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Wound Healing and Antimicrobial Effects of Chitosan-hydrogel/Honey Compounds in a Rat Full-thickness Wound Model
In this study, the effects of the incorporation of high molecular weight chitosan hydrogel on antibacterial, antifungal, and wound healing properties of honey were investigated.
Abstract
Introduction. Honey and chitosan have shown antimicrobial and wound healing effects. As a biocompatible and biodegradable biomaterial, chitosan has shown antimicrobial capabilities. Objective. In this study, the effects of the incorporation of high molecular weight chitosan hydrogel on antibacterial, antifungal, and wound healing properties of honey were investigated. Materials and Methods. The minimum inhibitory concentration of chitosan and honey were examined in pure and 3:1, 1:1, and 1:3 (v/v) compound ratios for Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. In addition, the inflammatory, granulation and fibrotic tissue formation, reepithelialization indices, and wound shrinkage effects of each treatment were evaluated and compared with saline and silver sulfadiazine. Results. Chitosan/honey 1:3 was found to be effective against all 5 aforementioned germs. Honey, chitosan/honey 1:1, and chitosan/honey 1:3 showed faster wound healing and shrinkage effects. Conclusions. Incorporation of chitosan hydrogel into honey can substantially enhance its antimicrobial and wound healing effects. Chitosan-hydrogel/honey (1:3) is an optimal wound dressing formulation with ample antimicrobial and healing properties.
Introduction
The use of honey as a traditional remedy has been documented for more than 3000 years.1 In the Quran, honey has been referred to as an important food containing healing properties.2,3 Rediscovery of honey has opened a new branch of apitherapy in modern medicine, contributing to fill the voids in wound treatment.1 The antibacterial and antifungal effects of honey have been reported in many studies.4-7 The antibacterial activity can vary in spectrum and potency with honeys of different sources.1 Antifungal effects against Candida albicans, C glabrata, C dubliniensis, C krusei, Trichosoporon spp, and Cryptococcus neoformans have been reported with various dilutions of honeys from different botanical origins.8-10
Honey at concentrations of 11-1.8% has been reported to inhibit large samples of infectious wounds while concentrations of 4-1% suppresses methicillin-resistant Staphylococcus aureus (MRSA).4,5
In addition, chitosan has drawn substantial attention in medical applications as a precious biomaterial because of its biocompatibility and biodegradability as well as antimicrobial and antitumoral capabilities.11 Scaffolds in tissue engineering and regenerative medicine,12,13 wound healing,14-16 and protein,17 gene,18 and drug19,20 delivery systems are some of the most famous applications of chitosan. Increased collagen production21 and enhanced reepithelialization22 resulting in accelerated wound healing have been reported with chitosan-containing multilayered hydrogel dressings applied to full-thickness wounds in diabetic rats.23 Enhancing the cell growth and decreasing the bacterial growth, acceleration of angiogenesis, formation of granulation and fibrotic tissues, and improvement in reepithelialization are additional wound healing effects of chitosan.24 Chitosan functions as an antibacterial and anti-inflammatory material through weakening bacteria-cell attachment, increasing bacterial membrane permeability, and activating the immune system.24,25
Research has documented chitosan as a biologic wound dressing material with healing effects due to its bacteriostatic and hemostatic effects.26-30 These chitosan-honey-gelatin films have shown 100% antibacterial efficacy and superiority to honey and chitosan when used separately. This tricompound sheet decreases the burn wound size significantly within 12 days.29,31 The present study was designed to assess the wound healing and antimicrobial effects of honey-containing chitosan hydrogel in a rat full-thickness wound model.
Materials and Methods
Chitosan hydrogel preparation
The chitosan hydrogel was prepared by gradual stir-dissolving 2 g of high molecular weight chitosan (92% degree of deacetylation) (Honeywell Fluka; Fisher Scientific Sweden, Göteborg, Sweden) in 1% (v/v) aqueous glacial acetic acid (Merck & Co, Kenilworth, NJ).32 The final volume of the solution was adjusted to 100 mL after completely dissolving.32 The resulting gel was kept at room temperature for 1 hour to remove air bubbles.
Intact comb waxy honey was procured in collaboration with Ardabil veterinary headquarters (Dasht-e-Moghan, Iran) with Ziziphora tenuior L and Thymus vulgaris as the dominant vegetation. Antibacterial, antifungal, and wound healing properties of both Z tenuior L and T vulgaris have been reported and traditionally used in practice.33,34 The quality of filtered honey (0.42-mm sieve) was approved by the Institute of Standards and Industrial Research of Iran.
