]

On the other hand, chitosan, a natural linear polysaccharide,[7] possesses a number of intrinsic biological properties including biodegradability, dissolution in weak organic acids, cationic properties, blood-clotting activity, antimicrobial activity, etc.[8],[9] Positive effects of chitosan in healing of skin wounds in relation to controlling inflammation have been reported.[10] Chitosan-based membranes were developed as wound dressings due to ease of production, maintenance, and promising bioactive properties of chitosan.[7],[9],[10]

With respect to the aforementioned potentials of aloe and chitosan in the healing of skin wounds, the synergistic association of aloe gel with chitosan was explored in this study in the form of a simple gel formulation which could be beneficial as wound healing agent. Our work is an example of an in vivo study to evaluate this combinational potential; hence, the objective of the current study was to examine the effects of mixed aloe/chitosan gel in comparison to each component alone on full-thickness skin wounds in experimental rats.


  Materials and Methods Top


Animals

Male Sprague-Dawley rats (200–250 g) were purchased from the experimental animal breeding center of Bushehr University of Medical Sciences (BPUMS), Iran. To make the animals get familiar with the new living condition, they were taken to the experimental room 1 week before the experiments while placed in cages with free access to water and standard pellet food. The room temperature was adjusted to 22°C with 12-h light-dark intervals to control physiological diurnal changes. All animals were handled in accordance with guidelines for animal care and with the approval of the Ethics Committee of BPUMS.

Preparation of topical formulations

Aloe topical gel

A. vera leaves were purchased from the Aloe Cultivation Farmland, Bushehr, Iran. Whole leaves were fully washed with distilled water up to the removal of superficial impurities. The leaf parenchymal tissues were carefully detached from crusts using a clean knife. They were washed with distilled water, cut into smaller pieces, homogenized, and finally filtered to obtain a transparent gel. Subsequently, the gel was well mixed with carboxymethyl cellulose (CMC) 1% (Sigma, USA) at a ratio of 4:1 (v/v) to produce the final aloe topical gel (80% [v/v], pH = 6.3 ± 0.2). CMC 1% served as both vehicle and plasticizing agent in all formulations used in the study. Aloe gel and other formulations were freshly prepared on a daily basis under clean conditions and sterilized under ultraviolet-C light for 30 min before use.

Chitosan topical gel

Chitosan powder (molecular weight: >310,000 Da; deacetylation degree: >75%) produced from shrimps of the Persian Gulf was purchased from Lian Kimia Azma Co. (Bushehr, Iran). Chitosan 2% solution was prepared by dissolving 2 g chitosan powder in glacial acetic acid 0.2 M up to 100 mL final volume. To make chitosan topical gel, chitosan 2% solution was well mixed with CMC 1% at a 4:1 (v/v) ratio (pH = 6.1 ± 0.1).

Mixed aloe/chitosan topical gel

Equal volumes of aloe crude gel and chitosan 2% solution were mixed and blended until a soft homogenous mixture was formed. Subsequently, the mixture and CMC 1% were mixed at 4:1 (v/v) ratio to produce aloe/chitosan gel (pH = 6.0 ± 0.2).

Skin wound model

Animals were anesthetized by intraperitoneal injection of a mixture of ketamine (60 mg/kg) and xylazine (10 mg/kg). The interscapular region was shaved and rubbed clean with alcohol 70% pad. Subsequently, a circular excisional full-thickness wound (1 cm in diameter) was made by means of a sharp curved-tip surgical pair of scissors. Excess care was taken to excise the skin smoothly and not to injure the subcutaneous muscular layer, namely, panniculus carnosus. The interscapular region was selected for wound excisions so that animals could not manipulate the dressings easily.

Study design

A number of 35 rats were randomly and evenly distributed in five experimental groups. Wounds in Group 1 received no treatment. Group 2 wounds were topically treated with CMC 1%. Groups 1 and 2 were negative controls in the study. Wounds in Groups 3, 4, and 5 received aloe, chitosan, and aloe/chitosan topical gels, respectively. All wounds were treated with an equal volume of gel (0.5 mL). After applying the appropriate gel (except for Group 1), wounds were dressed with paraffin-embedded gauze to reduce adhesion of the dressing to the wounded tissue. Rats received topical treatments once daily for 14 days. Wounds were photographed using a digital camera on days 0 (surgical day), 3, 7, and 14 to measure wound areas. When photographing, a ruler was put beside each wound to compensate for variations in calculated wound areas caused by variable distances between the camera and the wounds. On day 14, the wounded area plus a surrounding marginal intact skin was excised and stored in formalin 10% for histopathologic analysis. Finally, animals were sacrificed by high-dose ketamine/xylazine intracardiac injection.

