INTRODUCTION
Skin plays a significant role in normal barrier homeostasis and preventing the invasion of microorganisms.1 Skin lesions are traumatic events that lead to an increase of infections, fluid loss, hypothermia, locally immunocompromised regions, scarring, and a change of body image.2,3 Among these, the growing incidence of infections remains the leading problem in wound management.1,4 The accumulation of fluid at wound site is one of the major contributions to the bacterial invasion. Wound infections not only elicit a systemic infection (sepsis) but also inhibit the multiple processes involved in the orchestrated progression of normal wound healing. Each process involved in wound healing is affected when bacteria proliferate in a wound.5,6
For protection, wounds generally need to be covered with dressings immediately.7 Effective wound dressings require not only protection of the wound from environmental irritants but also effective removal of excessive wound exudate, thus providing an optimal microenvironment to allow continuous tissue reconstruction processes.7 In the past decades, many skin substitutes such as biologic, synthetic, and biologic–synthetic dressings have been used for the treatment of deep partial- and full-thickness wounds, but none of them is without disadvantages.3,8 The prime requirement of an ideal dressing is that it should have the following properties: antibacterial, biocompatible, exudate absorptive, easy to apply, and promote wound healing.9–11 These properties are primarily attributed to the functional characteristic of wound healing constituents of the dressing.
Current wound care strategies focus on the improvement of wound repair by developing better materials for the wound dressings. Different polymers, when blended together, usually achieve the functions of their constituting components in a synergistic way.11,12 Blending of two or more polymers has become an increasingly important approach to developing new materials with new properties which could not be achieved by the individual component polymer. Chitin’s derivative, chitosan, is an attractive biomaterial for wound care owing to its antibacterial, wound healing promoting, nontoxic, biocompatible, and biodegradable properties.7,13 Chitosan has been widely used to develop dressings for wound care applications.11,14 Vaseline (petroleum jelly), known as a lubricant, can also be used as a moisture insulator for local skin conditions characterized by tissue dehydration. Vaseline gauze (VG), which is widely used in clinics, possesses the moisture-retention ability and can reduce the pain and prevent synechia formation.15,16 However, the disadvantage of VG is obvious by lacking the antibacterial activity, and thus limiting its clinical application.
In view of these, we aimed to create a dressing with antibacterial and wound-healing properties. Our previous work had proved that chitosan mixture could give full play to the performance of chitosan, which had obtained the China National Patent (patent number: ZL 2011100512896, date of authorization announcement: November 28, 2012). In this study, chitosan mixture was blended with Vaseline to prepare a chitosan–Vaseline gauze (CVG) dressing. The miscibility of the blend system and functional group interaction were investigated by electron microscopy and infrared (IR) spectroscopy. Characteristics of the dressings, including the surface morphology, cytotoxicity, antibacterial activity, and water retention properties, were evaluated. The wound-healing properties of dressings were also investigated in murine models through macroscopic and histological analyses.
MATERIALS AND METHODS
Materials.
High molecular weight (MW ∼ 800,000) and low (MW ∼ 2000) molecular weight chitosan (deacetylation degree of 95%) were obtained from Golden-Shell Pharmaceutical Co (Zhejiang, China). Mixed chitosan solution was made in our own laboratory. Sterile gauzes were obtained from Hainuo Medical Technologies Co (Qingdao, China). All other materials were obtained from Gibco Co (Thermo Fisher Scientific, Waltham, MA) unless otherwise noted.
Fabrication of wound dressings.
A 3.0% mixed chitosan solution was prepared by dissolving chitosan powder (MW ∼ 2000 and ∼ 800,000, 1:1, w/w) in 1.0% glacial acetic acid solution (v/v), followed by vigorous stirring overnight to achieve dissolution and then filtered to sterilize. To fabricate chitosan gauze (CG), sterile gauze was soaked in chitosan solution for 24 hours and dried at 60–65°C for 1 hour to evaporate the excess water. To fabricate VG and CVG, the medical Vaseline and paraffin oil were mixed in advance (2:1, w/w) and sterilized by autoclaving. Then dried sterile gauze or CG was soaked in mixed vaseline–paraffin oil for 5 minutes.