Chitosan/honey (CH) mixtures were prepared with 3 different v/v ratios: CH1 (3:1) = 75% chitosan + 25% honey; CH2 (1:1) = 50% chitosan + 50% honey; and CH3 (1:3) = 25% chitosan + 75% honey. All compounds were sterilized by low-temperature hydrogen peroxide plasma sterilization at 37°C to 44°C for 75 minutes.
Sampling
Healthy male Wistar rats (250 g–350 g, aged 16–24 weeks old) were obtained from the Mashhad University of Medical Sciences (Khorasan Razavi, Iran) animal house. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals of the university animal care committee. The clearance to conduct this study was provided by the Mashhad University of Medical Sciences Committee of Animals and Ethics (project license No.: 3998). The rats were maintained with food and water ad libitum for 1 week for acclimatization to the laboratory environment in a temperature-controlled room. The animals were randomly divided into 7 groups, each containing 15 rats (Table 1).
Wound excision model
Fifteen animals in each group were anaesthetized by intraperitoneal injection of xylazine/ketamine 7.5/60 mg/kg-1. After prepping the backs of the rats, draping was done by povidone-iodine followed by 70% ethanol. A single full-thickness excision was performed by cutting a 15-mm punch of the skin from a predetermined unreachable area in between the animal shoulders. Each animal was kept in a solitary cage to prevent the wounds from being eaten by other animals. Each rat received 2 intramuscular injections of 22 000 IU of penicillin G procaine once every 24 hours, to prevent infection.
Saline and silver sulfadiazine creams were applied twice daily to the wounds in the negative and positive control groups, respectively. Antibacterial efficacy of silver sulfadiazine has been established.35,36 The test group’s wounds were treated twice daily by one of the following treatments: 2% chitosan hydrogel (group 1); honey (group 2); chitosan hydrogel/honey (3:1 v/v) (group 3); chitosan hydrogel/honey (1:1 v/v) (group 4); or chitosan hydrogel/honey (1:3 v/v) (group 5) (Table 1). Special care was taken to cover the entire wound surface with the proposed treatment in order to avoid variation in the dose.
Antimicrobial tests
Microbial limit test. The microorganisms used were S aureus (ATCC 6538P/PTCC 1112), Bacillus cereus (PTCC 1247), Escherichia coli (ATCC 8739/PTCC 1330), Pseudomonas aeruginosa (ATCC 9027/PTCC 1074), and C albicans (ATCC 10231/PTCC 5027), all procured from the Iranian Research Organization for Science and Technology.
As a quantitative test, 1 mL of 1% and 0.1% dilutions of each treatment solution in sterile Soybean Casein Digest Broth (SCDB) in a Petri dish was fully covered with molten SCDB (45°C) and incubated at 37°C, 5% CO2. The cultures were examined quantitatively for any microbial growth after 3 to 5 days.37
As the qualitative study, 1 mL of each treatment solution was diluted with 9 mL of SCDB and lactose broth (LB) separately, and examination for the presence of pathogenic microorganisms was carried out after overnight incubation at 37°C, 5% CO2.37
Antimicrobial activity. Serial dilutions of each treatment solution in SCDB were used for determination of minimum inhibitory concentration (MIC). Then, 106 CFU of one of the microorganisms was added to 200-µL serial dilutions of each treatment in a 48-well plate. Microorganism + SCDB, and SCDB with no microorganism were used as positive and negative controls, respectively. The MIC was defined as the lowest concentration of the extract that inhibited the visible growth on SCDB.38
Histopathology
The details of the histopathological study have been described in a previous study by the authors.39 Briefly, a skin specimen sample was isolated from the wound sites of each group of animals on days 3, 7, and 14 after wounding for histopathological examination. Specimens from the wounds and the adjacent healthy skin were precisely excised, fixed in 10% (v/v) buffered formalin, and sent to the pathology lab. Processing the specimens using a Tissue Tek Rx11a Rotary Tissue Processor (Sakura Finetek, Alphen aan den Rijn, Netherlands) was followed by paraffin embedding, preparation of thin sections, and hematoxylin and eosin staining. The slides were examined by 2 separate pathologists under a light microscope for inflammatory, fibrotic tissue formation, granulation tissue formation, and reepithelialization indices. Qualitative scoring was performed according to Table 2 and based on the guidelines described in the authors’ previous work.39,40
Wound surface area calculation
Digital images of the wound area were captured on days 0, 1, 3, 7, and 14. The images were processed with Scion Image Alpha (4.0.3.2) software (Meyer Instruments, Inc, Houston, TX) for surface area changes. Total reduction in the wound size was calculated by dividing the surface area at each time point to that of the original wound using the Formula.
Statistical analysis
Statistical analysis was performed using SPSS Statistics for Windows, version 20.0 (IBM, Armonk, NY). Data were expressed as the mean ± standard deviation for each group of animals. Comparison between the groups for pathologic indices was done by Kruskal-Willis test. The paired comparison between the groups was done by the Mann-Whitney U test. Changes in wound surface area were analyzed by repeated measure analysis of variance. Tukey’s test was used for paired comparison between groups.