Macroscopic (wound area) measurements

Wound photos were processed with ImageJ Version 1.47 Software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA) to calculate the area of wounds. For each skin wound, calculated areas at different days were normalized to its area at day 0. This would compensate for any variations in the primary size of each wound. Hence, macroscopic data were reported as area (%) at days 3, 7, and 14 postsurgery as calculated by the following equation:



Microscopic (histopathologic) measurements

Routine process for hematoxylin-eosin (H and E) and Masson's trichrome staining was performed for light microscopy. In brief, central cross-sections were done on the excised wounds. Samples were fixed in 3.7% formalin, embedded in paraffin, sectioned into 3–4 μm slices, and stained in accordance with a routine protocol of H and E and Masson's trichrome staining. Histopathologic parameters included the levels of reepithelialization, polymorphonuclear (PMN) leukocytes, fibroblasts, collagen fibers, new vessels (neovascularization), and granulation tissue. These variables were scored 0 (absence), 1 (mild), 2 (moderate), or 3 (severe or full). This semiquantitative scaling system for scoring histologic findings was partially adopted from previous works [11] with some modifications and finally confirmed by an expert pathologist. [Table 1] contains detailed information about the interpretation of the levels (scores) of histopathologic parameters.
Table 1: Explanation of the used scale in the assessment of histopathologic parameters in ten high-power fields

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Statistical analysis

Repeated-measures ANOVA with Tukey honestly significant difference post hoc test were used to analyze macroscopic (wound area) data (mean ± standard deviation). Mean ranks of histopathologic parameters were analyzed using the Kruskal–Wallis test with Bonferroni post hoc comparisons. Differences at the level of P < 0.05 were considered statistically significant. All data analyses were performed using SPSS ® Software (Version 21.0, IBM Corp., SPSS Statistics for Windows, Armonk, NY, USA).


  Results Top


Macroscopic (wound area) analysis

Representative wounds with either no topical treatment (Group 1) or treatment with CMC 1%, aloe, chitosan, and aloe/chitosan topical gels (Groups 2–5) at days 3, 7, and 14 postsurgery are shown in [Figure 1]. Moreover, the corresponding means of wound areas are summarized in [Table 2]. The maximum wound area was observed in CMC 1%-treated group with 88.15% ±5.1% at day 3, while the minimum was obtained in aloe/chitosan-treated group with 1.08% ±0.59% at day 14 postsurgery.
Figure 1: Representative photos of rat full-thickness skin wounds in different experimental groups receiving either no treatment or topical gels of carboxymethyl cellulose 1%, aloe, chitosan, and aloe/chitosan at different postsurgical days with the aid of a ruler for scaling purpose (n = 5–7 per group)

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Table 2: Wound areas (%) (mean±standard deviation) after no treatment or treatments with topical gels of carboxymethyl cellulose 1%, aloe, chitosan, and aloe/chitosan at days 3, 7, and 14 postsurgery (n=5–7 per group)

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Effects of time and time-group interaction on skin wound healing

Repeated-measures ANOVA analyzed data obtained from different experimental groups at different time intervals and reported estimated marginal means of wound areas. Irrespective to different treatments, it was found that wound areas decreased significantly in a chronological manner, i.e., from day 0 to 14 (F = 270.25, df = 2, P < 0.001) [Figure 2]. Besides, time and treatment groups revealed no statistically significant interaction as visually observed in [Figure 2]. In other words, trends of decrease in wound areas in different experimental groups were generally comparable and did not, for example, crossed each other significantly as confirmed by using the statistical analysis (F = 84.39, df = 6.52, P = 0.779).
Figure 2: Effects of time and experimental group as independent variables on macroscopic wound healing regarding estimated marginal wound area means (%) analyzed using repeated measures ANOVA

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Effects of topical gel treatment (group effect) on skin wound healing

[Figure 2] reveals some difference between wound areas of experimental groups on the average of all time points. This was statistically confirmed where significant difference among groups was detected (F = 4.42, df = 4, P = 0.007). Subsequently, intergroup comparisons by post hoc test revealed that aloe/chitosan-treated wounds had significantly smaller sizes than wounds with no treatment (P = 0.019) or under CMC 1% treatment (P = 0.021). Wounds in aloe/chitosan group did not show a significant difference compared to wounds treated with aloe or chitosan. However, they were averagely smaller in size by 14.1% and 16.4%, respectively. Wound mean area in aloe group was lower, although not significant than those in nontreated or CMC 1%-treated groups by 26.9% (P = 0.265) and 25.1% (P = 0.234), respectively. Moreover, wound areas in chitosan group showed almost similar results where falls by 24.7% (P = 0.194) and 25.9% (P = 0.175) were found compared to nontreated and CMC 1%-treated experimental groups, respectively. Treatment with CMC 1% topical gel did not produce significant changes in wound mean area in comparison to nontreated group (P = 0.9).