Scanning electron microscopy (SEM).
The surface characteristics of these dressings were investigated by using a scanning electron microscope (SEM, S-3000N; Hitachi, Tokyo, Japan). Thick layers of gold were used to provide the conduction for the dressings.
Infrared spectroscopy.
The IR spectra of membranes were recorded on a Perkin–Elmer IR System Spectrum GX in the range of 4,000–400 cm−1 with a resolution of 4 cm−1 and averaged over 32 scans.
Cytotoxicity.
National Institutes of Health (NIH) 3T3 mouse embryonic fibroblast cells were purchased from the cell bank of the Chinese Academy of Science. Approximately 10 × 103 cells were dispensed into each well of 96-well plates. Different kinds of dressings (CVG, VG, and untreated gauze) were then cut into designated sizes (1.0 × 1.0 cm2) and soaked in 10 mL of cell culture medium at 37°C for 24 hours to form the experimental extracts of chitosan and Vaseline. The cells were treated with different concentrations (10, 50, and 100%) of the extract of each dressing and incubated in the presence of 5% CO2 with 95% humidity at 37°C for 24 hours. Cells cultured in 100% cell culture medium without the study dressing (extracts of untreated gauze) were used as negative control group. Cell adhesion and proliferation was visualized using an inverted light microscope (CKX41SF, OLMPUS). Cell activity of NIH3T3 cells was measured after 1.5 hours with a Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan).
The cytotoxicity was evaluated by the relative growth rate (RGR), which was calculated according to the following formula: RGR = the value of the experimental group/the value of the blank control group × 100%. The relationship between RGR and cytotoxicity grade was classified by the GB/T 16175–2008 (the national standard of China about biological evaluation test methods for medical organic silicon materials). If RGR is greater than 100%, the toxicity level is 0. If RGR is between 75% and 99%, the toxicity level is 1. For RGR between 50% and 74%, the toxicity level is 2; level 3 for RGR between 25% and 49%, and level 4 for RGR between 1% and 24%. If RGR is 0, the toxicity level is 5. In the national standard, cytotoxicity evaluation can be considered to meet the criteria of no cytotoxicity at level 0 or 1.
Antibacterial studies.
Antibacterial properties of the dressings were evaluated by using the American Association of Textile Chemists and Colorists test method, with some modifications.17 Briefly, our methods were as follows: nutrient agar, Staphylococcus aureus, and Escherichia coli were supplied by the Clinical Laboratory Department, the First Affiliated Hospital, College of Medicine, Zhejiang University. Nutrient agar plates were prepared by dissolving 28 g of nutrient agar in 1 L distilled water, and the pH was adjusted to 7. The contents were sterilized by autoclaving at 15 lbs pressure (∼120°C) for 20 minutes. The medium when still hot, was poured in sterile plates (approximately 20 mL) allowed to cool so that the agar solidified. The composition of nutrient agar medium was 5 g/L of peptic digest of animal tissue, 5 g/L of sodium chloride, 1.5 g/L of beef extract, 1.5 g/L of yeast extract, and 15 g/L of agar.
Sterile gauzes soaked in penicillin (PE, 80 U in 10 mL of normal saline) and streptomycin (ST, 100 U in 10 mL of normal saline) were used as positive controls, whereas sterile gauzes soaked in 1% acetic acid and chitosan solution were used as negative and blank controls. Bacteria (0.5 MacFarland Standard) were streaked onto the agar surface with a swab. The plates were then divided into five equal sections, labeled for S. aureus and E. coli. Samples of different types of dressings were then placed at the center of each section and pressed lightly onto the agar. Then the plates were incubated in the inverted position at 37°C for 24 hours.18 After that, the zone of inhibition around each dressing was measured.