Results
Antimicrobial tests
Microbial limit test. The tested samples passed the quantitative test, and in qualitative tests, no sign of pathogenic microorganisms were observed.35
Minimal inhibitory concentration. The MICs of each chitosan and honey alone and in different formulations are shown in Figure 1. The MIC of chitosan was 0.125% for S aureus, and B cereus was dropped 8 times to 0.015625% when formulated with honey as CH₃ (1:3 v/v). In addition, chitosan alone was absolutely ineffective against P aeruginosa and C albicans, but it became effective in combination with honey in all 3 combined forms to the extent of an MIC of as low as 0.0625% and 0.03125% for P aeruginosa and C albicans, respectively, in CH₃ (1:3 v/v).
Honey also showed 25% less MIC for S aureus, B cereus, E coli, and P aeruginosa as well as 50%, 75%, and > 80% less MIC for C albicans in CH1 (3:1 v/v), CH2 (1:1 v/v), and CH3 (1:3 v/v) formulations, respectively, compared with its pure form.
Histopathology
Results of histopathological indices are depicted as boxplots in Figure 2.
Inflammatory index
The inflammatory index at days 3 and 7 showed significant improvement in all treatment groups compared with the negative control (saline [S]) group (P < .01). No significant difference was seen between the 5 treatment groups as well as the positive control at days 3 and 7 of treatment. At day 14, the inflammatory index was at chronic to mild stage in the honey alone (H), CH3, and CH2 groups (Figure 2A).
Granulation tissue formation index
The granulation tissue formation index was higher (grade 3) in the H, CH2, and CH3 groups compared with chitosan hydrogel (C) and CH1 (both at grade 2) and S (grade 1) at day 3. At day 7, it increased to grade 4 among all 5 treatment groups as well as the positive control (silver sulfadiazine 1% cream [SSD]) and was significantly higher than the negative control (S) group. By the end of week 2 of treatment, the H, CH3, and CH2 groups showed a significantly higher degree of granulation tissue formation (grade 5) (Figure 2C).
Fibrotic tissue formation index
The fibrotic tissue formation index was significantly higher (grade 2) among the C, CH2, and CH3 groups at day 3. At day 7, this index was significantly higher in the CH2 and CH3 groups compared with S (grade 2), C, H, CH1, and SSD (all 4 at grade 3). The H, CH3, and CH2 groups showed a significantly higher fibrotic tissue formation index (grade 5) at the end of week 2 of treatment (Figure 2B).
Reepithelialization index
The H, CH3, and CH2 groups showed the highest reepithelialization indices during week 1 of treatment, while at the end of the next week, C, H, CH2, and CH3 groups had the highest indices (Figure 2D).
Wound surface area reduction
Figure 3 depicts the representative serial digital images of the top view of full-thickness wounds at dorsal areas of the rats, treated with different treatments at various time points. Wound size changes during the 2-week period after induction of full-thickness skin excisions are shown in Figure 4. The graph is presented dimensionless due to the small variation in initial wound size induced by occasional subcutaneous muscle contractions. The C group showed a significantly faster wound size reduction among all groups. The H, CH3, and CH2 groups were placed second to C group with no significant difference among themselves and with significantly faster wound shrinkage compared with S, SSD, and CH1 groups after day 3.
Discussion
While antimicrobial and wound healing effects of honey and chitosan had already been reported in previous studies, this current research aimed to evaluate the efficacy of the chitosan/honey compounds. Although all 3 chitosan/honey formulations showed antibacterial and antifungal effects, CH3 formulation consisting 75% honey and 25% chitosan had the lowest MIC among all 3 studied formulations. All 4 histopathologic indices were higher among H, CH3, and CH2 groups by the end study week 2.
The antibiotic effects of honey have been reported to vary in the spectrum and potency and depend on the source and type of honey.1,8,10,41-45 Different MICs against gram-negative, gram-positive, and fungal strains have been reported with even New Zealand Manuka or Malaysian Tualang honeys,1,43,44,46 while raw and processed forms of honey have shown to be equally effective.41
The honey from the Dasht-e-Moghan area, derived from Z tenuior L and T vulgaris plantations, appeared to be superior to almost all other reported types of honey for its bacteriostatic effect on S aureus, B cereus, and E coli. Dasht-e-Moghan honey was more potent against P aeruginosa compared with many other known types of honey and still effective on C albicans. Mixing the honey with chitosan hydrogel (CH3: 25% chitosan + 75% honey) resulted in a 25% boost in the inhibitory effect against all 4 bacterial strains, and more than 80% increase in the efficacy against C albicans (Figure 51,10,41-44). It is worthy to mention that microbial inhibition in vitro does not always reflect what happens in real wounds where the materials are continually diluted with fresh exudate.