Microscopic (histopathologic) analysis

[Figure 3] depicts representative H and E-stained slides of skin wounds in different experimental groups 14 days after surgery. The corresponding tissues stained with Masson's trichrome are shown in [Figure 4] and provide a good measure for comparing groups in terms of collagen production and density. Wounds in control groups showed moderate reepithelialization, high edematous background and granulation tissue full of PMNs, new vessels with thin endothelia, and low proliferation of fibroblasts. Skin appendages were not present 14 days after skin wounding [Figure 3]a, [Figure 3]b and [Figure 4]a, [Figure 4]b. For wounds in aloe or chitosan group, lower levels of edematous stroma, granulation tissue, PMNs, and new vascularity were observed in comparison to control groups. Instead, fibroblasts and collagen fibers became more prominent. Furthermore, reepithelialization showed a relative improvement. However, skin appendages were still missing [Figure 3]c, [Figure 3]d and [Figure 4]c, [Figure 4]d. Wounds in mixed aloe/chitosan group showed reepithelialization almost completed; wounds were bridged with keratinized layer covering the wounded skin. Wound stroma had a fibrotic presentation with minimal levels of granulation tissue, neovascularity, and PMNs. Lymphocytes and to some extent macrophages partially replaced neutrophils. A good recovery of skin adnexa was observable after 14-day treatment with aloe/chitosan gel [Figure 3]e and [Figure 4]e.
Figure 3: Representative low-magnification (×35) H and E-stained slides of rat full-thickness skin wound 14 days after either no treatment (a) or treatments with topical gels of carboxymethyl cellulose 1% (b), aloe (c), chitosan (d), and aloe/chitosan (e). Arrow (→) and arrowhead (▴) marks demonstrate wound margins and re-epithelization edges, respectively. Wound beds beneath the re-epithelized area contain primarily granulation tissue and lack most of the skin appendages as in a, b, c, d but not e (aloe/chitosan group)

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Figure 4: Representative low-magnification (×35–50) photomicrographs of rat full-thickness skin wound tissues stained with Masson's trichrome on day 14 after either no treatment (a) or treatments with topical gels of carboxymethyl cellulose 1% (b), aloe (c), chitosan (d), and aloe/chitosan (e). More abundant dense collagen fibers can be observed in wounds treated with aloe/chitosan (e) in comparison to wounds in other experimental groups

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Analyzed data of different histopathologic parameters involved in skin wound healing process are depicted in [Figure 5]. In brief, the Kruskal–Wallis test revealed significant differences between groups regarding reepithelialization (χ2 = 19.87, df = 4, P = 0.001), fibroblast (χ2 = 24.22, df = 4, P < 0.001), collagen (χ2 = 25.92, df = 4, P < 0.001), PMN (χ2 = 21.24, df = 4, P < 0.001), neovascularization (χ2 = 26.10, df = 4, P < 0.001), and granulation tissue (χ2 = 25.04, df = 4, P < 0.001). The mean rank of reepithelialization in aloe/chitosan group was significantly higher compared to nontreated (P = 0.001) or CMC 1%-treated (P = 0.002) groups. Fibroblasts and collagen fiber levels in wounds treated with aloe/chitosan gel showed mean ranks significantly higher than nontreated (P < 0.001) or CMC 1%-treated wounds (P < 0.001). Mean ranks of new vessels and PMNs which mainly consisted of neutrophils showed significantly higher levels after 14 days in control groups compared to aloe/chitosan-treated wounds (P < 0.001). Accordingly, granulation tissue which is a transitional state between inflammatory and maturation phases of wound healing followed the same trend showing higher mean ranks in non-treated and CMC 1%-treated groups compared to aloe/chitosan group (P < 0.001). Of note, the mean rank of granulation tissue in wounds treated with chitosan was significantly lower compared to wounds treated with CMC 1% (P = 0.033).
Figure 5: Semiquantitative analysis of histopathologic parameters involved in wound healing after 14-day topical treatment of wounds in different experimental groups (n = 5–7). Data are presented as mean ranks of histopathologic parameters analyzed using the Kruskal–Wallis test