Animals.
Adult male Sprague–Dawley (SD) rats (weighing approximately 200–250 g, 8–14 weeks old) were provided by the Laboratory Animal Research Center of the First Affiliated Hospital of Zhejiang University, Hangzhou, China. The animal protocols were approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University, and were performed according to the eighth edition of the Guide for the Care and Use of Laboratory Animals (NIH Publications, revised 2011).
Water retention measurement.
Wound model.
A total of 16 male SD rats were used in this experiment, and they were randomly grouped by the random number table (N = 8 for each group). The skin from the back of each rat was prepared by shaving the desired area using electric clipper. After washing the area by 70% ethyl alcohol, two full-thickness round wounds of 2.0 cm diameter down to the panniculus carnosus were created on the skin at sites parallel and equidistant from the vertebral column using a surgical scissor and scalpel. The dimension of the wound site was measured by the digital caliper. Then the wounds of rats were covered with a 2.5 × 2.5-cm piece of CVG or VG. In half of the rats (group A), CVG was applied on the left side and VG on the right side, and in the other half (group B), the dressings were switched. The dressings were changed every 3 days until the wounds healed. The endpoint of healing was set at 100% re-epithelialization. On postoperative days 3, 7, and 14, unhealed wound dimensions were measured blindly, and wound tissues were sampled to allow pathological evaluation.
Gross observation of healing wounds.
On the 3rd, 7th, and 14th post-wounding days, gross observation was performed in each rat. After carefully removing the dressing, images were captured by a digital camera (Nikon D610, Japan) to record and analyze the healing and size changes of the wounds. Then the wound outline was first copied to a transparent sheet. After that, a grid was used to count the number of squares and calculate the sizes of the wound sites.
Histology.
A strip of tissue containing the wound center and normal skin was obtained. Tissue specimens at different time points were embedded in paraffin wax, sectioned at a thickness of 5 µm, and stained by hematoxylin–eosin (H&E) and Masson’s trichrome to visualize the neotissue formation, collagen deposition, and amount of neovasculature.
Immunohistochemical staining (IHC).
To investigate the formation of neovessels during the process of wound healing, IHC of CD31 was performed. The positive areas were observed under a microscope (DM2500, Leica) at a fixed magnification (×100) to identify the area with the highest vascular density. Then, five non-overlapping fields of fixed magnification (×400) were chosen randomly and images were saved. The relative area of stained neovessels in each image was measured and recorded automatically by the “Count/Size” tool of Image-Pro Plus 6.0.19,20
Statistical analysis.
SPSS version 21.0 statistical package (IBM Corp., Chicago, IL) was used for data analysis. Normality and homogeneity of the variables were checked first. Comparisons of means of several groups were performed using one-way Analysis of Variance (ANOVA). Comparisons of the differences between two groups were analyzed using Student’s t-test. A P-value < 0.05 indicated a significant difference between the two groups.
RESULTS AND DISCUSSION
The aim of our study was to develop chitosan and Vaseline–coated sterile gauze with improved properties for wound dressing application. Sterile gauze is used as a support layer for the chitosan–Vaseline mixture. The earlier studies showed that high molecular weight chitosan exerts more potent antibacterial activity against Gram-positive S. aureus, whereas low molecular weight chitosan exerts better antibacterial activity against Gram-negative E. coli as well as moisture retention.21,22 Thus, we used chitosan mixture as the basic material, blended with Vaseline. The effects of adding Vaseline to chitosan mixture, bioactive performance, and tissue compatibility of the dressing were investigated by various tests in this study.
Morphology of dressings.