Chitosan has shown to improve wound healing by shortening the inflammatory phase through accelerating the migration of macrophages and other active cells during the inflammatory reaction.16 Honey also has demonstrated the ability to suppress both chronic and acute inflammation47 by decreasing the inflammatory cells, stimulating T and B lymphocytes, and activating the phagocytes in the bloodstream.48 Equivalent inflammatory indices during week 1 of treatment with all 3 chitosan/honey compounds with no significant difference with chitosan or honey alone is indicative of no cumulative or synergistic effect on inflammation in mixture form. However, by the end of treatment week 2, H, CH3, CH2, and SSD groups were at chronic to mild stage (grade 4-5), showing a faster relief from inflammation.
Marginal migration and proliferation of fibroblasts followed by extracellular matrix formation are key factors in granular tissue formation.49 Stimulation of interleukin 8 and type III collagen production, and hence, fibroblast cell proliferation and fibrotic tissue formation, have been attributed to chitosan.16 However, the H, CH3, and CH2 groups were found to be significantly ahead in fibrotic tissue formation compared with both control groups, pointing out the predominance of honey over chitosan.
Completion of granulation tissue regression process in the H, CH3, and CH2 groups by day 14 is a sign of accelerated healing as honey can improve wound oxygenation through induction of angiogenesis and Bohr effect.48 Higher reepithelialization index in the C, H, CH3, and CH2 groups confirmed the results of former studies with honey48,50 and chitosan.32,51 Although the C group showed faster shrinkage in wound size, which was in line with previous studies,32,52 no significant difference in wound size was seen between the C, H, CH3, and CH2 groups at day 14. Although El-Kased et al53 used a different formulation for the chitosan-based hydrogel in a recent study, their findings on the efficacy of honey (75%)/chitosan-based hydrogel on wound healing and tissue regeneration and repair is in line with the present results.
Limitations
Rodents are believed to have faster wound contraction capabilities, and some other animals (eg, pig) might be more suitable for wound healing studies due to their higher skin similarity to human. Because of traditional and religious considerations regarding handling pig and porcine samples as well as cost considerations and financial limitation,the rat was selected as the experimental animal model in this study. For future studies, using guinea pigs instead of rats must be considered as guinea pig skin and hair growth patterns resemble human skin even more closely than pig skin does.
As a second limitation, quantification of wound healing has not yet been defined and described in previous studies and hence, the authors have introduced a new histopathological scoring system for evaluation of wound healing, which might need further improvements in future works.
Conclusions
Even though chitosan and honey have been known as antimicrobial agents, these results showed certain formulations of chitosan/honey compounds can augment the effect insofar, and even some insensitive germs can be affected. In addition, enhanced wound healing and shrinkage is a result of chitosan/honey compounds.
All in all, CH3 (chitosan hydrogel/honey [1:3 v/v]) is a potential wound dressing formulation with ample antimicrobial and healing properties. Further complementary tests with other possible formulations are suggested for future works to find the most effective compound.
Acknowledgments
Authors: Jebrail Movaffagh, PhD1; Bibi Seddigheh Fazly Bazzaz, PhD1; Abbas Tabatabaei Yazdi, PhD2; Abolghasem Sajadi-Tabassi, PhD1; Mohammad Azizzadeh, PhD3; Esmaeel Najafi, PharmD1; Nafise Amiri, PhD1; Hamidreza Bahrami Taghanaki, MD4; Mohammad Hossein Ebrahimzadeh, MD5; and Ali Moradi, PhD5,6
Affiliations: 1Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran; 2Department of Pathology, Qaem Hospital, Mashhad University of Medical Sciences; 3Department of Clinical Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran; 4Department of Chinese and Complementary Medicine, Faculty of Persian and Complementary Medicine, Mashhad University of Medical Sciences; 5Orthopedic Research Center, Mashhad University of Medical Sciences; and ⁶Clinical Research Unit, Qaem Hospital, Faculty of Medicine, Mashhad University of Medical Sciences
Correspondence: Ali Moradi, PhD, Assistant Professor, Mashhad University of Medical Sciences, Orthopedic Research Center, Bone and Joint Research Lab, Library Building, Ghaem Hospital, Ahmad-Abad Street, Shariati Square, 9176699199 Mashhad, Khorasan Razavi, Iran; ralimoradi@gmail.com
Disclosure: This work was financially supported the Medicinal Plants Pharmacological Research Center, Mashhad University of Medical Sciences (Mashhad, Iran). The authors disclose no financial or other conflicts of interest.