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  Discussion Top


Skin wound healing and approaches to enhance it with the least side effects have attracted the attention of researches in the field of dermatology and hence have been the main focus of investigation for many years. In this regard, animal wound models have been routinely utilized to evaluate the effects of various dressings, bandages, growth factors, and topical pharmaceuticals on skin wound healing. Animal model of excisional full-thickness wound has been widely used for this purpose.[12]

Topical effects of aloe, chitosan, or mixed aloe/chitosan gels were evaluated on rat full-thickness excisional wounds in a time span of 14 days. Two main criteria for evaluation were macroscopic (wound area) and microscopic (histopathologic) changes after treatments. Microscopic data included semiquantitative analysis of wound reepithelialization, levels of fibroblasts, collagen fibers, PMNs, new vessels, and granulation tissue. The main finding of the study was the better and significant improvement of wounds, both macroscopically and microscopically, treated with aloe/chitosan than with aloe or chitosan gels alone.

As a general rule, both the rate and quality of healing are the two main judgment indices in skin wound healing. Physiologically, stages of skin wound healing (clotting, inflammation, proliferation, and remodeling) successively take place to regenerate the damaged skin as perfectly as possible.[13] However, researchers are always trying new approaches to improve the extent as well as the rate of this physiological action. This would help in reaching a high-quality healing with the least adverse effects such as infections and esthetic issues. Accordingly and unlike other similar works,[14] our study aimed at simultaneously evaluating roles of time (healing rate), treatments (extent of healing), and their possible interactions on wound areas using repeated-measure ANOVA which is a well-suited analytical test for this purpose.

Irrespective of different topical treatments, it was found that wound areas decreased significantly as time passed on. This means that the passage of time independently acted to reduce wound areas after 14 days. This is in agreement with the findings by other studies. For example, Santos et al.[14] reported significantly lower rat wound areas in all experimental groups 14 days after topical treatments. This was observed either after 18 or 21 days postsurgery depending on the topical treatment. Similarly, Mohammadi et al.[15] who evaluated platelet-rich plasma in combination found that time had a significant impact on skin wound healing in all experimental groups. It seems that part of the independent time effect on healing can be explained by the aforementioned spontaneous physiologic healing stages after injury, especially in excisional deep wounds. Furthermore, selection of animal species for wound modeling may influence the rate of wound closure. Despite in humans, a muscular layer called panniculus carnosus exists subcutaneously in rats. It contracts to facilitate wound shrinkage and hence reduction in the wound area.[16] Although this occurs independently to the reepithelialization process, its final goal and outcome are to close the wound. The use of other animal species which lack panniculus carnosus can be suggested for the induction of experimental wounds as a solution to this limitation. Of note, the wound shrinkage due to panniculus carnosus was not considered as an influencing confounder in our data interpretation since skin wounds intrinsically experienced it equally in all experimental groups. Moreover, repeated-measures ANOVA being able to simultaneously analyze effects of time and experimental groups enabled us to distinguish independent effects of time (self-shrinkage or healing) and treatment groups on wound healing.

As the interaction term between time and treatment group was not significant, it means that changes in wound areas in different experimental groups followed the same trend (slope) on the averaged time of healing are shown in [Figure 2].

Even though treatments with aloe or chitosan gels did not result in significant difference in comparison to control groups, they decreased wound areas to some extent after 14 days. In this regard, a large number of studies have shown the healing properties of aloe or chitosan on skin wounds.[17],[18] However, some studies reported contrasting results. For example, a human study by Schmidt and Greenspoon showed that A. vera topical gel delayed wound healing.[19] In general, evidence that supports skin wound healing by aloe or chitosan overweigh the opposing findings. Therefore, the negative results found in our study probably lie in a number of confounding factors such as short duration of treatments, low power of analysis due to multiple treatment groups, low sample size, etc., However, topical treatment with mixed aloe/chitosan gel acted differently and yielded the best macroscopic wound healing among other groups. This observed feature of aloe/chitosan probably was due to direct and/or indirect interactions between them under the experimental condition. To the best of our knowledge, no similar study to evaluate actions of aloe/chitosan mixture in vivo on macroscopic wound healing was found in the literature. However, a number of studies reported other properties of aloe/chitosan combinations such as physicochemical, antimicrobial, and cell-modifying features which would be discussed in later sections.[20],[21],[22],[23]