As illustrated in Figure 1, surface morphologies of different dressings were evaluated using SEM micrographs. In the high-magnification image of CG, reticulation surrounding the sterile gauze was obviously visible compared with the sterile gauze. However, reticulation in CVG could not be clearly observed. Compared with VG, the structure of CVG showed an uneven morphology. So further, we used IR spectroscopy to investigate the miscibility of these dressings and functional group interaction in them.
Scanning electron microscopy images of dressings: (A) chitosan gauze, (B) sterile gauze, (C) chitosan–Vaseline gauze, and (D) Vaseline gauze.
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
The representative bands of chitosan were evident in IR spectroscopy, denoted by arrows, with reference to their pristine materials23 (Figure 2A). Compared with the sterile gauze, a peak at 2,871 cm−1 originating from the vibration of C–H in chitosan was detected in CG (Figure 2B), indicating the successful incorporation of chitosan into sterile gauze. Simultaneously, split peaks between 2,750 and 3,000 cm−1 were observed in VG. Compared with VG, the split peaks in CVG were much stronger. It could be inferred that chitosan and Vaseline co-contributed to the stronger split peaks in the CVG.
Infrared spectrum of (A) high and low molecular weight chitosan powders (B) samples of chitosan–Vaseline gauze, Vaseline gauze, chitosan gauze, and sterile gauze.
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
Cytotoxicity.
To investigate the applicability of our new dressings for biomedical applications, the in vitro studies were first performed to evaluate the cytocompatibility of these dressing extracts. NIH3T3 cells at the same initial density were seeded in the 96-well plates and added with different dressing extracts. After 24 hours, cell adhesion and proliferation were visualized using an inverted light microscope. It could be observed in all groups that fibroblast cells adhered to and grew in the vicinity of the dressings. Furthermore, the cell activity was measured by CCK-8 and all the RGR were calculated (Table 1, Supplemental Figure 1). The toxicity grades of all the experimental groups were at level 0 or 1, which can be considered to meet the criteria of non-cytotoxicity. These results obviously suggested that our new dressing extracts did not affect cell viability and this combination would not have an acute cytotoxic effect.
Cytotoxity detection in various concentrations of extracts (N = 6)
| Groups | Extract concentration (%) | 450 nm absorbance value (× ± s) | Relative growth rate(%) | Cytotoxicity grade |
|---|---|---|---|---|
| Chitosan–Vaseline gauze | 100 | 1.52 ± 0.20 | 96 | 1 |
| 50 | 1.60 ± 0.37 | 101 | 0 | |
| 10 | 1.78 ± 0.36 | 112 | 0 | |
| Vaseline gauze | 100 | 1.38 ± 0.15 | 87 | 1 |
| 50 | 1.40 ± 0.13 | 89 | 1 | |
| 10 | 1.53 ± 0.13 | 97 | 1 | |
| Untreated gauze | – | 1.58 ± 0.11 | 100 | 0 |
Antibacterial activity (inhibition zone test).
The dressings were placed in the plates which were inoculated by E. coli or S. aureus for 24 hours. Then, a distinct zone of inhibition was observed, which can show the antibacterial activities of the dressings. No zone of inhibition was observed around VG (control) for both E. coli and S. aureus. The acetic acid dressing (control) were also completely surrounded by bacterial colonies (Figure 3). However, CG, CVG, and the positive control showed obvious surrounding inhibition zones.
Zone of inhibition studies of dressings against Escherichia coli (left) and Staphylococcus aureus (right). Ace = acetic acid gauze; Chi = chitosan dressing; CVG = chitosan–Vaseline gauze; PG = penicillin gauze; SG = streptomycin gauze; VG = Vaseline gauze.
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
We consider that chitosan, which is known for its antibacterial nature, plays an important role in this test as it is one of the constituents of CG and CVG dressings. We did not see any bacterial growth on the surface of those dressings, suggesting that they exercised the bactericidal effects. These results of the antibacterial assay clearly indicated our new dressings’ antibacterial activities.
Water retention measurement.
It is well known that keeping wounds moist and clean has a beneficial effect on the repair of wound sites. Dressings should keep the wound moist to favor healing and ensure solubilization of antibacterial agents.24 Thus, we performed the water retention assay by applying dressings on the rat back skin and measuring the water content rate by a Corneometer. The results showed that all the moisture values of rat skin increased after applying the dressings compared with those at 0 hours time point (Figure 4). The increase in moisture in the CVG group was much faster and significantly higher than other groups (P < 0.05) at time points 3, 6, 12, and 24 hours. All the findings suggested that our new dressings had good moisture retention properties.
Increased moisture rate at different time points after the application of dressings. The increased moisture of chitosan–Vaseline gauze was statistically higher than other groups at 3, 6, 12, 24, and 48 hours (ANOVA, P < 0.05).
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
Gross observation of healing wounds.
The evaluation of wound healing was performed in rat transcutaneous full-thickness dermal wounds. Gross observation was used to directly compare the healing patterns in this study and the easiness of removing dressings from the wounds was also evaluated when changing the dressings. All 16 animals achieved 100% re-epithelialization by the end of the study. No infection or allergic reactions occurred in this study. Figure 5 shows a series changes of wound beds after the application of different dressings. The healing patterns (Figure 6, 12 wounds of six rats in each group) showed that topical application of CVG facilitated wound closure significantly (t-test, P < 0.05) from day 3 to day 14, comparing with the control group. Besides, the time for 100% re-epithelialization was significantly shorter in CVG groups than in the VG groups (14.83 ± 0.75 days versus 18.50 ± 0.55 days, t-test, P = 0.00)
Typical photographs of macroscopic appearance of wound repair covered with Vaseline gauze (left) and chitosan–Vaseline gauze (right) at different healing times.
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
Quantitative analysis in gross observation for healing wounds. * indicates a statistically significant difference, P < 0.05 (t-test).
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
The freshly excised wound surface was easy to be adhered by the wound dressings, and the dressings also have the ability to absorb the exudate from the wound surface.3,25 When we evaluated the easiness of removing dressings, we found it was hard to perform with quantitative analysis, as it was subjective. Thus, we just described qualitatively here. We found our new dressings were easy to remove from the wounds.
Hematoxylin–eosin and Masson’s trichrome staining.
At different time points, the tissue specimens treated with different dressings were harvested for histological analysis. During necropsy, there was no microorganism observed in skin lesions of the rats, and no pathological abnormalities were observed in their brains, lung, heart, liver, kidney, and spleen. The results of H&E and Masson’s staining are shown in Figures 7 and 8, respectively.
Hematoxylin–eosin (H&E) staining of wound tissues of chitosan–Vaseline gauze and Vaseline gauze group for 3, 7, and 14 days after operation. Red arrows indicate blood vessels.
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
Masson’s trichrome staining of wound tissues of chitosan–Vaseline gauze and Vaseline gauze group on postoperative days 3, 7, and 14. The blue areas indicate the sites of collagen deposition.
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
On the 3rd postoperative day, abundant inflammatory cells, capillary buds, and neovessels were observed in both the groups and the proliferation of fibroblasts was also obvious. On the 7th postoperative day, there were still visible inflammatory cells in CVG group, but relatively sparse. We observed an increase in new capillaries and fibroblasts in both groups. The VG group showed presence of angiogenesis and proliferation of fibroblasts to a small degree. On the 14th postoperative day, we observed that there were increased neovessels and fibroblast proliferation in the VG group, whereas there were decreased neovessels and fibroblast proliferation in the CVG group. In addition, re-epithelialization was found in the boundary area around the wounds.