Microscopic findings in wound healing studies are much important because they provide a more detailed view of the histopathologic events beneath the observed macroscopic changes. Aloe or chitosan gel each demonstrated more improved wound histologic profiles than control groups, although they were statistically insignificant. It seems that the same explanation for their insignificant macroscopic findings may be applied here as well. However, other studies reported positive effects of aloe or chitosan on histopathologic parameters of wound healing. For example, chitosan was associated with the followings: PMNs recruitment to the wounded area, facilitating coarse fibrin formation as a stimulator of fibroblast migration to wound, stimulating macrophages migration, fibroblast proliferation, and synthesis of collagen type 3.[24],[25] Similarly, aloe proved effective in modulating wound histopathologic parameters, especially in burn wound studies and to a lesser extent in excisional wound works.[26],[27]

In the present study, there was a relatively complete correspondence between the macroscopic and microscopic findings in each experimental group. For instance, wounds treated with aloe/chitosan mixture presented the best efficacy in wound closure which was in accordance with their positive histopathologic profiles of wound healing; reepithelialization was almost complete and bridged the wound margins with a keratinized layer formed normally after 14 days. Inflammatory phase was minimal since PMNs (mainly neutrophils), which were dominant in control groups, were replaced largely by lymphocytes and macrophages. More falls in edema, granulation tissue, and new vessels in wounds treated with aloe/chitosan gel compared to others were confirmative of alleviated levels of inflammation. In addition, more abundant fibroblasts and subsequently higher synthesis of collagen fibers indicated high ability of aloe/chitosan gel in transiting wounds from inflammatory to maturation (remodeling) phase.

The observed enhanced effects by the mixed aloe/chitosan gel might be due to changes in physicochemical and/or biologic properties of the components after mixing. In this regard, direct or indirect interactions between aloe constituents and chitosan may be involved. Silva et al. reported that aloe/chitosan-based membranes had high water absorbability and improved survival and metabolic activity of L929 fibroblast-like lineage in direct contact test.[23] Another work by this research group revealed that the addition of even small amount of aloe gel to chitosan solution resulted in membranes with more stability, convincing degradability rate, and desirable mechanical properties. This structure provided a suitable biomedia for attachment, spread, proliferation, and survival of human dermal fibroblasts and showed antibacterial activity against Staphylococcus aureus Scientific Name Search  in vitro.[20] Topical administration of microparticles containing chitosan/aloe/Vitamin E on second-degree burn wounds after a 14-day treatment period demonstrated a significant reepithelialization and collagen fibers synthesis with lower levels of neovascularity and exudate of albumin and leukocytes.[22] As the chitosan molecule possesses basic properties and adopts positive charge in physiologic pH, it may interact electrostatically with negatively-charged polysaccharides and proteins in aloe gel. Hence, it may provide the mixture with modulated and stabilized electrostatic features so that aloe/chitosan mixture can interact more efficiently with fibroblasts, other cells, and structures within extracellular space of skin tissue. Accordingly, it was reported that surface energy and wettability of bioactive polymers significantly affected biological processes at the cellular (attachment, spread, and proliferation) and subcellular (e.g., attachment to proteins) levels.[28] For example, modulation of chitosan surface with plasma resulted in improved attachment to fibroblasts and their enhanced proliferation.[29] Similarly, it may be postulated that bioactivity of aloe/chitosan topical gel, in the current study, was affected by a series of new physicochemical properties such as changes in surface energy, wettability, total electrostatic charges, etc., These new probable features, in turn, might have affected attachment of the gel components to proteins and cells involved in wound healing. Of note, this postulation awaits affirmation in the future studies.

One limitation of the current study was the lack of macroscopic and histopathologic data at shorter (e.g., daily) intervals. Relatively short treatment period of the study was another limitation due to budget restrictions. Another limitation was the use of only one ratio (50:50%) and concentration of aloe and chitosan gels. Examining other ratios would provide better understanding of the interactive effects of gels on wound healing and thus can be suggested in the future works. Moreover, the measurement of biochemical parameters involved in skin wound healing such as oxidative stress and/or nitric oxide synthesis pathways would be suggested in the future studies. This would provide a more detailed and mechanistic view of wound healing efficacy after topical treatment of aloe/chitosan gel.


  Conclusions Top


Promising wound healing efficacy of mixed aloe/chitosan gel in rat full-thickness skin wounds was the main finding of the present study. However, our data do not deny the promising wound healing activities of aloe or chitosan separately. All in all, the results confirmed the general conception of beneficial effects of bioactive natural materials in wound healing and supported the idea of combining compatible natural substances to yield additive or synergistic outcomes, especially in the research field of skin wound care.

Acknowledgment

This work was supported by a grant from the Bushehr University of Medical Sciences (Grant Code. IR.BPUMS.REC.1395.48). The authors would like to thank Professor Iraj Nabipour for his kind scientific guidance and generosity in providing us with the laboratory facilities.

Financial support and sponsorship

This study was financially supported by the Bushehr University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

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