In both groups, collagen deposition was distributed in the granulation tissue and became more abundant with prolongation of the time after surgery (Figure 7 and 8). The morphology of collagen was cotton-like, especially in the early stages (days 3 and 7), whereas on day 14, the arrangement of collagen was relatively thick, irregular, and twisted into spirals in the VG group and this was accompanied by a large number of neovessels. Meanwhile, in the CVG group, the newly formed collagen aggregations appeared as streaks or masses in the wounds on Days 3 and 7. By Day 14, collagen fibers in the CVG groups were thin and arranged in an orderly fashion. The morphology and distribution of new collagen fibers in the CVG group approximated those in the normal dermis.
Immunohistochemical staining.
The number of endothelial cells is one of the most significant part of the formation of neovessels during the process of wound healing, which could be measured by IHC of CD31.26 Thus, we performed IHC in these groups. Endothelial cells appeared brown by CD31 IHC staining (Figure 9), as seen by light microscopy. Figure 10 showed neovessel density as measured using image software (relative area), where the CVG group showed a declining trend, whereas the VG group showed a rising trend in a time-dependent manner. The vascular density in the CVG group was much higher than that in the VG group on Day 3. By Day 7, the number of neovessels in the CVG group declined, whereas it rose in the VG group. On Day 14, the blood vessels were abundantly distributed throughout the granulation tissue in the VG group compared with the CVG group (P < 0.05). The results of CD31 IHC were in agreement with those shown in H&E and Masson’s trichrome stainings.
CD31 immunohistochemical staining of tissues slices of the wounds treated by chitosan–Vaseline gauze and Vaseline gauze on postoperative days 3, 7, and 14. Black arrows indicate blood vessels.
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
Density of blood vessel measurement in the tissues slices of the wounds treated by chitosan–Vaseline gauze and Vaseline gauze for 3, 7, and 14 days after operation. * Indicates statistically significant difference, P < 0.05 (t-test).
Citation: The American Journal of Tropical Medicine and Hygiene 102, 2; 10.4269/ajtmh.19-0387
In rat model, the smaller wound beds and shorter 95% re-epithelialization time in the CVG groups demonstrated that the CVG dressing significantly promoted wound healing. During the healing period, histological evidence and the result of IHC staining revealed that in the CVG group, collagen fibers were thin and arranged in an orderly fashion, and the number of neovessels markedly decreased. A plausible explanation is as follows: after the wound dressings were applied to the wounds, the loaded constituents (chitosan and Vaseline) then gradually released. They might provide a nonprotein matrix for tissue growth, activate macrophages for antibacterial activity, and stimulate cell proliferation and histoarchitectural tissue organization. Furthermore, chitosan gradually depolymerized to release N-acetyl-β-D-glucosamine, which could initiate fibroblast proliferation, stimulate natural hyaluronic acid synthesis, and help in ordered collagen deposition at the wound site.7 Vaseline might act as a moisture insulator for local wound conditions. Chitin-based dressings can accelerate repair of different tissues by facilitating the contraction of wounds as well as regulating the secretion of some inflammatory mediators such as prostaglandin E, interleukin 1, and interleukin 8.7,27 We hypothesized that chitosan and Vaseline also accelerated the remodeling and expansion of the vascular network as the expression levels of blood vessels were significantly influenced by applying the CVG. The faster and functional angiogenesis in the CVG group improved the delivery of nutrients and oxygen to the cells at the wound sites. On the 14th day, the number of neovessels was much less in the CVG group possibly because the newly formed blood vessels become more mature. Thus, all the aforementioned factors together contributed to faster wound healing.
CONCLUSION
The observations of the study suggest that chitosan–Vaseline dressing is effective in animal models of wound healing and has the potential as a wound care material for future development. Considering the longer healing period of chronic wounds, patients should be provided the correct dressing, which is curative and cost-effective.28 It is also noteworthy that chronic wounds are dynamic in presentation, and dressings that can optimize each stage in the healing process should be further evaluated. Furthermore, studies will be required to focus on delineating the mechanisms promoting the wound healing and evaluating the effectiveness of the dressings in preventing hypertrophic scars or other skin conditions.
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